Pan-Coronavirus Vaccine

Abstract

The present specification discloses vaccine compositions comprising a pan-corona receptor binding domain (RBD)-encoding cassette and / or an MHC epitope-encoding cassette. Also disclosed are nucleotides, cells, and methods related to said compositions, including their use as vaccines.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 251,441, filed October 1, 2021, and U.S. Provisional Patent Application No. 63 / 374,664, filed September 6, 2022, which are hereby incorporated by reference in their entireties for all purposes.

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

[0003] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral strain responsible for the coronavirus disease 2019 (COVID-19) pandemic. As of December 21, 2021, the virus has infected over 275 million people worldwide, causing approximately 5.4 million deaths. In COVID-19, CD8+ T cell responses appear important in the context of coronaviruses for two reasons. First, SARS vaccines that stimulate only antibody responses are frequently associated with pulmonary inflammation, independent of viral clearance, a recurring observation in preclinical models. This has been observed in both rodents and non-human primates (NHPs) and the current consensus is that it is caused by an unbalanced immune response and is likely resolved by using vaccines that induce balanced antibody and CD8+ T cell (Th1) responses (Consensus considerations on the assessment of the risk of disease enhancement with COVID-19 vaccines: Outcome of a Coalition for Epidemic Preparedness Innovations (CEPI) / Brighton Collaboration (BC) scientific working meeting, March 12-13, 2020). Second, coronaviruses apparently mutate frequently, infect humans from animal reservoirs, and cause three epidemics / pandemics in the past 18 years (SARS in 2002, MERS in 2012, and now COVID-19). While antibody responses are directed against highly variable proteins that vary widely between strains and isolates (e.g., the spike protein of SARS-CoV-2), T cell epitopes are often derived from more evolutionarily conserved proteins. T cell memory is generally more durable than B cell memory, and therefore CD8+ T memory against SARS-CoV-2 is likely to confer better and longer-lasting protection against future SARS variants.Although many vaccines have been shown to be capable of inducing antibody responses in NHPs and humans, widely used modalities such as protein / peptide and mRNA vaccines do not stimulate effective CD8+ T cell responses in these species.

[0004] One of the further questions in antigen vaccine design for infectious diseases is which of the many proteins that exist will yield the "best" therapeutic antigens, ie, antigens capable of stimulating immunity.

[0005] In addition to the challenges of current antigen prediction methods, existing vector systems that can be used for antigen delivery in humans, many of which are of human origin, also present certain challenges. For example, many humans have pre-existing immunity to human viruses as a result of previous natural exposure, which can pose a major obstacle to the use of recombinant human viruses to deliver antigens in vaccination strategies, such as cancer treatment or vaccines against infectious diseases.

[0006] Although some progress has been made in vaccination strategies addressing the above challenges, there is still room for improvement, particularly in clinical use, such as improving vaccine potency and efficacy; for example, there is a need for a pan-coronavirus vaccine that stimulates balanced B and T cell immunity in humans against current and potentially future coronaviruses. [Non-Patent Document 1] Consensus considerations on the assessment of the risk of disease enhancement with COVID-19 vaccines:Outcome of a Coalition for Epidemic Preparedness Innovations(CEPI) / Brighton Collaboration(BC) scientific working meeting,March12-13,2020 Summary of the Invention

[0007] Provided herein is a composition for delivering an antigen expression system comprising an antigen expression system, wherein the antigen expression system comprises at least two different coronavirus receptor binding domain (RBD)-derived nucleic acid sequences encoding at least two different RBDs, respectively, wherein the at least two different RBD domains are collectively at least 70% identical in amino acid composition to RBD domains from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

[0008] Also provided herein is a composition for delivering an antigen expression system, the composition comprising: (a) a vector comprising a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) at least one polyadenylation (poly(A)) sequence; and (b) an antigen cassette inserted into the vector backbone so as to be operably linked to the at least one promoter nucleotide sequence, the antigen cassette comprising at least two different coronavirus receptor binding domain (RBD)-derived nucleic acid sequences encoding at least two different coronavirus RBDs, respectively, wherein the at least two different RBD domains are collectively at least 70% identical in amino acid composition to RBD domains derived from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

[0009] In some embodiments, the at least two different RBD domains are at least 70% identical in amino acid composition to the RBD domain from each of (A) a clade 3 sarbecovirus and (B) a clade 1 sarbecovirus and / or a clade 2 sarbecovirus. In some embodiments, the at least two different RBD domains are at least 80% identical in amino acid composition to the RBD domain from each of a clade 1 sarbecovirus and a clade 2 sarbecovirus. In some embodiments, the at least two different RBD domains are at least 85% identical in amino acid composition to the RBD domain from each of a clade 1 sarbecovirus and a clade 2 sarbecovirus. In some embodiments, the at least two different RBD domains are at least 90% identical in amino acid composition to the RBD domain from each of a clade 1 sarbecovirus and a clade 2 sarbecovirus.

[0010] In some embodiments, the at least two different RBD domains are selected from the group consisting of NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, MK211377.1, KJ473816.1, MK2113 76.1, AY572034.1, KP886809.1, MT072864.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, and KJ473814.1, and are at least 70% identical in amino acid composition to the RBD domains from each of these.

[0011] In some embodiments, the antigen expression system comprises at least three different coronavirus RBD-derived nucleic acid sequences encoding at least three different RBD domains, which in some embodiments are collectively at least 70% identical in amino acid composition to an RBD domain from each of a clade 1 sarbecovirus, a clade 2 sarbecovirus, and a clade 3 sarbecovirus. In some embodiments, the at least three different RBD domains are selected from the group consisting of NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, MK211377.1, KJ473816.1, MK211376.1, It is at least 70% identical in amino acid composition to the RBD domains from AY572034.1, KP886809.1, MT072864.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, KJ473814.1, and SARS-CoV-2.

[0012] In some embodiments, the antigen expression system comprises at least four different coronavirus RBD-derived nucleic acid sequences encoding at least four different RBD domains, which in some embodiments are collectively at least 70% identical in amino acid composition to an RBD domain from each of a clade 1 sarbecovirus, a clade 2 sarbecovirus, and a clade 3 sarbecovirus. In some embodiments, the at least four different RBD domains are selected from the group consisting of NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, MK211377.1, KJ473816.1, MK211376.1, It is at least 70% identical in amino acid composition to the RBD domains from AY572034.1, KP886809.1, MT072864.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, KJ473814.1, and SARS-CoV2.

[0013] In some embodiments, each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from the RBD nucleic acid sequence of a sarbecovirus sequence selected from the group consisting of KP886809, KJ473815, MK211376, DQ648856, GQ153542, NC_004718, JX993988, and SARS-CoV2. In some embodiments, each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from the RBD nucleic acid sequence of a sarbecovirus sequence from each of KP886809, KJ473815, MK211376, and SARS-CoV2. In some embodiments, each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from the RBD nucleic acid sequence of a sarbecovirus sequence from each of KJ473815, MK211376, DQ648856, and SARS-CoV2. In some embodiments, each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from the RBD nucleic acid sequence of a sarbecovirus sequence from each of GQ153542, NC_004718, JX993988, and SARS-CoV2.

[0014] In some embodiments, the at least two different RBD domains encode full-length RBD domains that are collectively at least 70% identical in amino acid composition to full-length RBD domains from at least two of a Clade 1 sarbecovirus, a Clade 2 sarbecovirus, or a Clade 3 sarbecovirus. In some embodiments, the at least two different RBD domains encode full-length RBD domains that are collectively at least 70% identical in amino acid composition to full-length RBD domains from at least two of a Clade 1 sarbecovirus, a Clade 2 sarbecovirus, or a Clade 3 sarbecovirus. In some embodiments, the at least two different coronavirus RBD-derived nucleic acid sequences comprise at least a beta coronavirus RBD-derived nucleic acid sequence.

[0015] In some embodiments, the at least two different coronavirus RBD-derived nucleic acid sequences each comprise a beta coronavirus RBD-derived nucleic acid sequence, hi some embodiments, the at least two different coronavirus RBD-derived nucleic acid sequences are selected from the group consisting of a beta coronavirus RBD-derived nucleic acid sequence, an alpha coronavirus RBD-derived nucleic acid sequence, and a combination thereof.

[0016] In some embodiments, each of the different RBD domains comprises a different respective receptor binding motif (RBM) domain, wherein the different RBM domains are collectively at least 30% identical in amino acid composition to RBM domains from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

[0017] In some embodiments, the amino acid sequences of at least two different RBD domains other than the amino acid sequence of their respective RBM domains are at least 70% identical in amino acid composition to the amino acid sequences of the RBD domain sequences other than the amino acid sequences of the RBMs from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

[0018] Also provided herein is a composition for delivering an antigen expression system, the composition comprising an antigen expression system, wherein the antigen expression system comprises at least two different coronavirus receptor binding domain (RBD)-derived nucleic acid sequences, each encoding at least two different RBDs, wherein the at least two different RBD domains are at least 70% identical in amino acid composition to RBD domains derived from at least two of a sarbecovirus RBD-derived nucleic acid sequence, a merbecovirus RBD-derived nucleic acid sequence, an enveloped virus RBD-derived nucleic acid sequence, and combinations thereof.

[0019] In some embodiments, the at least two different RBD domains comprise 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, or 2 to 20 different RBD domains. In some embodiments, the at least two different RBD domains comprise 3 to 8, 4 to 8, 3 to 8, 4 to 8, 3 to 9, 4 to 9, 3 to 10, or 4 to 10 different RBD domains.

[0020] In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are encoded by a single polynucleotide sequence. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are encoded by a single antigen cassette. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are each encoded by a separate polynucleotide sequence. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are each encoded by a separate antigen cassette. In some embodiments, each separate antigen cassette is encoded by a separate vector.

[0021] In some embodiments, each of the at least two different coronavirus RBD-derived nucleic acid sequences further comprises a different trimerization domain-derived nucleic acid sequence. In some embodiments, each of the at least two different coronavirus RBD-derived nucleic acid sequences comprises the same trimerization domain-derived nucleic acid sequence. In some embodiments, the trimerization domain is selected from the group consisting of a T4 trimerization domain, an MTQ trimerization domain, a GCN4 trimerization domain, and combinations thereof. In some embodiments, each of the at least two different coronavirus RBD-derived nucleic acid sequences comprises a T4 trimerization domain-derived nucleic acid sequence. In some embodiments, the at least two different coronavirus RBD-derived nucleic acid sequences independently comprise a T4 trimerization domain, an MTQ trimerization domain, or a GCN4 trimerization domain.

[0022] In some embodiments, the coronavirus RBD-derived nucleic acid sequence encodes a full-length RBD domain. In some embodiments, the coronavirus RBD-derived nucleic acid sequence encodes an RBD domain that (a) lacks a receptor binding motif (RBM) domain, (b) includes an RBM domain that is not derived from the coronavirus from which the remainder of the RBD is derived, or (c) includes an RBD sequence that is not derived from the coronavirus from which the RBM domain is derived. In some embodiments, the coronavirus RBD-derived nucleic acid sequence encodes only the RBM domain of the corresponding RBD domain. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are directly linked to each other. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are linked such that the sequences can be expressed as a single mRNA. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are linked to each other by a peptide linker-encoding nucleic acid sequence (a nucleic acid sequence encoding a peptide linker). In some embodiments, the peptide linker-encoding nucleic acid sequence encodes a 2A ribosome skipping sequence element optionally selected from the group consisting of an E2A ribosome skipping sequence element, a P2A ribosome skipping sequence element, an F2A ribosome skipping sequence element, a T2A sequence ribosome skipping sequence element, and combinations thereof. In some embodiments, the peptide linker-encoding nucleic acid sequence encodes a cleavable peptide linker optionally selected from a TEV cleavage site, a furin cleavage site, and combinations thereof. In some embodiments, the peptide linker-encoding nucleic acid sequence encodes a T2A sequence ribosome skipping sequence element and a furin cleavage site.

[0023] In some embodiments, each of the at least two different coronavirus RBD-derived nucleic acid sequences further comprises a signal peptide-encoding nucleic acid sequence (a nucleic acid sequence encoding a signal peptide). In some embodiments, the signal peptide comprises a coronavirus-derived signal peptide. In some embodiments, the signal peptide comprises a SARS-CoV-2-derived signal peptide.

[0024] In some embodiments, one or more of the at least two different coronavirus RBD-derived nucleic acid sequences are sequence-optimized.

[0025] In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are operably linked to a promoter. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences are operably linked to a single promoter. In some embodiments, each of the at least two different coronavirus RBD-derived nucleic acid sequences is operably linked to a separate promoter. In some embodiments, the promoter comprises a subgenomic promoter sequence, and optionally, the subgenomic promoter sequence comprises an alphavirus-derived subgenomic promoter.

[0026] In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences each independently encode a peptide in the following format: signal peptide-RBD-trimerization domain. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences encode a linked peptide in the following format: signal peptide-first RBD-first trimerization domain-signal peptide-second RBD-second trimerization domain-signal peptide-third RBD-third trimerization domain. In some embodiments, at least two different coronavirus RBD-derived nucleic acid sequences encode a linked peptide in the following format: signal peptide-first RBD-first trimerization domain-T2A-furin-signal peptide-second RBD-second trimerization domain-T2A-furin-signal peptide-third RBD-third trimerization domain.

[0027] In some embodiments, at least one of the at least two distinct RBD domains comprises a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein comprises: a) a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59, or an epitope-bearing fragment thereof, optionally wherein said spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein said spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87, or b) a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and a combination thereof relative to the spike polypeptide sequence set forth in SEQ ID NO:59, optionally comprising the polypeptide sequence set forth in SEQ ID NO:60 or SEQ ID NO:90, or an epitope-bearing fragment thereof.

[0028] In some embodiments, the antigen expression system comprises at least one coronavirus-derived nucleic acid sequence encoding an immunogenic polypeptide distinct from at least two different RBD domains, hi some embodiments, the at least one coronavirus-derived nucleic acid sequence comprises a β coronavirus-derived nucleic acid sequence.

[0029] In some embodiments, the antigen expression system comprises at least one SARS-CoV-2 derived nucleic acid sequence encoding an immunogenic polypeptide. In some embodiments, the antigen cassette comprises at least one SARS-CoV-2 derived nucleic acid sequence encoding an immunogenic polypeptide. In some embodiments, the at least one SARS-CoV-2 derived nucleic acid sequence comprises a SARS-CoV-2 spike derived nucleic acid sequence. In some embodiments, the SARS-CoV-2 spike-derived nucleic acid sequence comprises: a) a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59, or an epitope-bearing fragment thereof, optionally wherein said spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein said spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; or b) a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and a combination thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO:59, and optionally comprising the polypeptide sequence set forth in SEQ ID NO:60 or SEQ ID NO:90, or an epitope-bearing fragment thereof.

[0030] In some embodiments, at least one coronavirus-derived nucleic acid sequence and / or SARS-CoV-2-derived nucleic acid sequence comprises an MHC class I epitope-encoding sequence. In some embodiments, at least one coronavirus-derived nucleic acid sequence, SARS-CoV-2-derived nucleic acid sequence, and / or MHC class I epitope-encoding sequence comprises: - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A; - at least one MHC class II epitope comprising a polypeptide sequence set forth in Table B; - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table C, optionally present within a linked polypeptide sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58; - at least one polypeptide sequence set forth in Table 7, or an epitope-containing fragment thereof, optionally present within a linked polypeptide sequence set forth in SEQ ID NO: 92, - at least one polypeptide sequence set forth in Table 9A, Table 9B, or Table 9C, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 9A, Table 9B, or Table 9C, and optionally wherein said linked polypeptide comprises the order of the sequences set forth in Table 9A, Table 9B, or Table 9C; - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A and / or Table C, or an MHC class II epitope comprising a polypeptide sequence set forth in Table B, wherein the encoded SARS-CoV-2 immunogenic polypeptide is conserved between SARS-CoV-2 and a coronavirus species and / or subspecies other than SARS-CoV-2, optionally wherein the coronavirus species and / or subspecies other than SARS-CoV-2 is Severe Acute Respiratory Syndrome (SARS) and / or Middle East Respiratory Syndrome (MERS); - one or more validated epitopes and / or at least 4, 5, 6, or 7 predicted epitopes, wherein at least 85%, 90%, or 95% of the population carries at least one HLA validated to present at least one of said one or more validated epitopes and / or at least one HLA predicted to present each of said at least 4, 5, 6, or 7 predicted epitopes, - a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59 or an epitope-containing fragment thereof, optionally wherein the spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein the spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; - a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and combinations thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO: 59, and optionally comprising the polypeptide sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 90, or an epitope-containing fragment thereof; - a SARS-CoV-2 membrane protein comprising the membrane polypeptide sequence set forth in SEQ ID NO: 61 or an epitope-containing fragment thereof; - a SARS-CoV-2 nucleocapsid protein comprising the nucleocapsid polypeptide sequence set forth in SEQ ID NO: 62 or an epitope-containing fragment thereof; - a SARS-CoV-2 envelope protein comprising the envelope polypeptide sequence set forth in SEQ ID NO: 63 or an epitope-containing fragment thereof; - any of the above variants comprising a mutation found in 1% or more of SARS-CoV-2 subtypes, optionally including a SARS-CoV-2 variant set forth in Table 1, and / or optionally including a SARS-CoV-2 variant spike protein comprising the spike D614G mutation relative to the spike polypeptide sequence set forth in SEQ ID NO: 59, optionally a SARS-CoV-2 variant spike protein corresponding to the B.1.351 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO: 112, or optionally a SARS-CoV-2 variant spike protein corresponding to the B.1.1.7 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO: 110; - at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally wherein said linked polypeptide comprises the order of sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D; - or a combination of these Includes.

[0031] In some embodiments, the MHC class I epitope-encoding sequence comprises at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein the at least one polypeptide sequence is present within a concatenated polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, optionally wherein the concatenated polypeptide comprises the order of sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

[0032] In some embodiments, the at least one coronavirus-derived nucleic acid sequence, SARS-CoV-2-derived nucleic acid sequence, and / or MHC class I epitope-encoding sequence is selected from the group consisting of spike proteins, membrane proteins, nucleocapsid proteins, envelope proteins, replicase orf1a and orf1b proteins, and combinations thereof.

[0033] In some embodiments, the at least one coronavirus-derived nucleic acid sequence is encoded on a separate vector that is distinct from one or more vectors encoding at least two different coronavirus RBD-derived nucleic acid sequences, and optionally the encoded immunogenic polypeptide that is distinct from the at least two different coronavirus RBDs comprises an MHC class I epitope-encoding sequence that comprises at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein the at least one polypeptide sequence is present within a concatenated polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally wherein the concatenated polypeptide comprises the order of sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

[0034] In some embodiments, the composition, after administration to a subject, is capable of stimulating an immune response against multiple coronaviruses. In some embodiments, the composition, after administration to a subject, is capable of stimulating an immune response against at least each of the coronaviruses from which the at least two different coronavirus RBD-derived nucleic acid sequences are derived. In some embodiments, the composition, after administration to a subject, is capable of stimulating an immune response against at least a coronavirus different from the coronavirus from which the two different coronavirus RBD-derived nucleic acid sequences are derived. In some embodiments, the composition is capable of stimulating neutralizing antibody production. In some embodiments of the composition, stimulating neutralizing antibody production comprises a neutralizing antibody titer having an NT50 value, calculated as the minimum dilution of serum from an immunized subject that neutralizes the coronavirus by 50%. In some embodiments, stimulating neutralizing antibody production comprises a minimum neutralizing antibody titer for completely neutralizing the coronavirus. In some embodiments, the composition is capable of stimulating antibody production, and the antibodies produced are capable of antibody-mediated viral clearance, optionally including Fc-mediated viral clearance.

[0035] In some embodiments, the antigen expression system comprises one or more vectors, the one or more vectors comprising: (a) a vector backbone comprising a chimpanzee adenovirus (optionally a ChAdV68 vector) or an alphavirus vector (optionally a Venezuelan equine encephalitis virus vector); and (b) a cassette encoding at least two different RBD domains, optionally integrated between a native promoter nucleotide sequence naturally present in the vector backbone and a poly(A) sequence, optionally wherein the poly(A) sequence is naturally present in the vector backbone.

[0036] In some embodiments, the antigen expression system comprises one or more vectors comprising a vector backbone derived from a Venezuelan equine encephalitis virus vector.

[0037] Also provided herein is a composition for delivering an antigen expression system, comprising an antigen expression system, wherein the antigen expression system optionally comprises (a) one or more vectors, the one or more vectors comprising a vector backbone comprising (i) at least one promoter nucleotide sequence and (ii) at least one polyadenylation (poly(A)) sequence, and (b) an antigen cassette, optionally inserted into the vector backbone, if present, wherein the antigen cassette comprises at least one SARS-C polypeptide encoding an immunogenic polypeptide. Compositions are provided that comprise an antigen cassette comprising a oV-2-derived nucleic acid sequence, wherein the immunogenic polypeptide comprises at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, and optionally the at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally the linked polypeptide comprises the order of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

[0038] In some embodiments, the MHC class I epitope-encoding sequence comprises at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein the at least one polypeptide sequence is present within a concatenated polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, optionally wherein the concatenated polypeptide comprises the order of sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

[0039] Also provided are methods for treating a coronavirus infection or preventing a coronavirus infection in a subject, comprising administering to the subject any one of the compositions described herein.

[0040] Also provided herein is a method for inducing an immune response in a subject, comprising administering to the subject any one of the compositions described herein.

[0041] In some embodiments, the methods comprise a homologous priming / booster strategy. In some embodiments, the methods comprise a heterologous priming / booster strategy, optionally comprising (a) the same antigen cassette encoded by different vaccine platforms, (b) different antigen cassettes encoded by the same vaccine platform, and / or (c) different antigen cassettes encoded by different vaccine platforms.

[0042] In some embodiments, the methods include administering one or more vectors encoding at least two different coronavirus RBD-derived nucleic acid sequences, and administering one or more vectors encoding at least one coronavirus-derived nucleic acid sequence that encodes an immunogenic polypeptide that is different from the at least two different RBD domains.

[0043] In some embodiments, one or more vectors encoding at least two different coronavirus RBD-derived nucleic acid sequences and one or more vectors encoding at least one coronavirus-derived nucleic acid sequence are co-formulated. In some embodiments, one or more vectors encoding at least two different coronavirus RBD-derived nucleic acid sequences and the one or more vectors encoding at least one coronavirus-derived nucleic acid sequence are administered separately. In some embodiments, one or more vectors encoding at least two different coronavirus RBD-derived nucleic acid sequences and the one or more vectors encoding at least one coronavirus-derived nucleic acid sequence are administered simultaneously. In some embodiments, the at least one coronavirus-derived nucleic acid sequence is - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A; - at least one MHC class II epitope comprising a polypeptide sequence set forth in Table B; - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table C, optionally present within a linked polypeptide sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58; - at least one polypeptide sequence set forth in Table 7, or an epitope-containing fragment thereof, optionally present within a linked polypeptide sequence set forth in SEQ ID NO: 92, - at least one polypeptide sequence set forth in Table 9A, Table 9B, or Table 9C, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 9A, Table 9B, or Table 9C, and optionally wherein said linked polypeptide comprises the order of the sequences set forth in Table 9A, Table 9B, or Table 9C; - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A and / or Table C, or an MHC class II epitope comprising a polypeptide sequence set forth in Table B, wherein the encoded SARS-CoV-2 immunogenic polypeptide is conserved between SARS-CoV-2 and a coronavirus species and / or subspecies other than SARS-CoV-2, and optionally the coronavirus species and / or subspecies other than SARS-CoV-2 is Severe Acute Respiratory Syndrome (SARS) and / or Middle East Respiratory Syndrome (MERS); - one or more validated epitopes and / or at least 4, 5, 6, or 7 predicted epitopes, wherein at least 85%, 90%, or 95% of the population carries at least one HLA validated to present at least one of said one or more validated epitopes and / or at least one HLA predicted to present each of said at least 4, 5, 6, or 7 predicted epitopes, - a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59 or an epitope-containing fragment thereof, optionally wherein the spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein the spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; - a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and combinations thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO: 59, and optionally comprising the polypeptide sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 90, or an epitope-containing fragment thereof; - a SARS-CoV-2 membrane protein comprising the membrane polypeptide sequence set forth in SEQ ID NO: 61 or an epitope-containing fragment thereof; - a SARS-CoV-2 nucleocapsid protein comprising the nucleocapsid polypeptide sequence set forth in SEQ ID NO: 62 or an epitope-containing fragment thereof; - a SARS-CoV-2 envelope protein comprising the envelope polypeptide sequence set forth in SEQ ID NO: 63 or an epitope-containing fragment thereof; - any of the above variants comprising a mutation found in 1% or more of SARS-CoV-2 subtypes, optionally including a SARS-CoV-2 variant set forth in Table 1, and / or optionally including a SARS-CoV-2 variant spike protein comprising the spike D614G mutation relative to the spike polypeptide sequence set forth in SEQ ID NO: 59, optionally a SARS-CoV-2 variant spike protein corresponding to the B.1.351 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO: 112, or optionally a SARS-CoV-2 variant spike protein corresponding to the B.1.1.7 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO: 110; - at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally wherein said linked polypeptide comprises the order of sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D; - or a combination of these Includes.

