Self-amplifying RNA compositions and methods of use thereof

Abstract

The present disclosure specifically includes RNA polynucleotide compositions that (A) contain modified nucleosides and / or (B) are purified using a chromatography system and / or an affinity-based separation system. Methods of manufacture and treatment related to said compositions are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,166, filed November 8, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was made at ###, is named ### and is ### bytes in size. [Background technology]

[0003] background Messenger RNA (mRNA), which encodes physiologically important proteins for therapeutic applications, has significant advantages over DNA-based plasmid and viral vectors for delivering genetic material. However, mRNA-based therapies may also have potential drawbacks, such as off-target stimulation of the innate immune response that may hinder therapeutic efficacy.

[0004] Thus, there is a need in the industry for improved mRNA therapeutics and manufacturing methods. Summary of the Invention

[0005] overview Provided herein is a composition for delivering a self-amplifying expression system comprising a single-stranded RNA (ssRNA) vector, the ssRNA vector comprising 7-methylguanylate (mGUA), 7 The compositions include a polyadenylated (polyA) tail, a self-amplifying backbone, and one or more modified nucleosides, wherein the one or more modified nucleosides include m5C and are free of other modified nucleotides, optionally other than an m7G cap analog.

[0006] Provided herein is a composition for delivering a self-amplifying expression system comprising a single-stranded RNA (ssRNA) vector, the ssRNA vector comprising 7-methylguanylate (mGUA), 7 Also provided are compositions comprising a nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of ...

[0007] In some embodiments, the self-amplifying backbone comprises a polynucleotide selected from a self-replicating RNA virus. In some embodiments, the self-replicating RNA virus is selected from the group including alphavirus, flavivirus, measles virus, and rhabdovirus.

[0008] In some embodiments, the one or more modified nucleosides include methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof. In some embodiments, the one or more modified nucleosides include m5C. In some embodiments, the one or more modified nucleosides include m5C and are compatible with other modified nucleotides, optionally m 7 Contains no modified nucleotides other than the G-cap analog.

[0009] In some embodiments, the ssRNA vector is purified. In some embodiments, the ssRNA vector is purified by chromatography. In some embodiments, the chromatography comprises a cellulose chromatography system or an affinity-based separation system. In some embodiments, the affinity-based separation system is a deoxythymidine (dT) oligonucleotide (oligo(dT)) system.

[0010] In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the adenine, guanine, cytidine, and / or uridine nucleosides of the ssRNA vector are modified nucleosides. In some embodiments, 25% of the adenosine, guanosine, cytidine, and / or uridine nucleosides of the ssRNA vector comprise modified nucleosides. In some embodiments, 100% of the adenine, guanine, cytidine, and / or uridine nucleosides of the ssRNA vector comprise modified nucleosides. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the cytidine nucleosides contained in the ssRNA vector are modified nucleosides.In some embodiments, more than 25% of the cytidine nucleosides of the ssRNA vector are modified nucleosides.In some embodiments, 100% of the cytidine nucleosides contained in the ssRNA are modified nucleosides.

[0011] In some embodiments, the composition comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of contaminants. In some embodiments, the contaminants comprise salts, detergents, and / or double-stranded RNA (dsRNA). In some embodiments, the contaminants comprise dsRNA.

[0012] In some embodiments, ssRNA comprises 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the total RNA present in the composition. In some embodiments, ssRNA comprises 97% or more, 98% or more, 99% or more, or 99.5% or more of the total RNA present in the composition. In some embodiments, the ssRNA vector is the only RNA species present in the composition. In some embodiments, the total RNA, dsRNA, and / or ssRNA content is assessed by dot blot or ELISA, and optionally, the dot blot or ELISA assessment includes detection with an antibody specific for RNA, dsRNA, and / or ssRNA. In some embodiments, the total RNA, dsRNA, and / or ssRNA content is assessed by capillary electrophoresis. In some embodiments, the total RNA, dsRNA, and / or ssRNA content is assessed by liquid chromatography. In some embodiments, the total RNA, dsRNA, and / or ssRNA content is quantified as the area under the curve (AUC).

[0013] In some embodiments, the ssRNA vector is generated by in vitro transcription. 7 G cap is m 7 In some embodiments, m 7 G-cap analogues are trinucleotides 7 G-ppp-AU cap analog or dinucleotide m 7 Contains the G-ppp-A cap analog.

[0014] In some embodiments, the ssRNA vector comprises a cassette comprising at least one nucleic acid sequence to be delivered, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the cassette is operably linked to or operably inserted into a self-amplifying backbone.

[0015] In some embodiments, the self-amplifying backbone has the following formula in the 5' to 3' direction: m 7G-ppp-N1-N2-N V and a polynucleotide selected from a self-replicating RNA virus comprising at least one nucleic acid sequence represented by During the ceremony, m 7 G is 7-methylguanylate (m 7 G) Cap; ppp is a triphosphate bridge, N1 is the first nucleotide of a self-amplifying backbone corresponding to the first endogenous 5' nucleotide of the self-replicating RNA virus; N2 is the second nucleotide of the self-amplifying backbone that corresponds to the second endogenous 5′ nucleotide of the self-replicating RNA virus; and N V comprises (1) one or more additional nucleic acid sequences of a self-amplifying backbone; and (2) a cassette comprising at least one nucleic acid sequence for delivery, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the cassette is operably linked to or operably inserted into the self-amplifying backbone.

[0016] In some embodiments, the composition further comprises a nanoparticle delivery vehicle. In some embodiments, the nanoparticle delivery vehicle is a lipid nanoparticle (LNP).

[0017] Also provided herein is a pharmaceutical composition comprising any one of the compositions described herein and a pharma- ceutically acceptable carrier.

[0018] Also provided herein is a method for treating a subject having a disease, comprising administering to the subject an amount of any one of the compositions described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the disease is cancer or an infectious disease. In some embodiments, the infectious disease is caused by a virus selected from the group including HPV, influenza, TB, CMV, HMPV, PIV, Chikungunya virus, Zika virus, SARS-CoV-2, and pan-coronavirus.

[0019] In some embodiments, the amount of ssRNA in the composition is 1000 μg or less, 100 μg or less, 50 μg or less, 30 μg or less, 10 μg or less, 5 μg or less, or 1 μg or less.

[0020] In some embodiments, the natural immune response of the subject is reduced compared to a control subject that is administered the composition comprising the nucleic acid sequence that does not contain modified nucleosides.In some embodiments, the natural immune response is IFN response.In some embodiments, the IFN response is IRF-3 expression, IRF-7 expression, or a combination thereof.

[0021] In some embodiments, replication of the ssRNA vector is increased as compared to a control composition comprising an identical ssRNA vector except that it does not contain the modified nucleosides.

[0022] Also provided herein is a method for purifying a self-amplifying expression system from a nucleic acid mixture, the method comprising purifying the self-amplifying expression system by a cellulose chromatography system or an affinity-based separation system, the self-amplifying expression system comprising a single-stranded RNA (ssRNA) vector, and the ssRNA vector is a nucleic acid mixture comprising a nucleic acid sequence that is ... 7 Also provided are methods comprising a G-cap, a polyA tail, and a self-amplifying backbone.

[0023] Also provided herein is a method for reducing double-stranded RNA (dsRNA) in a nucleic acid mixture, comprising: The method further comprising: (A) purifying the self-amplifying expression system by a cellulose chromatography system or an affinity-based separation system, and / or (B) generating a self-amplifying expression system, such that the self-amplifying expression system comprises a modified nucleoside. Including, the self-amplifying expression system comprises a single-stranded RNA (ssRNA) vector, the ssRNA vector comprising an m7G cap, a polyA tail, and a self-amplifying backbone; A method as described above is also provided.

[0024] In some embodiments, the affinity-based separation system comprises a deoxythymidine (dT) oligonucleotide (oligo(dT)) system. In some embodiments, the ssRNA vector comprises a modified nucleoside. In some embodiments, the modified nucleoside comprises methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof. In some embodiments, the modified nucleoside consists of methyl-5-cytosine (m5C).

[0025] Also provided herein is a composition mixture, the composition comprising a self-amplifying expression system, the self-amplifying expression system comprising: (a)m 7 a single-stranded RNA (ssRNA) vector comprising a G-cap, a polyadenylated (polyA) tail, and a self-amplifying backbone; (b) double-stranded RNA (dsRNA) and and dsRNA comprises less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the total RNA present in the mixture; Mixtures of said compositions are also provided.

[0026] Also provided herein is a composition mixture, the composition comprising a self-amplifying expression system, the self-amplifying expression system comprising: (a)m7 a single-stranded RNA (ssRNA) vector comprising a G-cap, a polyadenylated (polyA) tail, and a self-amplifying backbone; (b) double-stranded RNA (dsRNA) and Including, the ssRNA vector constitutes 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more of the total RNA present in the mixture; Mixtures of said compositions are also provided.

[0027] In some embodiments, the only detectable RNA comprises ssRNA vector. In some embodiments, total RNA, dsRNA, and / or ssRNA content is assessed by dot blot or ELISA, and optionally, the dot blot or ELISA assessment includes detection with an antibody specific for RNA, dsRNA, and / or ssRNA. In some embodiments, total RNA, dsRNA, and / or ssRNA content is assessed by capillary electrophoresis. In some embodiments, total RNA, dsRNA, and / or ssRNA content is assessed by liquid chromatography. In some embodiments, total RNA, dsRNA, and / or ssRNA content is quantified as area under the curve (AUC).

[0028] In some embodiments, the ssRNA is generated by in vitro transcription. 7 G cap is m 7 In some embodiments, m 7 G-cap analogues are trinucleotides 7 G-ppp-AU cap analog or dinucleotide m 7 Contains the G-ppp-A cap analog.

[0029] In some embodiments, the ssRNA vector comprises a cassette comprising at least one nucleic acid sequence to be delivered, optionally the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and the cassette is operably linked to or operably inserted into a self-amplifying backbone. In some embodiments, the self-amplifying backbone comprises the following structure in the 5' to 3' direction: m 7 G-ppp-N1-N2-N V and a polynucleotide selected from a self-replicating RNA virus comprising at least one nucleic acid sequence represented by During the ceremony, m 7 G is 7-methylguanylate (m 7 G) Cap; ppp is a triphosphate bridge, N1 is the first nucleotide of a self-amplifying backbone corresponding to the first endogenous 5' nucleotide of the self-replicating RNA virus; N2 is the second nucleotide of the self-amplifying backbone that corresponds to the second endogenous 5′ nucleotide of the self-replicating RNA virus; and N V comprises (1) one or more additional nucleic acid sequences of a self-amplifying backbone; and (2) a cassette comprising at least one nucleic acid sequence for delivery, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the cassette is operably linked to or operably inserted into the self-amplifying backbone.

[0030] In some embodiments, the ssRNA vector comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides comprise methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof. In some embodiments, the one or more modified nucleosides comprise m5C. In some embodiments, the one or more modified nucleosides comprise m5C and do not comprise other modified nucleotides, optionally other than a cap analog.

[0031] In some embodiments, the ssRNA vector is purified. In some embodiments, the ssRNA vector is purified by chromatography. In some embodiments, the chromatography comprises a cellulose chromatography system or an affinity-based separation system. In some embodiments, the affinity-based separation system is a deoxythymidine (dT) oligonucleotide (oligo(dT)) system.

[0032] In some embodiments, a composition for delivering a self-amplifying expression system comprises: (A) A self-amplifying expression system comprising one or more self-amplifying mRNA (SAM) vectors, the one or more SAM vectors comprising: (a) a self-amplifying backbone, the self-amplifying backbone comprising the nucleic acid sequence set forth in SEQ ID NO:6, the self-amplifying backbone sequence comprising a subgenomic promoter nucleotide sequence and a poly(A) sequence, the subgenomic promoter sequence being endogenous to the self-replicating RNA virus and the poly(A) sequence being endogenous to the self-replicating RNA virus backbone; (b) a cassette integrated between the subgenomic promoter nucleotide sequence and the poly(A) sequence, the cassette being operably linked to the subgenomic promoter nucleotide sequence, and optionally the cassette comprising at least one antigen-encoding nucleic acid sequence, the at least one antigen-encoding nucleic acid sequence comprising: a. an epitope-encoding nucleic acid sequence, optionally comprising: (1) at least one alteration that renders the encoded epitope sequence different from a corresponding peptide sequence encoded by a wild-type nucleic acid sequence; or (2) a nucleic acid sequence that encodes an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide; b. optionally a 5' linker sequence, and c. an optional 3' linker sequence The cassette comprising: The self-amplifying expression system, (B) Optionally, lipid nanoparticles (LNPs) encapsulating a self-amplifying expression system. Includes.

[0033] In some embodiments, the ordered sequence of each element of a cassette in a composition for delivering a self-amplifying expression system is, from 5' to 3', as follows: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g It is represented by a formula including During the ceremony, P comprises a second promoter nucleotide sequence, and a=0 or 1; N comprises one of the epitope-encoding nucleic acid sequences, the epitope-encoding nucleic acid sequence comprises an MHC class I epitope-encoding nucleic acid sequence, and c=1; L5 comprises a 5' linker sequence and b=0 or 1; L3 comprises a 3' linker sequence and d=0 or 1; G5 comprises at least one nucleic acid sequence encoding a GPGPG amino acid linker, and e=0 or 1; G3 comprises at least one nucleic acid sequence encoding a GPGPG amino acid linker, and g=0 or 1; U comprises one of at least one MHC class II epitope-encoding nucleic acid sequence, and f=1; X = 1 to 400, and for each X, the corresponding N c is an MHC class I epitope-encoding nucleic acid sequence, and Y=0, 1, or 2, and for each Y, the corresponding U f is an MHC class II epitope-encoding nucleic acid sequence.

[0034] In some embodiments, for each X, a corresponding N c are distinct MHC class I epitope-encoding nucleic acid sequences. In some embodiments, for each Y, a corresponding U f are different MHC class II epitope-encoding nucleic acid sequences.

[0035] In some embodiments, a=0, b=1, d=1, e=1, g=1, h=1, X=10, Y=2, the at least one promoter nucleotide sequence is a single subgenomic promoter nucleotide sequence provided by a self-amplifying backbone, the at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 80 consecutive A nucleotides provided by a self-amplifying backbone, the cassette is integrated between the subgenomic promoter nucleotide sequence and the poly(A) sequence, the cassette is operably linked to the subgenomic promoter nucleotide sequence and the poly(A) sequence, and each N is an MHC class I epitope of 7-15 amino acids in length. L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of an MHC I epitope, which 5' linker sequence encodes a peptide at least 3 amino acids in length, L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of an MHC I epitope, which 3' linker sequence encodes a peptide at least 3 amino acids in length, U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence, the self-amplifying backbone is the sequence shown in SEQ ID NO:6, and each of the MHC class I epitope-encoding nucleic acid sequences encodes a polypeptide 13 to 25 amino acids in length.

[0036] In some embodiments, the at least one nucleic acid sequence for delivery comprises a polypeptide-encoding nucleic acid sequence. In some embodiments, the polypeptide-encoding nucleic acid sequence encodes an antigen-encoding nucleic acid sequence. In some embodiments, the antigen-encoding nucleic acid sequence comprises an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B-cell response, or a combination thereof. In some embodiments, the antigen-encoding nucleic acid sequence comprises a full-length protein, a protein subunit, a protein domain, or a combination thereof. In some embodiments, the polypeptide-encoding nucleic acid sequence encodes a full-length protein or a functional portion thereof. In some embodiments, the full-length protein or a functional portion thereof is selected from the group consisting of an antibody, a cytokine, a chimeric antigen receptor (CAR), a T-cell receptor, and / or a genome editing system nuclease.

[0037] In some embodiments, the at least one nucleic acid sequence for delivery comprises at least one nucleic acid sequence that comprises a non-coding nucleic acid sequence. In some embodiments, the non-coding nucleic acid sequence is an RNA interference (RNAi) polynucleotide or a polynucleotide of a genome editing system.

[0038] In some embodiments, the LNP comprises a lipid selected from the group consisting of an ionizable amino lipid, a phosphatidylcholine, cholesterol, a PEG-based coat lipid, or a combination thereof. In some embodiments, the LNP comprises an ionizable amino lipid, a phosphatidylcholine, cholesterol, and a PEG-based coat lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the LNP encapsulated expression system has a diameter of 60-140 nm.

[0039] In some embodiments, the composition for delivering the self-amplifying expression system is formulated for intramuscular (IM), intradermal (ID), subcutaneous (SC), intravitreal (IVT), intrathecal, or intravenous (IV) administration. In some embodiments, the composition for delivering the self-amplifying expression system is formulated for intramuscular (IM) administration.

[0040] In some embodiments, the cassette is integrated between at least one promoter nucleotide sequence and at least one poly(A) sequence. In some embodiments, the at least one promoter nucleotide sequence is operably linked to the cassette.

[0041] In some embodiments, the ssRNA vector comprises a positive strand RNA vector. In some embodiments, the ssRNA vector comprises a negative strand RNA vector. In some embodiments, the negative strand RNA vector comprises at least one polynucleotide sequence of measles virus or rhabdovirus. In some embodiments, the ssRNA vector self-replicates in mammalian cells. In some embodiments, the self-replicating RNA virus is selected from the group consisting of alphavirus, flavivirus, measles, and rhabdovirus. In some embodiments, the self-amplifying backbone comprises at least one polynucleotide sequence of alphavirus, optionally the alphavirus is selected from the group consisting of aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the self-amplifying backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus. In some embodiments, the self-amplifying backbone comprises at least sequences for nonstructural protein-mediated amplification, a subgenomic promoter sequence, a poly(A) sequence, nonstructural protein 1 (nsP1) gene, nsP2 gene, nsP3 gene, and nsP4 gene encoded by an Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus nucleotide sequence. In some embodiments, the self-amplifying backbone comprises at least sequences for nonstructural protein-mediated amplification, a subgenomic promoter sequence, and a poly(A) sequence encoded by an Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus nucleotide sequence. In some embodiments, the sequence for nonstructural protein-mediated amplification is selected from the group consisting of an alphavirus 5' UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3' UTR, or a combination thereof.In some embodiments, the self-amplifying backbone does not encode the structural virion proteins capsid, E2, and E1, and optionally E1 is full-length E1 or does not encode the structural virion proteins capsid, E3, E2, 6K. In some embodiments, the cassette is inserted in place of a structural virion protein in the polynucleotide sequence of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus. In some embodiments, the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5 further comprising a deletion between base pairs 7544 and 11175. In some embodiments, the self-amplifying backbone comprises the sequence shown in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the cassette is inserted at position 7544 of SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, insertion of the cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and at least one nucleic acid sequence, wherein the nsP1-4 genes and the at least one nucleic acid sequence to be delivered are in separate open reading frames.

[0042] In some embodiments, the ssRNA vector comprises at least one promoter nucleotide sequence. In some embodiments, the at least one promoter nucleotide sequence comprises a natural promoter nucleotide sequence encoded by an autonomously replicating RNA virus, and optionally the natural promoter nucleotide sequence is a subgenomic promoter nucleotide sequence. In some embodiments, the at least one promoter nucleotide sequence is an exogenous RNA promoter. In some embodiments, the ssRNA vector comprises a second promoter sequence, and optionally the second promoter nucleotide sequence is a subgenomic promoter nucleotide sequence. In some embodiments, the second promoter nucleotide sequence comprises a plurality of subgenomic promoter nucleotide sequences, and each subgenomic promoter nucleotide sequence causes the transcription of one or more of the separate open reading frames.

[0043] In some embodiments, the ssRNA vector is at least 300nt in size. In some embodiments, the ssRNA vector is at least 1kb in size. In some embodiments, the ssRNA vector is at least 2kb in size. In some embodiments, the ssRNA vector is less than 5kb in size.

[0044] In some embodiments, at least one antigen-encoding nucleic acid sequence comprises two or more antigen-encoding nucleic acid sequences. In some embodiments, each antigen-encoding nucleic acid sequence within each antigen-encoding nucleic acid sequence is directly linked to each other. In some embodiments, each antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker. In some embodiments, the linker links two MHC class I epitope-encoding nucleic acid sequences or one MHC class I epitope-encoding nucleic acid sequence to one MHC class II epitope-encoding nucleic acid sequence. In some embodiments, the linker is selected from the group consisting of: (1) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive glycine residues in length; (2) a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by the mammalian proteasome; and (6) one or more naturally occurring sequences adjacent to an antigen derived from a protein of cognate origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. In some embodiments, the linker comprises one or more naturally occurring sequences flanking the antigen from a protein of cognate origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. In some embodiments, the linker connects two MHC class II epitope-encoding nucleic acid sequences or one MHC class II sequence with one MHC class I epitope-encoding nucleic acid sequence. In some embodiments, the linker comprises the sequence GPGPG.

[0045] In some embodiments, the antigen-encoding nucleic acid sequence is operably or directly linked to a separate or contiguous sequence that enhances expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the epitope-encoding nucleic acid sequence. In some embodiments, the separate or contiguous sequence comprises at least one of a ubiquitin sequence, a ubiquitin sequence modified to increase proteasomal targeting (e.g., the ubiquitin sequence contains a Gly to Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal associated membrane protein (LAMP)-1, a human dendritic cell lysosomal associated membrane protein, and a major histocompatibility class II sequence, optionally wherein the ubiquitin sequence modified to increase proteasomal targeting is A76.

[0046] In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences, optionally where each antigen-encoding nucleic acid sequence encodes a different antigen-encoding nucleic acid sequence. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences. In some embodiments, the at least one antigen-encoding nucleic acid sequence comprises at least 2-400 antigen-encoding nucleic acid sequences, and at least two of the antigen-encoding nucleic acid sequences encode epitope sequences or portions thereof presented by MHC class I on the cell surface. In some embodiments, each antigen-encoding nucleic acid sequence independently comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitope-encoding nucleic acid sequences, optionally where each epitope-encoding nucleic acid sequence encodes a different epitope-encoding nucleic acid sequence. In some embodiments, each antigen-encoding nucleic acid sequence independently comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 epitope-encoding nucleic acid sequences, optionally where each epitope-encoding nucleic acid sequence encodes a different epitope-encoding nucleic acid sequence.In some embodiments, each antigen-encoding nucleic acid sequence independently comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 epitope-encoding nucleic acid sequences. In some embodiments, each antigen-encoding nucleic acid sequence independently comprises at least 2-400 epitope-encoding nucleic acid sequences, and at least two of the epitope-encoding nucleic acid sequences encode epitope sequences or portions thereof that are presented by MHC class I on the cell surface. In some embodiments, at least two of the MHC class I epitopes are presented by MHC class I on the cell surface, optionally on the tumor cell surface or on the infected cell surface.

