Modified adenovirus

Abstract

Disclosed herein are compositions comprising a modified adenovirus. Also disclosed are nucleotides, cells and methods related to the compositions, including their use as vaccines. Also disclosed herein are viral vectors using the TET promoter systems and methods of producing viruses having the vectors.

Description

[0001] This application is a divisional application of the invention patent application with application date of June 1, 2020, application number 202080047889.2, and invention name “Modified Adenovirus”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 854,865, filed May 30, 2019, which is hereby incorporated by reference in its entirety for all purposes.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 30, 2019, is named GSO_033PR_Sequence_Listing.txt and is 422,136 bytes in size. Background Art

[0006] Therapeutic vaccines based on tumor-specific antigens hold great promise as the next generation of personalized cancer immunotherapy. 1-3 For example, cancers with high mutational burdens, such as non-small cell lung cancer (NSCLC) and melanoma, are particularly attractive targets for such therapies given the relatively high likelihood of neoantigen generation. 4，5 Early evidence suggests neoantigen-based vaccination can elicit T cell responses 6 , and cell therapy targeting neoantigens can induce tumor regression in some cases in selected patients 7 .

[0007] One issue in antigen vaccine design in the context of cancer and infectious disease is which of the many coding mutations that exist produces the "best" therapeutic antigen, e.g., an antigen that can elicit immunity.

[0008] In addition to the challenges of current antigen prediction methods, there are certain challenges with available vector systems for antigen delivery in humans, many of which are derived from humans. For example, many humans have pre-existing immunity to human viruses due to previous natural exposure, and this immunity can be a major obstacle to the use of recombinant human viruses for antigen delivery in vaccination strategies, such as in cancer treatment or vaccination against infectious diseases. Although some progress has been made in vaccination strategies that address the above issues, improvements are still needed, especially for clinical applications, such as improved vaccine efficacy and efficacy. Summary of the invention

[0009] An adenoviral vector, comprising: an adenoviral backbone comprising one or more genes or regulatory sequences of an adenoviral genome, and wherein the adenoviral backbone comprises a partially deleted E4 gene relative to the adenoviral genome, wherein the partially deleted E4 gene comprises a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region, and optionally, wherein the adenoviral vector further comprises a cassette, the cassette comprising: (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, and optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and / or a 3′ linker sequence, and optionally wherein; (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, (3) optionally, at least one universal nucleic acid sequence encoding an MHC class II antigen; (4) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (5) optionally, at least one polyadenylation sequence.

[0010] Also disclosed herein is a chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene with a partial deletion of the E4 gene sequence shown in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; and (b) one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence shown in SEQ ID NO: 1; and optionally wherein the chimpanzee adenovirus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

[0011] Also disclosed herein is a chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; (b) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion; and (c) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO: 1, corresponding to an E2 deletion. NO: 1, and wherein the partially deleted E4 gene is 5′ of nucleotides 35,643 to 36,518, and optionally, wherein the chimpanzee adenosine virus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

[0012] The present invention also discloses a chimpanzee adenovirus vector, comprising: a. a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (i) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO: 1, and it lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; (ii) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to the E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to the E3 deletion; and (iii) and nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, corresponding to the E4 deletion. NO: 1, wherein the partially deleted E4 gene is 5′ of the nucleotides 35,643 to 36,518, and; b. a CMV-derived promoter sequence; c. an SV40 polyadenylation signal nucleotide sequence; and d. a box comprising at least one payload nucleic acid sequence encoding: at least one MHC class I epitope, optionally wherein the at least one MHC class I epitope comprises at least 2 different MHC class I epitopes, the at least 2 different MHC class I epitopes being linearly linked to each other and each optionally comprising: (A) at least one change that makes the encoded peptide sequence different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, wherein the different MHC I epitopes are 7-15 amino acids in length, (B) an N-terminal linker comprising a native N-terminal amino acid sequence of the different MHC I epitope having a length of at least 3 amino acids, (C) a C-terminal linker comprising the different MHC (D) a native C-terminal acid sequence of at least 3 amino acids in length of an MHC class II epitope, or (D) a combination thereof, at least one MHC class II epitope, optionally wherein the at least one MHC class II epitope comprises at least 2 different MHC class II epitopes, at least one epitope capable of stimulating a B cell response, or a combination thereof, and wherein the cassette is inserted into the deleted region of ChAdV68 and the CMV-derived promoter sequence is operably linked to the cassette.