[0044] In some embodiments, at least one coronavirus-derived nucleic acid sequence comprises an MHC class I epitope-encoding sequence comprising at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein the at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, optionally wherein the linked polypeptide comprises the order of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

[0045] In some embodiments, the composition further comprises a nanoparticulate delivery vehicle. In some embodiments, the nanoparticulate delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticle delivery vehicle encapsulates an antigen expression system.

[0046] In some embodiments, one or more vectors comprise one or more positive-strand RNAs. In some embodiments, one or more positive-strand RNA vectors have a 5' 7-methylguanosine (m7g) cap. In some embodiments, one or more positive-strand RNA vectors are generated by in vitro transcription. In some embodiments, one or more vectors are autonomously replicating in mammalian cells.

[0047] In some embodiments, the backbone comprises at least one nucleotide 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 backbone comprises at least one nucleotide sequence of Venezuelan equine encephalitis virus. In some embodiments, the backbone comprises at least sequences for nonstructural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, nonstructural protein 1 (nsP1) gene, nsP2 gene, nsP3 gene, and nsP4 gene encoded by nucleotide sequences 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 backbone comprises at least a sequence for nonstructural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by a nucleotide 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 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 backbone does not encode the structural virion proteins capsid E2 and E1. In some embodiments, the antigen cassette is inserted in place of a structural virion protein within the nucleotide 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 having a deletion between base pairs 7544 and 11175. In some embodiments, the backbone comprises the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.In some embodiments, the antigen cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11175 set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, insertion of the antigen cassette results in transcription of a polycistronic RNA comprising the nsP1-4 genes and at least one coronavirus-derived nucleic acid sequence, wherein the nsP1-4 genes and the at least one coronavirus-derived nucleic acid sequence are in separate open reading frames. In some embodiments, the at least one promoter nucleotide sequence is the native 26S promoter nucleotide sequence encoded by the scaffold.

[0048] In some embodiments, the backbone comprises at least one nucleotide sequence of a chimpanzee adenoviral vector, optionally the chimpanzee adenoviral vector is a ChAdV68 vector. In some embodiments, the ChAdV68 backbone comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO: 1, except that at least one gene selected from the group consisting of chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO: 1 is completely or functionally deleted from the sequence, optionally the sequence is completely or functionally deleted from (1) E1A and E1B, (2) E1A, E1B, and E3, or (3) E1A, E1B, E3, and E4 of the sequence set forth in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector backbone comprises genes or regulatory sequences derived from the sequence of SEQ ID NO: 1, optionally wherein the genes are selected from the group consisting of the inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of chimpanzee adenovirus of the sequence set forth in SEQ ID NO: 1. In some embodiments, the ChAdV68 vector backbone comprises a deleted E4 gene comprising a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region. In some embodiments, the ChAdV68 vector backbone comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO: 1, and further comprises (1) an E1 deletion of at least nucleotides 577-3403 of the sequence set forth in SEQ ID NO: 1, (2) an E3 deletion of nucleotides 27,125-31,825 of the sequence set forth in SEQ ID NO: 1, and (3) an E4 deletion of nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO: 1, optionally with an antigen cassette inserted within the E1 deletion. In some embodiments, the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO: 75, and optionally with an antigen cassette inserted within the E1 deletion.In some embodiments, the ChAdV68 vector backbone has one or more deletions between base pairs 577 and 3403 or between base pairs 456 and 3014 of the sequence set forth in SEQ ID NO: 1, and optionally, the vector further has one or more deletions between base pairs 27,125 and 31,825 or between base pairs 27,816 and 31,333. In some embodiments, the ChAdV68 vector backbone has one or more deletions between base pairs 3957 and 10346, between base pairs 21787 and 23370, and between base pairs 33486 and 36193 of the sequence set forth in SEQ ID NO: 1. In some embodiments, the cassette is inserted into the ChAdV backbone in the E1 region, the E3 region, and / or any deleted AdV region that allows for cassette integration. In some embodiments, the ChAdV backbone is generated from one of a first-generation, second-generation, or helper-dependent adenoviral vector.

[0049] In some embodiments, the at least one promoter nucleotide sequence is selected from the group consisting of CMV, SV40, EF-1, RSV, PGK, HSA, MCK, and EBV promoter sequences. In some embodiments, the at least one promoter nucleotide sequence is a CMV promoter sequence. In some embodiments, the at least one promoter nucleotide sequence is an exogenous RNA promoter. In some embodiments, the second promoter nucleotide sequence is a 26S promoter nucleotide sequence or a CMV promoter nucleotide sequence. In some embodiments, the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences or multiple CMV promoter nucleotide sequences, each 26S promoter nucleotide sequence or CMV promoter nucleotide sequence directing transcription of one or more of the separate open reading frames.

[0050] In some embodiments, each MHC class I or MHC class II epitope-encoding coronavirus-derived nucleic acid sequence is selected by: (a) obtaining at least one of exome, transcriptome, and whole genome coronavirus nucleotide sequencing data from a coronavirus or a coronavirus-infected cell, wherein the coronavirus nucleotide sequencing data is used to obtain data representing the peptide sequence of each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on the surface of a coronavirus-infected cell, wherein the set of numerical likelihoods was determined based at least on the received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens used to generate the MHC class I or MHC class II epitope-encoding coronavirus-derived nucleic acid sequence.

[0051] In some embodiments, each MHC class I or MHC class II epitope-encoding coronavirus-derived nucleic acid sequence is selected by: (a) obtaining at least one of exome, transcriptome, and whole genome coronavirus nucleotide sequencing data from a coronavirus virus or coronavirus-infected cells, wherein the coronavirus nucleotide sequencing data is used to obtain data representing each peptide sequence of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on the surface of coronavirus-infected cells, wherein the set of numerical likelihoods is determined based at least on received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens used to generate the at least 18 coronavirus-derived nucleic acid sequences. In some embodiments, the number of sets of selected antigens is between 2 and 20. In some embodiments, the presentation model represents a dependency between (a) the presence of a pairing of a particular one of the MHC alleles with a particular amino acid at a particular position in a peptide sequence, and (b) the likelihood of presentation of such a peptide sequence containing a particular amino acid at a particular position on the surface of a coronavirus-infected cell by a particular one of the paired MHC alleles. In some embodiments, selecting a set of selected antigens includes selecting antigens that have an increased likelihood of being presented on the surface of the coronavirus-infected cell relative to antigens not selected based on the presentation model, and optionally, the selected antigens have been validated as being presented by one or more specific HLA alleles.In some embodiments, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of inducing a coronavirus-specific immune response in a subject relative to antigens not selected based on the presentation model. In some embodiments, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented to naive T cells by professional antigen-presenting cells (APCs) relative to antigens not selected based on the presentation model, optionally where the APCs are dendritic cells (DCs). In some embodiments, selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of being inhibited by central or peripheral tolerance relative to antigens not selected based on the presentation model. In some embodiments, selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of inducing an autoimmune response against normal tissue in a subject relative to antigens not selected based on the presentation model. In some embodiments, the exome or transcriptome coronavirus nucleotide sequencing data is obtained by performing sequencing on coronavirus viruses or coronavirus-infected tissues or cells. In some embodiments, the sequencing is next-generation sequencing (NGS) or any massively parallel sequencing approach.

[0052] In some embodiments, the antigen cassette comprises a junction epitope sequence formed by adjacent sequences within the antigen cassette. In some embodiments, at least one or each junction epitope sequence has an affinity for MHC greater than 500 nM. In some embodiments, each junction epitope sequence is non-self.

[0053] In some embodiments, each of the MHC class I and / or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele that is present in at least 5% of the population. In some embodiments, each of the MHC class I and / or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency in the population of at least 0.01%. In some embodiments, each of the MHC class I and / or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, and each antigen / HLA pair has an antigen / HLA frequency in the population of at least 0.1%.

[0054] In some embodiments, the antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence that comprises a wild-type nucleic acid sequence after translation, and the non-therapeutic epitope is predicted to be presented on an MHC allele of interest. In some embodiments, the predicted non-therapeutic MHC class I or class II epitope sequence is a junction epitope sequence formed by adjacent sequences within the antigen cassette.

[0055] In some embodiments, the prediction is based on a presentation likelihood generated by inputting the sequence of the non-therapeutic epitope into a presentation model. In some embodiments, the order of the at least one coronavirus-derived nucleic acid sequence within the antigen cassette is determined by a series of steps including: (a) generating a set of candidate antigen cassette sequences corresponding to different orders of the at least one coronavirus-derived nucleic acid sequence; (b) determining, for each candidate antigen cassette sequence, a presentation score based on the presentation of non-therapeutic epitopes within the candidate antigen cassette sequence; and (c) selecting candidate antigen cassette sequences associated with a presentation score below a predetermined threshold as antigen cassette sequences for an antigen vaccine.

[0056] Also provided herein are pharmaceutical compositions comprising any of the compositions provided herein and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition further comprises an immunomodulator. In some embodiments, the immunomodulator 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, or an anti-OX-40 antibody or antigen-binding fragment thereof.

[0057] Also provided herein is a vector or set of vectors comprising any of the isolated nucleotide sequences or sets of isolated nucleotide sequences provided herein.

[0058] Also provided herein is an isolated cell comprising an isolated nucleotide sequence or set of isolated nucleotide sequences provided herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or a mutant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell.

[0059] Also provided herein are kits comprising any of the compositions provided herein and instructions for use.

[0060] Also provided herein are methods for treating or preventing a coronavirus infection in a subject, comprising administering to the subject any of the compositions or pharmaceutical compositions provided herein. In some embodiments, the coronavirus-derived nucleic acid sequence encodes at least one immunogenic polypeptide corresponding to a polypeptide encoded by a coronavirus subtype with which the subject is infected or at risk of infection.

[0061] In some embodiments, any of the methods described herein comprises a homologous prime / booster strategy. In some embodiments, any of the methods described herein comprises a heterologous prime / booster strategy. In some embodiments, the heterologous prime / booster strategy comprises the same antigen cassette encoded by different vaccine platforms. In some embodiments, the heterologous prime / booster strategy comprises different antigen cassettes encoded by the same vaccine platform. In some embodiments, the heterologous prime / booster strategy comprises different antigen cassettes encoded by different vaccine platforms. In some embodiments, the different antigen cassettes comprise a spike-encoding cassette and a separate T-cell epitope-encoding cassette. In some embodiments, the different antigen cassettes comprise cassettes encoding different epitopes and / or antigens derived from different isolates of coronavirus.

[0062] Also provided herein are methods for inducing an immune response in a subject, comprising administering to the subject any of the compositions or pharmaceutical compositions provided herein. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I or MHC class II epitope encoded by the at least one coronavirus-derived nucleic acid sequence. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class I epitope encoded by the at least one coronavirus-derived nucleic acid sequence, wherein the MHC class I epitope comprises at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A. In some embodiments, the subject expresses at least one HLA allele predicted or known to present an MHC class II epitope encoded by the at least one coronavirus-derived nucleic acid sequence, wherein the MHC class II epitope comprises at least one MHC class II epitope comprising a polypeptide sequence set forth in Table B. In some embodiments, the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some embodiments, the composition is administered intramuscularly.

[0063] In some embodiments, the method further comprises administering a second vaccine composition to the subject. In some embodiments, the second vaccine composition is administered before administration of the first composition or pharmaceutical composition. In some embodiments, the second vaccine composition is administered after administration of any of the compositions or pharmaceutical compositions provided herein. In some embodiments, the second vaccine composition is the same as the first composition or pharmaceutical composition administered. In some embodiments, the second vaccine composition is different from the first composition or pharmaceutical composition administered. In some embodiments, the second vaccine composition comprises a chimpanzee adenoviral vector encoding at least one coronavirus-derived nucleic acid sequence. In some embodiments, the at least one coronavirus-derived nucleic acid sequence encoded by the chimpanzee adenoviral vector is the same as at least one coronavirus-derived nucleic acid sequence of any of the compositions provided herein.

[0064] Also provided herein are methods for producing one or more vectors of any of the preceding composition claims, the methods comprising: (a) obtaining a linearized DNA sequence comprising a backbone and an antigen cassette; (b) in vitro transcribing the linearized DNA sequence by adding the linearized DNA sequence to an in vitro transcription reaction containing all components necessary for transcribing the linearized DNA sequence into RNA, optionally including in vitro addition of an m7g cap to the resulting RNA; and (c) isolating one or more vectors from the in vitro transcription reaction. In some embodiments, the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR. In some embodiments, the plasmid sequence is generated using one of bacterial recombination or total genome DNA synthesis or total genome DNA synthesis with amplification of DNA synthesized in bacterial cells. In some embodiments, isolating one or more vectors from the in vitro transcription reaction comprises one or more of phenol-chloroform extraction, purification using a silica column, or similar RNA purification methods.

[0065] Also provided herein is a method of making a composition according to any of the preceding composition claims for delivering an antigen expression system, the method comprising: (a) providing components of a nanoparticulate delivery vehicle; (b) providing an antigen expression system; and (c) providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to form a composition for delivering the antigen expression system. In some aspects, such conditions are provided by microfluidic mixing.

[0066] Also disclosed herein is a method for producing an adenoviral vector disclosed herein, comprising obtaining a plasmid sequence comprising the at least one promoter sequence and the antigen cassette, transfecting the plasmid sequence into one or more host cells, and isolating the adenoviral vector from the one or more host cells.

[0067] In some embodiments, the isolating comprises lysing the host cells to obtain a cell lysate comprising the adenoviral vector, and purifying the adenoviral vector from the cell lysate.

[0068] In some embodiments, the plasmid sequences are generated using one of bacterial recombination or total genomic DNA synthesis or total genomic DNA synthesis with amplification of DNA synthesized in bacterial cells. In some embodiments, the one or more host cells are at least one of CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, and AE1-2a cells. In some embodiments, purifying the adenoviral vector from the cell lysate involves one or more of chromatographic separation, centrifugation, virus precipitation, and filtration.

[0069] In some embodiments, any of the above compositions further comprises a nanoparticulate delivery vehicle. In some embodiments, the nanoparticulate delivery vehicle can be a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable amino lipid. In some embodiments, the ionizable amino lipid comprises an MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecule. In some embodiments, the nanoparticle delivery vehicle encapsulates an antigen expression system.

[0070] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising an antigen expression system, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of the LNPs, wherein at least about 95% of the LNPs have either a non-lamellar morphology or are electron-dense.

[0071] In some embodiments, the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.

[0072] In some embodiments, the conjugated lipid that inhibits aggregation of LNPs is a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is selected from the group consisting of PEG-diacylglycerol (PEG-DAG) conjugates, PEG-dialkyloxypropyl (PEG-DAA) conjugates, PEG-phospholipid conjugates, PEG-ceramide (PEG-Cer) conjugates, and mixtures thereof. In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate, PEG-distearyloxypropyl (C 18 ) complexes, and mixtures thereof.

[0073] In some embodiments, the antigen expression system is fully encapsulated in the LNP.

[0074] In some embodiments, the non-lamellar morphology of the LNPs is an inverted hexagonal (H II ) or cubic phase structure.

[0075] In some embodiments, the cationic lipids comprise about 10 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the cationic lipids comprise about 20 mol% to about 40 mol% of the total lipids present in the LNP.

[0076] In some embodiments, non-cationic lipids comprise about 10 mol% to about 60 mol% of the total lipids present in the LNP. In some embodiments, cationic lipids comprise about 20 mol% to about 55 mol% of the total lipids present in the LNP. In some embodiments, cationic lipids comprise about 25 mol% to about 50 mol% of the total lipids present in the LNP.

[0077] In some embodiments, complex lipids comprise about 0.5 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 2 mol% to about 20 mol% of the total lipids present in the LNP. In some embodiments, complex lipids comprise about 1.5 mol% to about 18 mol% of the total lipids present in the LNP.

[0078] In some embodiments, greater than 95% of the LNPs have a non-lamellar morphology. In some embodiments, greater than 95% of the LNPs are electron-dense.

[0079] In some embodiments, any of the above compositions further comprises a plurality of LNPs, the LNPs comprising a cationic lipid comprising 50 mol% to 65 mol% of the total lipids present in the LNPs, a complex lipid that inhibits aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and a non-cationic lipid, the complex lipid being a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises 4 mol% to 10 mol% of the total lipids present in the LNPs and the cholesterol or a derivative thereof comprises 30 mol% to 40 mol% of the total lipids present in the LNPs. The mixture comprises the non-cationic lipid, which is either a mixture of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids constitute 3 mol% to 15 mol% of the total lipids present in the LNP and the cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipids present in the LNP, or a mixture which is 49.5 mol% or less of the total lipids present in the LNP, comprises a mixture of phospholipids and cholesterol or a derivative thereof, and wherein the cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipids present in the LNP.

[0080] In some embodiments, any of the above compositions further comprise a plurality of LNPs, the LNPs comprising cationic lipids comprising 50 mol% to 85 mol% of the total lipids present in the LNPs, complex lipids that inhibit aggregation of the LNPs comprising 0.5 mol% to 2 mol% of the total lipids present in the LNPs, and non-cationic lipids that comprise 13 mol% to 49.5 mol% of the total lipids present in the LNPs.

[0081] In some embodiments, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0082] In some embodiments, the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof. In some embodiments, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearoyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In some embodiments, the PEG portion of the conjugate has an average molecular weight of about 2,000 daltons.

[0083] In some embodiments, complex lipids comprise between 1 mol% and 2 mol% of the total lipids present in the LNP.

[0084] In some embodiments, the LNP has the structure of Formula I: TIFF2024537791000002.tif50128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, -C(=0)-, -0-, -S(0) x -, -SS-, -C(=0)S-, -SC(=0)-, -R a C(=0)-, -C(=0)R a -, -R a C(=0)R a -, -OC(=0)R a -, -R a C(=0)0- or a direct bond, and G 1 is Ci to C2 alkylene, -(C=0)-, -0(C=0)-, -SC(=0)-, -R a C(=0)-, or a direct bond, -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)R a - or a direct bond, G is a Ci-C6 alkylene, and R a is H or C1-C12 alkyl, and R 1a and R 1b is, in each case independently: (a) H or C1-C 12 alkyl, or (b) R1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b are, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b are, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b forms a carbon-carbon double bond with the carbon atom to which it is attached, and with the adjacent R and the carbon atom to which it is attached, and R 4a and R 4b is, independently in each occurrence: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently H or methyl, and R 7 is a C4 to C20 alkyl, and R 8 and R 9 are each independently C1-C12 alkyl, or R 8 and R 9together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are each independently an integer from 1 to 24; and x is 0, 1, or 2; or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0085] In some embodiments, the LNP has the structure of Formula II: TIFF2024537791000003.tif38128[where L 1 and L 2 are each independently -0(C=0)-, -(C=0)0-, or a carbon-carbon double bond; R 1a and R 1b are, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 2a and R 2b are, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1~C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 3a and R 3b are, in each case independently: (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1~C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 4a and R 4b In each case, (a) H or C1~C12 alkyl, or (b) R 4a is H or C1~C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and form a carbon-carbon double bond together with the carbon atom to which it is attached, R 5 and R 6 are each independently H or methyl, and R 7 is, in each case, independently H or C1-C 12 alkyl, and R 8 and R 9 are each independently unsubstituted C1 to C 12 alkyl or R 8 and R 9 and together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom, a and d each independently represent an integer of 0 to 24, b and c each independently represent an integer of 1 to 24, and e is 1 or 2, with the proviso that R 1a , R 2a , R 3a , or R 4a At least one of the groups is C1-C12 alkyl, or L 1 or L 2 At least one of is -0(C=0)- or -(C=0)0-, and R 1a and R 1b is not isopropyl when a is 6 and is not n-butyl when a is 8; or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0086] In some embodiments, any of the above compositions further comprises one or more excipients, including a neutral lipid, a steroid, and a polymer-bound lipid. In some embodiments, the neutral lipid comprises at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the neutral lipid is DSPC.

[0087] In some embodiments, the molar ratio of compound to neutral lipid ranges from about 2:1 to about 8:1.

[0088] In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0089] In some embodiments, the polymer-bound lipid is a PEGylated lipid. In some embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 25:1. In some embodiments, the PEGylated lipid is PEG-DAG, PEG polyethylene (PEG-PE), PEG-succinoyl-diacylglycerol (PEG-S-DAG), PEG-cer, or PEG dialkyloxypropylcarbamate. In some embodiments, the PEGylated lipid has the following structure III: TIFF2024537791000004.tif26128[where, R 10 and R 11are each independently a straight or branched, saturated or unsaturated alkyl chain having from 10 to 30 carbon atoms, said alkyl chain optionally interrupted by one or more ester bonds, and z has an average value in the range of 30 to 60, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. 10 and R 11 are each independently a linear saturated alkyl chain having 12 to 16 carbon atoms. In some embodiments, the average z is about 45.

[0090] In some embodiments, the LNPs self-assemble into non-bilayer structures when mixed with polyanionic nucleic acids. In some embodiments, the non-bilayer structures have diameters of 60 nm to 120 nm. In some embodiments, the non-bilayer structures have diameters of about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, the nanoparticulate delivery vehicles have diameters of about 100 nm.

[0091] Also provided herein is a vector or set of vectors comprising any of the above-described nucleotide sequences. Also disclosed herein are vectors comprising the isolated nucleotide sequences disclosed herein.

[0092] Also provided herein is an isolated cell comprising a nucleotide sequence or set of isolated nucleotide sequences described herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or mutant thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a cell.

[0093] Also provided herein are kits comprising any of the compositions described herein and instructions for use. Also disclosed herein are kits comprising a vector or composition disclosed herein and instructions for use.

[0094] Also provided herein is a method for treating a subject suffering from Covid-19, comprising administering to the subject any of the compositions described herein or any of the pharmaceutical compositions described herein.

[0095] Also provided herein is a method for treating a subject infected with or at risk of infection with a coronavirus, comprising administering to the subject any of the compositions described herein or any of the pharmaceutical compositions described herein.

[0096] Also provided herein is a method for stimulating an immune response in a subject, comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein.

[0097] Also disclosed herein is a method for treating a subject, comprising administering to the subject a vector disclosed herein, or a pharmaceutical composition disclosed herein.

[0098] Also provided herein are methods of producing one or more vectors of any of the above compositions.

[0099] Also provided herein are methods of making any of the compositions disclosed herein. [Brief explanation of the drawings]

[0100] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings.

[0101] [Figure 1]A schematic diagram of the genome structure of SARS-CoV-2 showing at least 14 open reading frames (ORFs) identified in a diagram adapted from Zhou et al. (2020) [A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579 (January)].

[0102] [Figure 2] This figure shows the 16 cleavage products of replicase ORF1ab and related information. The figure is adapted from Wu et al. (2020). [A new coronavirus associated with human respiratory disease in China. Nature, 579 (January)].

[0103] [Figure 3] This paper presents a general vaccination strategy to generate a balanced immune response that induces both neutralizing antibodies (from B cells) and effector and memory CD8+ T cell responses for maximum efficacy. The genome structure of SARS-CoV-2 is adapted from Zhou et al. (2020) [A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579 (January)].

[0104] [Figure 4] Figure 1 shows the known prevalence of wild-type and D614G variant SARS-CoV-2 spike proteins over time in different geographic locations.

[0105] [Figure 5]The figure shows the coverage of cassettes encoding spike alone, or spike plus additional predicted concatenated T cell epitopes, across the four populations shown. Column 1 shows the number of SARS-CoV-2 epitopes predicted to be presented, and column 2 shows the expected number of epitopes presented based on 0.2 PPV. Each row shows the protective coverage for each population using a specific number of epitopes.

[0106] [Figure 6A] The number of epitopes predicted to be presented by each MHC class II for the spike protein or additional predicted linked T cell epitopes is shown separately.

[0107] [Figure 6B] The number of SARS-CoV-2 epitopes predicted to be presented across the four populations is shown, shown from cassettes encoding spike only (top panel) or spike plus additional predicted concatenated T cell epitopes (bottom panel).

[0108] [Figure 7A] The number of training samples containing class I alleles is indicated (including at least 10 samples).

[0109] [Figure 7B] A histogram showing the number of alleles versus the number of training samples for each class I allele is shown.

[0110] [Figure 8A] Western blots using anti-spike S2 ​​antibody are shown for spike expression in vectors encoding different spike variations.

[0111] [Figure 8B] Western blots using anti-spike S1 antibody are shown for spike expression in vectors encoding different spike variations.

[0112] [Figure 8C] Western blots using anti-spike S1 antibody are shown for spike expression in vectors encoding full-length spike, spike S1 only, or spike S2 ​​only.

[0113] [Figure 8D] Western blots using anti-spike S2 ​​antibody are shown for spike expression in vectors encoding full-length spike, spike S1 only, or spike S2 ​​only.

[0114] [Figure 9] Western blots using anti-spike S2 ​​antibody are shown for spike expression in vectors encoding various sequence-optimized spike variations.

[0115] [Figure 10A] Schematic diagram of a PCR-based assay for assessing RNA splicing of SARS-CoV-2 transcripts.

[0116] [Figure 10B] PCR amplicons of the encoded spike protein are shown. The left panel shows amplicons from a cDNA template ("ChAd-Spike (IDT) cDNA") from infected 293 cells or a plasmid encoding the SARS-CoV-2 spike cassette ("Spike Plasmid"). The right panel shows amplicons from cDNA from 293 cells infected with vectors encoding spike only ("Spike S1") or full-length spike ("Spike").