[0047] In some embodiments, the epitope-encoding nucleic acid sequence comprises at least one MHC class I epitope-encoding nucleic acid sequence, each antigen-encoding nucleic acid sequence encoding a polypeptide sequence 8-35 amino acids in length, optionally 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length. In some embodiments, at least one MHC class II epitope-encoding nucleic acid sequence is present. In some embodiments, the at least one MHC class II epitope-encoding nucleic acid sequence comprises at least one MHC class II epitope-encoding nucleic acid sequence that is present and has at least one alteration, which alteration causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence.

[0048] In some embodiments, the epitope-encoding nucleic acid sequences include MHC class II epitope-encoding nucleic acid sequences, and each antigen-encoding nucleic acid sequence encodes a polypeptide sequence 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length.

[0049] In some aspects, the epitope-encoding nucleic acid sequence comprises an MHC class II epitope-encoding nucleic acid sequence, wherein at least one MHC class II epitope-encoding nucleic acid sequence is present and wherein the at least one MHC class II epitope-encoding nucleic acid sequence comprises at least one universal MHC class II epitope-encoding nucleic acid sequence, and optionally, the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0050] In some embodiments, at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible. In some embodiments, at least one promoter nucleotide sequence or the second promoter nucleotide sequence is non-inducible. In some embodiments, the poly(A) sequence comprises a poly(A) sequence native to the autonomously replicating virus. In some embodiments, the at least one poly(A) sequence comprises a poly(A) sequence exogenous to the autonomously replicating virus. In some embodiments, the at least one poly(A) sequence is operably linked to at least one of the at least one nucleic acid sequence. In some embodiments, the at least one poly(A) sequence is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, or at least 120 consecutive A nucleotides. In some embodiments, the at least one poly(A) sequence is at least 80 consecutive A nucleotides.

[0051] In some embodiments, the epitope-encoding nucleic acid sequence comprises an MHC class I epitope-encoding nucleic acid sequence, the MHC class I epitope-encoding nucleic acid sequence being (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor, an infected cell, or an infectious pathogenic organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences for each of a set of epitopes; (b) inputting the peptide sequence of each epitope into a presentation model to generate a set of numerical likelihoods that each of said epitopes will be presented by one or more of the MHC alleles on a cell surface, and optionally on a tumor cell surface or an infected cell surface, said set of numerical likelihoods having been determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes used to generate MHC class I epitope-encoding nucleic acid sequences; The selection is made by performing the following:

[0052] In some embodiments, each of the MHC class I epitope-encoding nucleic acid sequences comprises: (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor, an infected cell, or an infectious pathogenic organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences for each of a set of epitopes; (b) inputting the peptide sequence of each epitope into a presentation model to generate a set of numerical likelihoods that each of said epitopes will be presented by one or more of the MHC alleles on a cell surface, and optionally on a tumor cell surface or an infected cell surface, said set of numerical likelihoods having been determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes used to generate at least 20 of the MHC class I epitope-encoding nucleic acid sequences; The selection is made by performing the following:

[0053] In some embodiments, the number of sets of selected epitopes is between 2 and 20. In some embodiments, the presentation model comprises: (a) the presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position of the peptide sequence; (b) the possibility of presentation, by said particular one of said MHC alleles of said pair, of such peptide sequence comprising said particular amino acid at said particular position on the cell surface, optionally on a tumor cell or an infected cell surface; It represents the degree of dependence between

[0054] In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being presented on a cell surface, optionally on a tumor cell surface or an infected cell surface, based on a presentation model, compared to non-selected epitopes. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being able to stimulate a tumor-specific or infectious pathogen-specific immune response in a subject, based on a presentation model, compared to non-selected epitopes. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have an increased likelihood of being able to be presented to naive T cells by professional antigen-presenting cells (APCs), based on a presentation model, compared to non-selected epitopes, optionally the APCs are dendritic cells (DCs). In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a decreased likelihood of being inhibited by central or peripheral tolerance, based on a presentation model, compared to non-selected epitopes. In some embodiments, selecting the set of selected epitopes comprises selecting epitopes that have a reduced likelihood of stimulating autoimmune response against target normal tissues compared to non-selected epitopes based on the presented model.In some embodiments, the nucleotide sequencing data of exome or transcriptome is obtained by sequencing tumor cells or tissues, infected cells, or infectious pathogenic organisms.In some embodiments, sequencing is next generation sequencing (NGS) or any massively parallel processing sequencing approach.

[0055] In some embodiments, the composition for delivering the self-amplifying expression system is administered as a priming vaccine. In some embodiments, the method further comprises administering a second composition, optionally the second composition is a vaccine composition. In some embodiments, the second composition is administered prior to the composition for delivering the self-amplifying expression system. In some embodiments, the second composition is administered after the administration of the composition for delivering the self-amplifying expression system. In some embodiments, the second composition is the same as the composition for delivering the self-amplifying expression system. In some embodiments, the second composition is different from the composition for delivering the self-amplifying expression system. In some embodiments, the second composition comprises a cassette of the self-amplifying expression system, optionally the second composition comprises a chimpanzee adenoviral vector encoding the cassette of the self-amplifying expression system. In some embodiments, more than one second composition is administered, optionally the composition for delivering the self-amplifying expression system is administered as a priming vaccine.

[0056] In some embodiments, the composition for delivering the self-amplifying expression system is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), intravitreally (IVT), intrathecally, or intravenously (IV). In some embodiments, the method further comprises administering an immunomodulatory agent, optionally an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, an anti-OX-40 antibody or antigen-binding fragment thereof, or a cytokine, optionally an cytokine being at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or a variant thereof. In some embodiments, the method further comprises administering an adjuvant. [Brief description of the drawings]

[0057] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings. [Figure 1] Transcription of SAM vectors using the canonical T7 promoter or a modified ("minimal") T7 promoter is shown. [Diagram 2] A schematic diagram of a representative AU-SAM vector is shown. [Diagram 3] Figure 1 shows RNA profiles of nucleoside-modified SAMs encoding GFP or β-spike by capillary electrophoresis. Three ng of in vitro transcribed SAM-GFP or β-spike containing no modified nucleosides or Ψ, m1Ψ, and m5C modified nucleosides were separated by capillary electrophoresis. The U*SAM backbone contains catalytically inactive nsP4. [Figure 4] SAM activity measured by RT-qPCR after transfection is shown. BHK-21 cells were transfected with 11 ng of the indicated SAM. RT-qPCR was performed on total RNA isolated 20 hours after transfection to measure RNA copy number. Results are shown as the mean ± standard deviation for each group from two independent experiments. [Figure 5A] Figure 5 shows FPLC-based oligo(dT) affinity chromatography of in vitro transcribed SAMs. A total of 5 mg or 11,760 nt in length of U-containing IVT SAMs was purified by FPLC-based oligo(dT) affinity chromatography. Representative UV chromatograms of affinity purification experiments performed with β-spike-encoding SAMs are shown. Collected fractions containing IVT contaminants are shown as "flow-through" and "wash". Oligo(dT)-bound full-length SAM eluted with water is shown as "eluate". [Figure 5B]Figure 5 shows FPLC-based oligo(dT) affinity chromatography of in vitro transcribed SAMs. A total of 5 mg or 11,760 nt in length of U-containing IVT SAMs was purified by FPLC-based oligo(dT) affinity chromatography. Analysis of fractions collected by capillary electrophoresis is shown. [Figure 6] Assessment of SAM purity by dot blot. (A) dsRNA by-products in silica- and oligo(dT)-purified SAM-GFP or β-spike with no modified nucleosides or with m5C- or m1Ψ-modified nucleosides analyzed by dot blotting with J2 dsDNA-specific monoclonal antibody and (B) relative quantification of dsRNA content in 1000 ng dots of each group. [Figure 7A] Induction of innate immune responses in SAM-transfected MoDCs. Human MoDCs were transfected with LNPs (10 μg) containing the indicated SAMs prior to oligo(dT) purification. The mean fluorescence intensity (MFI) of IRF7 was measured by flow cytometry 24 hours after transfection. Results are shown as the mean ± standard deviation for each group from four independent experiments. [Figure 7B] Induction of innate immune responses in SAM-transfected MoDCs. Human MoDCs were transfected with LNPs (10 μg) containing the indicated SAMs after oligo(dT) purification. The mean fluorescence intensity (MFI) of IRF7 was measured by flow cytometry 24 hours after transfection. Results are shown as the mean ± standard deviation of each group from four independent experiments. [Figure 7C] Induction of innate immune responses in SAM-transfected MoDCs. Human MoDCs were transfected with LNPs (10 μg) containing the indicated SAMs before ("β spike") and after ("β spike-ssRNA") purification using both oligo(dT)-mediated affinity purification (left panel) or cellulose-based purification (right panel). Representative flow cytometry 24 hours after transfection is shown. [Figure 8] Translation of SAM encoding GFP in MoDCs. Human MoDCs were transfected with SAM (10 μg) encapsulated in LNPs. GFP expression was measured by flow cytometry 48 h after transfection. Results are shown as the mean ± standard deviation for each group from three to four independent experiments. [Figure 9] Antigen-specific cellular immune responses against the β-spike epitope of SARS-CoV-2 in Balb / c mice 6 or 14 days after immunization with 1 or 10 μg of SAM-LNP. IFNγ ELISpot responses are shown. The cellulose-based purification is referred to as "purification A" and the oligo(dT)-based purification is referred to as "purification B." [Figure 10] Intracellular cytokine staining of splenocytes from Balb / c mice after stimulation with overlapping peptide pools spanning the β-spike epitope of SARS-CoV-2 in Balb / c mice 5 or 12 days after immunization with 1 mg of samRNA-LNP. Boxes represent interquartile range (IQR) (25-75%), midline, whiskers represent range. [Figure 11] Serum pseudovirus neutralization titers (50% inhibition) in Balb / c mice 4 or 8 weeks after immunization with 1 mg of samRNA-LNP are shown. Geometric mean and geometric SD of n=6 independent animals in each group. [Figure 12] Intracellular cytokine staining of splenocytes from Balb / c mice after stimulation with tetrameric AH1 antigen in Balb / c mice 3, 7, 10, and 14 days after immunization with 1 mg of samRNA-LNP. Boxes represent interquartile range (IQR) (25-75%), midline, whiskers represent range. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description definition As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharma- ceutically acceptable, non-toxic acid addition salts include salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or using other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of the salts include phonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0059] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharma-ceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium; and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0060] The recitation of a list of chemical groups in any definition of a variable herein includes that definition of that variable as any single group or combination of listed groups. The recitation of an embodiment of a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0061] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof; biopsies or extracts thereof obtained from mammals; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof.

[0062] As used herein, a "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that stimulates a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered as part of a dosing regimen to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance delivered, and the target cell or tissue. For example, an effective amount of a provided compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the occurrence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a "therapeutically effective amount" is at least a minimal amount of a provided compound, or a composition containing a provided compound, sufficient to treat one or more symptoms of a disease or disorder.

[0063] Disease, disorder, and condition are used interchangeably herein.

[0064] As used herein, the terms "treatment," "treat," and "therapeutic" refer to partially or completely alleviating, inhibiting, delaying the onset of, preventing, ameliorating, and / or alleviating a disease or condition described herein, or one or more symptoms of a disease or condition. In some embodiments, treatment can be administered after one or more symptoms have developed. In some embodiments, the term "therapeutic" includes preventing or arresting the progression of a disease or disorder. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of the condition and / or in light of genetic or other susceptibility factors). Treatment may be continued after symptoms have resolved, e.g., to prevent or delay recurrence. In some embodiments, the term "therapeutic" includes preventing the recurrence or relapse of a disease or disorder.

[0065] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent), or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)), and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. "Patient" and "subject" are used interchangeably herein.

[0066] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound(s) with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the compositions of the compounds disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, partial glyceride mixtures of saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, protamine sulfate, water, salts or electrolytes such as protamic acid sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0067] Alternative Embodiments In alternative embodiments, the compounds described herein may contain one or more isotopic substitutions. For example, hydrogen is 2 H (D or deuterium) or 3 H (T or tritium), carbon can be e.g. 13 C or14 C, and oxygen can be, for example 18 O, the nitrogen may be e.g. 15 N, etc. In other embodiments, a particular isotope (e.g., 3 H, 13 C. 14 C. 18 O, or 15 N) may be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the total isotopic abundance of the element occupying a particular site on the compound.

[0068] Pharmaceutical Compositions In some embodiments, the present disclosure provides a composition comprising any of the compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein) and a pharma- ceutically acceptable carrier, adjuvant, or vehicle. The compounds of the present disclosure are preferably formulated in unit dosage form for ease of administration and uniformity of dosage.

[0069] Methods of Using the Disclosed Compounds - Synthesis of RNA Nucleotides In general, the methods described herein may be useful for preparing in vitro transcribed RNA. In some embodiments, the methods described herein may be useful for preparing single-stranded RNA (ssRNA). In some embodiments, the methods described herein may be useful for preparing a self-amplifying expression system, such as a self-amplifying expression system comprising ssRNA (e.g., an ssRNA vector derived from a self-replicating RNA virus). In some embodiments, the methods described herein may be useful for preparing modified RNA, such as RNA (e.g., ssRNA) incorporating modified nucleosides. In some embodiments, the methods described herein may be useful for preparing purified ssRNA, e.g., ssRNA incorporating modified nucleosides.

[0070] In some embodiments, the methods described herein may be useful for preparing 5'-capped RNA. Methods and compositions contemplated herein for preparing 5'-capped RNA include, but are not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Cajal body-specific small RNA (scaRNA). In some embodiments, the methods involve the use of capped oligonucleotide primers, nucleoside 5' triphosphates (NTPs), and RNA polymerases for promoter-controlled RNA synthesis using DNA templates. In certain aspects, the methods use an initiating capped oligonucleotide primer, which confers utility in RNA synthesis, particularly in the synthesis of capped mRNA.

[0071] In some embodiments, the methods described herein may be useful in methods for preparing RNA, including but not limited to, mRNA, snRNA, snoRNA, scaRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), and transfer-messenger RNA (tmRNA), that have modifications at or near the 5' end of the molecule. In some embodiments, the methods involve the use of an initiating oligonucleotide primer, with or without a cap, nucleoside '-triphosphates (NTPs), and an RNA polymerase to perform promoter-controlled RNA synthesis using a DNA template. In certain aspects, the methods use modified initiating capped oligonucleotide primers that have structural modifications that confer utility in RNA synthesis, particularly the synthesis of 5' modified RNA.

[0072] The initiating capped oligonucleotide primer has a reactive 3'OH group that allows the initiation of RNA synthesis by RNA polymerase on a DNA template by the addition of a nucleoside unit to the 3' end of the primer. The initiating capped oligonucleotide primer is substantially complementary to the transcription initiation site of the template DNA (i.e., the initiation site is located near the 3' end of the promoter sequence and may overlap with the promoter sequence), and in certain embodiments, the initiating capped oligonucleotide primer primarily directs RNA synthesis in one direction ("forward") starting from the 3' end of the primer. In certain aspects and embodiments, the initiating capped oligonucleotide primer predominates over any nucleoside 5'-triphosphate in initiating RNA synthesis, thereby maximizing the production of RNA initiated by the initiating capped oligonucleotide primer and minimizing the production of RNA initiated by a 5'-triphosphate nucleoside (usually GTP).

[0073] The initiating capped oligonucleotide primer of the present disclosure has a hybridization sequence that can be complementary to a sequence at the initiation site of the DNA template. Due to the presence of the hybridization sequence, the initiating capped oligonucleotide primer will align primarily with the complementary sequence of the DNA template at the initiation site only in the desired orientation (i.e., the "forward" orientation). In the forward orientation, the RNA transcript will begin with an inverted guanosine residue (i.e., 7m G(5')ppp(5')N...). The preference for the forward alignment of the primer to the DNA template compared to the incorrect "reverse" orientation is maintained by the thermodynamics of the hybridization complex. The thermodynamics of the hybridization complex is determined by the length of the hybridization sequence of the initiating cap oligonucleotide primer and the type of bases involved in hybridization with the DNA template. The desired forward hybridization may also depend on the temperature and reaction conditions used to hybridize the DNA template and the initiating cap oligonucleotide primer during in vitro transcription.

[0074] The initiation capped oligonucleotide primer of the present disclosure improves the efficiency of transcription initiation compared to the efficiency of initiation with standard GTP, ATP, CTP or UTP. In some embodiments, transcription initiation is considered improved when the synthesis of RNA begins primarily from the initiation capped oligonucleotide primer and not from any NTP in the transcription mixture. The improved efficiency of transcription initiation leads to a higher yield of RNA transcripts. The improved efficiency of transcription initiation can be increased by about 10%, about 20%, about 40%, about 60%, about 80%, about 90%, about 100%, about 150%, about 200% or about 500% compared to synthesis of RNA by conventional methods without the initiation capped primer. In certain embodiments, the "initiation capped oligonucleotide primer" outperforms any NTP (including GTP) in initiating transcription. One of skill in the art can easily determine the level and efficiency of substrate activity of the initiation capped oligonucleotide primer. In certain embodiments, initiation occurs from the capped oligonucleotide primer rather than from the NTP, resulting in a higher level of capping of the transcribed mRNA.

[0075] In some embodiments, a method is provided for synthesizing RNA using an initiating capped oligonucleotide primer with substitution or modification. In some embodiments, the substitution and modification of the initiating capped oligonucleotide primer does not significantly interfere with the synthesis of RNA. Routine test synthesis can be performed to determine whether the modified initiating capped oligonucleotide primer can be used to obtain the desired synthesis result. Those skilled in the art can perform such routine experiments to determine whether the desired result can be obtained. Substitutions or modifications of the initiating capped oligonucleotide primer can include, for example, one or more modified nucleoside bases, one or more modified sugars, one or more modified internucleotide bonds, and / or one or more modified triphosphate bridges.

[0076] The modified initiating capped oligonucleotide primer may contain one or more modification groups provided herein and can be extended by RNA polymerase on a DNA template by incorporation of NTPs onto the reactive 3'OH group. The initiating capped oligonucleotide primer may contain natural RNA and DNA nucleosides, modified nucleosides, or nucleoside analogs. The initiating capped oligonucleotide primer may contain natural internucleotide phosphodiester bonds or modifications thereof, or combinations thereof.

[0077] Methods of Use of the Disclosed Compounds - Methods of Treatment In some embodiments, the disclosure provides a method for treating or reducing the severity of a disease or condition (e.g., cancer or an infectious disease) in a patient, comprising administering to the patient an RNA polynucleotide. In some embodiments, the disclosure provides a prophylactic method for treating a disease or condition, such as a prophylactic vaccination.

[0078] In general, the methods described herein may be useful for preparing in vitro transcribed RNA for use in pharmaceutical compositions for treating or reducing the severity of a condition or disease. In some embodiments, the methods described herein may be useful for preparing single-stranded RNA (ssRNA) for use in pharmaceutical compositions. In some embodiments, the methods described herein may be useful for preparing a self-amplifying expression system for use in pharmaceutical compositions, such as a self-amplifying expression system comprising ssRNA (e.g., an ssRNA vector derived from a self-replicating RNA virus). In some embodiments, the methods described herein may be useful for preparing modified RNA for use in pharmaceutical compositions, such as RNA (e.g., ssRNA) incorporating modified nucleosides. In some embodiments, the methods described herein may be useful for preparing purified ssRNA for use in pharmaceutical compositions, such as ssRNA incorporating modified nucleosides.

[0079] In some embodiments, the compounds and compositions according to the disclosed methods can be administered in any amount and by any route of administration effective in treating or reducing the severity of cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0080] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of microsatellite-stable colorectal cancer (MSS-CRC), non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDA), and gastroesophageal adenocarcinoma (GEA). In some embodiments, the cancer is selected from the group consisting of MSS-CRC, NSCLC, and PDA.

[0081] In some embodiments, an RNA polynucleotide composition described herein, such as any of the nucleoside modified and / or purified RNA compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein), is administered to a patient having a cancer selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, rectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, bladder cancer, brain cancer, B cell lymphoma, acute myeloid leukemia, adult acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0082] In some embodiments, any of the RNA polynucleotide compositions described herein, such as any of the nucleoside modified and / or purified RNA compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein) are administered to a patient having an infection. In some embodiments, the infection is a viral infection, a fungal, or a bacterial infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is an infection by a virus, and the virus is HPV. In some embodiments, the viral infection is an infection by a virus, and the virus is an influenza virus. In some embodiments, the viral infection is an infection by a virus, and the virus is a coronavirus (e.g., species associated with Severe Acute Respiratory Syndrome (SARS) 2002, isolates and subisolates of SARS-CoV-2, species associated with Middle East Respiratory Syndrome (MERS) 2012, and / or other coronavirus species). In some embodiments, any of the compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein) are administered to a subject having COVID-19. In some embodiments, any of the compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein) is administered to a subject at risk for infection with COVID-19. In some embodiments, any of the compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein) is administered to a subject as a prophylactic vaccination against COVID-19.

[0083] In some embodiments, the disclosure provides methods of contacting a biological sample with any of the RNA polynucleotide compositions described herein, such as any of the nucleoside modified and / or purified RNA compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein).

[0084] In some embodiments, one or more additional therapeutic agents can also be administered in combination with any of the RNA polynucleotide compositions described herein, such as any of the nucleoside modified and / or purified RNA compositions described herein, such as any of the nucleoside modified and / or purified RNA compositions described herein (e.g., any of the ssRNA SAM vaccine compositions described herein). In some embodiments, the RNA polynucleotide compositions described herein and one or more additional therapeutic agents can be administered as part of a multiple dosing regimen. In some embodiments, the RNA polynucleotide compositions described herein and one or more additional therapeutic agents can be administered simultaneously, sequentially, or within a given time period. In some embodiments, the RNA polynucleotide compositions described herein and one or more additional therapeutic agents can be administered within 5 hours of each other. In some embodiments, the RNA polynucleotide compositions described herein and one or more additional therapeutic agents can be administered within 24 hours of each other. In some embodiments, the RNA polynucleotide compositions described herein and one or more additional therapeutic agents can be administered within one week of each other.

[0085] Self-amplifying mRNA vector Generally, all self-replicating mRNA (SAM) vectors contain a self-amplifying backbone derived from a self-replicating virus. The term "self-amplifying backbone" refers to the minimal sequence(s) of a self-replicating virus that allows for the autonomous replication of the viral genome. For example, the minimal sequence that allows for the autonomous replication of an alphavirus can include conserved sequences for nonstructural protein-mediated amplification (e.g., the nonstructural protein 1 (nsP1) gene, the nsP2 gene, the nsP3 gene, the nsP4 gene, the 5' untranslated region (UTR), the 3' UTR, and / or a polyA sequence). The self-amplifying backbone can also include sequences for the expression of subgenomic viral RNA (e.g., a subgenomic promoter such as the 26S promoter element in alphaviruses). The SAM vector can be a (+)-sense RNA polynucleotide or a (-)-sense RNA polynucleotide, such as a vector with a backbone derived from a (+)-sense or (-)-sense autonomous replicating virus. Self-replicating viruses include, but are not limited to, alphaviruses, flaviviruses (e.g., Kunjin virus), measles virus, and rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus). Examples of SAM vector systems derived from self-replicating viruses are described in Lundstrom (Molecules.2018 Dec 13;23(12).pii:E3310.doi:10.3390 / molecules23123310), which is incorporated by reference for all purposes.

[0086] In vitro self-amplifying generation Methods well known in the art for generating RNA include in vitro transcription (IVT), in which a DNA template of the desired vector is first generated by methods well known in the art, including standard molecular biology methods such as cloning, restriction digestion, ligation, gene synthesis (e.g., chemical and / or enzymatic synthesis), and polymerase chain reaction (PCR).

[0087] The DNA template has an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA (e.g., SAM). Promoters include, but are not limited to, promoters for bacteriophage polymerases such as T3, T7, SP6, or K11. Depending on the specific RNA polymerase promoter sequence selected, additional 5' nucleotides may be transcribed beyond the desired sequence. For example, the canonical T7 promoter can be referenced by the sequence TAATACGACTCACTATAGG, and the DNA template TAATACGACTCACTATAGGN is used to generate the desired sequence N. V The IVT reaction was performed using the mRNA sequence GG-N V Generally, and without wishing to be bound by theory, T7 polymerase can more efficiently transcribe RNA transcripts that begin with a guanosine. However, additional 5' nucleotides may be undesirable and / or detrimental. Thus, the RNA polymerase promoter included in the DNA template can be a sequence that results in a transcript that contains only the 5' nucleotides of the desired sequence (e.g., a SAM having the endogenous (also called "native" or "genomic") 5' sequence of the self-replicating virus from which the SAM vector is derived, which refers to the native genomic sequence of the self-replicating virus (e.g., having endogenous 5'VEEV nucleotides AU, also called "AU-SAM"). For example, a minimal T7 promoter can be referenced by the sequence TAATACGACTCACTATA (5' to 3' orientation; φ6.5 T7 promoter), and the DNA template TAATACGACTCACTATAN1N2N is used to generate the desired sequence N. V IVT reaction using the mRNA sequence N1N2N VAnother minimal T7 promoter can be referenced by the sequence TAATACGACTCACTATT (5' to 3' orientation; φ2.5 T7 promoter). Similarly, a minimal SP6 promoter can be used to generate transcripts with no additional 5' nucleotides, which can be referenced by ATTTAGGTGACACTATA. Similarly, a minimal K11 promoter can be used to generate transcripts with no additional 5' nucleotides, which can be referenced by AATTAGGGCACACTATA. In a typical IVT reaction, a DNA template is incubated with the appropriate RNA polymerase enzyme, buffering agents, and nucleotides (NTPs).

[0088] The resulting RNA polynucleotide can optionally be further modified, including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or related structures, and optionally modifying the 3' end to include a polyadenylated (polyA) tail. In a modified IVT reaction, the RNA is co-transcriptionally capped with a 5' cap structure in the IVT by the addition of a cap analog. Cap analogs include the dinucleotide ((m 7 G-ppp-N) cap analog or trinucleotide (m 7 G-ppp-N1-N2) cap analogs, where N represents a nucleotide or a modified nucleotide, such as, but not limited to, adenosine, guanosine, cytidine, and uridine. Modified nucleotides include modified adenosines, such as N6 methylguanosine, where the 2'OH is methylated. Trinucleotides (m 7In an exemplary non-limiting example, including a G-ppp-N1-N2) cap analog, N1 can be an N6 methyladenosine with a methylated 2'OH. The cap analog can include any of the structures or formulas described herein. Exemplary cap analogs and their use in IVT reactions are also described in more detail in U.S. Pat. No. 10,519,189, which is incorporated by reference herein for all purposes. As noted above, T7 polymerase can more efficiently transcribe RNA transcripts that begin with a guanosine. Trinucleotide cap analogs (mPPP-N1-N2) can be used to increase the transcription efficiency of templates that do not begin with a guanosine. 7 G-ppp-NN) can be used. Trinucleotide cap analogs can be used with dinucleotide cap analogs (m 7 The efficiency of transcription can be increased by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold or more compared to an IVT reaction using a 5'-G-ppp-N.

[0089] A 5' cap structure can also be added post-transcriptionally, such as by using the Vaccinia capping system, which includes mRNA 2'-O-methyltransferase and S-adenosylmethionine (eg, NEB catalog no. M2080).

[0090] Modified Nucleosides Nucleoside triphosphates (ATP, CTP, UTP, GTP) with modified nucleosides can be added to the in vitro transcription reaction to generate the SAM vector. Without wishing to be bound by theory, RNA incorporating modified nucleosides can improve the stability of the RNA vector after transfection of a target cell, reduce stimulation of the innate immune pathway of the RNA vector, and / or improve viral protein-mediated RNA replication / amplification of the RNA vector.

[0091] Modified nucleosides include, but are not limited to, hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alken ...(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycy Cytosine, 5-(C2-C6)alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (non-basic residue). Modified nucleotides may include methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), or N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ). Modified nucleotides may include m5C. Modified nucleotides may include m6A. Modified nucleotides may include 2'-O-Me. Modified nucleotides may include s-thiouridine. Modified nucleotides may include m5U. Modified nucleotides may include m1Ψ. Modified nucleotides may include Ψ.

[0092] A single different modified nucleoside can be added to the in vitro transcription reaction together with unmodified nucleosides. For example, as an illustrative, non-limiting example, only mC modified nucleosides can be added to the in vitro transcription reaction (e.g., together with unmodified ATP, UTP, and GTP) and no other modified nucleosides can be added. Multiple modified nucleosides can also be added to the in vitro transcription reaction. Multiple modified nucleosides can be added to the in vitro transcription reaction together with unmodified nucleosides. Unmodified nucleosides include mC modified nucleosides that include chemical modifications. 7 It may or may not contain a G-cap analog.

[0093] In ssRNA incorporating modified nucleosides, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the adenosine, guanosine, cytidine, and / or uridine nucleosides of the ssRNA may be modified nucleosides. In ssRNA incorporating modified nucleosides, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In an ssRNA incorporating modified nucleosides, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or 100% of the uridine nucleosides of the ssRNA can be modified nucleosides.

[0094] In the ssRNA incorporating modified nucleosides, more than 25% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 50% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 75% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 95% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 99% of the adenosine nucleosides of the ssRNA may be modified nucleosides. In an ssRNA incorporating modified nucleosides, 100% of the adenosine nucleosides of the ssRNA can be modified nucleosides.

[0095] In the ssRNA incorporating modified nucleosides, more than 25% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 50% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 75% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 95% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 99% of the guanosine nucleosides of the ssRNA may be modified nucleosides. In an ssRNA incorporating modified nucleosides, 100% of the guanosine nucleosides of the ssRNA can be modified nucleosides.

[0096] In the ssRNA incorporating modified nucleosides, more than 25% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 50% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 75% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 95% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 99% of the cytidine nucleosides of the ssRNA may be modified nucleosides. In an ssRNA incorporating modified nucleosides, 100% of the cytidine nucleosides of the ssRNA may be modified nucleosides.

[0097] In the ssRNA incorporating modified nucleosides, more than 25% of the uridine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 50% of the uridine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 75% of the uridine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 95% of the uridine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, more than 99% of the uridine nucleosides of the ssRNA may be modified nucleosides. In the ssRNA incorporating modified nucleosides, 100% of the uridine nucleosides of the ssRNA may be modified nucleosides.

[0098] In the ssRNA incorporating modified nucleosides, more than 25% of the cytidine nucleosides of the ssRNA may be m5C. In the ssRNA incorporating modified nucleosides, more than 50% of the cytidine nucleosides of the ssRNA may be m5C. In the ssRNA incorporating modified nucleosides, more than 75% of the cytidine nucleosides of the ssRNA may be m5C. In the ssRNA incorporating modified nucleosides, more than 95% of the cytidine nucleosides of the ssRNA may be m5C. In the ssRNA incorporating modified nucleosides, more than 99% of the cytidine nucleosides of the ssRNA may be m5C. In the ssRNA incorporating modified nucleosides, 100% of the cytidine nucleosides of the ssRNA may be m5C.

[0099] Purification of ssRNA Following in vitro transcription of the SAM vector with or without the addition of modified nucleosides, the RNA can be purified. Without wishing to be bound by theory, the desired SAM vector is a m 7It is a single-stranded RNA (ssRNA) containing a G-cap and a polyadenylated (polyA) tail. Furthermore, without wishing to be bound by theory, purification removes all unwanted double-stranded RNA by-products of the in vitro transcription reaction, including those that may result in unwanted immune stimulation of the RNA vector after transfection of target cells and / or reduced viral protein-mediated RNA replication / amplification. For example, stimulation of the innate immune system by the RNA IVT products can be evaluated, for example, by assessing IFN response. IFN response can be evaluated by analysis of interferon expression, IRF-3 expression, IRF-7 expression, or a combination thereof (e.g., RT-qPCR, flow cytometry, Western blot analysis, or a combination thereof). Improvements in replication of a SAM ssRNA vector can be assessed, such as by comparing SAM ssRNA, which (A) contains modified nucleosides and / or (B) has been purified using a chromatography system and / or an affinity-based separation system, to a control composition that contains an otherwise identical ssRNA vector that does not contain modified nucleosides and / or is unpurified.

[0100] Purification can use a chromatography-based system, such as an FPLC or HPLC system. In general, FPLC is advantageous for the general SAM ssRNA vector size described herein. Those skilled in the art will recognize applicable FPLC and HPLC systems that can be used, such as the Akta avant 25 FPLC system (Cytiva), and its use (e.g., washing, equilibration, elution, etc.).

[0101] Chromatography-based systems include cellulose-based chromatography systems. Chromatography-based systems include affinity-based separation systems. An illustrative, non-limiting example of an affinity-based separation system is a deoxythymidine (dT) oligonucleotide (oligo(dT))-based system that binds to the polyA tail of RNA, such as the OROS OligodT(25) GoPure Column (Thermo Fisher Scientific).

[0102] Purification techniques such as chromatography-based techniques can be used to produce pure or essentially pure ssRNA that is free of contaminants such as IVT reaction by-products (e.g., dsRNA, salts, detergents, etc.). Purification techniques such as chromatography-based techniques can be used to produce SAM ssRNA-containing compositions or mixtures in which contaminants make up less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the composition or mixture. Purification techniques can be used to produce mixtures that contain both SAM ssRNA and dsRNA, but in which dsRNA makes up less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the composition or mixture.

[0103] Using purification techniques, the desired ssRNA vector (e.g., 7 A SAM ssRNA-containing composition or mixture can be produced in which the desired ssRNA vector (full-length ssRNA vector containing both a G-cap and a polyA tail) constitutes 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the total RNA present in the composition or mixture. Purification techniques can be used to produce a SAM ssRNA-containing composition or mixture in which the desired ssRNA vector constitutes 97% or more, 98% or more, 99% or more, or 99.5% or more of the total RNA present in the composition or mixture.

[0104] Purification techniques can be used to produce mixtures that contain both SAM ssRNA and dsRNA, but where ssRNA represents 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the RNA present in the mixture. Purification techniques can be used to produce mixtures that contain both SAM ssRNA and dsRNA, but where ssRNA represents 97% or more, 98% or more, 99% or more, or 99.5% or more of the RNA present in the mixture.

[0105] Purification techniques can be used to generate ssRNA-containing compositions or mixtures in which the desired ssRNA vector is the only detectable RNA species present in the composition of the composition or mixture. Purification techniques can be used to generate ssRNA-containing compositions or mixtures in which the desired ssRNA vector is the only detectable RNA species present in the composition of the composition or mixture.

[0106] The purity of ssRNA after purification, such as after a chromatography-based technique, can be assessed. The quality and concentration of RNA (e.g., total RNA, dsRNA, and / or ssRNA) can be assessed using methods including, but not limited to, capillary electrophoresis, liquid chromatography (e.g., FPLC or HPLC), dot blot, and / or ELISA. For dot blot and ELISA, detection methods including antibodies specific for RNA, dsRNA, and / or ssRNA can be used. Quantification can include determining the area under the curve (AUC), band intensity, and / or dot intensity, such as peaks in the FPLC spectrum.

[0107] The RNA purification process may include other purification techniques well known in the art, such as phenol chloroform extraction.

[0108] Alphavirus biology Alphaviruses are members of the Togaviridae family and are single-stranded positive-sense RNA viruses. Members are generally classified as Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aula, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbrial Review 1994). Natural alphavirus genomes are usually about 12 kb in length, the first two-thirds of which contain genes encoding nonstructural proteins (nsPs) that form an RNA replication complex for autonomous replication of the viral genome, and the last third contains subgenomic expression cassettes encoding structural proteins for the production of virions (Frolov RNA 2001).

[0109] The model life cycle of alphaviruses involves several distinct steps (Strauss Microbrial Review 1994, Jose Future Microbiol 2009). After viral adsorption to the host cell, the virion fuses with the membrane in the intracellular compartment, eventually releasing the genomic RNA into the cytosol. The genomic RNA, which has a positive-strand orientation, a 5'-end methylguanylic acid cap and a 3'-end polyA tail, is translated to generate nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive strand is replicated by this complex onto the negative strand template. In the current model, the replication complex is further processed as infection progresses, and the resulting processed complex switches to transcribe the negative strand into the full-length positive strand genomic RNA and the 26S subgenomic positive strand RNA that contains the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified with potential roles in various steps of RNA synthesis, including the complement of the 5' UTR in replication of positive-strand RNA from a negative-strand template, a 51-nt CSE in replication of negative-strand synthesis from a genomic template, a 24-nt CSE within the junction region of nsP and 26S RNA in transcription of subgenomic RNA from the negative strand, and a 19-nt CSE at the 3' end in negative-strand synthesis from a positive-strand template.

[0110] In the natural life cycle of viruses, after replication of different RNA species, viral particles are usually assembled. The 26S RNA is translated and the resulting proteins are further processed to generate structural proteins including capsid proteins, glycoproteins E1 and E2, and two small polypeptides E3 and 6K (Strauss 1994). After encapsidation of the viral RNA occurs and capsid proteins, which are usually specific only to the genomic RNA, are packaged, virions are assembled and budded onto the membrane surface.

[0111] Alphavirus Delivery Vectors Alphaviruses (alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also called alphavirus vectors, alphavirus virus vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying mRNA (SAM) vectors). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses have several advantages in vaccine settings where expression of heterologous antigens may be desirable. Due to their ability to replicate autonomously in the host cytosol, alphaviruses can generally achieve high copy numbers of the expression cassette within the cell, thus achieving high levels of heterologous antigen production. Furthermore, the vectors are generally transient, resulting in high biosafety and low induction of immune tolerance to the vector. Additionally, the general public generally does not have pre-existing immunity to alphaviruses compared to other standard viral vectors such as human adenoviruses. Alphavirus-based vectors also generally produce cytotoxic responses to infected cells. Cytotoxicity may have some importance in a vaccine setting to adequately stimulate an immune response to the expressed heterologous antigen. However, the degree of desired cytotoxicity is a matter of balance, and therefore several attenuated alphaviruses have been developed, including the TC-83 strain of VEEV. Thus, one example of a neoantigen expression vector described herein uses an alphavirus backbone that allows high levels of antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response to the vector itself, and can be used safely. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimizing which alphavirus sequences the vector uses, including but not limited to sequences from VEEV or its attenuated derivative TC-83.

[0112] Several expression vector design strategies using alphavirus sequences have been developed (Pushko 1997). In one strategy, the design of the alphavirus vector involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene followed by insertion of the heterologous gene (Frolov 1993). This results in the production of a subgenomic RNA that expresses an additional heterologous protein in addition to the native nonstructural and structural proteins. In this system, all the factors are present to produce infectious virions, and therefore repeated rounds of infection of the expression vector in uninfected cells can be carried out.

[0113] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Thus, after autonomous replication of the viral RNA mediated by the still intact nonstructural genes, the 26S subgenomic RNA provides expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is given in trans, for example by co-transfection of a cell line, resulting in an infectious virus. One system is described in U.S. Pat. No. 8,093,021, the entire contents of which are incorporated herein by reference for all purposes. Helper vector systems offer the advantage of limiting the possibility of forming infectious particles, thus improving biosafety. In addition, helper vector systems may reduce the overall vector length and improve the efficiency of replication and expression. Thus, in one example of an antigen expression vector described herein, an alphavirus backbone may be used in which the structural proteins are replaced by an antigen cassette, and the resulting vector promotes efficient expression due to reduced biosafety concerns as well as reduced overall expression vector size.

[0114] Lipid Nanoparticle (LNP) Delivery One important aspect to consider in the design of vaccine vectors is immunity to the vector itself (Riley 2017). This can be in the form of pre-existing immunity to the vector itself, for example certain human adenovirus systems, or immunity to the vector that arises after administration of the vaccine. The latter is an important consideration when multiple doses of the same vaccine are administered, for example separate priming and booster doses, or when the same vaccine vector system is used to deliver different antigen cassettes.

[0115] In the case of alphavirus vectors, the standard delivery method is the helper virus system mentioned above, which generates infectious viral particles by delivering the capsid, E1, and E2 proteins in trans. However, it is important to note that E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, the efficacy of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if the infectious particles are targeted by neutralizing antibodies.

[0116] An alternative to viral particle-mediated gene delivery is the delivery of expression vectors using nanoparticles (Riley 2017). Importantly, nanomaterial carriers can be formed of non-immunogenic materials, generally avoiding the induction of immunity against the delivery vector itself. These materials can include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials. Lipids can be cationic, anionic, or neutral. Such materials can be synthetic or naturally derived, and in certain instances, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid complexes including, but not limited to, polyethylene glycol (PEG) complexes (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0117] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids that allows for the formation of membranes and vesicle-like structures (Riley 2017). Typically, these vesicles deliver expression vectors by absorbing into the membrane of target cells and releasing the nucleic acid into the cytosol. Furthermore, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. As an illustrative example, selective and targeted delivery of LNPs can be achieved by 1) incorporating lipid-binding ligands (e.g., mannose) for cell type-specific receptors into the LNPs, and / or 2) incorporating membrane-linked lipoproteins (anchors) that interact with targeting antibodies. The anchors can be protein A / G and any structural form of antibody, including scFv, Fab, and VHH single domain antibodies or nanobodies with exogenous lipid modification signals (e.g., palmitoylation, prenylation, and / or myristoylation) encoded at their N-terminus or their C-terminus. Another consideration in the design of LNPs is the balance between targeting efficiency and cytotoxicity. Lipid compositions generally include defined mixtures of cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent aggregation of the LNP, to prevent lipid oxidation, or to provide chemical functional groups that facilitate attachment of additional moieties. The lipid composition can affect the size and stability of the overall LNP. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as, for example, PEG or PEG-conjugated lipids, phosphocholine, phosphoethanolamine, sterols, or neutral lipids.

[0118] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable effects, including degradation of the nucleic acid by nucleases in serum, or stimulation of off-targets of the immune system by free nucleic acid. Thus, encapsulation of the alphavirus vector can be used to prevent degradation while also preventing potential off-target effects. In certain examples, the alphavirus vector is fully encapsulated within the delivery vehicle, such as the aqueous interior of the LNP. Encapsulation of the alphavirus vector within the LNP can be performed by methods well known in the art, such as microfluidic mixing and droplet generation performed in a microfluidic droplet generator. Such devices include, but are not limited to, standard T-junction devices or flow focusing devices. In one example, a desired lipid formulation, such as an MC3 or MC3-like molecule-containing composition, is fed into the droplet generator in parallel with the alphavirus delivery vector and other desired substances, resulting in the delivery vector and desired substances being fully encapsulated within the MC3 or MC3-like molecule-based LNP. In one example, the droplet generator can control the particle size range and size distribution of the generated LNP. For example, LNPs can have particle sizes ranging from 1 to 1000 nm in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nm in diameter. After droplet generation, the delivery vector encapsulating the expression vector can be further processed or modified in preparation for administration.

[0119] Other Vectors The self-amplifying mRNA (SAM)-based compositions described herein can be used with other compositions having different (e.g., non-SAM) vector backbones. For example, SAM compositions can be used as part of a vaccine strategy that also uses a chimpanzee-derived vector backbone to encode an antigen cassette. The nucleotide sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions to deliver antigens (see SEQ ID NO: 1). The use of C68 adenovirus-derived vectors is further described in U.S. Patent No. 6,083,716, U.S. Patent Application Publication No. US20200197500A1, and International Patent Application Publication No. WO2020 / 243719, each of which is incorporated herein by reference in its entirety for all purposes.

[0120] antigen Antigens can include nucleotides or polypeptides. For example, antigens can be RNA sequences that code for polypeptide sequences. Thus, antigens useful in vaccines include nucleotide sequences or polypeptide sequences.

[0121] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Neoantigen peptides can be described in terms of their coding sequences, and neoantigens include nucleotide sequences (e.g., DNA or RNA) that encode the relevant polypeptide sequences.

[0122] The present specification also discloses peptides derived from any polypeptide known or found to be altered in expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database. COSMIC is a curated comprehensive information on somatic mutations in human cancers. Peptides can include tumor-specific mutations. Tumor antigens (e.g., shared tumor antigens and tumor neoantigens) include, but are not limited to, those described in U.S. Patent Application No. 17 / 058,128, which is incorporated by reference herein for all purposes.

[0123] Also disclosed herein are peptides derived from any polynucleotide associated with an infectious pathogenic organism, an infectious disease in a subject, or an infected cell in a subject. The antigen may be derived from a nucleotide sequence or a polypeptide sequence of an infectious pathogenic organism. Polypeptide sequences of infectious pathogenic organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacteria-derived peptides, fungi-derived peptides, and / or parasite-derived peptides. Infectious pathogenic organisms include, but are not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae virus, and tuberculosis.

[0124] Disclosed herein are isolated peptides comprising infectious disease-causing organism-specific antigens or epitopes identified by the methods disclosed herein, peptides comprising known infectious disease-causing organism-specific antigens or epitopes, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. Antigenic peptides can be described in terms of their coding sequences, and antigens include nucleotide sequences (e.g., DNA or RNA) that encode the relevant polypeptide sequence.

[0125] The vectors and related compositions described herein can be used to deliver antigens from any organism, including their toxins or other by-products, to prevent and / or treat infectious diseases or other adverse reactions associated with the organism or its by-products.