[0117] [Figure 11] PCR amplicons of the encoded spike protein from cDNA of 293 cells infected with vectors encoding different spike variants are shown.

[0118] [Figure 12]Estimated coverage is shown for the proportion of the indicated ancestry population with at least one HLA predicted to receive at least one immunogenic epitope encoded by TCE5. Reception of immunogenic peptide presentation is considered to occur if an individual's HLA is either (1) known to present the encoded epitope ("validated epitopes") or (2) predicted to present at least four (column 1), five (column 2), six (column 3), or seven (column 4) encoded epitopes ("predicted epitopes"; EDGE score >.01). FA = African American, API = Asian or Pacific Islander, EUR = European, HIS = Hispanic.

[0119] [Figure 13A] T cell responses (left panel), spike-specific IgG antibodies (middle panel), and neutralizing antibodies (right panel) after administration of ChAdV platforms containing spike-coding cassettes with different sequence optimization (IDT spike g ("spike V1" or "v1") or "CT spike g" ("spike V2" or "v2"). Balb / c mice were immunized with 1 x 10 VP of the ChAdV-based vaccine platform.

[0120] [Figure 13B] Figure 1 shows T cell responses (left panel), spike-specific IgG antibodies (middle panel), and neutralizing antibodies (right panel) after administration of the SAM platform containing spike-coding cassettes with different sequence optimization (IDT spike g ("spike V1" or "v1") or "CT spike g" ("spike V2" or "v2"). Balb / c mice were immunized with 10 μg of the SAM-based vaccine platform.

[0121] [Figure 14]Spike-specific IgG antibody production after administration of ChAdV platforms (left panel) or SAM platforms (right panel) containing unmodified or modified (CTSpikeF2Pg, denoted as "SpikeF2P") spike-coding cassettes (both vectors use spike sequence v2). Balb / c mice were immunized with 1 x 10 VP of the ChAdV-based vaccine platform or 10 μg of the SAM-based vaccine platform, as indicated.

[0122] [Figure 15A] Figure 1 shows T cell responses to spike (left panel) and the encoded T cell epitopes (right panel) after administration of ChAdV platforms containing a cassette encoding only the modified spike ("CTspikeF2Pg" denoted as "spike") and TCE5 encoding the modified spike and an additional non-spike T cell epitope (denoted as "spikeTCE"). Balb / c mice were immunized with 1x10 VP of the ChAdV-based vaccine platform. IFNγ ELISpots are shown 2 weeks after immunization. T cell responses to overlapping peptide pools spanning spike, nucleocapsid, or Orf3a.

[0123] [Figure 15B] Figure 1 shows T cell responses to spike (left panel) and the encoded T cell epitopes (right panel) after administration of SAM platforms containing cassettes encoding only the modified spike ("CTspikeF2Pg" shown as "spike") and TCE5 encoding the modified spike and additional non-spike T cell epitopes (shown as "TCEspike"). Balb / c mice were immunized with 10 μg of the SAM-based vaccine platform. IFNγ ELISpots are shown 2 weeks after immunization. T cell responses to overlapping peptide pools spanning spike, nucleocapsid, or Orf3a.

[0124] [Figure 16A]Following immunization with SAM constructs containing IDT spike g alone (left column), IDT spike g expressed from the first subgenomic promoter followed by TCE5 expressed from the second subgenomic promoter (center column), or TCE5 expressed from the first subgenomic promoter followed by IDT spike g expressed from the second subgenomic promoter (right column), T cell responses to the spike (top panel, IFN g ELISpot; sum of responses to eight overlapping peptide pools across the spike antigen), T cell responses to the encoded T cell epitopes (center panel, IFN g ELISpot; sum of responses to three overlapping peptide pools across nucleocapsid, membrane, and Orf3a), and spike-specific IgG antibodies (bottom panel, IgG binding of S1 measured by MSD ELISA; endpoint titers interpolated; geometric mean, geometric SD) are shown. For T cell responses, Balb / c mice were immunized with 10 μg of each vaccine (n = 6 per group). Splenocytes were isolated two weeks after immunization. For IgG responses, Balb / c mice were immunized with 10 μg of each vaccine (n=4 / group). Serum was collected and analyzed four weeks after immunization.

[0125] [Figure 16B]Shown are T cell responses to the spike (top panel, IFNg ELISpot; sum of responses to eight overlapping peptide pools across the spike antigen), T cell responses to the encoded T cell epitopes (middle panel, IFNg ELISpot; sum of responses to three overlapping peptide pools across nucleocapsid, membrane, and Orf3a), and spike-specific IgG antibodies (bottom panel, IgG binding of S1 measured by MSD ELISA; endpoint titers interpolated; geometric mean, geometric SD) after immunization with SAM constructs containing IDT spike g alone (column 1), IDT spike g expressed from the first subgenomic promoter followed by TCE6 or TCE7 expressed from the second subgenomic promoter (columns 2 and 4, respectively), or TCE6 or TCE7 expressed from the first subgenomic promoter followed by IDT spike g expressed from the second subgenomic promoter (columns 3 and 5, respectively). For T cell responses, Balb / c mice were immunized with 10 μg of each vaccine (n=6 / group). Splenocytes were isolated two weeks after immunization. For IgG responses, Balb / c mice were immunized with 10 μg of each vaccine (n=4 / group). Serum was collected and analyzed four weeks after immunization.

[0126] [Figure 16C]Shown are T cell responses to the spike (top panel, IFNg ELISpot; sum of responses to two overlapping peptide pools spanning the spike antigen), T cell responses to the encoded T cell epitopes (middle panel, IFNg ELISpot; sum of responses to two overlapping peptide pools spanning nucleocapsid and Orf3a), and spike-specific IgG antibodies (bottom panel, IgG binding of S1 measured by MSD ELISA; endpoint titers interpolated; geometric mean, geometric SD) after immunization with SAM constructs containing CT spike g alone (column 1), CT spike g expressed from the first subgenomic promoter followed by TCE5 or TCE8 expressed from the second subgenomic promoter (columns 2 and 4, respectively), or TCE5 or TCE8 expressed from the first subgenomic promoter followed by CT spike g expressed from the second subgenomic promoter (columns 3 and 5, respectively). For T cell responses, Balb / c mice were immunized with 10 μg of each vaccine (n=6 / group). Splenocytes were isolated two weeks after immunization. For IgG responses, Balb / c mice were immunized with 10 μg of each vaccine (n=4 / group). Serum was collected and analyzed four weeks after immunization.

[0127] [Figure 17A] 1 shows a map of the sequence contained in TCE10 of the nucleocapsid, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0128] [Figure 17B] 1 shows a map of the sequence contained in TCE10 of ORF3a, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0129] [Figure 17C] 1 shows a map of the sequence contained in TCE10 of nsp3, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0130] [Figure 17D] A map of the sequence contained in membrane TCE10 is shown, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlap between frames and mutations.

[0131] [Figure 17E] 1 shows a map of the sequence contained in TCE10 of nsp4, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0132] [Figure 17F] 1 shows a map of the sequence contained in TCE10 of nsp12, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0133] [Figure 18A] 1 shows a map of the sequence contained in TCE9 of nsp12, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0134] [Figure 18B] 1 shows a map of the sequence contained in TCE9 of nsp4, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0135] [Figure 18C] 1 shows a map of the sequence contained in membrane TCE9, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlap between frames and mutations.

[0136] [Figure 18D]1 shows a map of the sequence contained in TCE9 of nsp3, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0137] [Figure 18E] 1 shows a map of the sequence contained in TCE9 of ORF3a, including flanking sequences, validated epitopes, predicted epitopes, mutations, and in-frame with overlaps between the mutations.

[0138] [Figure 18F] 1 shows a map of the sequence contained in TCE9 of the nucleocapsid, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0139] [Figure 18G] 1 shows a map of the sequence contained in TCE9 of nsp6, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0140] [Figure 19A] 1 shows a map of the sequence contained in TCE11 of nsp12, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0141] [Figure 19B] A map of the sequence contained in membrane TCE11 is shown, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlap between frames and mutations.

[0142] [Figure 19C] 1 shows a map of the sequence contained in TCE11 of nsp4, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0143] [Figure 19D] 1 shows a map of the sequence contained in TCE11 of nsp3, including flanking sequences, validated epitopes, predicted epitopes, mutations, and frames with overlaps between frames and mutations.

[0144] [Figure 20] The percentage of shared candidate 9-mer epitope distribution between SARS-CoV-2 and SARS-CoV (left panel) and SARS-CoV-2 and MERS (right panel) is shown.

[0145] [Figure 21] A general outline of the selection of RBD sequences for inclusion in a vaccine is shown. BLOSUM62 is a function of (AA,AA) → R and is an expression of homology, a proxy for functional similarity, with higher values ​​indicating higher similarity. The average BLOSUM62 similarity, a and b, for a pair of aligned sequences is the sum of BLOSUM62 (ai,bi) divided by the length of a. The average BLOSUM62 distance across two aligned sequences is the negation of the negation of the average BLOSUM62 similarity.

[0146] [Figure 22A] Shown are the viral isolates selected (highlighted) for inclusion of their respective RBDs in the vaccine based on analysis of the phylogenetic tree and full-length RBD sequences.

[0147] [Figure 22B] Phylogenetic tree and viral isolates selected (highlighted) for inclusion of each RBD in the vaccine based on analysis of RBDΔRBM sequences are shown.

[0148] [Figure 22C] Phylogenetic tree and viral isolates selected (highlighted) for inclusion of their respective RBDs in the vaccine based on analysis of RBM sequences only are shown.

[0149] [Figure 23] Sarbecovirus clade designations of representative analyzed isolates, including those with selected RBDs, are shown.

[0150] [Figure 24] The amino acid similarity of the RBDs for representative isolates analyzed, including those with selected RBDs, is shown.

[0151] [Figure 25] Schematic diagram of the "mixed" (top panel) and single-vector (bottom panel) pan-coronavirus vaccine strategies.

[0152] [Figure 26] Western blots using anti-SARS-CoV-2 polyclonal antibodies run under the indicated conditions are shown.

[0153] [Figure 27A] Anti-RBD antibody responses to RBD-matched (gray or black bars) and mismatched vaccines (colored bars) measured 4 weeks after priming with group 1 vaccine (MK / KP / KJ) are shown.

[0154] [Figure 27B] Anti-RBD antibody responses to RBD-matched (gray or black bars) and mismatched vaccines (colored bars) measured 4 weeks after priming with the group 2 vaccine (DQ / KJ / MK) are shown.

[0155] [Figure 27C] Anti-RBD antibody responses to RBD-matched (gray or black bars) and mismatched (colored bars) vaccines measured 4 weeks after priming with the group 3 vaccine (GQ / JX / NC) are shown.

[0156] [Figure 28]Pseudovirus neutralization assays (PNAs) are shown for the indicated samRNA vectors in naive mice 4 weeks after priming (upper panel) and 4 weeks after boosting (lower panel).

[0157] [Figure 29] Pseudovirus neutralization assay (PNA) against the indicated "mixed" samRNA vectors in naive mice.

[0158] [Figure 30A] Anti-RBD antibody responses to samRNA vectors expressing full-length spike and three RBDs on the same SAM backbone in a homologous prime / booster strategy are shown.

[0159] [Figure 30B] Neutralizing antibody titers assessed by PNA for samRNA vectors expressing full-length spike and three RBDs on the same SAM backbone in a homologous prime / booster strategy are shown.

[0160] [Figure 31] Neutralizing antibody titers assessed by PNA after booster (lower panel) and after priming (upper panel) are shown.

[0161] [Figure 32] Neutralizing antibody titers assessed by PNA for vectors expressing full-length spike and three RBDs on the same SAM backbone in a heterologous prime / booster strategy are shown.

[0162] [Figure 33] Relative TCE RNA expression levels measured by RT-qPCR and normalized to another non-coronavirus TCE vector are shown.

[0163] [Figure 34] Figure 1 shows T cell responses induced by different TCE-encoding vaccine vectors measured by ELISpot.

[0164] [Figure 35] Figure 1 shows T cell responses induced by different TCE12-encoding samRNAs and ChAdV vaccine vectors as measured by ELISpot.

[0165] [Figure 36] We present homologous and heterologous priming / booster regimens in Indian rhesus macaques evaluating ChAdV and SAM platforms encoding different isolates of the SARS-CoV-2 spike protein.

[0166] [Figure 37A] For Group 1, T cell responses across multiple spike T cell epitope pools (top panel, mean ± SE for each pool), T cell responses over time in individual NHPs induced against a single large spike T cell epitope pool (middle panel), and spike-specific IgG antibody titers over time (bottom panel) (n = 5 NHPs) are shown.

[0167] [Figure 37B] For Group 2, T cell responses across multiple spike T cell epitope pools (top panel, mean ± SE for each pool), T cell responses over time in individual NHPs induced against a single large spike T cell epitope pool (middle panel), and spike-specific IgG antibody titers over time (bottom panel) (n = 5 NHPs) are shown.

[0168] [Figure 37C] For Group 5, T cell responses across multiple spike T cell epitope pools (top panel, mean ± SE for each pool), T cell responses over time in individual NHPs induced against a single large spike T cell epitope pool (middle panel), and spike-specific IgG antibody titers over time (bottom panel) (n = 5 NHPs) are shown.

[0169] [Figure 37D] For Group 6, T cell responses across multiple spike T cell epitope pools (top panel, mean ± SE for each pool), T cell responses over time in individual NHPs induced against a single large spike T cell epitope pool (middle panel), and spike-specific IgG antibody titers over time (bottom panel) (n = 5 NHPs) are shown.

[0170] [Figure 38] For group 1, T cell responses over time in individual NHPs induced against a single large spike T cell epitope pool (top panel), T cell responses against TCE5-encoded epitopes (middle panel), and spike-specific IgG antibody titers over time (bottom panel) are shown (n=5 NHPs).

[0171] [Figure 39] For each NHP group, neutralizing antibody production against D614G pseudovirus (left panel) and B.1.351 pseudovirus (right panel) after booster 1 (left column) and booster 2 (right column) is shown.

[0172] [Figure 40] Neutralizing antibody production is shown comparing relative Nab titer levels against each pseudovirus after booster 1 (top panel) and booster 2 (bottom panel).

[0173] [Figure 41] Dosing regimens are shown in rhesus macaques immunized twice with specific doses of 30 μg or 300 μg of SAM encoding the spike antigen of SARS-CoV-2. Spike-specific overlapping peptide pools measured in serum at week 8 of the study by pseudovirus neutralization assay (left panel) and PBMCs assessed by ex vivo IFNγ ELISpot after overnight stimulation with neutralizing antibodies (right panel).

[0174] [Figure 42]Anti-RBD IgG titers against different RBD domains are shown, assessed by MSD ELISA, for a single SAM vector expressing group 1 ("V1"), group 2 ("V2"), or group 3 ("V3") antigens (SARS-CoV2 spike + three sarbecovirus RBDs) compared to the SAM-SARS-CoV2 vaccine alone ("CoV-2 spike"). Arrows indicate vaccine components. Anti-RBD or spike data 4 weeks after vaccination. Each vaccine was administered to Balb / c mice at a dose of 5 μg.

[0175] [Figure 43] Anti-RBD IgG titers against different RBD domains are shown by MSD ELISA for a single SAM vector expressing group 2 ("V2") antigens (SARS-CoV2 spike + three sarbecovirus RBDs) compared to the SAM-SARS-CoV-2 vaccine alone ("CoV-2 spike"). Arrows indicate vaccine components. A homologous prime / booster strategy with a booster 4 weeks after priming is shown. Anti-RBD or spike data 4 weeks after booster vaccination (week 8). Each vaccine was administered to Balb / c mice at a dose of 5 μg.

[0176] [Figure 44] Neutralizing antibody titers assessed by PNA for a single SAM vector expressing group 2 ("V2") antigens (SARS-CoV-2 spike + three sarbecovirus RBDs) compared to the SAM-SARS-CoV2 vaccine alone ("CoV-2 spike"). Arrows indicate vaccine components. A homologous prime / booster strategy with a booster 4 weeks after priming is shown. Neutralizing antibody titers assessed 4 weeks after booster vaccination (week 8). Each vaccine was administered to Balb / c mice at a dose of 5 μg.

[0177] [Figure 45]Figure 1 shows anti-RBD IgG titers against different RBD domains assessed by MSD ELISA for a single SAM vector expressing group 2 ("V2") antigens (SARS-CoV2 spike + three sarbecovirus RBDs), a combination of a vector expressing group 2 antigens with a vector expressing the TCE12 epitope cassette ("mix"), compared to the SAM-SARS-CoV2 vaccine alone ("CoV-2 spike"). Arrows indicate vaccine components. A homologous prime / booster strategy with a booster 4 weeks after priming is shown. Anti-RBD or spike data 8 weeks after vaccination. Each vaccine was administered to Balb / c mice at a dose of 5 μg, with 5 μg of each individual vector administered as a single dose in the combined composition.

[0178] [Figure 46] Neutralizing antibody titers assessed by PNA are shown for a single SAM vector expressing group 2 ("V2") antigens (SARS-CoV-2 spike + three sarbecovirus RBDs), a combination of a vector expressing a group 2 antigen with a vector expressing the TCE12 epitope cassette ("mix"), compared to the SAM-SARS-CoV2 vaccine alone ("CoV-2 spike"). Arrows indicate vaccine components. A homologous prime / booster strategy with a booster 4 weeks after priming is shown. Neutralizing antibody titers assessed 8 weeks after vaccination. Each vaccine was administered to Balb / c mice at a dose of 5 μg, with 5 μg of each individual vector administered as a single dose in the combined composition.

[0179] [Figure 47]Figure 1 shows T cell responses induced by a TCE12-encoding vaccine vector against Nsp13, as measured by ELISpot, following administration of a single SAM vector expressing group 2 ("3xRBD") antigens (SARS-CoV2 spike + three sarbecovirus RBDs), a combination of a vector expressing a group 2 antigen with a vector expressing a TCE12 epitope cassette administered as a single dose in a mixed composition ("3xRBD+TCE12"), a combination of a vector expressing a group 2 antigen with a vector expressing a TCE12 epitope cassette administered as separate doses in the left and right legs ("3xRBD+TCE12(R+L)"), and a vector expressing a TCE12 epitope cassette ("TCE12"), compared to SARS-CoV-2 vaccine alone ("Spike-Delta"). Splenocytes were harvested 14 days after vaccination and T cell responses were measured by ELISpot assay using overlapping peptide pools specific for NSP13. Each vaccine contained 3 μg of each individual vector when combined and was administered to Balb / c mice at a dose of 3 μg. DETAILED DESCRIPTION OF THE INVENTION

[0180] Detailed Description I. Definition Generally, terms used in the claims and the specification shall be interpreted as having their ordinary meaning as understood by one of ordinary skill in the art. Certain terms are defined below to provide further clarity. If there is a conflict between the ordinary meaning and a given definition, the given definition shall control.

[0181] As used herein, the term "antigen" refers to a substance that stimulates an immune response. The antigen may be a "shared antigen," which is an antigen found among a particular population, for example, a particular population of SARS-CoV-2 patients with or at risk of infection.

[0182] As used herein, the term "antigen-based vaccine" refers to a vaccine composition based on one or more antigens, e.g., multiple antigens. The vaccine may be nucleotide-based (e.g., virus-based, RNA-based, or DNA-based), protein-based (e.g., peptide-based), or a combination thereof.

[0183] As used herein, the term "candidate antigen" refers to a mutation or other abnormality that gives rise to a sequence that is a potential antigen.

[0184] As used herein, the term "coding region" refers to the portion or portions of a gene that encodes a protein.

[0185] As used herein, the term "coding mutation" refers to a mutation that occurs in the coding region.

[0186] As used herein, the term "ORF" means open reading frame.

[0187] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0188] As used herein, the term "nonsense mutation" is a mutation that results in the substitution of an amino acid with a stop codon or the removal of the canonical start codon.

[0189] As used herein, the term "frameshift mutation" is a mutation that causes an alteration in the frame of a protein.

[0190] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0191] As used herein, the term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences in which a certain percentage of nucleotides or amino acid residues are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art), or by visual inspection. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the full length of the two sequences being compared.

[0192] In sequence comparison, generally, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the sequence identity (%) of the test sequence to the reference sequence based on the designated program parameters.Alternatively, sequence similarity or difference can also be established by the combination of the presence or absence of a specific nucleotide at a selected sequence position (e.g., sequence motif) or an amino acid in a translated sequence.

[0193] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0194] One example of an algorithm that is suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0195] As used herein, the term "non-stop or read-through" refers to a mutation that results in the removal of the natural stop codon.

[0196] As used herein, the term "epitope" refers to a specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0197] As used herein, the term "immunogenic" refers to the ability to stimulate an immune response, for example, via T cells, B cells, or both.

[0198] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity of binding between a specific antigen and a specific MHC allele.

[0199] As used herein, the term "bait" refers to a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.

[0200] As used herein, the term "mutation" is a difference between the nucleic acid of a subject and a reference human genome used as a control.

[0201] As used herein, the term "variant calling" is the algorithmic determination, typically from sequencing, of the presence of a mutation.

[0202] As used herein, the term "polymorphism" refers to a germline mutation, ie, a mutation found in all DNA-bearing cells of an individual.

[0203] As used herein, the term "somatic mutation" is a mutation that occurs in a non-germline cell of an individual.

[0204] As used herein, the term "allele" refers to one version of a gene or one version of a gene sequence or one version of a protein.

[0205] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0206] As used herein, the term "nonsense-mediated decay" or "NMD" refers to the degradation of mRNA by the cell due to a premature stop codon.

[0207] As used herein, the term "exome" refers to the subset of the genome that encodes proteins. The exome can be the collection of exons of the genome.

[0208] As used herein, the term "logistic regression" is a regression model for binary data from statistics in which the logit of the probability that the dependent variable is equal to 1 is modeled as a linear function of the dependent variable.

[0209] As used herein, the term "neural network" refers to a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinear transformations typically trained by stochastic gradient descent and backpropagation.

[0210] As used herein, the term "proteome" refers to the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0211] As used herein, the term "peptidome" refers to the set of all peptides presented by MHC-I or MHC-II on the cell surface. Peptidome can also refer to the properties of a cell or a collection of cells (e.g., infectious disease peptidome refers to the collection of peptidomes of all cells infected with an infectious disease).

[0212] As used herein, the term "ELISPOT" refers to enzyme-linked immunosorbent spot assay, a common method for monitoring immune responses in humans and animals.

[0213] As used herein, the term "dextramer" refers to a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0214] As used herein, the term "tolerance or immune tolerance" refers to a state of immune unresponsiveness to one or more antigens, eg, self-antigens.

[0215] As used herein, the term "central tolerance" is tolerance conferred in the thymus by either deleting autoreactive T cell clones or promoting their differentiation into immunosuppressive regulatory T cells (Tregs).

[0216] As used herein, the term "peripheral tolerance" refers to tolerance conferred in the peripheral system by downregulating or anergizing autoreactive T cells that survive central tolerance or by promoting the differentiation of these T cells into Tregs.

[0217] The term "sample" can include a single cell, or multiple cells, or fragments of cells, or an aliquot of bodily fluid obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention, or other means known in the art.

[0218] The term "subject" includes cells, tissues, or organisms, human or non-human, whether male or female, in vivo, ex vivo, or in vitro. The term subject includes mammals, including humans.

[0219] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0220] The term "clinical factor" refers to a measure of a subject's condition, e.g., disease activity or severity. "Clinical factor" encompasses all markers of a subject's health status, including non-sample markers, and / or other characteristics of the subject, such as, but not limited to, age and sex. A clinical factor can be a score, value, or set of values ​​that can be obtained from assessing a subject or a sample (or a population of samples) from a subject under a given condition. Clinical factors can also be predicted by other parameters, such as markers and / or gene expression surrogates. Clinical factors can include the type of infection (e.g., coronavirus species), the subtype of infection (e.g., SARS-CoV-2 variant), and disease history.

[0221] "Antigen-encoding nucleic acid sequences from an infectious agent" refers to nucleic acid sequences obtained from infected cells or infectious organisms, e.g., by RT-PCR, or sequence data obtained by sequencing infected cells or infectious organisms and then synthesizing nucleic acid sequences using the sequencing data, e.g., by various synthetic or PCR-based methods known in the art. The resulting sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and / or otherwise expression-optimized), that encode the same polypeptide sequence as the corresponding native infectious disease organism nucleic acid sequence. The resulting sequences can include nucleic acid sequence variants that encode modified infectious disease organism peptide sequences that have one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to the native infectious disease organism polypeptide sequence. For example, the modified polypeptide sequence can have one or more missense mutations relative to the native polypeptide sequence of the infectious disease organism protein.

[0222] A "coronavirus nucleic acid sequence encoding an immunogenic polypeptide" refers to a nucleic acid sequence obtained from a coronavirus virus, e.g., by RT-PCR, or sequence data obtained by sequencing a coronavirus virus or a coronavirus-infected cell and then synthesizing the nucleic acid sequence using the sequencing data, e.g., by various synthetic or PCR-based methods known in the art. The resulting sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and / or otherwise expression-optimized) that encode the same polypeptide sequence as a corresponding naturally occurring coronavirus nucleic acid sequence. The resulting sequences can include nucleic acid sequence variants that encode modified coronavirus peptide sequences that have one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to a naturally occurring coronavirus polypeptide sequence. For example, a modified spike polypeptide sequence can have one or more mutations, such as one or more missense mutations at R682, R815, K986P, or V987P, relative to the naturally occurring spike polypeptide sequence of a SARS-CoV-2 protein.