[0126] Antigens that can be incorporated (e.g., encoded in a cassette) into a vaccine include immunogens useful for immunizing humans or non-human animals against viruses, such as pathogenic viruses that infect humans and non-human vertebrates. Antigens can be selected from various virus families. Examples of desired virus families against which an immune response is desired include the genus Rhinovirus, which is responsible for approximately 50% of cases of the common cold; the genus Enterovirus, which includes human enteroviruses such as poliovirus, coxsackievirus, echovirus, and hepatitis A virus; and the genus Aphthovirus, which is primarily responsible for hand, foot, and mouth disease in non-human animals. Among the viruses of the Picornaviridae family, target antigens include VP1, VP2, VP3, VP4, and VPG. Another virus family is the Caliciviridae, which includes the Norwalk virus group, which is an important pathogen of epidemic gastroenteritis. Yet another virus family desirable for use in targeting antigens to stimulate immune responses in humans and non-human animals is the Togaviridae, which includes the genus Alphavirus, which includes Sindbis virus, Ross River virus, and the Venezuelan, Eastern and Western equine encephalitis viruses, as well as the Rubiviruses, including the rubella virus. The Flaviviridae family includes the dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick-borne encephalitis viruses. Other target antigens may be generated from the Hepatitis C and Coronaviridae families, which include many non-human viruses, such as infectious bronchitis virus (poultry), transmissible porcine gastroenteritis virus (pigs), porcine hemagglutinating encephalomyelitis virus (pigs), feline infectious peritonitis virus (felines), feline enteric coronavirus (felines), canine coronavirus (dogs), and human respiratory coronaviruses, which may cause the common cold and / or non-A, B or C hepatitis. Within the coronavirus family, target antigens include the E1 (also called M or matrix protein), E2 (also called S or spike protein), E3 (also called HE or hemagglutinin-erutero) glycoproteins (not present in all coronaviruses) or N (nucleocapsid).Still other antigens can be targeted to the Rhabdoviridae family, which includes the genus Vesiculovirus (e.g., Vesicular Stomatitis Virus) and the genus Rabies virus (e.g., Rabies). Within the Rhabdoviridae family, suitable antigens can be derived from the G protein or N protein. The Filoviridae family, which includes hemorrhagic fever viruses such as Marburg virus and Ebola virus, can be a source of suitable antigens. The Paramyxoviridae family includes the pneumoviruses, including parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubulavirus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, morbillivirus (including measles and canine distemper), and respiratory syncytial virus (as well as the glyco (G) protein and fusion (F) protein, sequences of which are available from GenBank). Influenza virus is classified within the Orthomyxoviridae family and can be a source of suitable antigens (e.g., HA protein, N1 protein). The family Bunyaviridae includes the genera Bunyavirus (California encephalitis, La Crosse), Phlebovirus (Rift Valley fever), Hantavirus (Puremala is a Hemahagin fever virus), Nairovirus (Nairobi sheep disease) and various non-designated Bunyaviruses. The family Arenaviridae provides the source of antigens for LCM and Lassa fever viruses. The family Reoviridae includes the genera Reovirus, Rotavirus (causing acute gastroenteritis in children), Orbivirus and Calcivirus (Colorado tick fever, Lebombo (human), equine encephalitis, bluetongue). The family Retroviridae includes the subfamily Oncovirinae, which encompasses human and veterinary diseases such as feline leukemia virus, HTLV-I and HTLV-II, the subfamily Lentivirinae (which includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus and the subfamily Spumavirinae). Among lentiviruses, many suitable antigens have been described and can be readily selected.The Papovaviridae family includes the Polyomavirinae (BKU and JCU viruses) and Papillomavirinae (associated with malignant progression of cancer or papillomas). The Adenoviridae family includes viruses that cause respiratory disease and / or enteritis (EX, AD7, ARD, OB). The Parvoviridae family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The Herpesviridae family includes the Alphaherpesvirinae, which includes the genera Simplexvirus (HSVI, HSVII), Varicellovirus (pseudorabies, varicella zoster), and the Betaherpesvirinae, which includes the genus Cytomegalovirus (human CMV, Muromegalovirus), as well as the Gammaherpesvirinae, which encompasses the genus Lymphocryptovirus, EBV (Burkitt's lymphoma), infectious rhinotracheitis, Marek's disease virus, and Rhadinovirus. The family Poxviridae includes the genera Orthopoxvirus (variola (smallpox) and Vaccinia (cowpox)), the subfamily Vertebropoxvirinae, which includes Parapoxvirus, Avipoxvirus, Capripoxvirus, Rabbitpoxvirus, Suipoxvirus, and the subfamily Entomopoxvirinae. The family Hepadnaviridae includes Hepatitis B virus. One unclassified virus that may be a suitable source of antigen is the hepatitis delta virus. Further viral sources may include chicken infectious bursal disease virus and porcine respiratory and reproductive syndrome virus. The family Alphaviridae includes equine arteritis virus and various encephalitis viruses.

[0127] Antigens that can be incorporated (e.g., encoded in a cassette) into a vaccine also include immunogens useful for immunizing humans or non-human animals against pathogens including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates. Examples of bacterial pathogens include pathogenic gram-positive cocci including pneumococci, staphylococci, and streptococci. Pathogenic gram-negative cocci include Neisseria meningitidis and Neisseria gonorrhoeae. Pathogenic enteric gram-negative bacilli include Enterobacteriaceae; Pseudomonas, Acinetobacteria, and Eikenella; Melioidosis; Salmonella; Shigella; Haemophilus influenzae, Haemophilus somnus; Moraxella; H. ducreyi (causing chancroid); Brucella; Franisella tularensis (causing tularemia); Yersinia (Pasteurella); Streptobacillus moniliformis, and Spirillum. Gram-positive bacilli include Listeria monocytogenes; Erysipelothrix rhusiopathia; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); donovanosis (granuloma venereum); and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus, botulism, other clostridial diseases, tuberculosis, leprosy, and other mycobacteria. Specific examples of bacterial species include, but are not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Moraxella catarrhalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia pneumoniae.pneumoniae, Chlamydia psittaci, Bordetella pertussis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris vulgaris, Staphylococcus aureus, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.gallisepticum). Pathogenic spirochetal diseases include syphilis; treponematosis; yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by higher pathogenic bacteria and fungi include actinomycosis; nocardiosis; cryptococcosis (Cryptococcus), blastomycosis (Blastomyces), histoplasmosis (Histoplasma), and coccidioidomycosis (Coccidioides); candidiasis (Candida), aspergillosis (Aspergillus), and mucormycosis; sporotrichosis; paracoccidioidomycosis, petriellidiosis, torulopsis, mycetoma, and chromomycosis; and dermatophytosis. Rickettsial infections include typhus, Rocky Mountain spotted fever, Q fever, and rickettsialpox. Examples of mycoplasma and chlamydia infections include Mycoplasma pneumoniae, lymphogranuloma venereum, psittacosis, and perinatal chlamydia infections. Pathogenic eukaryotes include pathogenic protozoa and helminths, including infections caused by amebiasis, malaria, leishmaniasis (e.g., caused by Leishmania major), trypanosomiasis, toxoplasmosis (e.g., caused by Toxoplasma gondii), Pneumocystis carinii, Trichans, Toxoplasma gondii, babesiosis, giardiasis (e.g., caused by Giardia), trichinosis (e.g., caused by Trichomonas), filariasis, schistosomiasis (e.g., caused by Schistosoma blood fluke), nematodes, trematode or fluke, and tapeworm (tapeworm) infections. Other parasitic infections include those caused by Ascaris, Trichuris, Cryptosporidium, and Pneumocystis carinii, among others.

[0128] Also disclosed herein are peptides derived from any polynucleotide associated with an infectious pathogenic organism, an infectious disease in a subject, or an infected cell in a subject. The antigen may be derived from a nucleic acid sequence or a polypeptide sequence of an infectious pathogenic organism. Polypeptide sequences of infectious pathogenic organisms include, but are not limited to, pathogen-derived peptides, virus-derived peptides, bacteria-derived peptides, fungi-derived peptides, and / or parasite-derived peptides. Infectious pathogenic organisms include, but are not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae virus, and tuberculosis.

[0129] Antigens can be selected that are predicted to be presented on the cell surface of cells such as tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic.

[0130] The one or more polypeptides encoded by the antigen nucleotide sequence can include at least one of the following: binding affinity to MHC with an IC50 value of less than 1000 nM, a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I peptides, the presence of a sequence motif within or near the peptide that promotes proteasomal cleavage, and the presence of a sequence motif that promotes TAP transport, a length of 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids for MHC class II polypeptides, the presence of a sequence motif within or near the peptide-promoted cleavage site by extracellular or lysosomal proteases (e.g., cathepsins) or the HLA binding site catalyzed by HLA-DM.

[0131] The one or more antigens may be present on the surface of a tumor. The one or more antigens may be present on the surface of an infected cell.

[0132] One or more antigens may be immunogenic in a subject having a tumor (e.g., capable of stimulating a T cell and / or B cell response in the subject). One or more antigens may be immunogenic in a subject having or suspected of having an infectious disease (e.g., capable of stimulating a T cell and / or B cell response in the subject). One or more antigens may be immunogenic in a subject at risk for an infectious disease (e.g., capable of stimulating a T cell and / or B cell response in the subject that confers immunological defense (i.e., immunity) against an infectious disease, such as stimulating the production of memory T cells, memory B cells, and / or antibodies specific for the infectious disease).

[0133] One or more antigens may be able to stimulate a B cell response, such as the production of antibodies that recognize one or more antigens (e.g., antibodies that recognize infectious disease antigens). Antibodies may recognize linear polypeptide sequences or recognize secondary and tertiary structures. Thus, B cell antigens may include linear polypeptide sequences or polypeptides with secondary and tertiary structures, including but not limited to full-length proteins, protein subunits, protein domains, or any polypeptides known or predicted to have secondary and tertiary structures. Antigens that can induce a B cell response to infection are found on the surface of infectious disease organisms. Antigens that can induce a B cell response to infection may be intracellular antigens expressed in infectious disease organisms.

[0134] The one or more antigens can include a combination of an antigen capable of stimulating a T cell response (e.g., a peptide containing a predicted T cell epitope sequence) and a different antigen capable of stimulating a B cell response (e.g., a full-length protein, a protein subunit, a protein domain).

[0135] One or more antigens that stimulate an autoimmune response in a subject can be excluded from consideration in the context of generating a vaccine for a subject.

[0136] The size of the at least one antigenic peptide molecule (e.g., epitope sequence) can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102 In a specific embodiment, the antigenic peptide molecule is 50 or less amino acid residues.

[0137] Antigenic peptides and polypeptides can be 15 residues or less in length, usually between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; and can be 6 to 30 residues for MHC class II.

[0138] If desired, longer peptides can be designed in several ways. In one example, where the presentation potential of the peptides on HLA alleles is predicted or known, the longer peptides can consist of either (1) individual presented peptides with 2-5 amino acid extensions towards the N-terminus and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the presented peptides with the extended sequence for each. In another case, where sequencing reveals the presence of long (more than 10 residues) neoepitope sequences in the tumor (e.g., due to frameshift, read-through, or intron introduction resulting in a novel peptide sequence), the longer peptides can consist of (3) the entire stretch of novel tumor-specific or infection-specific amino acids (which avoids the need to select the most strongly HLA-presented shorter peptides based on computational or in vitro test selection). In either case, the longer peptides can allow for endogenous processing by the patient's cells, resulting in more effective antigen presentation and stimulation of T cell responses. Longer peptides can include full-length proteins, protein subunits, protein domains, and combinations of peptides thereof, such as those expressed in infectious pathogenic organisms. Inclusion of longer peptides (e.g., full-length proteins, protein subunits, or protein domains) and combinations thereof can stimulate a B cell response.

[0139] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, antigenic peptides or polypeptides are presented on HLA proteins with higher affinity than wild-type peptides. In some embodiments, antigenic peptides or polypeptides can have an IC50 of at least 5000nM or less, at least 1000nM or less, at least 500nM or less, at least 250nM or less, at least 200nM or less, at least 150nM or less, at least 100nM or less, at least 50nM or less.

[0140] In some aspects, the antigenic peptides and polypeptides do not stimulate an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0141] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the composition comprises at least two different peptides. The at least two different peptides may be derived from the same polypeptide. Different peptides means that the peptides differ in length, amino acid sequence, or both. The tumor-specific peptides can be derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, for example, any polypeptide known or found to contain tumor-specific mutations or peptides derived from any polypeptide known or found to be aberrantly expressed in tumor cells or cancerous tissues compared to normal cells or tissues. The peptides can be derived from any polypeptide known or suspected to be associated with infectious disease organisms, or the peptides can be derived from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., infectious disease polynucleotides or polypeptides including infectious disease polynucleotides or polypeptides whose expression is restricted to host cells). Suitable polypeptides from which antigenic peptides can be derived can be found, for example, in the COSMIC database or the AACR GENIE (Genomics Evidence Neoplasia Information Exchange) database. COSMIC curates comprehensive information on somatic mutations in human cancers. AACR GENIE aggregates and links clinical-grade cancer genomic data with clinical outcomes from tens of thousands of cancer patients. The peptides can include tumor-specific mutations. In some embodiments, the tumor-specific mutations are driver mutations for a particular cancer type.

[0142] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desired attributes, e.g., improved pharmacological characteristics, while at least retaining substantially all or augmenting the biological activity of the unmodified peptides to bind to desired MHC molecules and activate appropriate T cells. By way of example, antigenic peptides and polypeptides can be further subjected to various modifications, such as either conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitution means replacing an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue. Substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

[0143] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in increasing the stability of peptides and polypeptides in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Peptide half-life can be conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows: Pooled human serum (type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, small aliquots of the reaction solution are removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4° C.) for 15 minutes and then spun to precipitate the precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatographic conditions.

[0144] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. As an example, the ability of a peptide to stimulate CTL activity can be enhanced by linkage to a sequence containing at least one epitope capable of stimulating a T helper cell response. The immunogenic peptide / T helper conjugate can be linked by a spacer molecule. The spacer is typically composed of relatively small neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacer is typically selected, for example, from Ala, Gly, or other neutral spacers of non-polar amino acids or neutral polar amino acids. It will be understood that the spacer, if present, need not be composed of the same residues and thus may be a hetero- or homo-oligomer. If present, the spacer will usually be at least 1 or 2 residues, more usually 3-6 residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0145] The antigenic peptide can be linked to the T helper peptide either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides include 830-843 of tetanus toxoid, 307-319 of influenza, and around 382-398 and 378-389 of malaria sporozoites.

[0146] Proteins or peptides can be produced by any technique known to those skilled in the art, including expressing the protein, polypeptide, or peptide through standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0147] In further embodiments, the antigen comprises a nucleic acid (e.g., polynucleotide) encoding an antigenic peptide or a portion thereof. The polynucleotide can be, for example, a single-stranded and / or double-stranded polynucleotide, such as DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), e.g., a polynucleotide having a phosphorothioate backbone, or in either a natural or stabilized form, or a combination thereof, and may or may not contain introns. The polynucleotide sequence encoding the antigen can be sequence-optimized to improve expression, such as by improving transcription, translation, post-transcriptional processing, and / or RNA stability. For example, the polynucleotide sequence encoding the antigen can be codon-optimized. As used herein, "codon optimization" refers to the replacement of low-frequency codons with high-frequency synonymous codons with respect to the codon bias of a particular organism. The polynucleotide sequence can be optimized to improve post-transcriptional processing, for example, by removing splicing motifs (e.g., canonical and / or cryptic / non-canonical splice donor, branch, and / or acceptor sequences) and / or by introducing exogenous splicing motifs (splice donor, branch, and / or acceptor sequences) to bias toward favorable splicing events, to reduce unintended splicing. Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., SV40 mini-intron) and / or immunoglobulins (e.g., human β-globulin gene). Exogenous intron sequences can be incorporated between the promoter / enhancer sequence and the antigen(s) sequence. Exogenous intron sequences for use in expression vectors are described in detail in Callendret et al. (Virology. 2007 Jul. 5;363(2):288-302), each of which is incorporated by reference in its entirety for all purposes.The polynucleotide sequence can be optimized to improve transcript stability, for example, by removing RNA stability motifs (e.g., AU-rich elements and / or 3'UTR motifs) and / or repetitive nucleotide sequences. The polynucleotide sequence can be optimized to improve accurate transcription, for example, by removing cryptic transcription initiators and / or terminators. The polynucleotide sequence can be optimized to improve translation and translation accuracy, for example, by removing cryptic AUG start codons, premature polyA sequences, and / or secondary structure motifs. The polynucleotide sequence can be optimized to improve nuclear export of the transcript, such as by adding a constitutive transport element (CTE), an RNA transport element (RTE), or a woodchuck posttranscriptional regulatory element (WPRE). Nuclear export signals for use in expression vectors are described in detail by Callendret et al. (Virology. 2007 Jul 5;363(2):288-302), each of which is incorporated herein by reference in its entirety for all purposes. A polynucleotide sequence can be optimized for GC content, for example, to reflect the average GC content of a particular organism. Sequence optimization can balance one or more sequence properties, such as transcription, translation, post-transcriptional processing, and / or RNA stability. Sequence optimization can generate an optimal sequence that balances each of transcription, translation, post-transcriptional processing, and RNA stability. Sequence optimization algorithms are well known to those skilled in the art, such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), Cool Tool (University of Singapore), SGI-DNA (La Jolla California). One or more regions of the antigen-encoding protein can be sequence-optimized separately. Yet a further aspect provides an expression vector capable of expressing a polypeptide or a portion thereof. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation.Generally, the DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, and such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0148] Vaccine Compositions Also disclosed herein are immunogenic compositions, e.g., vaccine compositions, that can generate a specific immune response, e.g., a tumor-specific immune response or an infectious pathogen-specific immune response. Vaccine compositions typically include one or more antigens selected using the methods described herein or selected from pathogen-, virus-, bacteria-, fungus-, and / or parasite-derived peptides. Vaccine compositions can also be referred to as vaccines.

[0149] The vaccine can include 1-30 peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different peptides, or 12, 13, or 14 different peptides. The peptides may have post-translational modifications. The vaccines contain 1 to 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different nucleotide sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences, or 12, 13, or 14 different antigen-encoding nucleic acid sequences.The vaccine contains antigen sequences 1-30, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, The antigen sequences may include 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different antigen sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen sequences, or 12, 13, or 14 different antigen sequences.

[0150] The vaccines comprise 1-30 antigen-encoding nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 109, The nucleic acid sequence may include 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different antigen-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences, or 12, 13, or 14 different antigen-encoding nucleic acid sequences. An antigen-encoding nucleic acid sequence may also be referred to as the antigen-encoding portion of an antigen "cassette." The characteristics of an antigen cassette are described in more detail below. An antigen-encoding nucleic acid sequence can include one or more epitope-encoding nucleic acid sequences (eg, an antigen-encoding nucleic acid sequence that encodes linked T cell epitopes).

[0151] The vaccines may comprise one to 30 different epitope-encoding nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more different epitope-encoding nucleic acid sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different epitope-encoding nucleic acid sequences, or 12, 13, or 14 different epitope-encoding nucleic acid sequences. An epitope-encoding nucleic acid sequence may also refer to a sequence of individual epitope sequences, such as each of the T cell epitopes in an antigen-encoding nucleic acid sequence that encodes linked T cell epitope-encoding nucleic acid sequences.

[0152] The vaccine may comprise at least two repeats of an epitope-encoding nucleic acid sequence. As used herein, "repeated sequence" refers to two or more repeats of the same nucleic acid epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence described herein) within an antigen-encoding nucleic acid sequence. In one example, the antigen-encoding nucleic acid sequence portion of the cassette encodes at least two repeats of an epitope-encoding nucleic acid sequence. In a further non-limiting example, the antigen-encoding nucleic acid sequence portion of the cassette encodes a plurality of different epitopes, at least one of the different epitopes being encoded by at least two repeats of a nucleic acid sequence encoding a different epitope (i.e., at least two different epitope-encoding nucleic acid sequences). In an illustrative non-limiting example, the antigen-encoding nucleic acid sequence comprises an epitope-encoding nucleic acid sequence A(E A ), epitope coding sequence B (E B), and epitope coding sequence C (E C ) and having repeats of at least one of the different epitopes is shown, without limitation, by the formula: - Repeats of one different epitope (repeats of epitope A): E A -E B -E C -E A ,or E A -E A -E B -E C - Repeats of several different epitopes (repeats of epitopes A, B and C): E A -E B -E C -E A -E B -E C ,or E A -E A -E B -E B -E C -E C - multiple repeats of multiple different epitopes (repeats of epitopes A, B and C): E A -E B -E C -E A -E B -E C -E A -E B -E C ,or E A -E A -E A -E B -E B -E B -E C -E C -E C .

[0153] The above examples are not limiting, and an antigen-encoding nucleic acid sequence having at least one repeat of different epitopes can encode each of the different epitopes in any order or frequency. For example, the order and frequency can be, for example, a sequence of the formula E A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B It can be a random arrangement of different epitopes, such as in the example with epitopes A, B, and C according to the formula:

[0154] As used herein, the amino acid sequence is represented in the 5' to 3' direction by the formula: (E x -(E N n ) y ) z An antigen-encoding cassette is provided having at least one antigen-encoding nucleic acid sequence represented by During the ceremony, E represents a nucleotide sequence comprising at least one different epitope-encoding nucleic acid sequence; n represents the number of distinct epitope-encoding nucleic acid sequences and is any integer including 0; E N represents a nucleotide sequence that contains a separate distinct epitope-encoding nucleic acid sequence for each corresponding n, For each repetition of z, at each n, x=0 or 1, y=0 or 1, and at least one of x or y is 1; and z=2 or more, and the antigen-encoding nucleic acid sequence is E, a specific E N or a combination thereof.

[0155] Each E or E Ncan independently comprise an epitope-encoding nucleic acid sequence described herein (e.g., a peptide encoding an infectious disease T cell epitope and / or a neoantigen epitope). For example, each E or E N is the formula (L5 b -N c -L3 d ), wherein N is each E or E N where L5 comprises a 5' linker sequence and b = 0 or 1, and L3 comprises a 3' linker sequence and d = 0 or 1. Epitopes and linkers that can be used are further described herein.

[0156] Repeats of epitope-encoding nucleic acid sequences (including any 5' linker sequence and / or any 3' linker sequence) may be directly linked to each other (e.g., as shown above, E A -E A -...). Repeated sequences of epitope-encoding nucleic acid sequences may be separated by one or more additional nucleotide sequences. Generally, repeated sequences of epitope-encoding nucleic acid sequences may be separated by nucleotide sequences of any size applicable to the compositions described herein. In one example, repeated sequences of epitope-encoding nucleic acid sequences may be separated by distinct and different epitope-encoding nucleic acid sequences (e.g., as shown above, E A -E B -E C -E A ...). In examples where the repeats are separated by single distinct and different epitope-encoding nucleic acid sequences, and each epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) encodes a peptide 25 amino acids in length, the repeats can be, for example, A -E B -E A …(E AIn one illustrative example, an antigen-encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT encoding repeats of the 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQ) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT), where the repeats of Trp1 are separated by the 25-mer Trp2, and thus the repeats of the Trp1 epitope-encoding nucleic acid sequence are separated by 75 nucleotides of the Trp2 epitope-encoding nucleic acid sequence. In examples where the repeat sequences are separated by 2, 3, 4, 5, 6, 7, 8, or 9 separate and distinct epitope-encoding nucleic acid sequences, each of which (including any 5' linker sequence and / or any 3' linker sequence) encodes a peptide 25 amino acids in length, the repeat sequences may be separated by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides, respectively.

[0157] In one embodiment, the different peptides and / or polypeptides, or the nucleotide sequences encoding them, are selected such that the peptides and / or polypeptides are capable of binding to different MHC molecules, such as different MHC class I molecules and / or different MHC class II molecules. In some aspects, one vaccine composition comprises coding sequences for peptides and / or polypeptides capable of binding to the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, the vaccine composition can comprise different fragments capable of binding to at least two preferred, at least three preferred, or at least four preferred MHC class I molecules and / or different MHC class II molecules.

[0158] The vaccine composition can stimulate a specific cytotoxic T cell response and / or a specific helper T cell response. The vaccine composition can stimulate a specific cytotoxic T cell response and a specific helper T cell response.

[0159] The vaccine composition is capable of stimulating a specific B cell response (eg, an antibody response).

[0160] The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition can stimulate a specific cytotoxic T cell response and a specific B cell response. The vaccine composition can stimulate a specific helper T cell response and a specific B cell response. The vaccine composition can stimulate a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response.

[0161] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The composition may be accompanied by a carrier, such as an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting the protein or peptide to a T cell.

[0162] An adjuvant is any substance that enhances or otherwise modifies the immune response to an antigen when mixed into a vaccine composition. The carrier may be a scaffold structure, such as a polypeptide or polysaccharide, to which the antigen can associate. Optionally, the adjuvant is attached by covalent or non-covalent bonds.

[0163] The ability of adjuvants to increase immune response to antigens is generally manifested as a significant or large increase in immune-mediated reactions or a reduction in disease symptoms.For example, the increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against antigens, and the increase in T cell activity is generally manifested as an increase in cell proliferation, or cytotoxicity, or cytokine secretion.Adjuvants can also change immune response, for example, by changing a predominantly humoral or Th response to a predominantly cellular or Th response.

[0164] Suitable adjuvants include, but are not limited to, 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA206, Montanide ISA 50V, Montanide Adjuvants include ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterium extracts and synthetic bacterial wall mimics, as well as other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are useful. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their formulations have been described (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11). Cytokines can also be used. Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0165] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in a vaccine environment. Other TLR binding molecules, such as RNA binding TLR 7, TLR 8 and / or TLR 9, can also be used.

[0166] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), Poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies that may have a therapeutic effect and / or act as an adjuvant, such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. The amounts and concentrations of adjuvants and additives can be readily determined by one of skill in the art without undue experimentation. Further adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0167] Vaccine compositions can include multiple different adjuvants. Additionally, therapeutic compositions can include any adjuvant material, including any of the above or a combination thereof. Vaccines and adjuvants can be administered together or separately in any suitable order.

[0168] A carrier (or excipient) may be present independent of the adjuvant. The function of the carrier may be to enhance activity or immunogenicity, confer stability, enhance biological activity, or extend serum half-life, for example by increasing the molecular weight of a particular variant. Additionally, the carrier may aid in the presentation of the peptide to T cells. The carrier may be any suitable carrier known to those skilled in the art, for example, a protein or an antigen-presenting cell. Carrier proteins may be, but are not limited to, keyhole limpet hemocyanin; serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin; immunoglobulins; hormones such as insulin or palmitic acid. For human immunization, the carrier is generally a safe, physiologically acceptable carrier that is tolerated by humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be a dextran, for example, sepharose.

[0169] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Thus, activation of CTLs is possible when a trimeric complex of peptide antigen, MHC molecule and APC is present. Correspondingly, CTLs can enhance immune responses when APCs bearing the corresponding MHC molecules are further added, rather than when only peptides are used to activate CTLs. Thus, in certain embodiments, the vaccine composition further comprises at least one antigen-presenting cell.

[0170] Antigens may also be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including but not limited to second, third, or hybrid second / third generation lentiviruses, and any generation of recombinant lentiviruses designed to target a specific cell type or receptor (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human The ubiquitin C promoter, Nucl.AcidsRes.(2015)43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J.Virol.(1998)72(12):9873-9880) can also be included in a viral vector-based vaccine platform. Depending on the packaging capacity of the above-mentioned viral vector-based vaccine platform, this approach can deliver one or more nucleotide sequences encoding one or more antigen peptides.The sequence may be flanked by non-mutated sequences, separated by linkers, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of antigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4): 433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291): 1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13): 3401-10). Upon introduction into the host, the infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration of antigens or immunization, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0171] Antigen cassette The methods used to select one or more antigens, the cloning and construction of an "antigen cassette" and its insertion into a viral vector are within the skill of the art in view of the teachings provided herein. By "antigen cassette" is meant a combination of a selected antigen or antigens (e.g., an antigen-encoding nucleic acid sequence) with other regulatory elements required to transcribe the antigen(s) and express the transcript. The selected antigen or antigens may refer to different epitope sequences (e.g., an antigen-encoding nucleic acid sequence in a cassette may encode an epitope-encoding nucleic acid sequence (or epitope-encoding nucleic acid sequences) such that the epitopes are transcribed and expressed). The antigen or antigens may be operably linked to a regulatory element in a manner that allows transcription. Such elements include conventional regulatory elements that can induce expression of the antigen(s) in cells transfected with the viral vector. Thus, the antigen cassette may also include a selected promoter linked to the antigen(s) and located with other optional regulatory elements within the selected viral sequence of the recombinant vector. The cassette can include one or more antigens, such as one or more pathogen-derived, virus-derived, bacteria-derived, fungal-derived, parasite-derived, and / or tumor-derived peptides. The cassette can have one or more antigen-encoding nucleic acid sequences, such as a cassette that includes multiple antigen-encoding nucleic acid sequences, each independently operably linked to a separate promoter and / or linked to each other using 2A ribosome skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or other multicistronic systems, such as internal ribosome entry site (IRES) sequence elements. The linker can also have a cleavage site, such as a TEV or furin cleavage site. Linkers with cleavage sites can also be combined with other elements, such as elements in multicistronic systems.In a non-limiting illustrative example, a furin protease cleavage site can be used in combination with a 2A ribosomal skipping sequence element to configure the furin protease cleavage site to facilitate removal of the 2A sequence after translation. In a cassette comprising multiple antigen-encoding nucleic acid sequences, each antigen-encoding nucleic acid sequence can include one or more epitope-encoding nucleic acid sequences (e.g., antigen-encoding nucleic acid sequences encoding linked T cell epitopes).

[0172] Useful promoters may be constitutive promoters or regulated (inducible) promoters that allow the amount of antigen(s) to be expressed to be controlled. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer [see, for example, Boshart et al, Cell, 41:521-530 (1985)]. Another desirable promoter is the Rous sarcoma virus LTR promoter / enhancer. Yet another promoter / enhancer sequence is the chicken β-actin promoter [TAKost et al, Nucl. Acids Res., 11 (23): 8287 (1983)]. Those skilled in the art can also select other suitable or desirable promoters.

[0173] The antigen cassette may also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences that provide signals for efficient polyadenylation of the transcript (poly(A), polyA or pA) and introns containing functional splice donor and acceptor sites. A common polyA sequence used in the exemplary vectors herein is derived from the papovavirus SV-40. The polyA sequence can be inserted into the cassette after the antigen-based sequence and before the viral vector sequence. A common intron sequence may also be derived from SV-40 and is referred to as the SV-40T intron sequence. The antigen cassette may contain an intron located between the promoter / enhancer sequence and the antigen(s). The selection of these and other common vector elements is conventional [see, for example, Sambrook et al, "Molecular Cloning. A Laboratory Manual.", 2d edit., Cold Spring Harbor Laboratory, New York (1989) and references cited therein], and many such sequences are available from commercial and industrial sources, as well as Genbank.

[0174] An antigen cassette can have one or more antigens. For example, a particular cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens. The antigens may be directly linked to each other. The antigens may be linked to each other by a linker. The antigens can be in any orientation relative to each other, including N-C or C-N.

[0175] As noted elsewhere herein, the antigen cassette can be placed within any selected deletion site within the viral vector, such as, for example, the site of the deleted structural proteins of the VEEV backbone or the deleted E1 gene region or deleted E3 gene region of ChAd-based vectors, among other choices.

[0176] The antigen cassette has the following formula, which describes the sequence of each element in the order from 5' to 3': (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g and Wherein P and P2 comprise a promoter nucleotide sequence, N comprises an MHC class I epitope-encoding nucleic acid sequence, L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequence encoding an amino acid linker, U comprises an MHC class II antigen-encoding nucleic acid sequence, and for each X, the corresponding Nc is an epitope-encoding nucleic acid sequence, and for each Y, the corresponding Uf is a universal MHC class II epitope-encoding nucleic acid sequence. The universal sequence can comprise at least one of tetanus toxoid and PADRE. The universal sequence can comprise a tetanus toxoid peptide. The universal sequence can comprise a PADRE peptide. The universal sequence can comprise a tetanus toxoid and a PADRE peptide. The compositions and ordered sequences can be further defined by selecting the number of elements present, e.g., a=0 or 1, b=0 or 1, c=1, d=0 or 1, e=0 or 1, f=1, g=0 or 1, h=0 or 1, X=1-400, Y=0, 1, 2, 3, 4 or 5, Z=1-400, and W=0, 1, 2, 3, 4 or 5.

[0177] In one example, the elements present are a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, where the case is described where there is no additional promoter (e.g., only the promoter nucleotide sequence provided by the vector backbone, such as an RNA alphavirus backbone, is present), there are 10 MHC class I epitopes, there is a 5' linker for each N, there is a 3' linker for each N, there are 2 MHC class II epitopes, there is a linker connecting the 2 MHC class II epitopes, there is a linker connecting the 5' ends of the 2 MHC class II epitopes to the 3' linker of the last MHC class I epitope, and there is a linker connecting the 3' ends of the 2 MHC class II epitopes to the vector backbone (e.g., an RNA alphavirus backbone). Examples of linking the 3' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include linking directly to a 3'UTR element provided by the vector backbone, such as the 3' 19 nt CSE. Examples of linking the 5' end of the antigen cassette to a vector backbone (e.g., an RNA alphavirus backbone) include linking directly to a promoter or 5'UTR element of the vector backbone, such as a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence), an alphavirus 5'UTR, a 51 nt CSE, or a 24 nt CSE.

[0178] Other examples include when a=1, describing the presence of a promoter other than the promoter nucleotide sequence provided by the vector backbone (e.g., an RNA alphavirus backbone); when a=1 and Z is greater than 1, describing the presence of multiple promoters other than the promoter nucleotide sequence provided by the vector backbone, each of which results in expression of one or more different MHC Class I epitope-encoding nucleic acid sequences; when h=1, describing the presence of another promoter that results in expression of an MHC Class II epitope-encoding nucleic acid sequence; and when g=0, describing the presence of an MHC Class II epitope-encoding nucleic acid sequence (if present) that is directly linked to the vector backbone (e.g., an RNA alphavirus backbone).

[0179] Other examples include where each MHC class I epitope present has a 5' linker, a 3' linker, neither, or both. In examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have both a 5' linker and a 3' linker, while other MHC class I epitopes may have a 5' linker or a 3' linker, or neither. In other examples where multiple MHC class I epitopes are present in the same antigen cassette, some MHC class I epitopes may have either a 5' linker or a 3' linker, while other MHC class I epitopes may have a 5' linker or a 3' linker, or neither.

[0180] In instances where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have both a 5' linker and a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither. In other instances where multiple MHC class II epitopes are present within the same antigen cassette, some MHC class II epitopes may have either a 5' linker or a 3' linker, while other MHC class II epitopes may have a 5' linker, a 3' linker, or neither.

[0181] Other examples include cases where each antigen present has a 5' linker, a 3' linker, neither, or both. In examples where multiple antigens are present in the same antigen cassette, some antigens may have both a 5' linker and a 3' linker, while other antigens may have a 5' linker or a 3' linker, or neither. In other examples where multiple antigens are present in the same antigen cassette, some antigens may have either a 5' linker or a 3' linker, while other antigens may have a 5' linker or a 3' linker, or neither.

[0182] The promoter nucleotide sequence P and / or P2 may be the same as the promoter nucleotide sequence provided by the vector backbone, such as the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the vector backbone may each comprise a subgenomic promoter sequence (e.g., a 26S subgenomic promoter) or a CMV promoter. The promoter nucleotide sequence P and / or P2 may be different from the promoter nucleotide sequence provided by the vector backbone (e.g., the RNA alphavirus backbone) and may be different from each other.

[0183] The 5' linker L5 may be a natural sequence or a non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 may also be a natural sequence or a non-natural sequence. Furthermore, L5 and L3 may both be natural sequences, both may be non-natural sequences, or one may be natural and the other non-natural. For each X, the amino acid linker may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The lengths may be 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. For each X, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0184] For each Y, the amino acid linker G5 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. For each Y, the amino acid linker may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0185] The amino acid linker G3 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 amino acids. , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more lengths. G3 may also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0186] For each X, each N can code for an MHC class I epitope, an MHC class II epitope, an epitope / antigen capable of stimulating a B cell response, or a combination thereof. For each X, each N can code for a combination of an MHC class I epitope, an MHC class II epitope, and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for a combination of an MHC class I epitope and an MHC class II epitope. For each X, each N can code for a combination of an MHC class I epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for a combination of an MHC class II epitope and an epitope / antigen capable of stimulating a B cell response. For each X, each N can code for an MHC class II epitope. For each X, each N can code for an epitope / antigen capable of stimulating a B cell response. For each X, each N can encode an MHC class I epitope 7-15 amino acids in length. For each X, each N may encode an MHC class I epitope 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. For each X, each N may also encode an MHC class I epitope that is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.

[0187] The cassette encoding one or more antigens may be 700 nucleotides or less. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode two different epitope-encoding nucleic acid sequences (e.g., encoding two different infectious disease- or tumor-derived nucleic acid sequences encoding immunogenic polypeptides). The cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 700 nucleotides or less and may include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0188] The cassette encoding one or more antigens may be 375-700 nucleotides in length. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-700 nucleotides in length and may include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0189] The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be no more than 600, 500, 400, 300, 200, or 100 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be up to 600, 500, 400, 300, 200, or 100 nucleotides in length and can encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be up to 600, 500, 400, 300, 200, or 100 nucleotides in length and can include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0190] The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode at least two different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens may be 375-600, 375-500, or 375-400 nucleotides in length and may encode at least three different epitope-encoding nucleic acid sequences. The cassette encoding one or more antigens can be 375-600, 375-500, or 375-400 nucleotides in length and can contain 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.

[0191] immunomodulatory factors A vector as described herein, such as a C68 vector as described herein, or an alphavirus vector as described herein, can include a nucleic acid encoding at least one antigen, and the same or another vector can include a nucleic acid encoding at least one immunomodulator. The immunomodulator can include a binding molecule (e.g., an antibody such as an scFv) that binds to and blocks the activity of an immune checkpoint molecule. The immunomodulator can include a cytokine such as IL-2, IL-7, IL-12 (including IL-12 p35, p40, p70, and / or p70 fusion constructs), IL-15, or IL-21. The immunomodulator can include a modified cytokine (e.g., pegylated IL-2). The vector can include an antigen cassette and one or more nucleic acid molecules encoding an immunomodulator.

[0192] Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on all NK (gamma delta) and memory CD8+ (alpha beta) T cells), CD160 (also called BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, or other binding proteins that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). The antibody coding sequence can be engineered into a vector using ordinary skill in the art. One exemplary method is described in Fang et al., Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005 May;23(5):584-90. Epub 2005 Apr 17, incorporated herein by reference for all purposes.

[0193] Payload Code SAM Composition Also disclosed herein are SAM vectors having endogenous 5' sequences of the self-replicating virus from which the SAM vector is derived, e.g., encoding one or more payload nucleic acid sequences within the cassette (e.g., having endogenous 5'VEEV nucleotides AU, also referred to as "AU-SAM"). By "cassette" is meant a combination of a selected polynucleotide(s) (e.g., an antigen-encoding nucleic acid sequence) and other regulatory elements required to transcribe the polynucleotide(s) and, generally in the case of a coding sequence, to express a transcript. Also disclosed herein are SAM vector delivery compositions capable of delivering one or more payload nucleic acid sequences. The payload nucleic acid sequence can be any nucleic acid sequence that is desired to be delivered to a cell of interest. Generally, the payload is a nucleic acid sequence linked to a promoter or any translation tool (e.g., any 2A self-cleaving peptide sequence, such as IRES, P2A, E2A, F2A, and T2A) to drive expression of the nucleic acid sequence. The payload nucleic acid sequence can encode a polypeptide (i.e., a nucleic acid sequence that can be transcribed and translated into a protein). Generally, the payload nucleic acid sequence encoding the peptide can encode any protein that is desired to be expressed in a cell. Examples of proteins include, but are not limited to, antigens (e.g., MHC class I epitopes, MHC class II epitopes, or epitopes capable of stimulating a B-cell response), antibodies, cytokines, chimeric antigen receptors (CARs), T-cell receptors, or components of genome editing systems (e.g., nucleases used in genome editing systems). Genome editing systems include, but are not limited to, CRISPR systems, zinc finger systems, or TALEN systems. The payload nucleic acid sequence may be a non-coding sequence (i.e., a nucleic acid sequence that can be transcribed but not translated into a protein). Generally, the non-coding payload nucleic acid sequence can encode any non-coding polynucleotide that is desired to be expressed in a cell.Examples of non-coding polynucleotides include, but are not limited to, RNA interference (RNAi) polynucleotides (e.g., antisense oligonucleotides, shRNA, siRNA, miRNA, etc.) or polynucleotides of genome editing systems (e.g., guide [gRNA], single guide RNA [sgRNA], transactivating CRISPR [tracrRNA], and / or CRISPR RNA [crRNA] with various / different lengths). The payload nucleic acid sequence can encode two or more (e.g., 2, 3, 4, 5 or more) different polypeptides (e.g., two or more different epitope sequences linked together) or can include two or more different non-coding nucleic acid sequences (e.g., two or more different RNAi polypeptides). The payload nucleic acid sequence may have a combination of polypeptide-encoding and non-coding nucleic acid sequences.

[0194] Identification of antigens Research models for NGS analysis of tumor and normal exomes and transcriptomes have been previously described and applied in the antigen-specific space. 6,14,15 Specific optimizations can be considered to increase the sensitivity and specificity of antigen identification in clinical situations. These optimizations can be divided into two areas: those related to laboratory processes and those related to NGS data analysis. The described approaches can also be applied to antigen identification in other situations, such as identification from infectious pathogenic organisms, infectious diseases of subjects, or infected cells of subjects. Examples of optimization are well known to those skilled in the art, and such methods are described in more detail in, for example, U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, and International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0195] Methods for identifying antigens (e.g., antigens derived from a tumor or infectious pathogenic organism) include identifying antigens that are likely to be presented on a cell surface (e.g., presented by MHC on tumor cells, infected cells, or immune cells, including professional antigen-presenting cells such as dendritic cells) and / or that are likely to be immunogenic. By way of example, one such method includes obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing and / or expression data from a tumor, an infected cell, or an infectious pathogenic organism, where the nucleotide sequencing and / or expression data is used to obtain data representing peptide sequences for each of a set of antigens (e.g., antigens from the tumor or the infectious pathogenic organism); inputting the peptide sequences for each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens will be presented by one or more MHC alleles on a cell surface, such as a tumor cell or an infected cell, of the subject, where the set of numerical likelihoods has been determined based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens.

[0196] Truncal peptides, meaning peptides presented by all or most of the subclones, can be prioritized for inclusion in the vaccine. Optionally, if there are no trunk peptides that are predicted to be highly presented and immunogenic, or if the number of trunk peptides that are predicted to be highly presented and immunogenic is small enough that additional non-trunk peptides can be included in the vaccine, additional peptides can be prioritized by estimating the number and type of subclones and selecting peptides that maximize the number of subclones encompassed by the vaccine.

[0197] After all of the above antigen filters have been applied, there may still be more candidate antigens available for vaccine inclusion than vaccine technology can accommodate. Additionally, there may remain uncertainties about various aspects of antigen analysis, and trade-offs may exist between various properties of candidate vaccine antigens. Therefore, instead of pre-determined filters at each stage of the selection process, an integral multidimensional model can be considered, placing the candidate antigens in a space with at least the following axes, and optimizing the selection using an integral approach: 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmune risk is typically favorable) 2. Probability of sequencing artifacts (lower artifact probability is typically preferred) 3. Probability of immunogenicity (a higher probability of immunogenicity is typically preferred) 4. Probability of presentation (higher probabilities of presentation are typically preferred) 5. Gene Expression (higher expression is typically preferred) 6. HLA gene coverage (a higher number of HLA molecules involved in the presentation of a set of antigens may reduce the probability that tumors, infections, and / or infected cells will evade immune attack through downregulation or mutation of HLA molecules) 7. HLA class coverage (covering both HLA-I and HLA-II may increase the chances of a treatment response and decrease the chances of avoiding tumors or infections)

[0198] In addition, optionally, an antigen can be deprioritized (e.g., excluded) from vaccination if it is expected to be presented by HLA alleles that are lost or inactivated in all or part of the patient's tumor or infected cells. Loss of HLA alleles can occur either by somatic mutation, loss of heterozygosity, or homozygous deletion of loci. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic LOH and homozygous deletion (including HLA loci) have also been described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). An antigen can be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0199] Methods of Treatment and Manufacturing Also provided are methods of stimulating a tumor-specific immune response in a subject, vaccinating against a tumor, and treating and / or alleviating symptoms of cancer in a subject by administering to the subject one or more antigens, such as a plurality of antigens identified using the methods disclosed herein.

[0200] Also provided are methods for stimulating an infectious pathogen-specific immune response in a subject, vaccinating against an infectious pathogen, and treating and / or alleviating symptoms of infection associated with an infectious pathogen in a subject by administering to the subject one or more antigens, such as a plurality of antigens identified using the methods disclosed herein.