[0223] The term "alphavirus" refers to members of the Togaviridae family, which are single-stranded, positive-sense RNA viruses. Alphaviruses 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, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83. Alphaviruses are generally self-replicating RNA viruses.

[0224] The term "alphavirus backbone" refers to the minimal sequence(s) of an alphavirus that allows for autonomous replication of the viral genome. The minimal sequences can include conserved sequences for nonstructural protein-mediated amplification, the nonstructural protein 1 (nsP1), nsP2, nsP3, nsP4 genes, and polyA sequences, as well as sequences for expression of subgenomic viral RNA, including the subgenomic promoter (e.g., the 26S promoter element).

[0225] The term "sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) known in the art, including, but not limited to, the alphavirus 5' UTR, a 51-nt CSE, a 24-nt CSE, a subgenomic promoter sequence (e.g., a 26S subgenomic promoter sequence), a 19-nt CSE, and an alphavirus 3' UTR.

[0226] The term "RNA polymerase" includes polymerases that catalyze the production of an RNA polynucleotide from a DNA template, including, but not limited to, bacteriophage-derived polymerases, including T3, T7, and SP6.

[0227] The term "lipid" includes hydrophobic and / or amphipathic molecules. Lipids may be cationic, anionic, or neutral. Lipids may be synthetic or naturally derived, and in certain instances may be biodegradable. Lipids may include cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins. Lipids may also include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0228] The term "lipid nanoparticle" or "LNP" includes vesicle-like structures, also known as liposomes, formed with a lipid-containing membrane surrounding an aqueous interior. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by surfactants. The core lipid can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants. Biological membrane lipids, such as phospholipids, sphingomyelin, bile acids (taurocholate), and sterols (cholesterol), can be used as stabilizers. Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids. Lipid nanoparticles can encapsulate molecules within their outer membrane shell and then be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, such as their surface. Lipid nanoparticles can be monolamellar (unilamellar) or multilamellar (multilamellar). Lipid nanoparticles can be complexed with nucleic acids. Unilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acids, where the nucleic acid is in the aqueous interior, or forming or sandwiched between them.

[0229] Abbreviations: MHC: major histocompatibility complex; HLA: human leukocyte antigen, or human MHC locus; NGS: next-generation sequencing; PPV: positive predictive value; TSNA: tumor-specific neoantigen; FFPE: formalin-fixed, paraffin-embedded; NMD: nonsense-mediated decay; NSCLC: non-small cell lung cancer; DC: dendritic cell.

[0230] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0231] Unless otherwise specified or clear from the context, the term "about" used herein is understood to be within the range of normal tolerance in the art, for example, within two standard deviations from the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​set forth herein are modified by the word "about."

[0232] Terms not directly defined herein should be understood to have the meanings generally associated with them as understood within the technical field of the present invention. Certain terms are discussed herein to provide further guidance to the practitioner in describing the compositions, devices, methods, etc. of embodiments of the present invention, as well as how to make or use them. It will be recognized that multiple ways of saying the same thing may be used. Accordingly, alternative terms and synonyms may be used for any one or more of the terms discussed herein. No weight should be placed on whether a term is detailed or discussed herein. Several synonyms or alternative methods, materials, etc. are provided. The recitation of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents, unless expressly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the inventive embodiments herein.

[0233] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0234] II. Antigen Identification Research models for NGS analysis of tumor and normal exomes and transcriptomes have been previously described and applied in the antigen-specific space. 6,14,15Specific optimizations can be considered to enhance the sensitivity and specificity of antigen identification in clinical settings. 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 contexts, such as identification of infectious disease organisms (e.g., coronaviruses), infections in subjects, or from infected cells in subjects. Examples of optimization are well known to those skilled in the art; for example, such methods are described in more detail in U.S. Pat. No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1, International Patent Application Publication Nos. WO / 2018 / 195357 and WO / 2018 / 208856, U.S. Patent Application No. 16 / 606,577, and International Patent Application No. PCT / US2020 / 021508, each of which is incorporated by reference in its entirety for all purposes.

[0235] Methods for identifying antigens (e.g., antigens from infectious disease organisms) include identifying antigens that are likely to be presented on cell surfaces (e.g., presented by MHC on 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 an infected cell or infectious disease organism (e.g., a coronavirus), wherein the nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequence of each of a set of antigens (e.g., antigens from the infectious disease organism); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each antigen will be presented by one or more MHC alleles on the surface of a cell, such as an infected cell, of the subject, wherein 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.

[0236] IV. Antigen Antigens can include nucleotides or polypeptides. For example, antigens can be RNA sequences that encode polypeptide sequences. Thus, antigens useful in vaccines include nucleotide sequences or polypeptide sequences.

[0237] Disclosed herein are peptides and nucleic acid sequences encoding peptides derived from any polypeptide associated with coronaviruses, including combinations of peptides and nucleic acid sequences encoding peptides derived from any polypeptide associated with different coronaviruses. Coronaviruses may include, but are not limited to, clade 1 sarbecoviruses, clade 2 sarbecoviruses, or clade 3 coronaviruses. Coronaviruses may include, but are not limited to, Genbank databank accession numbers NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, and MK21137. Examples of coronaviruses include coronaviruses related to 7.1, KJ473816.1, MK211376.1, AY572034.1, KP886809.1, MT072864.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, or KJ473814.1. Coronaviruses can include beta coronaviruses or alpha coronaviruses. Coronaviruses can include beta coronaviruses. Coronaviruses can include alpha coronaviruses. Coronaviruses can include merbecoviruses. Coronaviruses can include enveloped viruses.

[0238] Disclosed herein are peptides and nucleic acids encoding peptides derived from any polypeptide associated with coronavirus, coronavirus infection in a subject, or coronavirus-infected cells in a subject. Antigens can be derived from nucleotide or polypeptide sequences of a coronavirus virus. Coronavirus polypeptide sequences include, but are not limited to, predicted MHC class I epitopes listed in Table A, predicted MHC class II epitopes listed in Table B, predicted MHC class I epitopes listed in Table C, coronavirus spike peptides (peptides derived from SARS-CoV-2, such as SEQ ID NO: 59), coronavirus membrane peptides (peptides derived from SARS-CoV-2, such as SEQ ID NO: 61), coronavirus nucleocapsid peptides (peptides derived from SARS-CoV-2, such as SEQ ID NO: 62), coronavirus envelope peptides (peptides derived from SARS-CoV-2, such as SEQ ID NO: 63), coronavirus replicase orf1a and orf1b peptides (e.g., one or more of nonstructural proteins (nsp) 1-16), or any other peptide sequence encoded by a coronavirus virus. The peptides and nucleic acid sequences encoding the peptides can be derived from the Wuhan-Hu-1 SARS-CoV-2 isolate, sometimes referred to as the SARS-CoV-2 reference sequence (SEQ ID NO: 76, NC_045512.2, incorporated herein by reference for all purposes). The peptides and nucleic acid sequences encoding the peptides can be derived from an isolate different from the Wuhan-Hu-1 SARS-CoV-2 isolate, such as an isolate having one or more mutations in the protein relative to the Wuhan-Hu-1 isolate (also referred to as a protein variant). A vaccination strategy can include multiple vaccines comprising nucleic acid sequences encoding peptides and peptides from different isolates.For example, as an illustrative, non-limiting example, a vaccine encoding a spike protein from the Wuhan-Hu-1 SARS-CoV-2 isolate can be administered, followed by a vaccine encoding a spike protein from the B.1.351 ("South Africa") SARS-CoV-2 isolate (e.g., SEQ ID NO: 112) or a B.1.1.7 ("UK") SARS-CoV-2 isolate (e.g., SEQ ID NO: 110). The one or more variants include, but are not limited to, mutations in coronavirus spike proteins, coronavirus membrane proteins, coronavirus nucleocapsid proteins, coronavirus envelope proteins, coronavirus replicase orf1a and orf1b proteins (e.g., one or more of nonstructural proteins (nsp) 1-16), or any other protein encoded by a coronavirus. Variants can be selected based on the frequency of mutations among coronavirus subtypes / isolates, such as mutations / variants present in 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of coronavirus subtypes / isolates. Examples of mutations present in more than 1% of isolates are shown in Table 1. Variants can be selected based on the frequency of mutations among coronavirus subtypes / isolates present in a particular population, such as a particular demographic or geographic population. An illustrative, non-limiting example of a high-frequency variant / mutation is the spike D614G missense mutation, which is present in 60.05% of genomes sequenced worldwide and 70.46% and 58.49% in Europe and North America, respectively.Thus, for use in prophylactic vaccines for particular demographic or geographic populations at risk for infection with a particular coronavirus subtype / isolate, vaccines can be designed to encode at least one immunogenic polypeptide corresponding to a polypeptide encoded by a coronavirus subtype with which the subject is infected or at risk of infection, for example, for use in prophylactic vaccines for particular demographic or geographic populations at risk for infection with a particular coronavirus subtype / isolate. Vaccines can be designed to encode at least one immunogenic polypeptide corresponding to a polypeptide encoded by a coronavirus and at least one immunogenic polypeptide corresponding to a polypeptide encoded by a coronavirus species and / or subspecies other than SARS-CoV-2, such as, for example, species associated with Severe Acute Respiratory Syndrome (SARS) 2002 (NC_004718.3, incorporated by reference herein for all purposes) and / or species associated with Middle East Respiratory Syndrome (MERS) 2012 (NC_019843.3, incorporated by reference herein for all purposes). Vaccines can be designed to encode at least one immunogenic polypeptide corresponding to a polypeptide encoded by SARS-CoV-2 that is conserved (e.g., 100% amino acid sequence conservation between epitopes) between SARS-CoV-2 and other coronavirus species and / or subspecies, such as, for example, Severe Acute Respiratory Syndrome (SARS) and / or Middle East Respiratory Syndrome (MERS) species. SARS-CoV-2 epitopes conserved between SARS-CoV-2 and other coronavirus species and / or subspecies can include coronavirus spike protein, coronavirus membrane protein, coronavirus nucleocapsid protein, coronavirus envelope protein, coronavirus replicase orf1a and orf1b proteins (e.g., one or more of nonstructural proteins (nsp) 1-16), or any other protein sequence encoded by a coronavirus.

[0239] Antigens can be selected as part of a "pan-coronavirus" vaccine that confers broad immunogenicity against multiple coronaviruses, such as multiple coronaviruses of the Sarbecovirus subgenus, including the human pathogens SARS-CoV and SARS-CoV-2. Pan-coronavirus vaccines can include those encoding receptor binding domains (RBDs) from multiple coronaviruses. Pan-coronavirus vaccines can include at least two different coronavirus receptor binding domain (RBD)-derived nucleic acid sequences encoding at least two different RBD domains.

[0240] Selection of different RBD domains for inclusion in a pan-coronavirus vaccine can involve analysis of RBD domain sequence similarity to maximize coverage of diverse coronaviruses (e.g., coverage of multiple sarbecovirus clades). Sequence similarity analysis can be based on analysis of various subdomains of the RBD. While not wishing to be bound by theory, selecting RBDs based solely on the RBM allows selection based on the most variable domains, whereas selection based on RBD sequences other than the RBM (RBDΔRBM) allows selection based on conserved domains. Both selection criteria have potential advantages. More conserved regions may be more effective immunogens for antibodies that recognize multiple coronaviruses. Selection based on the RBM can provide greater coverage of diverse RBMs. Using the entire RBD cluster can combine the benefits of both approaches but may be biased toward conserved domains.

[0241] The pan-coronavirus vaccine can include at least two different RBD domains that are collectively at least 70% identical in amino acid composition to RBD domains from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, and a clade 3 sarbecovirus. The pan-coronavirus vaccine can include.

[0242] A pan-coronavirus vaccine can include at least two different RBD domains that are collectively at least 70%, 75%, 80%, 85%, or 90% identical in amino acid composition to the RBD domains from each of (A) a clade 3 sarbecovirus and (B) a clade 1 sarbecovirus and / or a clade 2 sarbecovirus. The pan-coronavirus vaccines are NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, MK211377.1, KJ473816.1, MK211376.1, AY572034.1, KP886809.1, MT0728 The RBD domains may comprise at least two different RBD domains that are collectively at least 70%, 75%, 80%, 85%, or 90% identical in amino acid composition to the RBD domains from each of the following: 64.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, and KJ473814.1.

[0243] A pan-COVID-19 vaccine can include selecting RBD domains based on analysis of sequence similarity between the respective RBM domains alone, such as at least two different RBD domains, including RBM domains that are collectively at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical in amino acid composition to RBM domains from at least two of clade 1 sarbecoviruses, clade 2 sarbecoviruses, or clade 3 sarbecoviruses.

[0244] A pan-COVID-19 vaccine can include selecting an RBD domain based on an analysis of sequence similarity between RBD sequences that do not include an RBM domain sequence (RBDΔRBM), such as at least two different RBDs, wherein the amino acid sequences of the different RBD domains, other than the amino acid sequence of each RBM domain, comprise at least two different RBD domains that are collectively at least 70%, 75%, 80%, 85%, or 90% identical in amino acid composition to RBM domains from at least two of clade 1 sarbecoviruses, clade 2 sarbecoviruses, or clade 3 sarbecoviruses.

[0245] A pan-coronavirus vaccine can comprise at least three different coronavirus RBD-derived nucleic acid sequences encoding at least three different RBD domains.A pan-coronavirus vaccine can comprise at least three different coronavirus RBD-derived nucleic acid sequences encoding at least three different RBD domains.

[0246] A pan-coronavirus vaccine can include at least four different RBD domains that are collectively at least 70% identical in amino acid composition to the RBD domains from each of clade 1 sarbecoviruses, clade 2 sarbecoviruses, and clade 3 sarbecoviruses.

[0247] A pan-coronavirus vaccine can include at least two, at least three, or at least four different coronavirus RBD-derived nucleic acid sequences, including at least one derived from a beta-coronavirus RBD-derived nucleic acid sequence. A pan-coronavirus vaccine can include at least two, at least three, or at least four different coronavirus RBD-derived nucleic acid sequences, each derived from a beta-coronavirus RBD-derived nucleic acid sequence. A pan-coronavirus vaccine can include at least two, at least three, or at least four different coronavirus RBD-derived nucleic acid sequences, including at least one derived from a beta-coronavirus RBD-derived nucleic acid sequence, an alpha-coronavirus RBD-derived nucleic acid sequence, an RBD-derived nucleic acid sequence, and combinations thereof.

[0248] The pan-coronavirus vaccine can include at least two, at least three, or at least four different coronavirus RBD-derived nucleic acid sequences, including at least one derived from a sarbecovirus RBD-derived nucleic acid sequence, a merbecovirus RBD-derived nucleic acid sequence, an envelope virus RBD-derived nucleic acid sequence, and combinations thereof.

[0249] A pan-coronavirus vaccine can include at least two different coronavirus RBD-derived nucleic acid sequences encoded by a single polynucleotide sequence. A pan-coronavirus vaccine can include at least two different coronavirus RBD-derived nucleic acid sequences encoded by a single antigen cassette (e.g., a multicistronic cassette). A pan-coronavirus vaccine can include at least two different coronavirus RBD-derived nucleic acid sequences encoded by separate polynucleotide sequences (e.g., when each RBD is encoded on a separate viral backbone).

[0250] The RBD domain can comprise a trimerization domain. Different RBD domains can comprise different trimerization domains. Different RBD domains may comprise the same trimerization domain. RBD trimerization domains include, but are not limited to, a T4 trimerization domain, an MTQ trimerization domain, and a GCN4 trimerization domain. The RBD domain can comprise a coronavirus-derived signal peptide, such as a SARS-CoV-2-derived signal peptide. The RBD domain can comprise an influenza hemagglutinin-, tissue plasminogen activator-, and / or Ag2 / PRA-derived signal peptide. The RBD-derived nucleic acid sequences can be linked to each other by a peptide linker-encoding nucleic acid sequence. The peptide linker-encoding nucleic acid sequence can comprise a 2A ribosomal skipping sequence element (e.g., an E2A ribosomal skipping sequence element, a P2A ribosomal skipping sequence element, an F2A ribosomal skipping sequence element, or a T2A sequence ribosomal skipping sequence element). The peptide linker-encoding nucleic acid sequence can include a cleavable peptide linker (eg, a TEV cleavage site or a furin cleavage site).

[0251] The encoded RBD domain may encode only the RBD domain, or may be encoded by a spike domain (e.g., a full-length SARS-CoV-2 spike) that encodes the RBD domain.

[0252] Antigens can be selected that are predicted to be presented on the cell surface of cells, such as infected cells or immune cells, including professional antigen-presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic. Exemplary antigens predicted to be presented on the cell surface by MHC using the methods described herein include the predicted MHC class I epitopes shown in Table A, the predicted MHC class II epitopes shown in Table B, and the predicted MHC class I epitopes shown in Table C.

[0253] Antigens can be selected that are presented by specific HLAs and / or have been validated to stimulate an immune response, such as those previously reported / validated in the literature (e.g., Nelde et al. [Nature Immunology volume 22, pages 74-85 2021], Tarke et al. 2021, or Schelien et al. [bioRxiv 2020.08.13.249433]). The magnitude of stimulation of the immune response can be used to guide epitope / antigen selection, such as with the goal of selecting epitopes that stimulate as strong an immune response as possible, including when the cassette has size constraints. As an illustrative, non-limiting example of selection based on the magnitude of stimulation of an immune response, the following can be used: (1) the magnitude of an individual's response is the sum of the magnitude of responses of all epitopes across each diplotype allele; and (2) the magnitude of response of each epitope = (magnitude of response) x (frequency of positive responses / 100) [e.g., using values ​​found in Tarke et al. (Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases. Cell Rep Med. 2021 Feb 16;2(2):100204. doi:10.1016 / j.xcrm.2021.100204. Epub 2021 Jan 16, 2021], which is incorporated by reference for all purposes. 26.)], (3) excluding epitopes other than those from the starting protein that span mutations at a frequency greater than 5% (variations in flanking regions are optionally tolerated), and / or (4) as described herein, cassettes are ordered to minimize unintended junction epitopes spanning adjacent frames and to minimize contiguous frames within the same protein to reduce the likelihood of functional protein fragments.

[0254] The cassette can be configured to encode one or more validated epitopes and / or at least four, five, six, or seven predicted epitopes, wherein at least 85%, 90%, or 95% of a population possesses at least one HLA validated to present at least one of the one or more validated epitopes and / or at least one HLA predicted to present each of the at least four, five, six, or seven predicted epitopes. The cassette can be configured to encode one or more validated epitopes and at least four, five, six, or seven predicted epitopes, wherein at least 85%, 90%, or 95% of a population possesses at least one HLA validated to present at least one of the one or more validated epitopes and at least one HLA predicted to present each of the at least four, five, six, or seven predicted epitopes.

[0255] The one or more polypeptides encoded by the antigen nucleotide sequence can comprise at least one of the following: a 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 an extracellular or lysosomal protease (e.g., cathepsin) or the HLA binding site catalyzed by HLA-DM.

[0256] The one or more antigens can be present on the surface of an infected cell (e.g., a coronavirus-infected cell).

[0257] One or more antigens can be immunogenic (e.g., capable of stimulating a T cell and / or B cell response in a subject having or suspected of having an infectious disease (e.g., a coronavirus infection). One or more antigens can be immunogenic (e.g., capable of stimulating a T cell and / or B cell response in a subject that confers immunological protection (i.e., immunity) against the infectious disease, such as stimulating the production of memory T cells, memory B cells, or antibodies specific for the infectious disease) in a subject at risk for the infectious disease (e.g., a coronavirus infection).

[0258] One or more antigens may be capable of stimulating a B cell response, such as the production of antibodies that recognize one or more antigens (e.g., antibodies that recognize coronavirus antigens and / or viruses). Antibodies can recognize linear polypeptide sequences or secondary and tertiary structures. Thus, B cell antigens can include linear polypeptide sequences or polypeptides having secondary and tertiary structures, including, but not limited to, full-length proteins, protein subunits, protein domains, or any polypeptide known or predicted to have secondary and tertiary structures. Antigens capable of stimulating a B cell response to infection can be found on the surface of infectious organisms (e.g., coronaviruses). Antigens capable of stimulating a B cell response to infection can be intracellular antigens expressed in infectious organisms. Coronavirus antigens capable of stimulating a B cell response to infection include, but are not limited to, coronavirus spike peptides, coronavirus membrane peptides, coronavirus nucleocapsid peptides, and coronavirus envelope peptides.

[0259] 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).

[0260] 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.

[0261] 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 The antigenic peptide molecule can include, but is not limited to, 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 60, about 70, about 80, about 90, about 100, about 110, about 120, or more amino acid residues, and any range derivable therein. In a specific embodiment, the antigenic peptide molecule is 50 or fewer amino acids.

[0262] Antigenic peptides and polypeptides can be 15 residues or less in length, typically between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; and 6 to 30 residues for MHC class II.

[0263] If desired, longer peptides can be designed in several ways. In one example, if the likelihood of peptide presentation on HLA alleles is predicted or known, longer peptides can consist of either (1) individual presented peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product; or (2) a concatenation of some or all of the presented peptides, each with its extended sequence. In another case, if sequencing reveals the presence of a long (more than 10 residues) epitope sequence, longer peptides can consist of (3) the entire novel infection-specific stretch of amino acids (thus eliminating the need to select the most strongly HLA-presented shorter peptides based on computational or in vitro selection). In either case, longer peptides allow for endogenous processing by patient cells, resulting in more effective antigen presentation and increased T cell responses. Longer peptides can also include full-length proteins, protein subunits, protein domains, and combinations of these peptides, such as those expressed in infectious disease organisms. Longer peptides (eg, full-length proteins, protein subunits, or protein domains) and combinations thereof can be included to stimulate B cell responses.

[0264] Antigenic peptides and polypeptides can be presented on HLA proteins. In some embodiments, the antigenic peptide or polypeptide is presented on HLA proteins with higher affinity than the wild-type peptide. In some embodiments, the antigenic peptide or polypeptide can have an IC50 of at least 5000 nM or less, at least 1000 nM or less, at least 500 nM or less, at least 250 nM or less, at least 200 nM or less, at least 150 nM or less, at least 100 nM or less, at least 50 nM or less, or less.

[0265] In some embodiments, the antigenic peptides and polypeptides do not stimulate an autoimmune response and / or do not induce immune tolerance when administered to a subject.

[0266] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the compositions comprise at least two different peptides. The at least two different peptides may be derived from the same polypeptide. Different peptides mean that the peptides differ in length, amino acid sequence, or both. The peptides may be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or the peptides may be derived from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., an infectious disease polynucleotide or polypeptide, including an infectious disease polynucleotide or polypeptide whose expression is restricted to host cells).

[0267] Antigenic peptides and polypeptides with desired activities or properties can be modified to confer certain desirable attributes, e.g., improved pharmacological characteristics, while enhancing or at least retaining substantially all of the biological activity of the unmodified peptide, which binds to desired MHC molecules and activates appropriate T cells. For example, antigenic peptides and polypeptides can be further subjected to various modifications, such as conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved MHC binding, stability, or presentation. Conservative substitutions refer to the replacement of 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 effects of single amino acid substitutions can 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), 2nd Ed. (1984).

[0268] Modification of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful for increasing peptide and polypeptide stability 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, e.g., 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 generally follows: Pooled human serum (type AB, non-heat-inactivated) is defatted 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.

[0269] Peptides and polypeptides can be modified to provide desirable attributes other than improved serum half-life. For example, the ability of a peptide to stimulate CTL activity can be enhanced by linking it 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 from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that the optional spacer need not be composed of the same residues and can therefore be a hetero- or homo-oligomer. If present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the peptide can be linked to the T helper peptide without a spacer.

[0270] Polypeptide-encoded antigens can be modified to alter polypeptide processing, such as protease cleavage and / or other post-translational processing. Polypeptide-encoded antigens can be modified to favor a particular conformation. Polypeptide-encoded antigens can be modified so that mutations (e.g., one or more missense mutations) prevent a particular conformation of the antigen, for example, by introducing a proline that disrupts secondary and tertiary structure (e.g., alpha helix or beta sheet formation). Altering, reducing, or eliminating processing or conformational changes can, in some cases, bias the antigen toward a state favorable for neutralizing antibody production. In one illustrative example, SARS-CoV-2 spike mutations at amino acids 682, 815, 987, and 988 are engineered to bias the spike protein toward remaining primarily in the pre-fusion state, a state potentially favorable for antibody-mediated neutralization. Specifically, without wishing to be bound by theory, a mutation at R682 (e.g., R682V) disrupts the furin cleavage site involved in processing the spike into S1 and S2, a mutation at R815 (e.g., R815N) disrupts the cleavage site within S2, and mutations at K986 and V987 (e.g., K986P and V987P, which introduce two prolines) disrupt the secondary structure of the spike, making it difficult for the spike to be processed from the pre-fusion state to the post-fusion state. Thus, the antigen cassette can encode a modified spike protein having a spike R682V mutation, a spike R815N mutation, a spike K986P mutation, a spike V987P mutation, and combinations thereof based on the Wuhan-Hu-1 isolate (see reference sequence SEQ ID NO: 59 and SEQ ID NO: 60 / SEQ ID NO: 90 with the mutations). The modified polypeptide sequence may be at least 60%, 70%, 80%, or 90% identical to a native coronavirus polypeptide sequence. The modified polypeptide sequence may be at least 91%, 92%, 93%, or 94% identical to a native coronavirus polypeptide sequence. The modified polypeptide sequence may be at least 95%, 96%, 97%, 98%, or 99% identical to a native coronavirus polypeptide sequence.The modified polypeptide sequence may be at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a native coronavirus polypeptide sequence.