[0201] In some embodiments, the subject has been diagnosed with cancer or is at risk of developing cancer. The subject can be a human, a dog, a cat, a horse, or any animal in which a tumor-specific immune response is desired. The tumor can be any solid tumor, such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck tumor, pancreatic cancer, brain tumor, melanoma, and other tissue organ tumors, as well as hematological tumors, such as lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0202] In some embodiments, the subject has been diagnosed with or is at risk for an infectious disease, or is at risk for seasonal and / or emerging disease infection, such as due to age, geographic / travel, and / or work-related high risk or predisposition to an infectious disease.

[0203] The antigen can be administered in an amount sufficient to stimulate a CTL response. The antigen can be administered in an amount sufficient to stimulate a T cell response. The antigen can be administered in an amount sufficient to stimulate a B cell response.

[0204] Antigens can be administered alone or in combination with other therapeutic agents, which may include those that target infectious pathogenic organisms, such as antivirals or antibiotics.

[0205] Additionally, the subject may be further administered an anti-immunosuppressant / immunostimulatory agent, such as a checkpoint inhibitor. For example, the subject may be further administered an anti-CTLA antibody or anti-PD-1 or anti-PD-L1. Blocking CTLA4 or PD-L1 with an antibody may enhance the patient's immune response to cancerous cells. In particular, blocking CTLA4 has been shown to be effective when following a vaccination protocol. The optimal dosage regimen of the checkpoint inhibitor may be determined. For example, the checkpoint inhibitor composition may be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Methods of injection include subcutaneous (sc), intradermal (id), intraperitoneal (ip), intramuscular (im), and intravenous (iv).

[0206] The optimal amount and optimal administration regimen of each antigen included in the vaccine composition can be determined. For example, the antigen or its variants can be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Methods of injection include subcutaneous (sc), intradermal (id), intraperitoneal (ip), intramuscular (im), and intravenous (iv). Methods of injection of DNA or RNA include intradermal (id), intramuscular (im), subcutaneous (sc), intraperitoneal (ip), and intravenous (iv). Other methods of administration of the vaccine composition are known to those skilled in the art.

[0207] Vaccines can be adapted so that the selection, number, and / or amount of antigens present in the composition are tissue, cancer, infection, and / or patient specific. For example, the exact selection of peptides can be guided by the expression pattern of a particular parent protein, or by the mutation or disease state of the patient. This selection can depend on the particular type of cancer, the particular infection (e.g., the particular infection isolate / strain with which the subject is infected or at risk of infection), the disease state, the purpose of vaccination (e.g., preventive or targeting an ongoing disease), the initial treatment regimen, the immune status of the patient, and, of course, the HLA haplotype of the patient. Furthermore, vaccines can include components that are personalized according to the personal needs of a particular patient. Examples include altering the selection of antigens according to the expression of antigens in a particular patient, or adjusting secondary treatments after a first round or scheme of treatment.

[0208] Patients for administering antigen vaccines can be identified by using various diagnostic methods, such as the patient selection methods described further below. Patient selection can include identifying mutations or expression patterns of one or more genes. Patient selection can include identifying ongoing infections. Patient selection can include identifying risk of infection from infections. Optionally, patient selection can include identifying haplotypes of patients. Various patient selection methods can be performed in parallel, for example, sequencing diagnostics can identify both mutations and haplotypes of patients. Various patient selection methods can also be performed sequentially, for example, one diagnostic test can identify mutations and another diagnostic test can identify haplotypes of patients, where each test can be the same (e.g., both high-throughput sequencing) or different (e.g., one high-throughput sequencing and the other Sanger sequencing) diagnostic method.

[0209] For compositions to be used as vaccines for cancer or infectious diseases, antigens including similar normal self-peptides that are expressed in high amounts in normal tissues may be avoided or present in low amounts in the compositions described herein. In contrast, if a patient's tumor or infected cells are known to express a particular antigen in high amounts, the respective pharmaceutical composition for the treatment of this cancer or infection may be present in high amounts and / or may include multiple antigens specific for this particular antigen or the pathway of this antigen.

[0210] Compositions containing antigens can be administered to individuals already suffering from cancer or infectious diseases. In therapeutic applications, compositions are administered to patients in amounts sufficient to stimulate effective CTLs against tumor antigens or infectious pathogenic organism antigens and cure or at least partially prevent symptoms and / or complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this purpose depend, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the weight and general condition of the patient, and the judgment of the prescribing physician. Compositions can generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized or infectious pathogenic organisms have induced organ damage and / or other immune pathologies. In such cases, it is believed that, given the minimization of the relative non-toxic properties of the foreign substances and antigens, it is possible and may be felt desirable by the treating physician to administer significant excesses of these compositions.

[0211] In therapeutic applications, administration can begin upon detection or surgical removal of a tumor, or upon detection or treatment of an infection, followed by booster doses at least until symptoms have substantially disappeared and for a predetermined period thereafter, or until immunity is deemed achieved (e.g., memory B or T cell populations, or antigen-specific B cells or antibodies are produced).

[0212] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Pharmaceutical compositions can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. The compositions can be administered at the site of surgical resection to stimulate a local immune response against the tumor. The compositions can be administered to target specific infected tissues and / or cells of the subject. Disclosed herein are compositions for parenteral administration that include a solution of the antigen, the vaccine composition dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. For example, a variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques, or sterile filtered. The resulting aqueous solutions can be packaged for use as is, or can be lyophilized, and the lyophilized formulation can be combined with a sterile solution prior to administration. The compositions may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0213] Antigens can also be administered via liposomes, which target the antigen to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these formulations, the antigen to be delivered is incorporated as part of the liposome, either alone or in combination with a molecule that binds to a receptor prevalent on lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes loaded with the desired antigen can be directed to the site of lymphoid cells, where they deliver the selected therapeutic / immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is typically guided by considerations, for example, of the size of the liposome, acid lability, and stability of the liposome in the bloodstream. There are various methods for preparing liposomes, for example, as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0214] For targeting to immune cells, ligands incorporated into the liposomes can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. The liposomal suspensions can be administered intravenously, locally, or topically in different doses depending, inter alia, on the method of administration, the peptide being delivered, and the stage of the disease being treated.

[0215] For therapeutic or immunization purposes, nucleic acids encoding peptides and optionally one or more of the peptides described herein may be administered to a patient. Many methods for administering nucleic acids to a patient are conveniently used. For example, the nucleic acid can be administered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and in U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acid can also be administered using ballistic delivery, for example, as described in U.S. Pat. No. 5,204,253. Particles composed of DNA alone can also be administered. Alternatively, the DNA can be attached to particles such as gold particles. Approaches for delivering nucleic acid sequences include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation.

[0216] Nucleic acids can also be delivered by complexing with cationic compounds, such as cationic lipids.Lipid-mediated gene delivery methods are described, for example, in 9618372 WOAWO96 / 18372, 9324640 WOAWO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691(1988), U.S. Patent No. 5,279,833 Rose, U.S. Patent No. 5,279,833, 9106309 WOAWO91 / 06309, and Felgner et al., Proc.Natl.Acad.Sci.USA 84:7413-7414(1987).

[0217] Antigens may be derived from vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or from second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C ( promoter, Nucl. Acids Res. (2015) 43(1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12): 9873-9880). Depending on the packaging capacity of the viral vector-based vaccine platform mentioned above, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be flanked by non-mutated sequences, separated by a linker, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22(4): 433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352(6291): 1337-41, Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13): 3401-10). Once introduced into the host, the infected cells express the antigen, thereby stimulating an immune (e.g., CTL) response of the host against the peptide(s). Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful in administering therapeutic agents or immunizing antigens, such as, for example, Salmonella typhi vectors, will be apparent to those skilled in the art from the disclosure herein.

[0218] One means of administering nucleic acids is to use a minigene construct that encodes one or more epitope-encoding nucleic acid sequences. To generate a DNA sequence (minigene) encoding a selected CTL epitope for expression in human cells, the amino acid sequence of the epitope is reverse translated. A human codon usage table is used to guide the codon selection for each amino acid. These epitope-encoding DNA sequences are directly linked to generate a contiguous polypeptide sequence. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse translated and incorporated into the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Additionally, MHC presentation of CTL epitopes can be improved by incorporating synthetic (e.g., polyalanine) or natural flanking sequences adjacent to the CTL epitopes. The minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified using well-known methods, and annealed under appropriate conditions. The ends of the oligonucleotides are joined together using T4 DNA ligase. This synthetic minigene encoding the CTL epitope polypeptide can then be cloned into a desired expression vector.

[0219] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is to reconstitute lyophilized DNA with sterile phosphate-buffered saline (PBS). A variety of methods have been described and new techniques may become available. As mentioned above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds collectively referred to as PINCs (protective, interactive, non-condensing) can be complexed with purified plasmid DNA to affect variables such as stability, distribution in muscle, or trafficking to specific organs or cell types.

[0220] Also disclosed is a method of producing a vaccine comprising carrying out each of the steps of the methods disclosed herein and producing a vaccine comprising a plurality of antigens or a subset of a plurality of antigens.

[0221] The antigens disclosed herein can be produced using methods well known in the art. For example, the method of producing the antigens or vectors disclosed herein (e.g., vectors comprising at least one sequence encoding one or more antigens) can include culturing a host cell comprising at least one polynucleotide encoding the antigen or vector under suitable conditions to express the antigen or vector, and purifying the antigen or vector. Standard purification methods include chromatography, electrophoresis, immunological methods, precipitation, dialysis, filtration, concentration, and chromatofocusing.

[0222] The host cell may comprise a Chinese Hamster Ovary (CHO) cell, an NS0 cell, yeast, or a HEK293 cell. The host cell may be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding an antigen or vector disclosed herein, and optionally, the isolated polynucleotide comprises a promoter sequence operably linked to at least one nucleic acid sequence encoding the antigen or vector. In certain embodiments, the isolated polypeptide may be a cDNA.

[0223] Use and Administration of Antigens Vaccination protocols can be used to administer one or more antigens to a subject. A subject can be administered a priming vaccine and a booster vaccine.

[0224] The priming vaccine can be based on the SAM vaccine composition described herein, where the SAM has an endogenous 5' sequence of the autonomously replicating virus from which the SAM vector is derived (e.g., the endogenous 5'VEEV nucleotides AU, also referred to as "AU-SAM").

[0225] Booster vaccines (including two or more booster doses) can be based on the SAM vaccine compositions described herein, where the SAM has an endogenous 5' sequence of the autonomously replicating virus from which the SAM vector is derived (e.g., the endogenous 5'VEEV nucleotide AU, also referred to as "AU-SAM").

[0226] Vaccination protocols can include both priming and booster vaccines, each based on the SAM vaccine described herein, where the SAM has an endogenous 5' sequence of the autonomously replicating virus from which the SAM vector is derived (e.g., the endogenous 5'VEEV nucleotide AU, also referred to as "AU-SAM").

[0227] Priming vaccines, including those used in combination with a SAM having an endogenous 5' sequence, may be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SAM (e.g., the sequence shown in SEQ ID NO: 3 or 4). Booster vaccines, including those used in combination with a SAM having an endogenous 5' sequence, may be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or SAM (e.g., the sequence shown in SEQ ID NO: 3 or 4).

[0228] Each vector in the priming / booster approach typically contains a cassette containing antigens. The cassette can contain about 1-50 antigens separated by spacers, such as the natural sequences that typically surround each antigen, or other non-natural spacer sequences such as AAY. The cassette can contain an MHCII antigen, such as a tetanus toxoid antigen, and a PADRE antigen, which is considered a universal class II antigen. The cassette can contain a targeting sequence, such as a ubiquitin targeting sequence. Additionally, each vaccine dose can be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) an immune modulator. Each vaccine dose can be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) a checkpoint inhibitor (CPI). The CPI can include an antibody or antigen-binding portion thereof that inhibits CTLA4, PD1, and / or PDL1. Such antibodies can include tremelimumab or durvalumab. The CPI can include an antibody or antigen-binding portion thereof that inhibits CTLA4, PD1, and / or PDL1. Such antibodies may include tremelimumab or durvalumab. CPIs may be administered via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Each vaccine dose may be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) a cytokine such as IL-2, IL-7, IL-12 (including IL-12 p35, p40, p70, and / or p70 fusion constructs), IL-15, or IL-21. Each vaccine dose may be administered to a subject in combination with (e.g., simultaneously with, prior to, or after) a modified cytokine (e.g., pegylated IL-2).

[0229] The priming vaccine can be injected (e.g., intramuscularly) into the subject. Unilateral or bilateral injections can be used per dose. For example, one or more injections of ChAdV68 (C68) can be used (e.g., a total dose of 1×10 12100 virions), one or more injections of a low vaccine dose of the SAM vector selected from the range of 0.001-1 ug of RNA can be used.

[0230] A vaccine boost (booster vaccine) can be injected (e.g., intramuscularly) after the prime vaccination. The booster vaccine can be administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the prime, e.g., every 4 weeks and / or every 8 weeks. Unilateral or bilateral injections can be used for each dose. For example, one or more injections of ChAdV68 (C68) can be used (e.g., a total dose of 1×10 12 100 virions), one or more injections of a low vaccine dose of the SAM vector selected from the range of 0.001-1 ug of RNA can be used.

[0231] The dose of SAM can be administered as a priming dose or one or more booster doses. The priming dose and booster dose can be the same amount of SAM or different amounts of SAM. Each booster dose can be the same amount of SAM or different amounts of SAM. A SAM dose of 10-30 μg, 10-100 μg, 10-300 μg, 30-100 μg, 30-300 μg, or 100-300 μg RNA can be administered. A SAM dose of 10-500 μg, 10-1000 μg, 30-500 μg, 30-1000 μg, or 500-1000 μg RNA can be administered. A SAM dose of 1-30 μg, 1-100 μg, or 1-300 μg RNA can be administered. A SAM dose of 1-500 μg or 1-1000 μg RNA can be administered. A SAM dose of at least 400 μg, at least 500 μg, at least 600 μg, at least 700 μg, at least 800 μg, at least 900 μg, at least 1000 μg RNA may be administered. A SAM dose of at least 1 μg, 3 μg, 10 μg, or 30 μg RNA may be administered. A SAM dose of 1 μg, 3 μg, 10 μg, or 30 μg RNA may be administered. A SAM dose of 1-3 μg, 1-10 μg, 1-30 μg, 3-10 μg, 3-30 μg, or 10-30 μg RNA may be administered. A SAM dose of 10 μg, 30 μg, 100 μg, or 300 μg RNA may be administered. A SAM dose of 300 μg RNA may be administered. A SAM dose of 100 μg RNA may be administered. A SAM dose of 30 μg RNA may be administered. A SAM dose of 10 μg RNA may be administered. A SAM dose of 3 μg RNA may be administered. A SAM dose of 1 μg RNA may be administered. A SAM dose of at least 300 μg RNA may be administered. A SAM dose of at least 100 μg RNA may be administered. A SAM dose of at least 30 μg RNA may be administered. A SAM dose of at least 10 μg RNA may be administered. A SAM dose of at least 3 μg RNA may be administered. A SAM dose of at least 1 μg RNA may be administered.SAM doses of up to 300 μg RNA can be administered.

[0232] Anti-CTLA-4 (e.g., tremelimumab) can also be administered to the subject. For example, anti-CTLA4 can be administered subcutaneously near the site of intramuscular vaccine injection (ChAdV68 prime or SAM low dose) to ensure delivery to the same lymph nodes. Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4. Targeted anti-CTLA-4 (tremelimumab) subcutaneous doses are typically 70-75 mg (specifically 75 mg), e.g., in the dose range of 1-100 mg or 5-420 mg.

[0233] In certain cases, anti-PD-L1 antibodies such as durvalumab (MEDI4736) can be used. Durvalumab is a selective, high-affinity human IgG1 mAb that blocks the binding of PD-L1 to PD-1 and CD80. Durvalumab is typically administered intravenously at 20 mg / kg every 4 weeks.

[0234] Immune monitoring can be performed before, during, and / or after vaccine administration. Such monitoring can provide information about safety and efficacy, among other parameters.

[0235] PBMCs are generally used for immune monitoring. PBMCs can be isolated before and after prime vaccination (e.g., 4 weeks and 8 weeks). PBMCs can be collected immediately before and after each boost vaccination (e.g., 4 weeks and 8 weeks).

[0236] Immune responses, such as T cell responses and B cell responses, can be evaluated as part of an immune monitoring protocol. For example, the ability of the vaccine compositions described herein to stimulate an immune response can be monitored and / or evaluated. As used herein, "stimulating an immune response" refers to any increase in an immune response, such as initiating an immune response (e.g., a priming vaccine that stimulates the initiation of an immune response in a naive subject) or enhancing an immune response (e.g., a booster vaccine that stimulates the enhancement of an immune response in a subject with a pre-existing immune response to an antigen, such as a pre-existing immune response initiated by a priming vaccine). T cell responses can be measured using one or more methods well known to those skilled in the art, such as ELISpot, intracellular cytokine staining, cytokine secretion, and cell surface capture, T cell proliferation, MHC multimer staining, or cytotoxicity assays. T cell responses to epitopes encoded in the vaccine can be monitored from PBMCs by measuring induction of cytokines, such as IFN-γ, using an ELISpot assay. Specific CD4 or CD8 T cell responses to epitopes encoded in the vaccine can be monitored from PBMCs by measuring the induction of intracellularly or extracellularly captured cytokines such as IFN-γ using flow cytometry. Specific CD4 or CD8 T cell responses to epitopes encoded in the vaccine can be monitored from PBMCs by measuring T cell populations expressing specific T cell receptors for the epitope / MHC class I complex using MHC multimer staining. Specific CD4 or CD8 T cell responses to epitopes encoded in the vaccine can be monitored from PBMCs by measuring ex vivo proliferation of T cell populations after incorporation of 3H-thymidine, bromodeoxyuridine, and carboxyfluorescein diacetate succinimidyl ester (CFSE). Antigen recognition and lytic activity of PBMC-derived T cells specific for epitopes encoded in the vaccine can be functionally assessed by chromium release assays or alternative colorimetric cytotoxicity assays.

[0237] B cell responses can be measured using one or more methods well known in the art, such as assays used to determine B cell differentiation (e.g., differentiation into plasma cells), B cell or plasma cell proliferation, B cell or plasma cell activation (e.g., increase in costimulatory markers such as CD80 or CD86), antibody class switching, and / or antibody production (e.g., ELISA). Antibodies can also be assessed for functionality, such as by assessing neutralizing capacity. EXAMPLES

[0238] In order that the disclosure described herein may be more fully understood, the following examples are set forth below. The synthetic and biological examples described in this application are presented to illustrate the compounds, pharmaceutical compositions, and methods provided herein, and should not be construed as in any way limiting the scope thereof.

[0239] Materials and Methods The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. Where general or preferred reaction conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, it will be recognized that other reaction conditions can also be used unless otherwise specified. Optimum reaction conditions will vary with the particular reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization.

[0240] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be required to prevent certain functional groups from undergoing undesired reactions. The selection of an appropriate protecting group for a particular functional group, as well as suitable conditions for protection and deprotection, are well known in the art. For example, many protecting groups and their introduction and removal are described in TW Greene and PG M Huts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0241] The compounds provided herein can be isolated and purified by standard techniques known in the art, including but not limited to trituration, column chromatography, HPLC, or supercritical fluid chromatography (SFC). The compounds provided herein can be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

[0242] Example 1 Self-amplifying expression system A. Self-replicating RNA virus backbone and SAM formation In one implementation of the invention, an RNA alphavirus backbone for the antigen expression system was generated from the self-replicating Venezuelan Equine Encephalitis Virus ("VEEV" Kinney; 1986, Virology 152:400-413) by deleting the structural proteins of VEEV located 3' to the 26S subgenomic promoter, except for the last 50 amino acids of E1 (VEEV deleted 7544-11176; numbering from Kinney et al 1986, SEQ ID NO: 6). The deleted sequences were replaced with payload sequences to generate the self-replicating mRNA ("SAM") ssRNA vectors. An exemplary SAM vector containing 20 model antigens is the "VEE-MAG25-mer" (SEQ ID NO: 4). A modified T7 RNA polymerase promoter lacking the canonical 3' dinucleotide GG ((TAATACGACTCACTATA)) was added to the 5' end of the SAM vector to generate template DNA for in vitro transcription (SEQ ID NO: 57, no cassette inserted, deletion of 7544-11,176). An additional template generation vector was generated by adding the PCR forward primer sequence and a 3' restriction site (SEQ ID NO: 58, no cassette inserted, deletion of 7544-11,176).

[0243] RNA generated using the above template was synthesized by direct ligation to the endogenous 5'VEEV nucleotide sequence. 7 It has a G cap. That is, m 7 Between the G-cap and the endogenous 5'VEEV nucleotide sequence, there are no additional intervening nucleotides, such as the dinucleotide GG, that are normally present when using canonical T7 RNA polymerase. ssRNA generation for SAM vectors beginning with the endogenous nucleotide AUG and having a backbone using a canonical or modified ("minimal") T7 promoter is shown in Figure 1. 7 SAM vectors that have no additional intervening nucleotides located between the G-cap and the endogenous 5'AU nucleotide are referred to herein as "AU-SAM" vectors. A schematic diagram of a representative AU-SAM vector is shown in FIG.

[0244] Capped AU-SAM ssRNA vectors containing cassettes encoding representative payload sequences (e.g., GFP or β-spike) were cotranscriptionally generated using the following steps. - The antigen cassette of interest was cloned into the in vitro transcription template DNA (SEQ ID NO: 57) to generate a DNA template. - Capped RNA was generated by in vitro transcription (IVT) as described below. The reaction contained: 1x T7 RNA polymerase mix (catalog no. E2040S, New England Biolabs), 0.025mg / mL DNA transcription template (linearized by restriction enzyme digestion), 8mM trinucleotide 7 1x transcription buffer (40 mM Tris (pH 7.9), 10 mM dithiothreitol, 2 mM spermidine, 0.002% Triton X-100, and 27 mM magnesium chloride) with G-ppp-AU cap analog (CleanCap Reagent AU (catalog no. N-7114, TriLink), and a final concentration of 10 mmol / l each of adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanine triphosphate (GTP), and uridine triphosphate (UTP) (HiScribe T7 Quick High Yield RNA Synthesis Kit; New England Biolabs). · SAMs containing modified nucleosides, transcription reactions were assembled by replacing one or two nucleotide triphosphates with the corresponding triphosphate derivatives of the following nucleosides: 5-methylcytidine (m5C), 5-methoxycytidine (mo5C), pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 5-methoxyuridine (mo5U), 2-thiouridine (s2U), or 6-methyladenine (m6A) (TriLink). ·IVT reaction conditions: Transcription reactions were incubated at 37°C for 2 h and then treated with DNase I (catalog no. AM2239, Thermo Fisher Scientific) at a final concentration of 2 U per 0.001 mg of DNA transcription template in DNase I buffer for 1 h at 37°C. Capped / modified AU-SAMs were purified using RNeasy Maxi (QIAGEN, 75162) or liquid chromatography following digestion with Turbo DNase (Thermo Fisher Scientific).

[0245] A model payload encoding green fluorescent protein (GFP) and the β-spike from a SARS-CoV-2 variant was inserted into the deleted region of the VEEV backbone. The capped AU-SAM RNA was inserted into the VEEV backbone using the trinucleotide m 7 Generated using the G-ppp-AU cap analog.

[0246] The quantity and quality of SAMs were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). All RNA samples were analyzed by capillary electrophoresis to confirm the structural integrity of the RNA and the representation of any SAMs.

[0247] All RNA samples were analyzed by capillary electrophoresis to confirm the structural integrity of the RNA and the representation of all SAMs used in the study (Figure 3). In a series of transcription reactions using protein-encoding plasmids and RNA polymerase, full-length transcripts were obtained, including Ψ, m1Ψ, and m5C. The modified SAMs analyzed by capillary electrophoresis were all intact and indistinguishable from their unmodified counterparts in that they migrated as expected based on their size.

[0248] B. Replication activity of SAM by RT-qPCR Replication activity of modified nucleoside SAM encoding green fluorescent protein (GFP) was measured by RT-qPCR after transfection into BHK-21 cells. BHK-21 cells were transfected with 11 ng of SAM complexed with Lipofectamine MessengerMAX transfection reagent (catalog number: LMRNA008, Thermo Fisher Scientific), after which total RNA was isolated using the RNeasy Micro Kit (catalog number: 74004, Qiagen) according to the manufacturer's protocol. To measure the replication activity of SAM (i.e., RNA self-replication), isolated RNA was reverse transcribed and analyzed by qPCR to measure RNA copy number of GFP transcripts 20 hours after transcription. SAM copy number is relative to a SAM control containing a catalytically inactive polymerase subunit nsP4 that cannot self-replicate. As shown in Figure 4, while all SAMs containing modified nucleosides were capable of replicating, SAMs containing m5C alone ("GFP-m5C") replicated more efficiently than their unmodified counterparts ("GFP-U"), yielding 24% more transcripts. GFP-m5C also replicated more efficiently than SAMs containing Ψ, m1Ψ, or m5C+m1Ψ.

[0249] C. Affinity purification of SAM The applicability of affinity chromatography techniques to synthetic SAMssRNA vectors, which are 3-4 times longer than typical non-replicating mRNAs, is unclear. Therefore, we investigated the performance of affinity chromatography purification methods to remove by-products (contaminants) generated during in vitro transcription reactions (IVT), reduce natural immunity, and / or improve the translation and vaccination efficacy of synthetic SAMs. Removal of IVT contaminants (e.g., salts, detergents, and / or dsRNA) from SAMs was performed using a POROS OligodT(25) GoPure column (Thermo Fisher Scientific) and a chromatography buffer containing 10 mM HEPES (pH 7.3), 1 mM EDTA, and 100 mM NaCl (buffer B) or 500 mM NaCl (buffer A). The column was connected to an Akta avant 25 FPLC system (Cytiva) and equilibrated with 5-fold column volume of buffer A at a flow rate of 2.5 mL / min. The SAM sample in Buffer A was then loaded onto the column at a flow rate of 1.5 mg / min and washed with 3 column volumes of Buffer A at a flow rate of 2.5 mL / min, followed by 5 column volumes of Buffer B. The chromatography buffer was changed to nuclease-free water at a flow rate of 1.5 mL / min to release the oligo(dT)-linked SAM. The UV absorbance of the flow-through and wash fractions was monitored at 260 nM, and the peak fractions corresponding to the eluted SAM were collected. Nucleic acids from the collected chromatography peaks were collected for further analysis by precipitation after adding 0.1 volume of 3M NaOAc (pH 5.5) and 1 volume of isopropanol.

[0250] FPLC chromatograms of SAMs encoding the β-spike showed a major peak eluting with water (FIG. 5A), which was collected and identified as the expected SAM product using capillary electrophoresis (FIG. 5B, "Eluate"). SAMs with or without nucleoside modifications encoding different sequences gave similar patterns with different relative heights for the preceding and following peaks. Additional UV-absorbing products with shorter and longer retention times compared to the main SAM product were also observed.

[0251] RNA quality and purification efficiency were assessed by capillary electrophoresis (3 ng of total RNA was loaded per lane and analyzed on an Agilent Fragment Analyzer). As shown in Figure 5B, a band or smear was observed below the expected ssRNA vector product. However, when comparing the purity of the SAM to the input SAM ("β-spiked input"), as quantified in Table A below, the oligo(dT)-purified ssRNA vector ("eluted") showed a 20% increase in expected product purity as measured by area under the curve (AUC) between nucleotide lengths 8500-13000 nt compared to the total RNA signal (100 nt-13000 nt).

[0252] (Table A) RNA quantification by capillary electrophoresis TIFF2024542126000002.tif33128

[0253] D. Effect of nucleoside modification and affinity purification on the natural immunogenicity of SAM We examined whether in vitro transcribed SAMs contained dsRNA using a dot blot assay with the J2 monoclonal antibody (mAb), which recognizes dsRNA. When in vitro transcripts encoding β-spike and GFP were tested that contained no nucleoside modifications, or m1Ψ, or m5C nucleoside modifications, all samples contained sequence-dependent dsRNA contamination (Figure 6A). Unpurified IVT m1Ψ or m5C SAMs contained significantly less dsRNA compared to the corresponding U-containing SAMs (Figure 6B), suggesting that m1Ψ or m5C suppresses the purification of dsRNA by-products. Affinity purification of both unmodified and nucleoside-modified SAMs further reduced staining by dsRNA-specific mAbs (Figure 6B).

[0254] Human monocyte-derived dendritic cells (MoDCs) were used to measure innate immune responses, particularly the RNA sensing pathway. Expression levels of IRF7 (an activation marker that regulates the transcription of interferons) were assessed by flow cytometry 24 h after transfection of SAM ssRNA vectors encapsulated in LNPs. Consistent with the results of quantification of dsRNA by dot blot (Figure 6B), FACS analysis showed that the expression of IRF7 induced by SAMs containing m1Ψ or m5C alone (Figure 7A), or in combination with their respective affinity purification (Figure 7B), was 54–77% less compared to unmodified SAMs. The m1Ψ modification reduced RNA-mediated MoDC activation to a greater extent than the m5C modification, which could be due to the generation of fewer dsRNA intermediates and the observed reduced SAM replication activity (Figure 4). Oligo(dT)-mediated affinity or cellulose-based purification alone of the SAM ssRNA vector encoding the β-spike induced approximately 70% less IRF7 expression compared with LNP-mediated transfection with unpurified SAM (Fig. 7C, left and right panels, respectively).

[0255] E. Translation efficiency of nucleoside-modified SAMs purified by oligo(dT) affinity purification FACS analysis of MoDCs 48 hours after LNP-mediated SAM delivery showed that the combination of m5C-modified SAM with oligo(dT) purification significantly improved the translational capacity of SAM-GFP compared to the corresponding control or m1Ψ-containing transcripts (Figure 8). Not only did more cells express GFP when m5C-modified SAM was used, but each cell also showed stronger staining, suggesting higher cellular levels of GFP. The level of GFP staining, both in number and intensity of positive cells, was proportional to the amount of delivered SAM-GFP added to MoDCs (data not shown).

[0256] F. Evaluation of self-amplifying mRNA viral vectors in mice The efficacy of a self-amplifying expression system containing a β spike-encoding ssRNA vector (SAM) having (A) modified nucleosides and / or (B) purified by a chromatography system and / or affinity-based separation system, as described herein, was evaluated in mice by monitoring T cell responses.

[0257] immunization Balb / c mice (n=12 / group) were immunized with SAM-LNP. SAM contained modified nucleosides and / or chromatographically purified SAM ssRNA. SAM-LNP complexes (1 μg and 10 μg) were administered in a volume of 100 μL by bilateral intramuscular injection (50 μL in each leg). Each immunization group is shown in Table B below (cellulose-based purification is referred to as "purification A" and oligo(dT)-based purification is referred to as "purification B").

[0258] Table B: In vivo evaluation of SAM containing modified nucleosides and / or chromatographically purified SAM ssRNA (Balb / c mice) TIFF2024542126000003.tif71161

[0259] Dissociation of splenocytes Splenocytes were isolated 6 days (n=6) and 12 days (n=6) after immunization. Spleens from each mouse were pooled in 3 mL of complete RPMI (RPMI, 10% FBS, penicillin / streptomycin). Mechanical dissociation was performed using a gentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer's protocol. Dissociated cells were filtered through a 40-micrometer filter and red blood cells were lysed with ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). Cells were filtered again through a 30-micrometer filter and then resuspended in complete RPMI. Cells were counted on a Cytoflex LX (Beckman Coulter) using propidium iodide staining to exclude dead and apoptotic cells. Cells were then adjusted to an appropriate viable cell concentration for subsequent analysis.

[0260] Ex vivo enzyme-linked immunospot (ELISpot) assay ELISPOT analysis was performed using the Mouse IFNγ ELISpotPLUS kit (MABTECH) according to the ELISPOT Harmonization Guidelines {DOI:10.1038 / nprot.2015.068}. 5 × 10 4 Splenocytes were stimulated ex vivo for 16 hours with 10 μM β-spike peptide in 96-well IFNγ antibody-coated plates. Spots were developed using alkaline phosphatase. The reaction time was 10 minutes and stopped by flushing the plate with tap water. Spots were counted using an AID vSpot Reader Spectrum. In the ELISPOT analysis, wells with saturation greater than 50% were recorded as "non-measurable majority." Samples with a deviation of greater than 10% in replicate wells were excluded from the analysis. Spot counts were then corrected for well confluency using the formula: spot count + 2 × (spot count × confluence (%) / [100% - confluence (%)]). Negative background was corrected by subtracting the spot counts of negative peptide-stimulated wells from antigen-stimulated wells. Finally, wells labeled as non-measurable majority were set to the highest observed corrected value and rounded to the nearest 100.

[0261] G. Immunogenicity Results in Mice Antigen-specific cellular immune responses against the SARS-CoV-2 β-spike epitope were measured in Balb / c mice 6 or 14 days after immunization with 1 or 10 μg of SAM-LNP. As shown in Figure 9, immune responses measured by IFNγ ELISpot showed consistent trends in epitope-specific T cell responses, suggesting that immunization with oligo(dT)-purified SAM improved T cell responses compared to other SAM compositions evaluated.

[0262] Antigen-specific immune responses against the SARS-CoV-2 β-spike epitope were further measured in Balb / c mice following immunization with 1 mg of SAM-LNPs generated and purified by standard "conventional" protocols or generated with m5C and purified with oligo(dT) ("oligo(dT)-purified m5C-β-spike samRNA"). Figure 10 shows immune responses measured by intracellular cytokine staining against overlapping peptide pools spanning the β-spike epitope of SARS-CoV-2 in Balb / c mice 5 or 12 days after immunization. Figure 11 shows serum pseudovirus neutralization titers (50% inhibition) in Balb / c mice 4 or 8 weeks after immunization.

[0263] Antigen-specific immune responses to the model antigen cassettes were further measured in Balb / c mice following immunization with 1 mg of SAM-LNPs generated and purified by standard "conventional" protocols, or generated in m5C and purified with oligo(dT) ("oligo(dT)-purified m5C-MAG samRNA"), or generated in m1Ψ and purified with oligo(dT) ("oligo(dT)-purified m1Ψ-MAG samRNA"). Figure 12 shows the immune responses measured by intracellular cytokine staining in splenocytes of Balb / c mice following stimulation with tetrameric AH1 antigen.

[0264] These results demonstrate that samRNA generated in m5C and purified with oligo(dT) or generated in m1Ψ and purified with oligo(dT) stimulated an immune response.

[0265] H. Evaluating Vaccine Efficacy in Nonhuman Primates The efficacy and safety of vaccines containing cassettes encoding immunogens are evaluated in non-human primates. In particular, self-amplifying expression systems containing ssRNA vectors (SAMs) that contain (A) modified nucleosides and / or (B) purified using chromatography and / or affinity-based separation systems, as described herein, are evaluated. Efficacy is evaluated by monitoring T cell and / or B cell responses.

[0266] immunization For SAM vaccines in Indian rhesus macaques (e.g., Mamu-A*01), SAM is administered as bilateral intramuscular injections into the quadriceps muscles, including but not limited to, at a dose of 1 mL per leg, totaling 1 mg per animal.

[0267] Immune monitoring in rhesus macaques For immune monitoring, 10–20 mL of blood is collected into heparinized vacutainer tubes and kept at room temperature until isolation. PBMCs are isolated by density gradient centrifugation using lymphocyte separation medium (LSM) and Leucosep separator tubes. PBMCs are stained with propidium iodide and viable cells are counted using a Cytoflex LX (Beckman Coulter). Samples are then diluted to 4 × 10 in RPMI complete medium (10% FBS). 6 Resuspend at 100 cells / mL.

[0268] IFNγ ELISPOT assays are performed using pre-coated 96-well plates (MAbtech, Monkey IFNγ ELISPOT PLUS, ALP (kit lot no. 36, plate lot no. 19)) according to the manufacturer's protocol. For each sample and stimulus, 1 x 10 cells were added per well. 5PBMCs are seeded in triplicate with 10 μg / mL peptide stimuli (GenScript) and incubated overnight in complete RPMI. Each sample is incubated overnight with 10 μM of the respective peptide encoded by the SAM ssRNA vector. DMSO alone is used as a negative control for each sample. Plates are washed with PBS and incubated with anti-monkey IFNγ MAb biotin (MAbtech) for 2 h, then washed again and incubated with streptavidin-ALP (MAbtech) for 1 h. After the final wash, plates are incubated with BCIP / NBT (MAbtech) for 10 min to develop the immunospots and dried overnight at 37°C. Spots are photographed and counted using an AID reader (Autoimmun Diagnostika).

[0269] Samples with replicate well variability (variability = variance / [median + 1]) > 10 and median > 10 are excluded. Spot values ​​are adjusted based on well saturation according to the following formula: Prepared spots = untreated spots + 2 x (untreated spots x saturation / [100 - saturation]). Wells with well saturation > 33% are considered "too many to measure" (TNTC) and excluded. Background correction is performed for each sample by subtracting the average of the negative control peptide wells. Add 1 x 10 to the corrected spot count. 6 Multiply the data by 1 x 10 / number of cells plated. 6 Normalize to spot forming colonies (SFC) per PBMC. For overall summary analysis, use 1 × 10 unless the sample is TNTC. 5 Using the calculated value obtained by seeding cells at 100 cells / well, 2.5 × 10 cells / well for TNTC 4 The calculated values ​​obtained by seeding cells at the cell / well are used for that particular sample / stimulus / time point. Data processing is performed using the R programming language.

[0270] Intracellular cytokine assays are also performed. PBMCs are plated at 1 × 10 per well in a V-bottom 96-well plate. 6Distribute with cells. Pellet cells and resuspend in 100 μl complete RPMI containing each of the encoded peptides above. DMSO is used as a negative control for each sample. After 1 hour, Brefeldin A (Biolegend) is added to a final concentration of 5 μg / mL and cells are incubated overnight. After viability staining, extracellular staining is performed in FACS buffer (PBS + 2% FBS + 2 mM EDTA). Cells are washed, fixed, and permeabilized using eBiosciences Fixation / Permeabilization Solution Kit. Intracellular staining is performed. Samples are evaluated for viability, CD3, CD4, CD8, IFNγ, TNFα, IL-2, perforin, CD107a, CCR7, and CD45RA.

[0271] Serum cytokine markers are also monitored. Serum cytokine and chemokine levels are measured by standard multiplex assays. Serum is collected and marker analysis is performed at 0 (baseline), 2, 8, 24, and 48 hours after vaccination. Cytokines evaluated are interleukin-1β (IL-1β), interleukin-1 (IL-10), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma-inducible protein 10 (Ip-10), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and IFN-α (IFN-α2a).

[0272] I. Immunogenicity Results in NHPs Self-amplifying expression systems comprising ssRNA vectors with modified nucleosides and / or purified using chromatography and / or affinity-based separation systems show improved vaccine efficacy as assessed by monitoring T cell and / or B cell responses.

[0273] array The vectors, cassettes, and antibodies referred to herein are described below and referenced by SEQ ID NO: Reference to SEQ ID NO: is also to the sequence listing found in U.S. Provisional Patent Application No. 63 / 277,116, which is incorporated by reference herein for all purposes. TIFF2024542126000004.tif216165TIFF2024542126000005.tif252165TIFF2024542126000006.tif25416 5TIFF2024542126000007.tif253165TIFF2024542126000008.tif253165TIFF2024542126000009.tif25316 5TIFF2024542126000010.tif253165TIFF2024542126000011.tif253165TIFF2024542126000012.tif25316 5TIFF2024542126000013.tif253165TIFF2024542126000014.tif253165TIFF2024542126000015.tif46165

[0274] Equivalence and Scope In the claims, the articles "a," "an," and "the" may mean one or more, unless otherwise indicated or otherwise clear from the context. A claim or description containing "or" between one or more elements of a group is deemed to be satisfied if one, more than one, or all of the group elements are present in, employed in, or otherwise relevant to a particular product or process, unless otherwise indicated or otherwise clear from the context. The disclosure includes embodiments in which exactly one element of the group is present in, employed in, or otherwise relevant to a particular product or process. The disclosure includes embodiments in which more than one, or all elements of the group are present in, employed in, or otherwise relevant to a particular product or process.

[0275] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms, etc., from one or more of the claims set forth herein are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are listed as a list, e.g., in Markush group format, each subgroup of elements is also disclosed, and any element(s) may be excluded from the group. In general, when the disclosure or aspects of the disclosure are said to comprise certain elements and / or features, it should be understood that certain embodiments of the disclosure or aspects of the disclosure consist of, or consist essentially of, such elements and / or features. For purposes of simplicity of description, these embodiments have not been specifically described in these terms herein. It should also be noted that the terms "comprising" and "containing" are intended to be open and allow for additional elements or steps to be included. When ranges are given, the endpoints are included. Additionally, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges can take any particular value or subrange within the ranges set forth in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0276] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Furthermore, any particular embodiment of the present disclosure that is within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed to be well known to those of ordinary skill in the art. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0277] Those skilled in the art will recognize or be able to ascertain, without more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications to the present specification can be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

1. A composition for delivering a self-amplifying expression system comprising a single-stranded RNA (ssRNA) vector, wherein the ssRNA vector is 7-methylguanylate (m ７ G cap), a polyadenylated (polyA) tail, a self-amplifying backbone, and one or more modified nucleosides, and optionally (a) the self-amplifying backbone comprises a polynucleotide selected from a self-replicating RNA virus, optionally wherein the self-replicating RNA virus is selected from the group comprising alphaviruses, flaviviruses, measles viruses, and rhabdoviruses; (b) the ssRNA vector is purified, optionally the ssRNA vector is purified by chromatography, optionally the chromatography comprises a cellulose chromatography system or an affinity-based separation system, optionally the affinity-based separation system is a deoxythymidine (dT) oligonucleotide (oligo(dT)) system; (c) more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or 100% of the adenine, guanine, cytidine, and / or uridine nucleosides of the ssRNA vector are modified nucleosides; (d) the composition comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of contaminants, optionally comprising salts, detergents, and / or double-stranded RNA (dsRNA); (e) the ssRNA vector constitutes 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more of the total RNA present in the composition; (f) the ssRNA vector is the only RNA species present in the composition; (g) the ssRNA vector is produced by in vitro transcription, and optionally, the m7G cap comprises an m7G cap analog, and optionally, the m7G cap analog comprises a trinucleotide m7G-ppp-A-U cap analog or a dinucleotide m7G-ppp-A cap analog; (h) the ssRNA vector comprises a cassette comprising at least one nucleic acid sequence to be delivered, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the cassette is operably linked to or operably inserted into the self-amplifying backbone; (i) the self-amplifying backbone has the following formula in the 5' to 3' direction: m 7 G-ppp-N 1 -N 2 -N V a polynucleotide selected from a self-replicating RNA virus comprising at least one nucleic acid sequence represented by During the ceremony, m 7 G is a 7-methylguanylate (m 7 G) cap; ppp is a triphosphate bridge, N 1 is the first nucleotide of a self-amplifying backbone corresponding to the first endogenous 5′ nucleotide of the self-replicating RNA virus; N2 is the second nucleotide of the self-amplifying backbone corresponding to the second endogenous 5' nucleotide of the self-replicating RNA virus; and N V comprises (1) one or more additional nucleic acid sequences of said self-amplifying backbone; and (2) a cassette comprising at least one nucleic acid sequence to deliver, optionally said at least one nucleic acid sequence comprising a polypeptide-encoding nucleic acid sequence, optionally said polypeptide-encoding nucleic acid sequence being an antigen-encoding nucleic acid sequence, and said cassette being operably linked to or operably inserted into said self-amplifying backbone; and / or (j) the composition further comprises a nanoparticle delivery vehicle, and optionally, the nanoparticle delivery vehicle is a lipid nanoparticle (LNP); The composition.

2. the one or more modified nucleosides are (a) comprising methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof; (b) comprising m5C; or (c) containing m5C and no other modified nucleotides, optionally modified nucleotides other than the m7G cap analog; The composition of claim 1.

3. The one or more modified nucleosides include mC, and m ７ The composition of claim 1, which does not contain any modified nucleotides other than a G-cap analog.

4. Total RNA, dsRNA, and / or ssRNA content (a) assessed by dot blot or ELISA, optionally wherein said dot blot or ELISA assessment comprises detection with antibodies specific for RNA, dsRNA, and / or ssRNA, and optionally wherein total RNA, dsRNA, and / or ssRNA content is quantified as area under the curve (AUC); (b) assessed by capillary electrophoresis; or (c) assessed by liquid chromatography, and optionally quantified as area under the curve (AUC) total RNA, dsRNA, and / or ssRNA content; The composition of claim 2.