[0271] 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 tetanus toxoid at 830-843, influenza at 307-319, and malaria sporozoites at around 382-398 and 378-389.

[0272] Proteins or peptides can be produced by any technique known to those of skill in the art, including expressing proteins, polypeptides, or peptides through standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. 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 of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on 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 of skill in the art. Alternatively, various commercial preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0273] In a further embodiment, the antigen comprises a nucleic acid (e.g., a 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), or a polynucleotide having a phosphorothioate backbone, in either a native 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 replacing low-frequency codons with high-frequency synonymous codons with respect to the codon bias of a particular organism. Polynucleotide sequences 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) to bias favorable splicing events and / or by introducing exogenous splicing motifs (splice donor, branch, and / or acceptor sequences). Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g., the SV40 mini-intron [SEQ ID NO: 88]) and / or immunoglobulins (e.g., the human β-globin 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.Polynucleotide sequences 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. Polynucleotide sequences can be optimized to improve accurate transcription, for example, by removing cryptic transcription initiators and / or terminators. Polynucleotide sequences 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. Polynucleotide sequences can be optimized to improve nuclear export of transcripts, for example, 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. Polynucleotide sequences 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 optimal sequences that balance 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, and SGI-DNA (La Jolla, California). One or more regions of an antigen-encoding protein can be sequence-optimized separately. As a non-limiting illustrative example, a coronavirus spike protein can be sequence-optimized (or not) in the S1 region of the protein, and the S2 region can be separately optimized (e.g., optimized using a different algorithm and / or optimized for one or more sequence properties specific to the S2 region).

[0274] The methods disclosed herein can also further include identifying one or more T cells that are antigen-specific for at least one of the antigens in the subset. In some embodiments, the identifying includes co-culturing one or more T cells with one or more of the antigens in the subset under conditions that expand the one or more antigen-specific T cells. In further embodiments, the identifying includes contacting one or more T cells with a tetramer comprising one or more of the antigens in the subset under conditions that allow binding of the T cells to the tetramer. In even further embodiments, the methods disclosed herein can also further include identifying one or more T cell receptors (TCRs) of the one or more identified T cells. In certain embodiments, identifying the one or more T cell receptors includes sequencing the T cell receptor sequences of the one or more identified T cells. The methods disclosed herein can further include genetically engineering a plurality of T cells to express at least one of the one or more identified T cell receptors, culturing the plurality of T cells under conditions that expand the plurality of T cells, and infusing the expanded T cells into a subject. In some embodiments, engineering the plurality of T cells to express at least one of the one or more identified T cell receptors comprises cloning the T cell receptor sequence of the one or more identified T cells into an expression vector and transfecting each of the plurality of T cells with the expression vector. In some embodiments, the methods disclosed herein further comprise culturing the one or more identified T cells under conditions that expand the one or more identified T cells and injecting the expanded T cells into a subject.

[0275] Also disclosed herein are isolated T cells that are antigen-specific for at least one selected antigen in the subset.

[0276] 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, 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 appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host; 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.

[0277] V. Vaccine Compositions Also disclosed herein are immunogenic compositions, e.g., vaccine compositions, that can generate specific immune responses, e.g., infectious disease organism-specific immune responses. Vaccine compositions typically contain one or more antigens selected, e.g., using the methods described herein. Vaccine compositions can also be referred to as vaccines.

[0278] The vaccine can comprise 1 to 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 or more different nucleotide sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14 different antigen-encoding nucleic acid sequences (nucleic acid sequences that encode antigens), or 12, 13, or 14 different antigen-encoding nucleic acid sequences.The vaccine contains 1–30 antigen 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, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, It may comprise 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.

[0279] The vaccines contain 1 to 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, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 The antigen-encoding 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, antigen-encoding nucleic acid sequences encoding linked T-cell epitopes).

[0280] The vaccines contain 1 to 30 different epitope-encoding nucleic acid sequences: , 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 a linked T cell epitope-encoding nucleic acid sequence.

[0281] The vaccine can 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 the 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 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 are shown by, but not limited to, the following 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 multiple 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

[0282] The above examples are not limiting, and an antigen-encoding nucleic acid sequence having repeats of at least one of the different epitopes can encode each of the different epitopes in any order or frequency. For example, the order and frequency can be determined by, for example, 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, as in the example with epitopes A, B, and C according to the formula:

[0283] Provided herein is an antigen-encoding cassette having at least one antigen-encoding nucleic acid sequence described in the 5' to 3' direction by the formula: (E x -(E N n ) y ) z wherein 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 comprising a 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; z=2 or more, and the antigen-encoding nucleic acid sequence is E, a specific E N or at least two repeats of any one of these combinations.

[0284] Each E or E Ncan independently comprise an epitope-encoding nucleic acid sequence described herein (e.g., a nucleotide sequence encoding a polynucleotide sequence described in Table A, Table B, and / or Table C). For example, each E or E N In the 5' to 3' direction, the formula (L5 b -N c -L3 d ), wherein N is each E or E N wherein c=1, L5 comprises a 5' linker sequence wherein b=0 or 1, and L3 comprises a 3' linker sequence wherein d=0 or 1. Epitopes and linkers that can be used are further described herein.

[0285] 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 repeat sequences 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 repeat sequences may be, for example, E A -E B -E A …(E AThe repeats of the Trp1 epitope-encoding nucleic acid sequence may be separated by 75 nucleotides, such as in an antigen-encoding nucleic acid represented by the sequence VTNTEMFVTAPDNLGYMYEVQWPGQ (SEQ ID NO: 116) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 117)), which encodes repeats of the 25-mer antigens Trp1 (VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 115), where the repeats of Trp1 are separated by a 25-mer of 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 an example 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 epitope-encoding nucleic acid sequence (including any 5' linker sequence and / or any 3' linker sequence) encoding a peptide 25 amino acids in length, the repeat sequences can be separated by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides, respectively.

[0286] 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 embodiments, a 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.

[0287] The vaccine composition is capable of stimulating a specific cytotoxic T cell response and a specific helper T cell response.

[0288] The vaccine composition can stimulate a specific B cell response (eg, an antibody response).

[0289] 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.

[0290] The combination of vaccine compositions 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 be homogeneous and can stimulate a combination of a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition can be homogeneous and can stimulate a combination of a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response. The vaccine composition can be heterogeneous and can stimulate a combination of a specific cytotoxic T cell response, a specific helper T cell response, and / or a specific B cell response. The vaccine composition can be heterogeneous and can stimulate a combination of a specific cytotoxic T cell response, a specific helper T cell response, and a specific B cell response. A heterogeneous vaccine comprises the same antigen cassette encoded by different vaccine platforms, such as a viral vaccine (e.g., a ChAdV-based platform) and an mRNA vaccine (e.g., a SAM-based platform). Heterologous vaccines comprise different antigen cassettes (e.g., a spike cassette and another T cell epitope-encoding cassette, or epitopes / antigens derived from different subtype isolates of SARS-CoV-2, such as spike protein variants from the Wuhan-Hu-1 subtype isolate and the B.1.351 subtype isolate) encoded by the same vaccine platform, e.g., either a viral vaccine (e.g., a ChAdV-based platform) or an mRNA vaccine (e.g., a SAM-based platform). Heterologous vaccines comprise different antigen cassettes (e.g., a spike cassette and another T cell epitope-encoding cassette, or epitopes / antigens derived from different isolates / subtypes of coronavirus, such as spike protein variants from the Wuhan-Hu-1 subtype isolate and the B.1.351 subtype isolate, and / or different sarbecovirus isolates) encoded by different vaccine platforms, e.g., a viral vaccine (e.g., a ChAdV-based platform) and an mRNA vaccine (e.g., a SAM-based platform).For example, as illustrative, non-limiting examples, viral vaccines (e.g., ChAdV-based platforms) are particularly capable of stimulating strong cytotoxic T cell responses, and mRNA vaccines (e.g., SAM-based platforms) are particularly capable of stimulating strong B cell responses.

[0291] 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, for example, an antigen-presenting cell, such as a dendritic cell (DC), capable of presenting a protein or peptide to a T cell.

[0292] An adjuvant is any substance that, when mixed into a vaccine composition, enhances or otherwise modifies the immune response to an antigen. A carrier can be a scaffold structure, such as a polypeptide or polysaccharide, to which an antigen can associate. Optionally, the adjuvant is attached by a covalent or non-covalent bond.

[0293] The ability of adjuvants to increase the immune response to antigens is generally manifested as a significant or substantial increase in immune-mediated reactions or a reduction in disease symptoms.For example, an increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against antigens, and an increase in T cell activity is generally manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion.Adjuvants can also change immune responses, for example, by changing a primarily humoral or Th response to a primarily cellular or Th response.

[0294] 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 (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, mycobacterium extracts and synthetic bacterial wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are also 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).

[0295] CpG immunostimulatory oligonucleotides have also been reported to enhance the effectiveness of adjuvants in a vaccine setting. Other TLR binding molecules, such as RNA binding TLR 7, TLR 8, and / or TLR 9, can also be used.

[0296] 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 adjuvants, 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 ordinary skill in the art without undue experimentation. Further adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0297] A vaccine composition can include multiple different adjuvants. Additionally, a therapeutic composition can include any adjuvant material, including any of the above or a combination thereof. The vaccine and adjuvant can be administered together or separately in any suitable order.

[0298] A carrier (or excipient) may be present independently 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. Furthermore, 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, such as 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; or 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 dextran, such as Sepharose.

[0299] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible in the presence of a trimeric complex of peptide antigen, MHC molecule, and APC. Correspondingly, CTLs can enhance immune responses not only when peptides alone are used to activate CTLs, but also when APCs bearing the corresponding MHC molecules are added. Thus, in certain embodiments, the vaccine composition further comprises at least one antigen-presenting cell.

[0300] Antigens can also be derived from vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (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 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 They can also be included in viral vector-based vaccine platforms, such as the 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 described 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 linkers, 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). Upon introduction into the host, the infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response to 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 (Bacille Calmette-Guerin). BCG vectors are described by 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.

[0301] VA antigen cassette The methods used to select one or more antigens, cloning and construction of a "cassette," and its insertion into a viral vector are within the skill of the art in light of the teachings provided herein. An "antigen cassette" or "cassette" refers to 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 transcripts. The selected antigen or antigens may refer to different epitope sequences (e.g., an antigen-encoding nucleic acid sequence within a cassette can encode an epitope-encoding nucleic acid sequence (or multiple epitope-encoding nucleic acid sequences) such that the epitopes are transcribed and expressed). The antigen or antigens can be operably linked to regulatory elements in a manner that allows transcription. Such elements include conventional regulatory elements capable of directing expression of the antigen(s) in cells transfected with the viral vector. Thus, the antigen cassette can also include a selected promoter linked to the antigen(s) and positioned, along with other optional regulatory elements, within the selected viral sequence of the recombinant vector. The cassette can have one or more antigen-encoding nucleic acid sequences, such as a cassette containing multiple antigen-encoding nucleic acid sequences, each operably linked independently to a separate promoter and / or linked to each other using a 2A ribosomal skipping sequence element (e.g., E2A, P2A, F2A, or T2A sequence) or other multicistronic system, such as an internal ribosome entry site (IRES) sequence element. 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 facilitate removal of the 2A sequence after translation.In cassettes containing 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). In illustrative examples of multicistronic formats, coronavirus antigen-encoding cassettes are configured as follows: (1) endogenous 26S promoter-spike protein-T2A-membrane protein, or (2) endogenous 26S promoter-spike protein-26S promoter-linked T cell epitope.

[0302] Useful promoters may be constitutive promoters or regulated (inducible) promoters that allow for control of the amount of antigen(s) expressed. For example, a desirable promoter is the cytomegalovirus immediate early promoter / enhancer [see, e.g., 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.

[0303] The antigen cassette may also contain nucleic acid sequences heterologous to the viral vector sequence, including sequences providing 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 also 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, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., 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.

[0304] 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.

[0305] As noted elsewhere, the antigen cassette can be placed within any selected deletion site within the viral vector backbone, such as, for example, the site of the deletion of the E1 gene region or the deletion of the E3 gene region of a ChAd-based vector or the deleted structural proteins of the VEE backbone, among other choices.

[0306] An antigen-encoding cassette (e.g., a cassette or one or more nucleic acid sequences encoding an immunogenic polypeptide within the cassette) can be described using the following formula, which describes the ordered sequence of each element in the 5' to 3' direction: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g wherein P comprises the second promoter nucleotide sequence, with the proviso that a=0 or 1 and c=1; N comprises one of the coronavirus-derived nucleic acid sequences described herein, optionally wherein each N encodes a polypeptide sequence described in Table A, Table B, and / or Table C; L5 comprises a 5' linker sequence, with the proviso that b=0 or 1; L3 comprises a 3' linker sequence, with the proviso that d=0 or 1; G5 comprises one of the at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO:56), with the proviso that e=0 or 1; G3 comprises one of the at least one nucleic acid sequence encoding a GPGPG amino acid linker (SEQ ID NO:56), with the proviso that g=0 or 1; U comprises one of the at least one MHC class II epitope-encoding nucleic acid sequences, with the proviso that f=1; X=1 to 400, with the proviso that for each X, c is a coronavirus-derived nucleic acid sequence, where Y=0, 1, or 2, where for each Y, the corresponding U f are (1) universal MHC class II epitope-encoding nucleic acid sequences, optionally wherein at least one universal sequence comprises at least one of tetanus toxoid and PADRE, or (2) an MHC class II coronavirus-derived epitope-encoding nucleic acid sequence. In some embodiments, for each X, a corresponding N c are different coronavirus-derived nucleic acid sequences. In some embodiments, for each Y, a corresponding U fare different universal MHC class II epitope-encoding nucleic acid sequences or different MHC class II coronavirus-derived epitope-encoding nucleic acid sequences. The antigen-encoding sequence formulas above, in certain cases, describe only a portion of an antigen cassette encoding linked epitope sequences, such as linked T cell epitopes. For example, in a cassette encoding linked T cell epitope sequences and one or more full-length coronavirus proteins, the antigen-encoding cassette formulas above describe the linked epitope sequences, and the cassette separately encodes one or more full-length coronavirus proteins, optionally linked using a multicistronic system such as a 2A ribosome skipping sequence element (e.g., an E2A, P2A, F2A, or T2A sequence), an internal ribosome entry site (IRES) sequence element, and / or independently operably linked to separate promoters.

[0307] In one example, the elements present include those where b=1, d=1, e=1, g=1, h=1, X=18, Y=2, and the vector backbone comprises a ChAdV68 vector, a=1, P is a CMV promoter, at least one second poly(A) sequence is present, the second poly(A) sequence is a poly(A) sequence exogenous to the vector backbone, optionally the exogenous poly(A) sequence comprises an SV40 poly(A) signal sequence or a BGH poly(A) signal sequence, and each N is an MHC class I epitope, an MHC class II epitope, or an MHC class II epitope, 7-15 amino acids in length. L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the epitope, wherein the 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 the epitope, wherein the 3' linker sequence encodes a peptide at least 3 amino acids in length; and U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence. The antigen coding sequence formula above describes only a portion of the antigen cassette, which in certain cases encodes linked epitope sequences, such as linked T cell epitopes.

[0308] In one example, the elements present include those where b=1, d=1, e=1, g=1, h=1, X=18, and Y=2; the vector backbone comprises a Venezuelan equine encephalitis virus vector; a=0; the antigen cassette is operably linked to an endogenous 26S promoter; and the at least one polyadenylation poly(A) sequence is a poly(A) sequence of at least 80 consecutive A nucleotides provided by the backbone (SEQ ID NO: 27940); each N is selected from the group consisting of an MHC class I epitope, an MHC class II ... L5 is a natural 5' linker sequence encoding the natural N-terminal amino acid sequence of the 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 the epitope, which 3' linker sequence encodes a peptide at least 3 amino acids in length; and U is each of the PADRE class II sequence and the tetanus toxoid MHC class II sequence.

[0309] The antigen coding sequence can be described using the following formula, which describes the ordered sequence of each element in the 5' to 3' direction: (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g wherein P and P2 comprise a promoter nucleotide sequence, N comprises one of the coronavirus-derived nucleic acids described herein (e.g., N encodes a polypeptide sequence described in Table A, Table B, Table C, and / or Table 7), 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 epitope-encoding nucleic acid sequence, and for each X, a corresponding N c is a coronavirus-derived nucleic acid sequence, and for each Y, the corresponding U f is (1) a universal MHC class II epitope-encoding nucleic acid sequence, optionally wherein at least one universal sequence comprises at least one of tetanus toxoid and PADRE, or (2) an MHC class II coronavirus-derived epitope-encoding nucleic acid sequence. The compositions and ordered sequences can be further defined by selecting the number of factors 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.

[0310] 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, and when describing the case where there is no additional promoter (e.g., only promoter nucleotide sequences provided by a vector backbone, such as an RNA alphavirus backbone, are present), there are 10 epitopes, there is a 5' linker for each N, there is a 3' linker for each N, there are two MHC class II epitopes, there is a linker connecting the two MHC class II epitopes, there is a linker connecting the 5' ends of the two 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 two MHC class II epitopes to the vector backbone. Examples of linking the 3' end of the antigen cassette to the vector backbone include directly linking it to a 3'UTR element provided by the vector backbone, such as the 3' 19nt CSE. Examples of linking the 5' end of the antigen cassette to the vector backbone include linking it directly to the promoter or 5' UTR element of the vector backbone, such as the 26S promoter sequence, the 5' UTR of an alphavirus, the 51 nt CSE, or the 24 nt CSE of an alphavirus backbone.

[0311] Other examples include when a=1, describing the presence of a promoter other than the promoter nucleotide sequence provided by the vector 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 drives expression of one or more different MHC Class I epitope-encoding nucleic acid sequences; when h=1, describing the presence of another promoter that drives 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, directly linked to the vector backbone.

[0312] Other examples include cases 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, 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, a 3' linker, or neither.

[0313] 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.

[0314] 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, 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, a 3' linker, or neither.

[0315] The promoter nucleotide sequences P and / or P2 can be the same as the promoter nucleotide sequences provided by the vector backbone, such as the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the vector backbone can comprise the 26S subgenomic promoter or the CMV promoter, respectively. The promoter nucleotide sequences P and / or P2 can be different from the promoter nucleotide sequences provided by the vector backbone and can also be different from each other.

[0316] The 5' linker L5 can be a natural or non-natural sequence. Non-natural sequences include, but are not limited to, AAY, RR, and DPP. The 3' linker L3 can also be a natural or non-natural sequence. Furthermore, L5 and L3 can both be natural sequences, both can be non-natural sequences, or one can be natural and the other can be 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.

[0317] 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.

[0318] 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 , 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.

[0319] For each X, each N can encode an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof. For each X, N can encode a combination of an MHC class I epitope, an MHC class II epitope, and an epitope capable of stimulating a B cell response. For each X, N can encode a combination of an MHC class I epitope and an MHC class II epitope. For each X, N can encode a combination of an MHC class I epitope and an epitope capable of stimulating a B cell response. For each X, N can encode a combination of an MHC class II epitope and an epitope capable of stimulating a B cell response. For each X, N can encode an MHC class I epitope 7 to 15 amino acids in length. For each X, each N may encode an MHC class I epitope that is 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 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. For each X, each N can encode an MHC class II epitope. For each X, each N can encode an epitope capable of stimulating a B cell response.

[0320] A cassette encoding one or more antigens may be 700 nucleotides or less. A 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 coronavirus-derived nucleic acid sequences encoding immunogenic polypeptides). A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least two different epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode three different epitope-encoding nucleic acid sequences. A cassette encoding one or more antigens may be 700 nucleotides or less and may encode at least three different epitope-encoding nucleic acid sequences. A 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.

[0321] 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.

[0322] 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 the 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 the 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.

[0323] 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.

[0324] Further considerations in VB vaccine design and manufacturing 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 predetermined filters at each stage of the selection process, one can consider an integral multidimensional model, in which candidate antigens are placed in a space with at least the following axes, and an integral approach is used to optimize selection: 1. Risk of autoimmunity or tolerance (germline risk) (lower autoimmune risk is typically preferred) 2. Probability of sequencing artifacts (lower artifact probabilities are typically preferred) 3. Probability of immunogenicity (higher probability of immunogenicity is typically preferred) 4. Probability of presentation (higher probability of presentation is typically preferred) 5. Gene Expression (higher expression is typically preferred) 6. HLA gene coverage (a greater number of HLA molecules involved in presenting a set of antigens may decrease the probability that 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 probability of a therapeutic response and decrease the probability of avoiding infection)

[0325] Furthermore, in some cases, antigens can be deprioritized (e.g., excluded) from vaccination if they are predicted to be presented by HLA alleles that are lost or inactivated in all or a portion of the patient's infected cells. Loss of HLA alleles can occur through somatic mutation, loss of heterozygosity, or homozygous deletion of the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art (e.g., Shukla et al., 2015). Methods for detecting somatic loss of heterozygosity and homozygous deletion (including HLA loci) have also been described (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). Antigens may be deprioritized if mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele.

[0326] VC self-replicating RNA vector Generally, all self-replicating (SAM) RNA vectors contain a self-replicating 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 the viral genome to replicate autonomously. For example, the minimal sequence that allows alphavirus replication 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, and / or a polyA sequence). The self-replicating backbone can also include sequences for the expression of subgenomic viral RNA (e.g., the 26S promoter element in alphaviruses). SAM vectors can be (+)-sense or (-)-sense RNA polynucleotides, such as vectors with backbones derived from (+)-sense or (-)-sense self-replicating viruses. 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 detail by Lundstrom (Molecules. 2018 Dec 13;23(12).pii:E3310.doi:10.3390 / molecules23123310), which is incorporated by reference herein for all purposes.

[0327] VC1. Alphavirus Biology Alphaviruses are members of the Togaviridae family and are single-stranded, positive-sense RNA viruses. Members are generally classified as either Old World types, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World types, such as Eastern equine encephalitis virus, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbrial Review 1994). Natural alphavirus genomes are typically approximately 12 kb in length, with the first two-thirds containing genes encoding nonstructural proteins (nsPs) that form the RNA replication complex for autonomous replication of the viral genome, and the last one-third containing subgenomic expression cassettes encoding structural proteins for virion production (Frolov RNA 2001).

[0328] The model life cycle of alphaviruses involves multiple distinct steps (Strauss Microbrial Review 1994, Jose Future Microbiol 2009). After viral adsorption to the host cell, the virion fuses with membranes within the intracellular compartment, ultimately releasing the genomic RNA into the cytosol. The genomic RNA, which has a positive-strand orientation, a 5'-methylguanylate cap, and a 3'-poly(A) tail, is translated to generate nonstructural proteins nsP1-4, which form a replication complex. Early in infection, the positive-strand RNA is replicated onto a negative-strand template by this complex. In the current model, the replication complex undergoes further processing as infection progresses, and the resulting processed complex switches to transcribing the negative-strand full-length positive-strand genomic RNA and the 26S subgenomic positive-strand RNA containing the structural genes. Several conserved sequence elements (CSEs) in alphaviruses have been identified as potentially playing roles in various steps of RNA synthesis, including the complement of the 5' UTR in replication of plus-strand RNA from a minus-strand template, a 51-nt CSE in replication of minus-strand synthesis from a genomic template, a 24-nt CSE within the junction region between nsP and 26S RNA in transcription of subgenomic RNA from the minus strand, and a 19-nt CSE at the 3' end in minus-strand synthesis from a plus-strand template.

[0329] In the natural life cycle of viruses, viral particles are typically assembled after replication of different RNA species. 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 particle occurs and capsid proteins, typically specific only to the genomic RNA, are packaged, the virion assembles and buds onto the membrane surface.

[0330] VC2. Alphaviruses as delivery vectors Alphaviruses (alphavirus sequences, characteristics, and other elements) can be used to generate alphavirus-based delivery vectors (also referred to as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors). Alphaviruses have traditionally been genetically engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages in vaccine settings, where expression of heterologous antigens may be desirable. Because of their ability to replicate autonomously in the host cytosol, alphaviruses typically achieve high intracellular copy numbers of the expression cassette, thereby enabling high levels of heterologous antigen production. Furthermore, vectors are generally transient, resulting in high biosafety and low induction of immune tolerance to the vector. The general public generally lacks pre-existing immunity to alphaviruses compared to other standard viral vectors, such as human adenoviruses. Alphavirus-based vectors also typically produce cytotoxic responses to infected cells. Cytotoxicity may be of some importance in vaccine settings to adequately elicit an immune response against the expressed heterologous antigen. However, the desired degree of cytotoxicity is a matter of balance, and for this reason, several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, one example of an antigen expression vector described herein utilizes an alphavirus backbone that allows for high levels of antigen expression, stimulates a strong immune response to the antigen, does not stimulate an immune response against the vector itself, and is safe to use. Furthermore, antigen expression cassettes can be designed to stimulate different levels of immune response through optimization of which alphavirus sequences the vector uses, including, but not limited to, sequences derived from VEE or its attenuated derivative, TC-83.