5. A pharmaceutical composition comprising the composition described in any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. 6. A pharmaceutical composition according to claim 5 for use in a method for treating a subject having a disease, optionally comprising: (a) the disease is cancer or an infectious disease, and optionally the infectious disease is caused by a virus selected from the group including HPV, influenza, TB, CMV, HMPV, PIV, Chikungunya virus, Zika virus, SARS-CoV-2, and a pan-coronavirus; (b) the amount of the ssRNA vector in the composition is 1000 μg or less, 100 μg or less, 50 μg or less, 30 μg or less, 10 μg or less, 5 μg or less, or 1 μg or less; (c) the subject's innate immune response is reduced compared to a control subject being administered a composition comprising a nucleic acid sequence that does not comprise modified nucleosides, and optionally the innate immune response is an IFN response, and optionally the IFN response is an increase in IRF-3 expression, an increase in IRF-7 expression, or a combination thereof. (d) increased replication of the ssRNA vector compared to a control composition comprising an identical ssRNA vector but without the modified nucleoside; and / or (e) the composition for delivering the self-amplifying expression system is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), intravitreally (IVT), intrathecally, or intravenously (IV), and optionally the method further comprises administering an immunomodulatory agent, wherein optionally the immunomodulatory agent is an anti-CTLA4 antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-4-1BB antibody or antigen-binding fragment thereof, an anti-OX-40 antibody or antigen-binding fragment thereof, or a cytokine, wherein optionally the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21, or a variant thereof, and optionally the method further comprises administering an adjuvant; The pharmaceutical composition.

7. A method for purifying a self-amplifying expression system from a nucleic acid mixture or reducing double-stranded RNA (dsRNA) in a nucleic acid mixture, comprising: The method comprises: (A) purifying the self-amplifying expression system by a cellulose chromatography system or an affinity-based separation system; and / or (B) generating the self-amplifying expression system such that the self-amplifying expression system comprises a modified nucleoside. Including, The self-amplifying expression system comprises a single-stranded RNA (ssRNA) vector, and the ssRNA vector is ７ comprising a G-cap, a polyA tail, and a self-amplifying backbone; Furthermore, optionally, (a) the affinity-based separation system comprises a deoxythymidine (dT) oligonucleotide (oligo(dT)) system; (b) the ssRNA vector comprises modified nucleosides; (c) the modified nucleoside comprises methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof; and / or (d) the modified nucleoside consists of methyl-5-cytosine (m5C); The method.

8. A composition mixture comprising: The composition comprises a self-amplifying expression system, the self-amplifying expression system comprising: (a) m ７ a single-stranded RNA (ssRNA) vector comprising a G-cap, a polyadenylated (polyA) tail, and a self-amplifying backbone; (b) double-stranded RNA (dsRNA); and (i) the dsRNA constitutes less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the total RNA present in the mixture; and / or (ii) the ssRNA vectors constitute 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more of the total RNA present in the mixture; The composition mixture. (a) the only detectable RNA comprises the ssRNA vector; (b) the total RNA, dsRNA, and / or ssRNA content is (i) assessed by dot blot or ELISA, optionally wherein the dot blot or ELISA assessment comprises detection with an antibody specific for RNA, dsRNA, and / or ssRNA; (ii) assessed by capillary electrophoresis; and / or (iii) as assessed by liquid chromatography; Optionally, total RNA, dsRNA, and / or ssRNA content is quantified as area under the curve (AUC). (c) the ssRNA is produced by in vitro transcription; (d) the m 7 G cap comprises an m 7 G cap analog, and optionally, the m 7 G cap analog comprises a trinucleotide m 7 G-ppp-A-U cap analog or a dinucleotide m 7 G-ppp-A cap analog; (e) the ssRNA vector comprises a cassette comprising at least one nucleic acid sequence to be delivered, optionally wherein the at least one nucleic acid sequence comprises a polypeptide-encoding nucleic acid sequence, optionally wherein the polypeptide-encoding nucleic acid sequence is an antigen-encoding nucleic acid sequence, and wherein the cassette is operably linked to or operably inserted into the self-amplifying backbone; (f) the self-amplifying backbone has the following formula in the 5' to 3' direction: m 7 G-ppp-N 1 -N 2 -N V a polynucleotide selected from a self-replicating RNA virus comprising at least one nucleic acid sequence represented by During the ceremony, m 7 G is a 7-methylguanylate (m 7 G) cap; ppp is a triphosphate bridge, N 1 is the first nucleotide of a self-amplifying backbone corresponding to the first endogenous 5′ nucleotide of the self-replicating RNA virus; N2 is the second nucleotide of the self-amplifying backbone corresponding to the second endogenous 5' nucleotide of the self-replicating RNA virus; and N V comprises (1) one or more additional nucleic acid sequences of said self-amplifying backbone; and (2) a cassette comprising at least one nucleic acid sequence to deliver, optionally said at least one nucleic acid sequence comprising a polypeptide-encoding nucleic acid sequence, optionally said polypeptide-encoding nucleic acid sequence being an antigen-encoding nucleic acid sequence, and said cassette being operably linked to or operably inserted into said self-amplifying backbone. (g) the ssRNA vector comprises one or more modified nucleosides, and optionally (i) the one or more modified nucleosides comprise methyl-5-cytosine (m5C), methyl-6-adenosine (m6A), ribose-methylated (2'-O-Me), s-thiouridine, (s2U), 5-methyluridine (m5U), N1-methylpseudouridine (m1Ψ), pseudouridine (Ψ), or any combination thereof; (ii) the one or more modified nucleosides comprise m5C, or (iii) the one or more modified nucleosides comprise m5C and no other modified nucleotides, optionally modified nucleotides other than a cap analog; and / or (h) the ssRNA vector is purified, optionally the ssRNA vector is purified by chromatography, optionally the chromatography comprises a cellulose chromatography system or an affinity-based separation system, optionally the affinity-based separation system is a deoxythymidine (dT) oligonucleotide (oligo(dT)) system; The mixture of claim 8.

10. (I) The composition for delivering the self-amplifying expression system comprises: (A) the self-amplifying expression system, comprising one or more self-amplifying mRNA (SAM) vectors, wherein the one or more SAM vectors comprise: (a) the self-amplifying backbone, wherein the self-amplifying backbone comprises the nucleic acid sequence set forth in SEQ ID NO:6, the self-amplifying backbone sequence comprising a subgenomic promoter nucleotide sequence and a poly(A) sequence, the subgenomic promoter sequence being endogenous to the self-replicating RNA virus and the poly(A) sequence being endogenous to the self-replicating RNA virus backbone; (b) a cassette integrated between the subgenomic promoter nucleotide sequence and the poly(A) sequence, the cassette being operably linked to the subgenomic promoter nucleotide sequence, optionally the cassette comprising at least one antigen-encoding nucleic acid sequence, the at least one antigen-encoding nucleic acid sequence comprising: a. an epitope-encoding nucleic acid sequence, optionally comprising: (1) at least one alteration that renders the encoded epitope sequence different from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence; or (2) a nucleic acid sequence encoding an infectious disease organism peptide selected from the group consisting of a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide; b. optionally a 5' linker sequence, and c. Optionally, a 3' linker sequence the cassette comprising: the self-amplifying expression system, (B) Optionally, a lipid nanoparticle (LNP) encapsulating the self-amplifying expression system. and / or (II) The ordered sequence of each element of the cassette in the composition for delivering the self-amplifying expression system is, in the 5' to 3' direction, as follows: P a -(L5 b -N c -L3 d) X -(G5 e -U f) Y -G3 g It is expressed by an equation containing During the ceremony, P comprises a second promoter nucleotide sequence and a=0 or 1; N comprises one of the epitope-encoding nucleic acid sequences, wherein the epitope-encoding nucleic acid sequence comprises an MHC class I epitope-encoding nucleic acid sequence, and c=1; L5 comprises a 5' linker sequence and b=0 or 1; L3 comprises a 3' linker sequence and d=0 or 1; G5 comprises one of the at least one nucleic acid sequences encoding a GPGPG (SEQ ID NO:56) amino acid linker, and e=0 or 1; G3 comprises one of the at least one nucleic acid sequences encoding a GPGPG (SEQ ID NO:56) amino acid linker, and g=0 or 1; U comprises one of at least one MHC class II epitope-encoding nucleic acid sequence, and f=1; X=1 to 400, and for each X, the corresponding N c is an MHC class I epitope-encoding nucleic acid sequence; and Y=0, 1, or 2, and for each Y, the corresponding U f is an MHC class II epitope-encoding nucleic acid sequence; Furthermore, optionally, (a) for each X, the corresponding N c is a different MHC class I epitope-encoding nucleic acid sequence; (b) for each Y, the corresponding U f is a different MHC class II epitope-encoding nucleic acid sequence; (c) a = 0, b = 1, d = 1, e = 1, g = 1, h = 1, X = 10, Y = 2; at least one of the promoter nucleotide sequences is a single subgenomic promoter nucleotide sequence provided by the self-amplifying backbone; at least one polyadenylated poly(A) sequence is a poly(A) sequence of at least 80 consecutive A nucleotides provided by the self-amplifying backbone; the cassette is integrated between the subgenomic promoter nucleotide sequence and the poly(A) sequence, and the cassette is operably linked to the subgenomic promoter nucleotide sequence and the poly(A) sequence; each N encodes an MHC class I epitope 7 to 15 amino acids in length; L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of an MHCI epitope, said 5' linker sequence encoding a peptide at least 3 amino acids in length; L3 is a natural 3' linker sequence encoding the natural C-terminal amino acid sequence of the MHCI epitope, wherein the 3' linker sequence encodes a peptide at least 3 amino acids in length; U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence; the self-amplifying backbone is the sequence set forth in SEQ ID NO: 6; and each of said MHC class I epitope-encoding nucleic acid sequences encodes a polypeptide 13 to 25 amino acids in length; Optionally, at least two of said MHC class I epitopes are presented by MHC class I on a cell surface, optionally on a tumor cell surface or an infected cell surface; and / or (III) the cassette is integrated between the at least one promoter nucleotide sequence and the at least one poly(A) sequence, and / or the at least one promoter nucleotide sequence is operably linked to the cassette; and / or (IV) The poly(A) sequence is (a) comprising a poly(A) sequence native to the autonomously replicating virus; (b) comprising a poly(A) sequence exogenous to the autonomously replicating virus; (c) operably linked to at least one of said at least one nucleic acid sequence; and / or (d) at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, or at least 120 consecutive A nucleotides, optionally at least 80 consecutive A nucleotides; A composition according to any one of claims 1 to 4, a pharmaceutical composition according to claim 6, a mixture according to claim 8 or 9, or a method according to claim 7.

11. wherein the at least one nucleic acid sequence for delivery comprises the polypeptide-encoding nucleic acid sequence, and optionally (a) the polypeptide-encoding nucleic acid sequence encodes the antigen-encoding nucleic acid sequence, and optionally the antigen-encoding nucleic acid sequence comprises an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B-cell response, or a combination thereof, and optionally the antigen-encoding nucleic acid sequence comprises a full-length protein, a protein subunit, a protein domain, or a combination thereof; or (b) the polypeptide-encoding nucleic acid sequence encodes a full-length protein or a functional portion thereof, and optionally, the full-length protein or functional portion thereof is selected from the group consisting of an antibody, a cytokine, a chimeric antigen receptor (CAR), a T-cell receptor, and / or a genome editing system nuclease. A composition according to any one of claims 1 to 4, a pharmaceutical composition according to claim 6, a mixture according to claim 8 or 9, or a method according to claim 7. (a) the at least one nucleic acid sequence for delivery comprises at least one nucleic acid sequence comprising a non-coding nucleic acid sequence, and optionally, the non-coding nucleic acid sequence is an RNA interference (RNAi) polynucleotide or a polynucleotide of a genome editing system; (b) the LNP is (i) comprising a lipid selected from the group consisting of an ionizable amino lipid, a phosphatidylcholine, cholesterol, a PEG-based coating lipid, or a combination thereof; and / or (ii) comprising an ionizable amino lipid, a phosphatidylcholine, cholesterol, and a PEG-based coating lipid; and (iii) optionally, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule; (c) the LNP-encapsulated expression system has a diameter of 60-140 nm; (d) the composition for delivering the self-amplifying expression system is formulated for intramuscular (IM), intradermal (ID), subcutaneous (SC), intravitreal (IVT), intrathecal, or intravenous (IV) administration, and optionally, is formulated for intramuscular (IM) administration; (e) the ssRNA vector comprises a positive-strand RNA vector or a negative-strand RNA vector, and optionally, the negative-strand RNA vector comprises at least one polynucleotide sequence of a measles virus or a rhabdovirus; (f) the ssRNA vector is self-replicating in mammalian cells; (g) the self-replicating RNA virus is selected from the group consisting of alphavirus, flavivirus, measles, and rhabdovirus. (h) the self-amplifying backbone comprises at least one polynucleotide sequence of an alphavirus, optionally wherein the alphavirus is selected from the group consisting of Aura virus, Fort Morgan virus, Venezuelan equine encephalitis virus, Ross River virus, Semliki Forest virus, Sindbis virus, or Mayaro virus, optionally wherein the self-amplifying backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus, and optionally (i) the self-amplifying scaffold comprises at least: A sequence for nonstructural protein-mediated amplification, a subgenomic promoter sequence, a poly(A) sequence, a nonstructural protein 1 (nsP1) gene, a nsP2 gene, a nsP3 gene, and a nsP4 gene encoded by a nucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus. Contains, or (ii) the self-amplifying scaffold comprises at least A sequence for nonstructural protein-mediated amplification, a subgenomic promoter sequence, and a poly(A) sequence encoded by a nucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus. Contains, and (iii) optionally, 1. the sequence for non-structural protein-mediated amplification is selected from the group consisting of an alphavirus 5'UTR, a 51 nt CSE, a 24 nt CSE, a 26S subgenomic promoter sequence, a 19 nt CSE, an alphavirus 3'UTR, or a combination thereof; and / or 2. the self-amplifying backbone does not encode the structural virion proteins capsid, E2, and E1, optionally wherein E1 is full-length E1 or does not encode the structural virion proteins capsid, E3, E2, 6K, and optionally wherein the cassette is inserted in place of a structural virion protein within the polynucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus; (iv) the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 5; (v) the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 5, further comprising a deletion between base pairs 7544 and 11175; and optionally 1. The self-amplifying backbone comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7; 2. The cassette is inserted at position 7544 of SEQ ID NO: 6 or SEQ ID NO: 7; (i) insertion of the cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and the at least one nucleic acid sequence, wherein the nsP1-4 genes and the at least one nucleic acid sequence to be delivered are in separate open reading frames; (j) the ssRNA vector comprises at least one promoter nucleotide sequence, and optionally (i) the at least one promoter nucleotide sequence comprises a native promoter nucleotide sequence encoded by the self-replicating RNA virus, and optionally the native promoter nucleotide sequence is a subgenomic promoter nucleotide sequence; or (ii) the at least one promoter nucleotide sequence is an exogenous RNA promoter; (k) the ssRNA vector comprises a second promoter sequence, optionally wherein the second promoter nucleotide sequence is a subgenomic promoter nucleotide sequence, and optionally wherein the second promoter nucleotide sequence comprises a plurality of subgenomic promoter nucleotide sequences, each subgenomic promoter nucleotide sequence directing transcription of one or more of the separate open reading frames; (l) the at least one antigen-encoding nucleic acid sequence comprises two or more antigen-encoding nucleic acid sequences, optionally each antigen-encoding nucleic acid sequence being directly linked to each other; (m) each antigen-encoding nucleic acid sequence is linked to a different antigen-encoding nucleic acid sequence by a nucleic acid sequence encoding a linker; (i) the linker links two MHC class I epitope-encoding nucleic acid sequences or one MHC class I epitope-encoding nucleic acid sequence with one MHC class II epitope-encoding nucleic acid sequence, and optionally 1. The linker is (1) a sequence of consecutive glycine residues at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (2) a sequence of consecutive alanine residues at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by the mammalian proteasome; and (6) one or more naturally occurring sequences adjacent to an antigen derived from a protein of cognate origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. or 2. The linker is one or more naturally occurring sequences flanking the antigen from a protein of cognate origin and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length Including, (ii) the linker links two MHC class II epitope-encoding nucleic acid sequences or one MHC class II sequence with one MHC class I epitope-encoding nucleic acid sequence, and optionally the linker comprises the sequence GPGPG (SEQ ID NO: 56); (n) the antigen-encoding nucleic acid sequence is operably or directly linked to a separate or contiguous sequence that improves expression, stability, cellular trafficking, processing and presentation, and / or immunogenicity of the epitope-encoding nucleic acid sequence, and optionally the separate or contiguous sequence comprises at least one of a ubiquitin sequence, a ubiquitin sequence modified to increase proteasomal targeting (e.g., the ubiquitin sequence contains a Gly to Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, a lysosomal-associated membrane protein (LAMP)-1, a human dendritic cell lysosomal-associated membrane protein, and a major histocompatibility class II sequence, and optionally the ubiquitin sequence modified to increase proteasomal targeting is A76. (o) the at least one antigen-encoding nucleic acid sequence comprises at least 2 to 10, 2, 3, 4, 5, 6, 7, 8, 9, or 10, antigen-encoding nucleic acid sequences, optionally each antigen-encoding nucleic acid sequence encoding a different antigen-encoding nucleic acid sequence. (p) the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences, optionally each antigen-encoding nucleic acid sequence encoding a different antigen-encoding nucleic acid sequence; (q) the at least one antigen-encoding nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 antigen-encoding nucleic acid sequences; (r) the at least one antigen-encoding nucleic acid sequence comprises at least 2 to 400 antigen-encoding nucleic acid sequences, and at least two of the antigen-encoding nucleic acid sequences encode epitope sequences or portions thereof that are presented by MHC class I on the cell surface; (s) each antigen-encoding nucleic acid sequence independently comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitope-encoding nucleic acid sequences, and optionally each epitope-encoding nucleic acid sequence encodes a different epitope-encoding nucleic acid sequence; (t) each antigen-encoding nucleic acid sequence independently comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 epitope-encoding nucleic acid sequences, optionally wherein each epitope-encoding nucleic acid sequence encodes a different epitope-encoding nucleic acid sequence; (u) each antigen-encoding nucleic acid sequence independently comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to 400 epitope-encoding nucleic acid sequences; (v) each antigen-encoding nucleic acid sequence independently comprises at least 2 to 400 epitope-encoding nucleic acid sequences, and at least two of the epitope-encoding nucleic acid sequences encode epitope sequences or portions thereof that are presented by MHC class I on the cell surface; (w) the epitope-encoding nucleic acid sequences comprise at least one MHC class I epitope-encoding nucleic acid sequence, each antigen-encoding nucleic acid sequence encoding a polypeptide sequence 8 to 35 amino acids in length, optionally 9 to 17, 9 to 25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length; (x) the at least one MHC class II epitope-encoding nucleic acid sequence is present, and optionally the at least one MHC class II epitope-encoding nucleic acid sequence comprises at least one MHC class II epitope-encoding nucleic acid sequence that is present and contains at least one alteration, which alteration causes the encoded epitope sequence to differ from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence; (y) the epitope-encoding nucleic acid sequences comprise MHC class II epitope-encoding nucleic acid sequences, and each antigen-encoding nucleic acid sequence encodes a polypeptide sequence 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length; (z) the epitope-encoding nucleic acid sequence comprises an MHC Class II epitope-encoding nucleic acid sequence, wherein the at least one MHC Class II epitope-encoding nucleic acid sequence is present and the at least one MHC Class II epitope-encoding nucleic acid sequence comprises at least one universal MHC Class II epitope-encoding nucleic acid sequence, and optionally the at least one universal sequence comprises at least one of tetanus toxoid and PADRE; (aa) the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible, and / or (bb) the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is non-inducible; A composition according to any one of claims 1 to 4, a pharmaceutical composition according to claim 6, a mixture according to claim 8 or 9, or a method according to claim 7.

13. The epitope-encoding nucleic acid sequence comprises an MHC class I epitope-encoding nucleic acid sequence, and the MHC class I epitope-encoding nucleic acid sequence is (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor, an infected cell, or an infectious pathogenic organism, wherein the nucleotide sequencing data is used to obtain data representing each peptide sequence of a set of epitopes; (b) inputting the peptide sequence of each epitope into a presentation model to generate a set of numerical likelihoods that each of the epitopes will be presented by one or more MHC alleles on a cell surface, optionally on a tumor cell surface or an infected cell surface, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; (c) selecting a subset of the set of epitopes based on the set of numerical likelihoods to generate a set of selected epitopes used to generate the MHC class I epitope-encoding nucleic acid sequences; and (d) optionally, the number of sets of selected epitopes is between 2 and 20; (e) optionally, the presentation model comprises: (i) the presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position in the peptide sequence; (ii) the possibility of presentation of such peptide sequences comprising said particular amino acids at said particular positions on the surface of cells, optionally on tumor cells or infected cells, by said particular one of said MHC alleles of said pair; represents the degree of dependence between (f) optionally, selecting said set of selected epitopes comprises: (i) selecting epitopes that have an increased likelihood of being presented on a cell surface, optionally on a tumor cell surface or an infected cell surface, compared to non-selected epitopes, based on said presentation model; (ii) selecting epitopes based on the presentation model that have an increased likelihood of being able to stimulate a tumor-specific or infectious pathogen-specific immune response in a subject compared to non-selected epitopes; and / or (iii) selecting epitopes that have an increased likelihood of being capable of being presented to naive T cells by professional antigen-presenting cells (APCs) compared to non-selected epitopes based on the presentation model, optionally wherein the APCs are dendritic cells (DCs); (g) optionally, selecting said set of selected epitopes comprises: (i) selecting epitopes based on said presentation model that have a reduced likelihood of being inhibited by central or peripheral tolerance compared to non-selected epitopes; and / or (ii) selecting, based on said presentation model, epitopes that have a reduced likelihood of being able to stimulate an autoimmune response against normal tissues in a subject compared to non-selected epitopes; and / or (h) optionally, the exome or transcriptome nucleotide sequencing data is obtained by sequencing tumor cells or tissues, infected cells, or infectious pathogenic organisms, and optionally, the sequencing is next generation sequencing (NGS) or any massively parallel sequencing method; A composition according to any one of claims 1 to 4, a pharmaceutical composition according to claim 6, a mixture according to claim 8 or 9, or a method according to claim 7.

14. (a) the composition for delivering the self-amplifying expression system is administered as a priming vaccine; and / or (b) the method further comprises administering a second composition, optionally wherein the second composition is a vaccine composition, and optionally, (i) the second composition is administered before or after the composition for delivering the self-amplifying expression system; (ii) the second composition is the same as or different from the composition for delivering the self-amplifying expression system, and optionally, if the second composition is different from the composition for delivering the self-amplifying expression system, the second composition comprises the cassette of the self-amplifying expression system, and optionally the second composition comprises a chimpanzee adenoviral vector encoding the cassette of the self-amplifying expression system; and / or (iii) two or more second compositions are administered, optionally the composition for delivering the self-amplifying expression system is administered as a priming vaccine; A composition according to any one of claims 1 to 4, a pharmaceutical composition according to claim 6, a mixture according to claim 8 or 9, or a method according to claim 7.

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