[0331] Several strategies for designing expression vectors using alphavirus sequences have been developed (Pushko 1997). In one strategy, alphavirus vector design involves inserting a second copy of the 26S promoter sequence element downstream of the structural protein gene followed by the insertion of a heterologous gene (Frolov 1993). This results in the production of subgenomic RNA that expresses additional heterologous proteins in addition to the native nonstructural and structural proteins. In this system, all factors for producing infectious virions are present, and therefore repeated rounds of infection of the expression vector in uninfected cells can be carried out.

[0332] 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 viral RNA mediated by the still-intact nonstructural genes, the 26S subgenomic RNA drives expression of the heterologous protein. Traditionally, an additional vector expressing the structural proteins is provided in trans, for example, by cotransfection of a cell line, to generate infectious virus. One system is described in U.S. Patent 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 potential for infectious particle formation, thus improving biosafety. Furthermore, helper vector systems may shorten the overall vector length and improve replication and expression efficiency. Thus, one example of an antigen expression vector described herein can use an alphavirus backbone in which structural proteins are replaced with an antigen cassette, and the resulting vector promotes efficient expression by reducing biosafety concerns while simultaneously reducing the overall expression vector size.

[0333] VC3. Generation of self-amplifying virus in vitro A conventional method known in the art for producing RNA is in vitro translation (IVT), in which a DNA template of the desired vector is first produced by methods well known in the art, including standard molecular biology methods such as cloning, restriction digestion, ligation, gene synthesis, and polymerase chain reaction (PCR).

[0334] This DNA template has an RNA polymerase promoter at the 5' end of the sequence (e.g., SAM) desired to be transcribed into RNA. Promoters include, but are not limited to, promoters for bacteriophage polymerases such as T3, T7, K11, or SP6. Depending on the specific RNA polymerase promoter sequence selected, additional 5' nucleotides may be transcribed in addition to the desired sequence. For example, the canonical T7 promoter can be referenced by the sequence TAATACGACTCACTATAGG (SEQ ID NO: 118), and an IVT reaction using the DNA template TAATACGACTCACTATAGGN (SEQ ID NO: 119) to generate the desired sequence N results in the mRNA sequence GG-N. Generally, and without wishing to be bound by theory, T7 polymerase can more efficiently transcribe RNA transcripts beginning with guanosine. If additional 5' nucleotides are undesirable (e.g., additional GG is undesirable), the RNA polymerase promoter contained in the DNA template can be a sequence that generates a transcript containing only the 5' nucleotide of the desired sequence (e.g., a SAM with the native 5' sequence of the self-replicating virus from which the SAM vector is derived). For example, a minimal T7 promoter can be referenced by the sequence TAATACGACTCACTATA, and an IVT reaction using the DNA template TAATACGACTCACTATAN to generate the desired sequence N will yield mRNA sequence N. Similarly, a minimal SP6 promoter can be referenced by ATTTAGGTGACACTATA to generate a transcript without additional 5' nucleotides. In a typical IVT reaction, a DNA template is incubated with the appropriate RNA polymerase enzyme, buffer, and nucleotides (NTPs).

[0335] The resulting RNA polynucleotides can optionally be further modified by methods including, but not limited to, the addition of a 5' cap structure, such as 7-methylguanosine or a related structure, and optionally modifying the 3' end to have a polyadenylation (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-NN) cap analogs (where N represents a nucleotide or modified nucleotide (e.g., ribonucleoside, including, but not limited to, adenosine, guanosine, cytidine, and uridine)). Exemplary cap analogs and their use in IVT reactions are also described in more detail in U.S. Pat. No. 10,519,189, incorporated herein by reference for all purposes. As noted above, T7 polymerase can more efficiently transcribe RNA transcripts that begin with guanosine. To increase the transcription efficiency of templates that do not begin with guanosine, trinucleotide cap analogs (m 7 G-ppp-NN) can be used. Trinucleotide cap analogs can be used in combination with dinucleotide cap analogs (m 7 The transfer efficiency 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 relative to an IVT reaction using a ribozyme (Ribozyme) containing ribozyme (Ribozyme).

[0336] A 5' cap structure can also be added post-transcriptionally, for example, by using the vaccinia capping system (eg, NEB catalog no. M2080) which includes mRNA 2'-O-methyltransferase and S-adenosylmethionine.

[0337] The resulting RNA polynucleotides can optionally be further modified separately or in addition to the capping methods described, including, but not limited to, modifying the 3' end to include a polyadenylate (polyA) tail.

[0338] The RNA can then be purified using methods well known in the art, such as phenol chloroform extraction.

[0339] VC4. Delivery via lipid nanoparticles One important aspect to consider in vaccine vector design 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 develops 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.

[0340] In the case of alphavirus vectors, the standard delivery method is the helper virus system described 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 effectiveness of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if the infectious particles are targeted by neutralizing antibodies.

[0341] An alternative to viral particle-mediated gene delivery is the delivery of expression vectors using nanoparticles (Riley 2017). Importantly, nanomaterial carriers can be formed from non-immunogenic materials, generally avoiding immune stimulation 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 conjugates including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0342] Lipid nanoparticles (LNPs) are attractive delivery systems due to the amphiphilic nature of lipids, which allows them to form membrane- and vesicular structures (Riley 2017). These vesicles typically 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. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions typically contain defined mixtures of cationic, neutral, anionic, and amphiphilic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide chemical functional groups that facilitate the 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 an MC3-like molecule. Compositions of MC3 and MC3-like lipids can be formulated to include one or more other lipids, such as, for example, PEG or PEG-conjugated lipids, sterols, or neutral lipids.

[0343] 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 acids. Therefore, encapsulation of alphavirus vectors 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 an LNP. Encapsulation of the alphavirus vector within the LNP can be achieved 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 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, thereby fully encapsulating the delivery vector and desired substances 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 LNPs. 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. After droplet generation, the delivery vector encapsulating the expression vector can be further processed or modified in preparation for administration.

[0344] VD chimpanzee adenovirus (ChAd) VD1. Viral delivery by chimpanzee adenovirus Vaccine compositions for delivering one or more antigens (e.g., via antigen cassettes) can be produced by providing chimpanzee-derived adenovirus nucleotide sequences, various novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleotide sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for delivering antigens (see SEQ ID NO: 1). The use of C68 adenovirus-derived vectors is described in further detail in U.S. Patent No. 6,083,716, the entire contents of which are incorporated herein by reference for all purposes.

[0345] In a further aspect, provided herein is a recombinant adenovirus comprising a DNA sequence of a chimpanzee adenovirus, such as C68, and an antigen cassette operably linked to a regulatory sequence that directs expression. The recombinant virus is capable of infecting mammalian, preferably human, cells and expressing the product of the neoantigen cassette in the cells. The vector can be deleted for the native chimpanzee E1 gene, E3 gene, and / or E4 gene. An antigen cassette can be inserted into any of these gene deletion sites. The antigen cassette can include an antigen against which a primed immune response is desired.

[0346] In another aspect, provided herein are mammalian cells infected with a chimpanzee adenovirus, such as C68.

[0347] In yet another embodiment, novel mammalian cell lines are provided that express chimpanzee adenovirus genes (eg, from C68) or functional fragments thereof.

[0348] In an even further aspect, provided herein is a method for delivering an antigen cassette into a mammalian cell, the method comprising introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, engineered to express the antigen cassette.

[0349] Yet another embodiment provides a method of stimulating an immune response in a mammalian host, which method can include administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens derived from the infection to which the immune response is targeted.

[0350] Yet another embodiment provides a method for stimulating an immune response in a mammalian host to treat or prevent a disease in a subject, such as an infectious disease, comprising administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, that includes an antigen cassette encoding one or more antigens, such as antigens from the infectious disease, to which the immune response is targeted.

[0351] Also disclosed are non-simian mammalian cells expressing chimpanzee adenovirus genes obtained from the sequence of SEQ ID NO: 1. The genes can be selected from the group consisting of adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 of SEQ ID NO: 1.

[0352] Also disclosed are nucleic acid molecules comprising a DNA sequence of a chimpanzee adenovirus comprising a gene obtained from the sequence of SEQ ID NO: 1. The gene can be selected from the group consisting of the chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 1. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, deleted for at least one gene selected from the group consisting of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of SEQ ID NO: 1.

[0353] Also disclosed are vectors comprising a chimpanzee adenoviral DNA sequence derived from SEQ ID NO: 1 and an antigen cassette operably linked to one or more regulatory sequences that direct expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenoviral DNA sequence comprises at least cis elements required for replication and encapsidation, the cis elements flanking the antigen cassette and regulatory sequences. In some embodiments, the chimpanzee adenoviral DNA sequence comprises genes selected from the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 gene sequences of SEQ ID NO: 1. In some embodiments, the vector may be deleted for the E1A and / or E1B genes.

[0354] Also disclosed herein are adenoviral vectors containing a partially deleted E4 gene, including a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region. The partially deleted E4 can include an E4 deletion of at least nucleotides 34,916-35,642 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can include an E4 deletion of at least a partial deletion of nucleotides 34,916-34,942 of the sequence set forth in SEQ ID NO: 1, at least a partial deletion of nucleotides 34,952-35,305 of the sequence set forth in SEQ ID NO: 1, and at least a partial deletion of nucleotides 35,302-35,642 of the sequence set forth in SEQ ID NO: 1, wherein the vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,980-36,516 of the sequence set forth in SEQ ID NO: 1, and the vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,979-35,642 of the sequence set forth in SEQ ID NO: 1, and the vector comprises at least nucleotides 2-36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E4Orf2, a completely deleted E4Orf3, and at least a partial deletion of E4Orf4. The partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3, and at least a partial deletion of E4Orf4. The partially deleted E4 may include an E4 deletion of at least a partial deletion of E4Orf1, a completely deleted E4Orf2, and at least a partial deletion of E4Orf3. The partially deleted E4 may include an E4 deletion of at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3. The partially deleted E4 may include an E4 deletion from the start site of E4Orf1 to the start site of E4Orf5. The partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf1. The partially deleted E4 may be an E4 deletion adjacent to the start site of E4Orf2.The E4 deletion may be adjacent to the start site of E4 Orf3. The E4 deletion may be adjacent to the start site of E4 Orf4. The E4 deletion may be at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 nucleotides. The E4 deletion may be at least 700 nucleotides. The E4 deletion may be at least 1500 nucleotides. The E4 deletion may be 50 or less, 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, 800 or less, 900 or less, 1000 or less, 1100 or less, 1200 or less, 1300 or less, 1400 or less, 1500 or less, 1600 or less, 1700 or less, 1800 or less, 1900 or less, or 2000 or less nucleotides. The E4 deletion may be 750 or less nucleotides. The E4 deletion may be at least 1550 nucleotides or less.

[0355] Also disclosed are host cells transfected with the vectors disclosed herein, such as the C68 vector engineered to express an antigen cassette. Also disclosed are human cells that express a selected gene introduced into the cell by introducing into the cell a vector disclosed herein.

[0356] Also provided is a method for delivering an antigen cassette to a mammalian cell, comprising introducing into said cell an effective amount of a vector disclosed herein, such as a C68 vector engineered to express the antigen cassette.

[0357] Also disclosed is a method for producing an antigen, comprising introducing a vector disclosed herein into a mammalian cell, culturing the cell under appropriate conditions, and producing the antigen.

[0358] Complementing cell lines expressing VD2.E1 To generate recombinant chimpanzee adenoviruses (Ad) having deletions in any of the genes described herein, the function of the deleted gene region (if essential for viral replication and infectivity) can be supplied to the recombinant virus by a helper virus or cell line (i.e., a complementation or packaging cell line). For example, to generate replication-deficient chimpanzee adenovirus vectors, cell lines expressing the E1 gene product of human or chimpanzee adenovirus can be used, and such cell lines can include HEK293 or variants thereof. Cell lines expressing any selected chimpanzee adenovirus gene can be generated according to the protocol for generating cell lines expressing the chimpanzee E1 gene (Examples 3 and 4 of U.S. Pat. No. 6,083,716).

[0359] AAV enrichment assay can be used to identify chimpanzee adenovirus E1-expressing cell lines. This assay is useful for identifying E1 function in cell lines created using E1 genes from other uncharacterized adenoviruses (e.g., from other species). This assay is described in Example 4B of U.S. Patent No. 6,083,716.

[0360] The selected chimpanzee adenovirus gene (e.g., E1) may be under the transcriptional control of a promoter for expression in the selected parent cell line. Inducible or constitutive promoters can be used for this purpose. Inducible promoters include the sheep metallothionine promoter, which is induced by zinc, or the mouse mammary tumor virus (MMTV) promoter, which is induced by glucocorticoids, particularly dexamethasone. Other inducible promoters, such as those identified in International Application No. WO 95 / 13392, incorporated herein by reference, can also be used to generate packaging cell lines. Constitutive promoters controlling the expression of chimpanzee adenovirus genes can also be used.

[0361] Parent cells can be selected to generate novel cell lines expressing any desired C68 gene. Such parent cell lines can be, but are not limited to, HeLa [ATCC Deposit No. CCL2], A549 [ATCC Deposit No. CCL185], KB [CCL17], Detroit [e.g., Detroit 510, CCL72], and WI-38 [CCL75] cells. Other suitable parent cell lines can be obtained from other sources. Parent cell lines can include CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AE1-2a.

[0362] E1-expressing cell lines may be useful in producing recombinant chimpanzee adenovirus E1-deleted vectors. Cell lines constructed using essentially the same procedures that express one or more other chimpanzee adenovirus gene products are useful in producing recombinant chimpanzee adenovirus vectors with deletions in the genes encoding these products. In addition, cell lines expressing other human Ad E1 gene products are also useful in producing chimpanzee recombinant Ad.

[0363] VD3. Recombinant viral particles as vectors The compositions disclosed herein can include a viral vector that delivers at least one antigen to cells. Such a vector includes a chimpanzee adenovirus DNA sequence, such as C68, and an antigen cassette operably linked to a regulatory sequence for directing expression of the cassette. The C68 vector is capable of expressing the cassette in infected mammalian cells. The C68 vector can have functional deletions in one or more viral genes. The antigen cassette includes at least one antigen under the control of one or more regulatory sequences, such as a promoter. Optional helper viruses and / or packaging cell lines can supply the chimpanzee viral vector with any necessary products of the deleted adenovirus genes.

[0364] The term "functionally deleted" means that a sufficient amount of the gene region has been removed or otherwise altered, for example, by mutation or modification, such that the gene region is no longer able to produce one or more functional products of gene expression. Mutations or modifications that can lead to functional deletions include, but are not limited to, nonsense mutations such as the introduction of premature stop codons and the removal of canonical and non-canonical start codons, mutations that alter mRNA splicing or other transcription processing, or combinations thereof. If necessary, the entire gene region can be removed.

[0365] Modifications of the nucleic acid sequences forming the vectors disclosed herein, including sequence deletions, insertions, and other mutations, can be produced using standard molecular biology techniques and are within the scope of the present invention.

[0366] VD4. Construction of Viral Plasmid Vectors Chimpanzee adenovirus C68 vectors useful in the present invention include recombinant defective adenoviruses, i.e., chimpanzee adenovirus sequences having functional deletions in the E1a or E1b genes and, optionally, other mutations, such as temperature-sensitive mutations or deletions in other genes. These chimpanzee sequences are also expected to be useful in forming hybrid vectors from other adenovirus and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses have been described in the published literature [see, e.g., Kozarsky I and II, cited above, and references cited therein, U.S. Patent No. 5,240,846].

[0367] A wide range of adenovirus nucleic acid sequences can be used in constructing chimpanzee adenovirus C68 vectors useful for delivering antigen cassettes into human (or other mammalian) cells. Vectors containing minimal chimpanzee C68 adenovirus sequences can be used in conjunction with a helper virus to generate infectious recombinant viral particles. The helper virus provides the essential gene products required for viral infectivity and propagation of the minimal chimpanzee adenovirus vector. When only one or more selected deletions of chimpanzee adenovirus genes are introduced into an otherwise functional viral vector, the deleted gene products can be supplied during the viral vector production process by propagating the virus that provides the deleted gene function in trans in a selected packaging cell line.

[0368] VD5. Recombinant Minimal Adenovirus The minimal chimpanzee Ad C68 virus is a viral particle that contains only the adenoviral cis elements necessary for replication and virion encapsidation. That is, the vector contains the adenoviral cis-acting 5' and 3' inverted terminal repeats (ITRs) (which function as origins of replication) and the native 5' packaging / enhancer domain (which contains sequences necessary for packaging the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the methods described for preparing a "minimal" human Ad vector in International Application No. WO 96 / 13597, incorporated herein by reference.

[0369] VD6. Other defective adenoviruses Recombinant replication-deficient adenoviruses may contain more than the minimal chimpanzee adenovirus sequences. These other Ad vectors can be characterized by deletion of different portions of the viral gene region and the infectious viral particles formed by the use of helper viruses and / or packaging cell lines as needed.

[0370] As one example, suitable vectors can be generated by deleting all or a sufficient portion of the immediate-early gene E1a and the delayed-early gene E1b of C68 adenovirus, thereby eliminating their normal biological function. Replication-deficient E1-deleted viruses are capable of replicating and generating infectious virus when grown in chimpanzee adenovirus-transformed complementation cell lines containing functional adenovirus E1a and E1b genes that provide the corresponding gene products in trans. Based on homology to known adenovirus sequences, the resulting recombinant chimpanzee adenovirus is capable of infecting many cell types and expressing antigen(s), as are known human recombinant E1-deleted adenoviruses, but is expected to be unable to replicate in many cells lacking chimpanzee E1 region DNA unless the cells are infected at an extremely high multiplicity of infection.

[0371] As another example, all or part of the C68 adenovirus immediate early gene E3 can be removed from the chimpanzee adenovirus sequences that form part of the recombinant virus.

[0372] Chimpanzee adenovirus C68 vectors can also be constructed with a deletion of the E4 gene. Yet another vector can have a deletion in the delayed-early gene E2a.

[0373] Deletions can be introduced into any of the late genes L1 through L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions within intermediate genes IX and IVa2 may be useful for certain purposes. Other deletions can also be introduced into other structural or nonstructural adenovirus genes.

[0374] The deletions described above can be used individually. That is, the adenoviral sequence can have a deletion of only E1. Also, any combination of whole or partial genes effective to disrupt or reduce their biological activity can be used. For example, in one exemplary vector, the adenoviral C68 sequence can have a deletion of the E1 and E4 genes, or the E1, E2a, and E3 genes, or the E1 and E3 genes, or the E1, E2a, and E4 genes, with or without a deletion of E3. As noted above, such deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve the desired results.

[0375] A cassette containing the antigen(s) is optionally inserted into any deleted region of the chimpanzee C68Ad virus, and, if desired, the cassette can be inserted within an existing gene region to disrupt the function of that region.

[0376] VD7. Helper virus Depending on the chimpanzee adenovirus gene content of the viral vector used to deliver the antigen cassette, helper adenovirus or non-replicating viral fragments can be used to provide sufficient chimpanzee adenovirus gene sequences to generate infectious recombinant viral particles containing the cassette.

[0377] Useful helper viruses contain selected adenoviral gene sequences that are not present in the adenoviral vector construct and / or are not expressed by the packaging cell line transfected with the vector. The helper virus may be replication-deficient and may contain various adenoviral genes other than those described above. Helper viruses can be used in combination with the E1-expressing cell lines described herein.

[0378] For C68, the "helper" virus can be a fragment formed by truncating the C-terminus of the C68 genome with SspI, which removes approximately 1300 bp from the left end of the virus. This truncated virus is then co-transfected with plasmid DNA into an E1-expressing cell line to form recombinant virus by homologous recombination with the C68 sequences in the plasmid.

[0379] The helper virus can also be formulated as a polycation complex, as described in Wu et al., J. Biol. Chem., 264:16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299:49 (Apr. 1, 1994). The helper virus may optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper virus that is different from the antigen cassette on the adenovirus vector allows for independent monitoring of the Ad vector and the helper virus. This second reporter gene allows for the separation of the resulting recombinant virus from the helper virus during purification.

[0380] VD8. Assembly of viral particles and infection of cell lines Assembly of selected DNA sequences of adenovirus, antigen cassettes, and other vector elements into various intermediate plasmids and shuttle vectors, as well as use of the plasmids and shuttle vectors to generate recombinant viral particles, can all be accomplished using conventional techniques, including traditional cDNA cloning, in vitro recombination (e.g., Gibson assembly), use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. Standard transfection and cotransfection techniques, such as CaPO precipitation or liposome-mediated transfection methods such as lipofectamine, can be used. Other conventional methods that can be used include homologous recombination of the viral genome, viral plaque formation in agar overlays, and signal generation assays.

[0381] For example, after construction and assembly of a viral vector containing a desired antigen, the vector can be transfected in vitro into a packaging cell line in the presence of a helper virus, and homologous recombination occurs between the helper and vector sequences, allowing the adenovirus-antigen sequences within the vector to be replicated and packaged into virion capsids, resulting in recombinant viral vector particles.

[0382] The resulting recombinant chimpanzee C68 adenovirus is useful for transferring antigen cassettes into selected cells. In vivo experiments using recombinant viruses propagated in packaging cell lines demonstrate the utility of the E1-deleted recombinant chimpanzee adenovirus for transferring cassettes into non-chimpanzee cells, preferably human cells.

[0383] VD9. Use of Recombinant Viral Vectors Thus, the resulting recombinant chimpanzee C68 adenovirus containing the antigen cassette (produced by combining an adenoviral vector and a helper virus, or an adenoviral vector and a packaging cell line, as described above) provides an efficient gene transfer vehicle capable of delivering antigen(s) to a subject in vivo or ex vivo.

[0384] The recombinant vectors described above are administered to humans according to published methods for gene therapy. The chimpanzee virus vector carrying the antigen cassette can be administered to patients preferably suspended in a biocompatible solution or pharmaceutically acceptable delivery vehicle. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known as pharmaceutically acceptable carriers and familiar to those skilled in the art can also be used for this purpose.

[0385] The chimpanzee adenoviral vector is administered in an amount sufficient to transform human cells and result in the introduction and expression of antigens at levels sufficient to provide a therapeutic effect without adverse effects or with a medically acceptable physiological effect, as can be determined by those skilled in the medical field. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intrahepatic, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Routes of administration can be combined as needed.

[0386] The dosage of the viral vector depends primarily on factors such as the condition being treated, the patient's age, weight, and health status, and may therefore vary between patients. The dosage is adjusted to balance the therapeutic effect against any side effects, and may vary depending on the therapeutic application for which the recombinant vector is used. The frequency of administration can be determined by observing the expression level of the antigen(s).

[0387] Recombinant replication-deficient adenovirus can be administered in a "pharmacologically effective amount," i.e., an amount of recombinant adenovirus effective by a given route of administration to transfect the desired cells and provide sufficient expression levels of the selected gene to produce a vaccine effect, i.e., a measurable level of protective immunity. The C68 vector containing the antigen can be co-administered with an adjuvant. The adjuvant may be a separate monomer from the vector (e.g., alum) or may be encoded within the vector, particularly if the adjuvant is a protein. Adjuvants are well known in the art.

[0388] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes of administration. Routes of administration can be combined as needed or adjusted depending on the immunogen or disease. For example, for the prevention of rabies, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration is primarily determined by the nature of the disease being treated.

[0389] By observing the expression level of the antigen(s), the need for boosters, if any, can be determined. For example, after evaluation of antibody titers in the serum, booster immunizations may be desirable, if necessary.

[0390] VI. Methods of Treatment and Manufacturing Also provided are methods for inducing an infectious disease organism-specific (e.g., coronavirus-specific) immune response in a subject, vaccinating against the infectious disease organism, and treating and / or alleviating symptoms of infection associated with the infectious disease organism in a subject by administering to the subject one or more antigens, such as a plurality of antigens identified using the methods disclosed herein.

[0391] In some embodiments, the subject has been diagnosed with or is at risk for infection (e.g., Covid-19 due to coronavirus infection), or is at risk for seasonal and / or novel disease infection, such as due to a high risk or predisposition for infection related to age, geography / travel, and / or work.

[0392] In some embodiments, the subject is immunocompromised, such as diagnosed with and / or suspected of having cancer. The subject may include a subject treated with a therapy that results in immunosuppression. For example, the subject may include a subject diagnosed with a hematopoietic malignancy, such as a B-cell malignancy treated with anti-CD20 therapy (e.g., rituximab), and being treated with a hematopoietic cell-targeted therapy. In another example, the subject may include a subject diagnosed with multiple sclerosis (e.g., relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), or primary progressive multiple sclerosis (PPMS)) and being treated with an anti-CD20 therapy.

[0393] 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.

[0394] Antigens can be administered alone or in combination with other therapeutic agents, which may include those that target infectious disease organisms, such as antivirals or antibiotics.

[0395] The optimal amount and optimal administration regimen of each antigen included in the vaccine composition can be determined. For example, the antigen or its variant can be formulated for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Injection methods include subcutaneous (sc), intradermal (id), intraperitoneal (ip), intramuscular (im), and intravenous (iv). Injection methods for DNA or RNA include intradermal (id), intramuscular (im), subcutaneous (sc), intraperitoneal (ip), and intravenous (iv). Other methods of administering vaccine compositions are known to those skilled in the art.

[0396] Vaccines can be tailored so that the selection, number, and / or amount of antigens present in the composition are tissue-, infection-, and / or patient-specific. For example, the precise selection of peptides can be guided by the expression pattern of a particular parent protein or by the patient's mutation or disease state. This selection can depend on the specific infection (e.g., the particular coronavirus isolate with which the subject is infected or at risk), the disease state, the purpose of vaccination (e.g., preventative or targeting ongoing disease), the initial treatment regimen, the patient's immune status, and, of course, the patient's HLA haplotype. Furthermore, vaccines can include components that are personalized according to the individual needs of a particular patient. Examples include altering the antigen selection according to the expression of antigens in a particular patient or tailoring secondary treatments after a first round or regimen of treatment.

[0397] Patients for administration of an antigen vaccine can be identified using various diagnostic methods, such as the patient selection methods described further below. Patient selection can involve identifying mutations or expression patterns of one or more genes. Patient selection can involve identifying an ongoing infection (e.g., coronavirus infection and / or the presence of a particular coronavirus isolate). Patient selection can involve identifying the risk of infection from an infectious disease. Optionally, patient selection can involve identifying the patient's haplotype. Various patient selection methods can be performed in parallel, for example, a sequencing diagnostic can identify both the patient's mutation and haplotype. Various patient selection methods can also be performed sequentially, for example, one diagnostic test can identify a mutation and another diagnostic test can identify the patient's haplotype, 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.

[0398] For compositions to be used as vaccines for infectious diseases, antigens containing similar normal self-peptides that are highly expressed in normal tissues may be avoided or present in low amounts in the compositions described herein. Conversely, if infected cells in a patient are known to express a particular antigen in high amounts, the respective pharmaceutical composition for treating this infection may be present in high amounts and / or multiple antigens specific for this particular antigen or pathway of this antigen may be included.

[0399] Compositions containing antigens can be administered to individuals already suffering from an infectious disease. In therapeutic applications, compositions are administered to patients in an amount sufficient to stimulate effective CTLs against infectious disease organism antigens and to 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 patient's weight and general condition, and the judgment of the prescribing physician. Compositions are generally used in severe disease states, i.e., life-threatening or potentially life-threatening situations, particularly when the infectious disease organism has induced organ damage and / or other immunopathology. In such cases, given the minimally induced relative non-toxic properties of the foreign material and antigen, it is believed that treating physicians may find it possible and desirable to administer significant excesses of these compositions.

[0400] For therapeutic applications, administration can begin upon detection or treatment of an infection, followed by booster doses at predetermined intervals thereafter until at least symptoms are substantially eliminated.

[0401] 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, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The compositions can be administered to target specific infected tissues and / or cells of a subject. Disclosed herein are compositions for parenteral administration, comprising a solution of an antigen, where the vaccine composition is dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various 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 immediate use or lyophilized, and the lyophilized formulation can be combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0402] Antigens can also be administered via liposomes, which target antigens 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, and lamellar layers. 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 commonly found on lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Liposomes loaded with the desired antigen can then be targeted to the site of lymphoid cells, where they deliver the selected therapeutic / immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, typically including neutral and negatively charged phospholipids and a sterol, such as cholesterol. The choice of lipid is generally guided by considerations such as liposome size, acid lability, and stability of the liposomes in the bloodstream. There are various methods for preparing liposomes, such as those 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.

[0403] For targeting immune cells, the ligand incorporated into the liposome can include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposome suspension can be administered intravenously, locally, or topically, at doses that vary depending, inter alia, on the method of administration, the peptide being delivered, and the stage of the disease being treated.

[0404] For therapeutic or immunization purposes, peptides, and optionally nucleic acids encoding 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, nucleic acids can be administered directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Patent No. 5,204,253. Particles composed solely of DNA can also be administered. Alternatively, 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.

[0405] 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).

[0406] Antigens can 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 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 (UbC) gene). The vectors may be included in a vaccine platform based on a viral vector, such as a nucleotide sequence encoding one or more antigenic peptides (see, for example, 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 described above, this approach can deliver one or more nucleotide sequences encoding one or more antigenic peptides.The sequence may be flanked by unmutated sequence, 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). Upon introduction into a host, infected cells express the antigen, thereby stimulating a host immune (e.g., CTL) response 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 by 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 Salmonella typhi vectors, will be apparent to those skilled in the art from the disclosure herein.

[0407] One means of administering nucleic acids is to use minigene constructs encoding one or more epitope-encoding nucleic acid sequences. To generate DNA sequences (minigenes) encoding selected CTL epitopes for expression in human cells, the amino acid sequences of the epitopes are reverse-translated. A human codon usage table is used to guide the codon selection for each amino acid. These epitope-encoding DNA sequences are then 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. Furthermore, 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 encoding the minigene's + and - strands. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well-known methods. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene encoding the CTL epitope polypeptide is then cloned into the desired expression vector.

[0408] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. Furthermore, purified plasmid DNA can be complexed with glycolipids, fusogenic liposomes, peptides, and compounds collectively known as PINCs (protective, interactive, non-condensing) to affect variables such as stability, intramuscular distribution, or trafficking to specific organs or cell types.

[0409] Also disclosed are methods of manufacturing a vaccine comprising carrying out each of the steps of the methods disclosed herein and producing a vaccine comprising multiple antigens or a subset of multiple antigens.

[0410] The antigens disclosed herein can be produced using methods well known in the art. For example, a method for producing an antigen or vector disclosed herein (e.g., a vector containing at least one sequence encoding one or more antigens) can include culturing a host cell containing at least one polynucleotide encoding the antigen or vector under conditions suitable for expressing the antigen or vector, and purifying the antigen or vector. Standard purification methods include chromatography, electrophoresis, immunological methods, precipitation, dialysis, filtration, concentration, and chromatofocusing.

[0411] The host cell may comprise a Chinese hamster ovary (CHO) cell, an NS0 cell, yeast, or an 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.

[0412] VII. Use and Administration of Antigens Vaccination protocols can be used to administer one or more antigens and / or epitopes to a subject. A subject can be administered using a priming vaccine and a booster vaccine.

[0413] The priming vaccine can be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or srRNA (e.g., the sequence shown in SEQ ID NO: 3 or 4), and the booster vaccine can be based on C68 (e.g., the sequence shown in SEQ ID NO: 1 or 2) or srRNA (e.g., the sequence shown in SEQ ID NO: 3 or 4). Each vector typically contains a cassette containing antigens and / or epitopes. The cassette can contain approximately 20 epitopes (or antigens from which the epitopes are derived) separated by spacers, such as the naturally occurring sequences that typically surround each epitope or other non-natural spacer sequences such as AAY. The cassette may also contain MHC II antigens / epitopes, such as tetanus toxoid antigens, and PADRE antigens, which are considered universal class II antigens. The cassette may also contain a targeting sequence, such as a ubiquitin targeting sequence.

[0414] The priming vaccine can be injected (e.g., intramuscularly) into the subject. 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 x 10 1210 viral particles), one or more injections of self-amplifying RNA (samRNA or SAM), e.g., 3 μg, 10 μg, 30 μg, 100 μg, or 300 μg of RNA, can be used. A priming dose of 30 μg or less of SAM can be used. A priming dose of 10 μg or less of SAM can be used. A priming dose of 3 μg or less of SAM can be used. For priming of ChAdV68, 1 × 10 12 For ChAdV68 priming, 3 × 10 viral particles or less can be administered. 11 For ChAdV68 priming, at least 1 × 10 viral particles can be administered. 11 For ChAdV68 priming, 1 × 10 viral particles can be administered. 11 ~1×10 12 pieces, 3×10 11 ~1×10 12 pieces, or 1 x 10 11 ~3×10 11 For ChAdV68 priming, 1 × 10 viral particles can be administered. 11 pieces, 3×10 11 pieces, or 1 x 10 12 For ChAdV68 priming, 5 x 10 viral particles can be administered. 11 It can be at a concentration of vp / mL.

[0415] After the prime vaccination, a vaccine boost (booster vaccine) can be injected (e.g., intramuscularly). The booster vaccine can be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after priming, e.g., every 4 weeks and / or every 8 weeks. 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 x 10 12One or more injections of self-amplifying RNA (samRNA or SAM), such as 3 μg, 10 μg, 30 μg, 100 μg, or 300 μg of RNA, can be used. A booster dose of SAM of 30 μg or less can be used. A booster dose of SAM of 10 μg or less can be used. A booster dose of SAM of 3 μg or less can be used. One or more injections of samRNA at a dose of 30 μg or less can be used. A dose of 30 μg or less can represent the total amount of RNA / samRNA administered. A dose of 30 μg or less can represent the total amount of RNA / samRNA administered and can include only a single different samRNA construct. A SAM booster of 10-30 μg, 10-100 μg, 10-300 μg, 30-100 μg, 30-300 μg, or 100-300 μg of RNA can be administered. A SAM booster of 10-500 μg, 10-1000 μg, 30-500 μg, 30-1000 μg, or 500-1000 μg RNA may be administered. A SAM booster 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, or at least 1000 μg RNA may be administered. A SAM booster of 10 μg, 30 μg, 100 μg, or 300 μg RNA may be administered. A SAM booster of 300 μg RNA may be administered. A SAM booster of 100 μg RNA may be administered. A SAM booster of 30 μg RNA may be administered. A SAM booster of 10 μg RNA may be administered. A SAM booster of 3 μg RNA may be administered. A SAM booster of at least 300 μg RNA may be administered. A SAM booster of at least 100 μg RNA may be administered. A SAM booster of at least 30 μg RNA may be administered. A SAM booster of at least 10 μg RNA may be administered. A SAM booster of at least 3 μg RNA may be administered. A SAM booster of up to 300 μg RNA may be administered. A SAM booster of up to 100 μg RNA may be administered.

[0416] The dose may refer to the total content of RNA / samRNA administered. The dose may refer to the total content of RNA / samRNA administered and may include only a single different samRNA construct.

[0417] Immune monitoring can be performed before, during, and / or after vaccine administration, and such monitoring can provide information about safety and efficacy, among other parameters.

[0418] PBMCs are commonly used for immune monitoring. PBMCs can be isolated before and after prime vaccination (e.g., 4 and 8 weeks). PBMCs can be collected immediately before and after each boost vaccination (e.g., 4 and 8 weeks).

[0419] T cell responses can be assessed 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 assessed. 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 an enhanced 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). Boosting an immune response can include stimulating an immune response in a convalescent subject (e.g., a booster vaccine that stimulates an enhanced immune response in a convalescent Covid-19 subject). The subject can include an HIV-positive subject. 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 the 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 the T cell population expressing T cell receptors specific 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 the ex vivo proliferation of T cell populations after incorporation of 3H-thymidine, bromodeoxyuridine, and carboxyfluorescein diacetate succinimidyl ester (CFSE).The antigen recognition and lytic activity of PBMC-derived T cells specific for the epitopes encoded in the vaccine can be functionally assessed by a chromium release assay or an alternative colorimetric cytotoxicity assay.

[0420] 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).

[0421] Pan-coronavirus vaccination methods include administering RBD-derived nucleic acid sequences encoded by a single antigen cassette (e.g., a multicistronic cassette). Pan-coronavirus vaccination methods can include administering RBD-derived nucleic acid sequences encoded by separate polynucleotide sequences (e.g., when each RBD is encoded on a separate viral backbone), such as a "mixed" vaccine strategy in which multiple different vaccines are administered, each encoding a distinct and different RBD, either alone (e.g., administered separately) or in combination (a single administration of a combination of different RBD-encoding delivery vectors). Pan-coronavirus vaccination methods can also include administering RBD-derived nucleic acid sequences encoded by a single antigen cassette (e.g., a multicistronic cassette) or as multiple different vaccines each encoding a distinct and different RBD, together with additional antigen-encoding nucleic acid sequences, e.g., encoding an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof. In an illustrative, non-limiting example, a method of pan-coronavirus vaccination may include administering a vaccine comprising an RBD-derived nucleic acid sequence encoded in a single antigen cassette together with a vaccine comprising a cassette encoding linked T cell epitopes (see, e.g., Tables 16A-D), either as separate doses or combined in a single dose in the same formulation.

[0422] VIII. HLA Peptide Isolation and Detection Isolation of HLA peptide molecules was performed using classical immunoprecipitation (IP) techniques after lysis and solubilization of tissue samples. (55-58) Examples and methods are described in more detail in International Patent Application Publication No. WO / 2018 / 208856, which is incorporated herein in its entirety for all purposes.

[0423] IX. Presented Model The presentation model can be used to identify the likelihood of peptide presentation in patients. Different presentation models are well known to those skilled in the art, and for example, such presentation models are described in more detail in U.S. Patent No. 10,055,540, U.S. Patent Application Publication No. US20200010849A1 and U.S. Patent Application Publication No. US20110293637, and International Patent Application Publication No. WO / 2018 / 195357, WO / 2018 / 208856, and WO2016187508, each of which is incorporated by reference in its entirety for all purposes.

[0424] X. Training Module A training module can be used to build one or more display models based on a training dataset, which generate the likelihood that a peptide sequence will be presented by the MHC allele associated with that peptide sequence. Various training modules are well known to those skilled in the art, and such display models 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. The training module can build a predictive model for predicting the likelihood of presentation of a peptide on an allele-by-allele basis. The training module can also build a display model for predicting the likelihood of presentation of a peptide in a multi-allele scenario where two or more MHC alleles are present.

[0425] XI. Prediction Module The prediction module can receive sequence data and use the proposed model to select candidate antigens within the sequence data. Specifically, the sequence data can be DNA, RNA, and / or protein sequences extracted from infected cells of a patient or from the infectious disease organism itself (e.g., coronavirus). The prediction module can identify candidate antigens that are pathogen-derived peptides (e.g., derived from a coronavirus), such as by comparing sequence data extracted from cells of the patient's normal tissue with sequence data extracted from the patient's infected cells to identify portions containing one or more infectious disease organism-associated antigens. The prediction module can identify candidate antigens that are expressed in infected cells or tissues relative to normal cells or tissues by comparing sequence data extracted from cells of the patient's normal tissue with sequence data extracted from cells of the patient's infected tissue to identify expressed candidate antigens (e.g., identify polynucleotides and / or polypeptides that are differentially expressed in infectious diseases).

[0426] The presentation module can apply one or more presentation models to the processed peptide sequences to estimate the presentation likelihood of the peptide sequences. Specifically, the prediction module can select one or more candidate antigen peptide sequences that are likely to be presented on HLA molecules of infected cells by applying the presentation model to the candidate antigens. In one implementation, the presentation module selects candidate antigen sequences with an estimated presentation likelihood above a predetermined threshold. In another implementation, the presentation model selects N candidate antigen sequences with the highest estimated presentation likelihood (N is generally the maximum number of epitopes that can be delivered in a vaccine). A vaccine containing the selected candidate antigens for a given patient can be injected into the patient to stimulate an immune response.

[0427] XI.B. Cassette Design Module XI.B.1 Overview The cassette design module can be used to generate vaccine cassette sequences based on the selected candidate peptides for injection into patients.For example, the cassette design module can be used to generate sequences encoding linked epitope sequences, such as linked T cell epitopes.Various cassette design modules are well known to those skilled in the art, and for example, such cassette design training modules are described in more detail in 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.

[0428] The set of therapeutic epitopes can be generated based on selected peptides determined by the prediction module that are associated with a presentation likelihood above a predetermined threshold (the presentation likelihood being determined by the presentation model). However, in other embodiments, the set of therapeutic epitopes can be generated based on any one or more (alone or in combination) of a number of methods, such as, for example, based on binding affinity or predicted binding affinity to the patient's HLA class I or class II alleles, binding stability or predicted binding stability to the patient's HLA class I or class II alleles, random sampling, etc.

[0429] The therapeutic epitope may correspond to the selected peptide itself. The therapeutic epitope may also include C- and / or N-terminal flanking sequences in addition to the selected peptide. The N- and C-terminal flanking sequences may be the natural N- and C-terminal flanking sequences of the therapeutic vaccine epitope in the context of its derived protein. The therapeutic epitope may represent an epitope of fixed length. The therapeutic epitope may represent an epitope of variable length, where the length of the epitope can vary, for example, depending on the length of the C- or N-terminal flanking sequences. For example, the C- and N-terminal flanking sequences may each have different lengths of 2 to 5 residues, thereby providing 16 possible choices of epitopes.

[0430] The cassette design module can also generate cassette sequences by considering the representation of junction epitopes spanning the junction between two therapeutic epitopes within a cassette. A junction epitope is a novel, non-self, but unrelated epitope sequence that arises within a cassette by the process of joining therapeutic epitopes and linker sequences within the cassette. The novel sequence of the junction epitope is different from the therapeutic epitopes of the cassette themselves.

[0431] The cassette design module can generate cassette sequences that reduce the likelihood that junction epitopes will be presented in the patient. Specifically, when the cassette is injected into a patient, the junction epitopes have the potential to be presented by the patient's HLA class I or HLA class II alleles, stimulating CD8 or CD4 T cell responses, respectively. Such responses are undesirable because T cells reactive to the junction epitopes have no therapeutic effect and may extinguish the immune response to the selected therapeutic epitope in the cassette due to antigen competition. 76 .

[0432] The cassette design module can iteratively process one or more candidate cassettes to determine a cassette sequence for which the presentation score of the junction epitope associated with that cassette sequence is below a numerical threshold. The junction epitope presentation score is a quantity related to the likelihood of presentation of the junction epitope within the cassette, with a higher junction epitope presentation score indicating a greater likelihood that the junction epitope of the cassette will be presented by HLA class I, HLA class II, or both.

[0433] In one embodiment, the cassette design module can determine the cassette sequence associated with the lowest junction epitope presentation score among the candidate cassette sequences.

[0434] The cassette design module can iteratively process one or more candidate cassette sequences, determine a junction epitope presentation score for each candidate cassette, and identify an optimal cassette sequence associated with a junction epitope presentation score below a threshold.

[0435] The cassette design module can further validate one or more candidate cassette sequences to identify whether any of the junction epitopes within the candidate cassette sequences are self-epitopes for the particular patient for whom the vaccine is being designed. To do this, the cassette design module validates the junction epitopes against a known database, such as BLAST. In one embodiment, the cassette design module can be configured to design cassettes that prevent junction self-epitopes.

[0436] The cassette design module can implement a brute force approach, iterating through all or most of the possible candidate cassette sequences to select the sequence with the lowest junction epitope presentation score. However, the number of such candidate cassettes can become prohibitively large as the vaccine volume increases. For example, for a vaccine volume of 20 epitopes, the cassette design module may need to iterate through approximately 10 candidate cassettes to determine the cassette with the lowest epitope presentation score. 18 The cassette design module must iterate over a number of possible candidate cassettes. This determination can be computationally intensive (in terms of required computing resources) and sometimes intractable for the cassette design module to generate a vaccine for a patient within a reasonable length of time. Furthermore, processing the possible junction epitopes for each candidate cassette can be even more intensive. Therefore, the cassette design module can select cassette sequences based on a method that iterates over a number of candidate cassette sequences that is significantly smaller than the number of candidate cassette sequences in a brute force approach.

[0437] The cassette design module can generate randomly or at least pseudo-randomly generated candidate cassettes and select as the cassette sequence a candidate cassette associated with a junction epitope presentation score below a predetermined threshold. Furthermore, the cassette design module can select as the cassette sequence a candidate cassette from the subset with the lowest junction epitope presentation score. For example, the cassette design module can generate a subset of approximately 1 million candidate cassettes for a set of 20 selected epitopes and select the candidate cassette with the lowest junction epitope presentation score. While generating a subset of random cassette sequences and selecting a cassette sequence with a low junction epitope presentation score from this subset is less optimal than a brute-force approach, it requires significantly fewer computational resources and is therefore technically feasible. Furthermore, performing a brute-force method against this more efficient approach may result in only a slight or even negligible improvement in the junction epitope presentation score, making it less worthwhile in terms of resource allocation. The cassette design module can determine improved cassette configurations by formulating the cassette's epitope sequences as an asymmetric traveling salesman problem (TSP). Given a list of nodes and the distance between each pair of nodes, the TSP determines the sequence of nodes associated with the minimum total distance required to visit each node exactly once and return to the original node. For example, given cities A, B, and C, each with a known distance between them, a solution to the TSP generates a closed sequence of cities such that the total distance traveled to visit each city exactly once among all possible routes is minimized. An asymmetric version of the TSP determines the optimal sequence of nodes when the distances between pairs of nodes are asymmetric. For example, the "distance" to travel from node A to node B may be different from the "distance" to travel from node B to node A.By solving for an improved optimal cassette using asymmetric TSP, the cassette design module can find a cassette sequence that provides a low presentation score across the junctions between each epitope in the cassette. The solution to the asymmetric TSP indicates the sequence of therapeutic epitopes corresponding to the order in which each epitope must be linked to minimize the junction epitope presentation score across each junction in the cassette. Cassette sequences determined by this approach may provide sequences with significantly lower presentation of junction epitopes while requiring significantly fewer computational resources than a random sampling approach, especially when a large number of candidate cassette sequences are generated. Illustrative examples of comparisons of different computational approaches and optimized cassette designs are described in more detail in 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.

[0438] The cassette design module can also generate cassette sequences by taking into account additional protein sequences encoded in the vaccine. For example, a cassette design module used to generate sequences encoding concatenated T cell epitopes can take into account T cell epitopes already encoded by additional protein sequences present in the vaccine (e.g., full-length protein sequences), such as by removing T cell epitopes already encoded by additional protein sequences from the list of candidate sequences.

[0439] The cassette design module can also generate cassette sequences by taking sequence size into consideration. While not wishing to be bound by theory, generally, large cassette sizes can adversely affect vaccine properties, such as vaccine production and / or vaccine efficacy. In one example, the cassette design module can take into consideration overlapping sequences, such as overlapping cellular epitope sequences. Generally, a single sequence (also referred to as a "frame") containing overlapping T cell epitope sequences is more efficient than concatenating individual T cell epitope sequences separately, as the sequence size required to encode multiple peptides is reduced. Thus, in one illustrative example, the cassette design module used to generate a sequence encoding a concatenated T cell epitope can take into consideration the cost / benefit of extending a candidate T cell epitope to encode one or more additional T cell epitopes, such as determining the benefit of additional population coverage of the MHC presenting the additional T cell epitope relative to the cost of increasing the sequence size.

[0440] The cassette design module can also generate cassette sequences by taking into account the magnitude of stimulation of the immune response brought about by the validated epitopes.

[0441] The cassette design module can also generate cassette sequences by taking into account the representation of the encoded epitope across a population, e.g., that at least one immunogenic epitope is represented by at least one HLA in a certain percentage of the population, e.g., at least 85%, 90%, or 95% of the population (e.g., HLA-A, HLA-B, and HLA-C across the four major ethnicities: European (EUR), African American (AFA), Asian or Pacific Islander (APA), and Hispanic (HIS)). As an illustrative, non-limiting example, the cassette design module can generate cassette sequences such that at least one HLA is present across at least 85%, 90%, or 95% of the population that represents at least one validated epitope or represents at least four, five, six, or seven predicted epitopes.

[0442] The cassette design module can also generate cassette sequences by taking into account other potential safety-enhancing properties, such as limiting the likelihood of encoding or encoding functional proteins, functional protein domains, functional protein subunits, or functional protein fragments that potentially pose safety risks. In some cases, the cassette design module can limit the sequence size of the encoded peptide to be less than 50%, 49%, 48%, 47%, 46%, 45%, 45%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, or 33% of the corresponding translated full-length protein. In some cases, the cassette design module can limit the sequence size of the encoded peptide to be less than 50% of the corresponding translated full-length protein, although multiple sequences may be derived from the same corresponding translated full-length protein and together encode more than 50%. In one illustrative example, if a single sequence ("frame") containing overlapping T cell epitope sequences is greater than 50% of the corresponding full-length protein after translation, the frame can be divided into multiple frames (e.g., f1, f2, etc.) such that each frame is less than 50% of the corresponding full-length protein after translation. The cassette design module can restrict a single contiguous sequence to be less than 49%, 48%, 47%, 46%, 45%, 45%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, or 33% of the corresponding full-length protein after translation. When multiple frames from the same gene are encoded, the multiple frames can overlap with each other, i.e., each frame can have sequences that separately encode the same sequence. When multiple frames from the same gene are encoded, two or more nucleic acid sequences from the same gene can be arranged in an order such that when a first nucleic acid sequence is followed directly or indirectly by a second nucleic acid sequence within the corresponding gene, the second nucleic acid sequence is not directly connected to or can be linked to the first nucleic acid sequence.For example, if there are three frames in the same gene (f1, f2, f3 in order of increasing amino acid position), - The following cassette sequences are not permitted: f2 immediately follows f1 f2 immediately followed by f3 f1 is immediately followed by f3 - The following cassette sequences are permitted: f3 immediately followed by f2 f2 is immediately followed by f1.

[0443] XIII. Exemplary Computer Any of the calculation methods described herein can use a computer.Those skilled in the art will recognize that computer can have different architectures.Examples of computers known to those skilled in the art are described in detail in, for example, United States Patent No. 10,055,540, United States Patent Application Publication No. US20200010849A1, and International Patent Application Publication No. WO / 2018 / 195357 and International Patent Application Publication No. WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes. [Example]

[0444] XIV. Examples Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0445] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature (e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0446] XIV.A. SARS-CoV-2 MHC Epitope Prediction and Vaccine Cassette Construction SARS-CoV-2 belongs to the Coronaviridae family, and its reference genome is a single-stranded RNA sequence of 29,903 base pairs. Its genome contains at least 14 open reading frames (ORFs), as shown in Figure 1. Among the encoded genes, essential genes are the replicase ORF1ab, spike (S), envelope (E), membrane (M), and nucleocapsid (N). The replicase ORF1ab (positions 266–21,555) encodes two proteins, orf1a and orf1b, the latter of which is translated by a -1 ribosomal frameshift at position 13,468. The two proteins collectively contain 16 nonstructural proteins (nsp1–nsp16), as shown in Figure 2; that is, ORF1a and ORF1b are cleaved into 16 nsp1s. The spike protein is thought to bind to the ACE2 receptor on human cells, allowing the virus to enter the cell and use its replication machinery to produce and spread further copies of the virus.

[0447] Because RNA viruses are known to have a high mutation rate, we analyzed multiple SARS-CoV-2 genomes to identify variable regions in their proteome. We obtained over 8,000 complete SARS-CoV-2 genomes, stored in the GISAID database [https: / / www.gisaid.org] as of April 19, 2020. Pairwise global alignments of each genome were performed against the SARS-CoV-2 reference genome (Genbank accession number NC_045512, SEQ ID NO: 76). The positions of sequences aligned to the coding regions of the reference genome were then identified. These sequences were then translated to obtain the protein sequences of these SARS-CoV-2 viruses. These protein sequences were then aligned to their respective reference protein sequences to identify variants.

[0448] This analysis identified 20 sites in the protein sequence with a variant rate greater than 1%, which are shown in Table 1. For T cell epitope selection, candidate epitopes that intersect with these variable sites were excluded.

[0449] We used our machine learning EDGE platform (see U.S. Patent No. 10,055,540, incorporated herein by reference for all purposes), which has been shown to be best-in-class [Bulik-Sullivan et al. (2018). Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification. Nature Biotechnology 2018, 37(1), incorporated herein by reference for all purposes], to predict CD8+ epitopes. This model for predicting class I epitopes was recently trained on 507,502 peptides represented by mass spectrometry across 398 samples and includes 116 identified alleles, 112 of which are shown in the haplotype distribution dataset (Table 2, Figure 7).

[0450] To generate a list of candidate CD8+ T cell epitopes, the orf1ab protein was split at the cleavage sites shown in Figure 2. Studies have shown that the spike protein contains a furin-like cleavage motif at positions 681–684, and that the cleavage event occurs after position 684 [Wrapp et al. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260–1263; Ou et al. (2020). Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature Communications, 11(1), 1620]. Cleavage of the spike protein into S1 and S2 is thought to facilitate cell entry and contribute to viral infectivity. Therefore, we split the spike protein at the furin cleavage site to generate candidate CD8+ T cell epitopes. All 8- to 11-mer peptides were generated from the cleaved proteins and other proteins flanked by their natural N- and / or C-terminal 5-mers.

[0451] The EDGE machine learning model was run on these candidate epitopes for each HLA class I allele. The presentation score of the candidate epitope is then given the EDGE score for each HLA allele. Generally, the probability of a peptide being presented is influenced by the family of the protein containing the peptide and the expression level of the protein. The EDGE model was also trained on the human peptidome dataset. Assuming there is no equivalent protein family for SARS-CoV-2, we assigned a random protein family to every peptide to predict the presentation of a specific SARS-CoV-2 peptide. Assigning the same, but random, protein family would have the same effect on all SARS-CoV-2 peptides. A high expression level was also used (tpm=10). A list of candidate epitopes with EDGE scores of 0.001 or greater for HLA alleles and allogeneic HLA alleles with predicted EDGE scores higher than 0.001 is shown in Table A , with each allogeneic pairing ranked as H (EDGE score >0.1), M (EDGE = 0.01–0.1), and L (EDGE score <0.01).

[0452] To account for different levels of expression of SARS-CoV-2 genes, we used the reported ratio of T cell responses between genes from the SARS-CoV-2 genome [Li et al. (2008) T Cell Responses to Whole SARS Coronavirus in Humans. The Journal of Immunology, 181(8), 5490-5500] as a proxy for the ratio of gene expression levels. The scores of all epitopes from SARS-CoV-2 genes were scaled so that the ratio of the 99th percentile of epitopes within selected genes to the 99th percentile of epitopes within the spike gene followed the ratio reported in [Li et al. (2008). T Cell Responses to Whole SARS Coronavirus in Humans. The Journal of Immunology, 181(8), 5490-5500].

[0453] Candidate CD8+ epitopes were then selected by selecting those with a scaled EDGE score equal to or greater than a threshold of t = 0.01. The threshold was chosen from an analysis of the HIV LANL dataset (data not shown) to estimate a PPV of 0.2 and a recall of 0.5 for T cell epitopes. Similar to the approach described by Grifoni et al. [(2020). A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. Cell Host & Microbe, 27(4), 671-680.e2], we also included sequences with 90% or greater homology to known SARS-CoV T cell epitopes reported in the IEDB [Vita et al. (2019). The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Research, 47(D1), D339-D343.].

[0454] Sequences containing at least one of the sites with a variability ratio higher than 0.01 as mentioned above and shown in Table 1 were excluded from the set of candidate epitopes.

[0455] To maximize vaccine ...

Claims

1. A composition for delivering an antigen expression system, said composition comprising said antigen expression system; The antigen expression system comprises at least two different coronavirus receptor binding domain (RBD)-derived nucleic acid sequences encoding at least two different RBDs, respectively, wherein the at least two different RBD domains are: (a) is at least 70% identical in amino acid composition to an RBD domain from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus; or (b) at least 70% identical in amino acid composition to an RBD domain derived from at least two of a sarbecovirus RBD-derived nucleic acid sequence, a merbecovirus RBD-derived nucleic acid sequence, an envecovirus RBD-derived nucleic acid sequence, and combinations thereof; The composition.

2. 2. The composition of claim 1, wherein the at least two different RBD domains are at least 70% identical in amino acid composition to an RBD domain from each of (A) a clade 3 sarbecovirus and (B) a clade 1 sarbecovirus and / or a clade 2 sarbecovirus.

3. The composition of claim 1, wherein: (A) the at least two different RBD domains are (a) is at least 80%, at least 85%, or at least 90% identical in amino acid composition to the RBD domain from each of a clade 1 sarbecovirus and a clade 2 sarbecovirus; or (b) NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ15354 0.1, MN996532.2, KJ473811.1, NC_014470.1, KC881005.1, MK211377.1, KJ473816.1, MK211376.1, AY572034 and / or is at least 70% identical in amino acid composition to the RBD domain from each of: KP886809.1, MT072864.1, KF569996.1, JX993987.1, MK211378.1, MK211374.1, KJ473815.1, JX993988.1, DQ071615.1, KT444582.1, MZ206298.1, and KJ473814.1; and / or (B) the antigen expression system comprises: (a) at least three different coronavirus RBD-derived nucleic acid sequences encoding at least three different RBD domains; optionally, the at least three different RBD domains are collectively at least 70% identical in amino acid composition to an RBD domain from each of (a) a clade 1 sarbecovirus, a clade 2 sarbecovirus, and a clade 3 sarbecovirus; or (b) NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_01447 or is at least 70% identical in amino acid composition collectively to the RBD domain from each of SARS-CoV-1, SARS-CoV-2, SARS-CoV-3, SARS-CoV-4, SARS-CoV-5, SARS-CoV-6, SARS-CoV-8, SARS-CoV-9, SARS-CoV-10, SARS-CoV-11, SARS-CoV-12, SARS-CoV-13, SARS-CoV-14, SARS-CoV-15, SARS-CoV-16, SARS-CoV-17, SARS-CoV-18, SARS-CoV-19, SARS-CoV-21, SARS-CoV-22, SARS-CoV-23, SARS-CoV-24, SARS-CoV-25, SARS-CoV-26, SARS-CoV-27, SARS-CoV-28, SARS-CoV-29, SARS-CoV-26, SARS-CoV-29, SARS-CoV-21, SARS-CoV-23, SARS-CoV-24, SARS-CoV-25, SARS-CoV-26, SARS-CoV-27, SARS-CoV-28, SARS-CoV-29, SARS-CoV-21, SARS-CoV-25, SARS-CoV-26, SARS-CoV-29, SARS-CoV-21, SARS-CoV-22, SARS-CoV-23, SARS-CoV-24, SARS-CoV-25, SARS-CoV-26, SARS-CoV-27, SARS-CoV-28, SARS-CoV-29, SARS-CoV-25, SARS-CoV-26, SARS-CoV-29, SARS-CoV-25, SARS-CoV-26, SA (b) at least four different coronavirus RBD-derived nucleic acid sequences encoding at least four different RBD domains; optionally, the at least four different RBD domains are collectively at least 70% identical in amino acid composition to an RBD domain from each of (a) a clade 1 sarbecovirus, a clade 2 sarbecovirus, and a clade 3 sarbecovirus, or (b) NC_045512.2, NC_004718.3, MT121216.1, DQ648857.1, GQ153542.1, DQ648856.1, AY278489.2, GQ153540.1, MN996532.2, KJ473811.1, NC_01447 and / or is at least 70% identical in amino acid composition to the RBD domain from each of SARS-CoV-1, SARS-CoV-2, SARS-CoV-3, SARS-CoV-4, SARS-CoV-5, SARS-CoV-6, SARS-CoV-7, SARS-CoV-8, SARS-CoV-9, SARS-CoV-10, SARS-CoV-11, SARS-CoV-12, SARS-CoV-13, SARS-CoV-14, SARS-CoV-15, SARS-CoV-16, SARS-CoV-17, SARS-CoV-18, SARS-CoV-19, SARS-CoV-20, SARS-CoV-21, SARS-CoV-22, SARS-CoV-23, SARS-CoV-24, SARS-CoV-25, SARS-CoV-26, SARS-CoV-27, SARS-CoV-28, SARS-CoV-29, SARS-CoV-30, SARS-CoV-31, SARS-CoV-32, SARS-CoV-33, SARS-CoV-34, SARS-CoV-35, SARS-CoV-36, SARS-CoV-37, SARS-CoV-38, SARS-CoV-41, SARS-CoV-42, SARS-CoV-43, SARS-CoV-44, SARS-CoV-45, SARS-CoV-46, SARS-CoV-47, SARS-CoV-48, SARS-CoV-49, SARS-CoV-51, SARS-CoV-52, SARS-CoV-53, SARS-CoV-54, SARS-CoV-55, SARS-CoV-56, SARS-CoV-57, SARS-CoV-58, SARS (C) the at least two different RBD domains comprise 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, or 2 to 20, 3 to 8, 4 to 8, 3 to 8, 4 to 8, 3 to 9, 4 to 9, 3 to 10, or 4 to 10 different RBD domains; and / or (D) the at least two different coronavirus RBD-derived nucleic acid sequences are (a) encoded by a single polynucleotide sequence, optionally wherein the at least two different coronavirus RBD-derived nucleic acid sequences are encoded by a single antigen cassette; or (b) each encoded by a separate polynucleotide sequence, optionally wherein the at least two different coronavirus RBD-derived nucleic acid sequences are each encoded by a separate antigen cassette, further optionally wherein the separate antigen cassettes are each encoded by a separate vector; and / or (E) the coronavirus RBD-derived nucleic acid sequence encodes: (a) Full-length RBD domain; (b) an RBD domain that (i) lacks a receptor binding motif (RBM) domain, or (ii) comprises an RBM domain that is not derived from the coronavirus from which the remainder of the RBD is derived, or (iii) comprises an RBD sequence that is not derived from the coronavirus from which the RBM domain is derived; and / or (c) only the RBM domain of the corresponding RBD domain; and / or (F) the at least two different coronavirus RBD-derived nucleic acid sequences are directly linked to each other; (a) the at least two different coronavirus RBD-derived nucleic acid sequences are linked such that the sequences can be expressed as a single mRNA; (b) the at least two different coronavirus RBD-derived nucleic acid sequences are linked to each other by a peptide linker-encoding nucleic acid sequence; Optionally, (i) the peptide linker-encoding nucleic acid sequence encodes a 2A ribosome skipping sequence element, optionally selected from the group consisting of an E2A ribosome skipping sequence element, a P2A ribosome skipping sequence element, an F2A ribosome skipping sequence element, a T2A ribosome skipping sequence element, and combinations thereof; (ii) the peptide linker-encoding nucleic acid sequence encodes a cleavable peptide linker optionally selected from a TEV cleavage site, a furin cleavage site, and combinations thereof; and / or (iii) the peptide linker-encoding nucleic acid sequence encodes a T2A sequence ribosomal skipping sequence element and a furin cleavage site; and / or (G) one or more of the at least two different coronavirus RBD-derived nucleic acid sequences are sequence-optimized; and / or (H) the at least two different coronavirus RBD-derived nucleic acid sequences are operably linked to a promoter, wherein: (a) the at least two different coronavirus RBD-derived nucleic acid sequences are operably linked to a single promoter; or (b) each of the at least two different coronavirus RBD-derived nucleic acid sequences is operably linked to a separate promoter; and Optionally, the promoter comprises a subgenomic promoter sequence, and optionally, the subgenomic promoter sequence comprises an alphavirus-derived subgenomic promoter sequence; and / or (I) the antigen expression system comprises one or more vectors, the one or more vectors comprising: (a) a vector backbone; wherein the vector backbone comprises: (i) a chimpanzee adenoviral vector, optionally wherein the chimpanzee adenoviral vector is a ChAdV68 vector; or (ii) an alphavirus vector, optionally wherein the alphavirus vector is a Venezuelan equine encephalitis virus vector; and (b) a cassette encoding at least two different RBD domains; optionally, the cassette is integrated between a native promoter nucleotide sequence naturally present in the vector backbone and a poly(A) sequence, optionally, the poly(A) sequence naturally present in the vector backbone.

4. The composition of claim 1, wherein: (i) each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from the RBD nucleic acid sequence of a sarbecovirus sequence selected from the group consisting of KP886809, KJ473815, MK211376, DQ648856, GQ153542, NC_004718, JX993988, and SARS-CoV2; and Optionally, each of the sarbecovirus RBD-derived nucleic acid sequences is independently derived from an RBD nucleic acid sequence of a sarbecovirus sequence derived from each of the following: (a) KP886809, KJ473815, MK211376, and SARS-CoV2; (b) KJ473815, MK211376, DQ648856, and SARS-CoV2; or (c) GQ153542, NC_004718, JX993988, and SARS-CoV2; and / or (ii) the at least two different RBD domains encode full-length RBD domains that are collectively at least 70% identical in amino acid composition to full-length RBD domains from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

5. The composition of claim 1, wherein: (i) the at least two different coronavirus RBD-derived nucleic acid sequences are: (a) comprising at least a beta coronavirus RBD-derived nucleic acid sequence, optionally each comprising a beta coronavirus RBD-derived nucleic acid sequence; and / or (b) is selected from the group consisting of a beta coronavirus RBD-derived nucleic acid sequence, an alpha coronavirus RBD-derived nucleic acid sequence, and a combination thereof; and / or (ii) each of the different RBD domains comprises a different receptor binding motif (RBM) domain, and the different RBM domains are collectively at least 30% identical in amino acid composition to RBM domains from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus; and optionally, The amino acid sequences of the at least two different RBD domains, other than the amino acid sequence of each RBM domain, are at least 70% identical in amino acid composition to the amino acid sequences of the RBD domain sequences other than the amino acid sequences of the RBMs derived from at least two of a clade 1 sarbecovirus, a clade 2 sarbecovirus, or a clade 3 sarbecovirus.

6. 2. The composition of claim 1, wherein each of the at least two different coronavirus RBD-derived nucleic acid sequences is: (a) further comprising a nucleic acid sequence derived from a different trimerization domain, optionally wherein the RBD trimerization domain is selected from the group consisting of a T4 trimerization domain, an MTQ trimerization domain, a GCN4 trimerization domain, and combinations thereof, and further optionally wherein the at least two different coronavirus RBD-derived nucleic acid sequences each independently comprise a T4 trimerization domain, an MTQ trimerization domain, or a GCN4 trimerization domain; or (b) comprise the same RBD trimerization domain-derived nucleic acid sequence, optionally wherein the RBD trimerization domain is selected from the group consisting of a T4 trimerization domain, an MTQ trimerization domain, a GCN4 trimerization domain, and combinations thereof, and further optionally wherein the at least two different coronavirus RBD-derived nucleic acid sequences comprise a T4 trimerization domain-derived nucleic acid sequence.

7. 2. The composition of claim 1, wherein each of the at least two different coronavirus RBD-derived nucleic acid sequences further comprises a signal peptide-encoding nucleic acid sequence, optionally wherein the signal peptide comprises a coronavirus-derived signal peptide, and further optionally wherein the signal peptide comprises a SARS-CoV-2-derived signal peptide.

8. The composition of claim 1, wherein: (a) each of the at least two different coronavirus RBD-derived nucleic acid sequences separately encodes a peptide of the following format: signal peptide-RBD-trimerization domain; (b) the at least two different coronavirus RBD-derived nucleic acid sequences encode a linked peptide in the following format: signal peptide-first RBD-first trimerization domain-signal peptide-second RBD-second trimerization domain-signal peptide-third RBD-third trimerization domain; or (c) the at least two different coronavirus RBD-derived nucleic acid sequences encode a linked peptide in the following format: signal peptide-first RBD-first trimerization domain-T2A-furin-signal peptide-second RBD-second trimerization domain-T2A-furin-signal peptide-third RBD-third trimerization domain.

9. Optionally, at least one of the at least two different RBD domains comprises a SARS-CoV-2 spike protein, wherein the SARS-CoV-2 spike protein comprises: (a) a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59, or an epitope-containing fragment thereof, optionally wherein said spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein said spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; or (b) a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and a combination thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO:59, and optionally comprising the polypeptide sequence set forth in SEQ ID NO:60 or SEQ ID NO:90, or an epitope-containing fragment thereof. Encoded by a SARS-CoV-2 spike-derived nucleic acid sequence comprising The composition of claim 1.

10. The composition of claim 1, wherein: (a) the antigen expression system comprises at least one coronavirus-derived nucleic acid sequence encoding an immunogenic polypeptide that is distinct from the at least two distinct RBD domains, optionally wherein the at least one coronavirus-derived nucleic acid sequence comprises a beta coronavirus-derived nucleic acid sequence; (b) the antigen expression system comprises at least one SARS-CoV-2 derived nucleic acid sequence encoding an immunogenic polypeptide; and / or (c) the antigen cassette comprises at least one SARS-CoV-2-derived nucleic acid sequence encoding an immunogenic polypeptide; and Further optionally, the at least one SARS-CoV-2 derived nucleic acid sequence comprises a SARS-CoV-2 spike derived nucleic acid sequence. Optionally, the SARS-CoV-2 spike derived nucleic acid sequence comprises: (i) a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59, or an epitope-containing fragment thereof; optionally, the spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally, the spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; or (ii) a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and a combination thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO:59; optionally, the modified spike protein comprises the polypeptide sequence set forth in SEQ ID NO:60 or SEQ ID NO:90, or an epitope-bearing fragment thereof.

11. Optionally, the at least one coronavirus-derived nucleic acid sequence and / or SARS-CoV-2-derived nucleic acid sequence comprises an MHC class I epitope-encoding sequence; the at least one coronavirus-derived nucleic acid sequence, SARS-CoV-2-derived nucleic acid sequence, and / or MHC class I epitope-encoding sequence is - at least one MHC class I epitope comprising a polypeptide sequence as set out in Table A, - at least one MHC class II epitope comprising a polypeptide sequence as set forth in Table B, - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table C, optionally present within a linked polypeptide sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58; - at least one polypeptide sequence according to Table 7, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide sequence according to SEQ ID NO: 92, - at least one polypeptide sequence set forth in Table 9A, Table 9B, or Table 9C, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 9A, Table 9B, or Table 9C, and optionally wherein said linked polypeptide comprises the order of the sequences set forth in Table 9A, Table 9B, or Table 9C; - at least one MHC class I epitope comprising a polypeptide sequence set forth in Table A and / or Table C, or an MHC class II epitope comprising a polypeptide sequence set forth in Table B, wherein the encoded SARS-CoV-2 immunogenic polypeptide is conserved between SARS-CoV-2 and a coronavirus species and / or subspecies other than SARS-CoV-2, and optionally, said coronavirus species and / or subspecies other than SARS-CoV-2 is Severe Acute Respiratory Syndrome (SARS) and / or Middle East Respiratory Syndrome (MERS); - one or more validated epitopes and / or at least 4, 5, 6, or 7 predicted epitopes, wherein at least 85%, 90%, or 95% of the population carries at least one HLA validated to present at least one of said one or more validated epitopes and / or at least one HLA predicted to present each of said at least 4, 5, 6, or 7 predicted epitopes, - spike protein, - membrane proteins, - nucleocapsid protein, - envelope proteins, - replicase orf1a and orf1b proteins, - a SARS-CoV-2 spike protein comprising the spike polypeptide sequence set forth in SEQ ID NO:59, or an epitope-containing fragment thereof, optionally wherein the spike polypeptide comprises a D614G mutation relative to SEQ ID NO:59, and optionally wherein the spike polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87; - a SARS-CoV-2 modified spike protein comprising a mutation selected from the group consisting of a spike R682 mutation, a spike R815 mutation, a spike K986P mutation, a spike V987P mutation, and combinations thereof, relative to the spike polypeptide sequence set forth in SEQ ID NO:59, and optionally comprising the polypeptide sequence set forth in SEQ ID NO:60 or SEQ ID NO:90, or an epitope-containing fragment thereof; - a SARS-CoV-2 membrane protein comprising the membrane polypeptide sequence set forth in SEQ ID NO: 61 or an epitope-containing fragment thereof; - a SARS-CoV-2 nucleocapsid protein comprising the nucleocapsid polypeptide sequence set forth in SEQ ID NO: 62 or an epitope-containing fragment thereof; - a SARS-CoV-2 envelope protein comprising the envelope polypeptide sequence set forth in SEQ ID NO: 63 or an epitope-containing fragment thereof; any of the above variants comprising a mutation found in 1% or more of SARS-CoV-2 subtypes, optionally including a SARS-CoV-2 variant set forth in Table 1, and / or optionally including a SARS-CoV-2 variant spike protein comprising a spike D614G mutation relative to the spike polypeptide sequence set forth in SEQ ID NO:59, optionally including a SARS-CoV-2 variant spike protein corresponding to the B.1.351 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO:112, or optionally including a SARS-CoV-2 variant spike protein corresponding to the B.1.1.7 SARS-CoV-2 isolate comprising the spike polypeptide sequence set forth in SEQ ID NO:110; at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, optionally wherein said at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally wherein said linked polypeptide comprises the order of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D; - or a combination of these, and Further optionally, the MHC class I epitope-encoding sequence comprises at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, and optionally, the at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally, the linked polypeptide comprises the order of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D. The composition of claim 10.

12. 11. The composition of claim 10, wherein the at least one coronavirus-derived nucleic acid sequence is encoded on a separate vector that is distinct from the one or more vectors encoding the at least two different coronavirus RBD-derived nucleic acid sequences, and optionally the encoded immunogenic polypeptides that are distinct from the at least two different coronavirus RBDs comprise an MHC class I epitope-encoding sequence comprising at least one polypeptide sequence set forth in Table 16A, Table 16B, Table 16C, or Table 16D, or an epitope-containing fragment thereof, and optionally the at least one polypeptide sequence is present within a linked polypeptide comprising each of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D, and optionally the linked polypeptide comprises the order of the sequences set forth in Table 16A, Table 16B, Table 16C, or Table 16D.

13. A pharmaceutical composition comprising the composition of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. An isolated nucleotide sequence, or a set of isolated nucleotide sequences, or a vector or a set of vectors, comprising the antigen expression system and / or the antigen cassette according to any one of claims 1 to 12.

15. 13. The composition of any one of claims 1 to 12 for use in a method for treating a coronavirus infection, preventing a coronavirus infection, or inducing an immune response in a subject, comprising: Optionally, the method comprises a composition: (a) a conspecific priming / booster strategy; or (b) a heterologous prime / booster strategy, optionally comprising: (a) the same antigen cassette encoded by different vaccine platforms; (b) different antigen cassettes encoded by the same vaccine platform; and / or (c) different antigen cassettes encoded by different vaccine platforms; and Further optionally, the method comprises administering one or more vectors encoding the at least two different coronavirus RBD-derived nucleic acid sequences, and administering one or more vectors encoding at least one coronavirus-derived nucleic acid sequence encoding an immunogenic polypeptide that is different from the at least two different RBD domains, and further optionally, (i) one or more vectors encoding the at least two different coronavirus RBD-derived nucleic acid sequences are co-formulated with one or more vectors encoding the at least one coronavirus-derived nucleic acid sequence; or (ii) the one or more vectors encoding the at least two different coronavirus RBD-derived nucleic acid sequences and the one or more vectors encoding the at least one coronavirus-derived nucleic acid sequence are administered separately; optionally, the one or more vectors encoding the at least two different coronavirus RBD-derived nucleic acid sequences and the one or more vectors encoding the at least one coronavirus-derived nucleic acid sequence are administered simultaneously.

Images