[0013] Also disclosed herein is a method for stimulating an immune response in a subject, the method comprising administering to the subject an adenoviral vector, the adenoviral vector comprising: an adenoviral backbone comprising one or more genes or regulatory sequences of an adenoviral genome, and wherein the adenoviral backbone comprises a partially deleted E4 gene relative to the adenoviral genome, wherein the partially deleted E4 gene comprises a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region, and wherein the adenoviral vector further comprises a cassette, the cassette comprising: (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, and optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and / or a 3′ linker sequence, and optionally wherein; (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, (3) optionally, at least one universal MHC encoding nucleic acid sequence (4) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (5) optionally, at least one polyadenylation sequence.

[0014] Also disclosed herein is a method for treating a subject having a disease, optionally wherein the disease is cancer or an infection, the method comprising administering to the subject an adenoviral vector, the adenoviral vector comprising: an adenoviral backbone comprising one or more genes or regulatory sequences of an adenoviral genome, and wherein the adenoviral backbone comprises a partially deleted E4 gene relative to the adenoviral genome, wherein the partially deleted E4 gene comprises a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region, and wherein the adenoviral vector further comprises a cassette comprising: (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, and optionally wherein the at least one payload nucleic acid sequence further comprises a 5' linker sequence and / or a 3' linker sequence, and optionally wherein; (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, (3) optionally, at least one universal nucleic acid sequence encoding an MHC class II antigen; (4) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (5) optionally, at least one polyadenylation sequence.

[0015] Also disclosed herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject an adenoviral vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence set forth in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO: 1; and (b) one or more genes or regulatory sequences of the ChAdV68 sequence set forth in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO: 1; and wherein the chimpanzee adenoviral vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

[0016] Also disclosed herein is a method for treating a subject having a disease, optionally wherein the disease is cancer or an infection, the method comprising administering to the subject an adenoviral vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence set forth in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO: 1; and (b) one or more genes or regulatory sequences of the ChAdV68 sequence set forth in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, LA, and L5 genes of the sequence set forth in SEQ ID NO: 1; and wherein the chimpanzee adenoviral vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

[0017] Also disclosed herein is a method for producing a virus, wherein the method comprises the following steps: a. providing a viral vector comprising a cassette, the cassette comprising: (i) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, and optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and / or a 3′ linker sequence, and optionally wherein; (ii) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, wherein the at least one promoter is a promoter controlled by a tetracycline (TET) repressor protein (TETr), (iii) optionally, at least one nucleic acid sequence encoding an MHC class II antigen; (iv) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (v) optionally, at least one polyadenylation sequence; b. providing a cell engineered to express the TETr protein; and c. contacting the viral vector with the cell under conditions sufficient to produce the virus.

[0018] In some aspects, the viral vector includes a chimpanzee adenovirus vector, and optionally wherein the chimpanzee adenovirus vector is a ChAdV68 vector. In some aspects, relative to the production of the virus produced by the vector without the promoter controlled by the TETr, the vector containing the promoter controlled by the TETr is used to increase the production of the virus. In some aspects, the increase in production is at least 1.5 times, at least 2 times, at least 2.5 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times relative to the production of the vector without the promoter controlled by the TETr. In some aspects, the increase in production is at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times or at least 100 times relative to the production of the vector without the promoter controlled by the TETr. In some respects, relative to the generation of the virus produced by the cell that is not engineered to express TETr protein, the vector that uses the promoter that comprises TETr control increases the generation of virus.In some respects, the generation increase is relative to the generation of the cell that is not engineered to express TETr protein and increases at least 1.5 times, at least 2 times, at least 2.5 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times.

[0019] Also provided herein is a viral vector, the viral vector comprising a cassette, the cassette comprising: (i) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, and optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and / or a 3′ linker sequence, and optionally wherein; (ii) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, wherein the at least one promoter is a promoter controlled by a tetracycline (TET) repressor protein (TETr), (iii) optionally, at least one nucleic acid sequence encoding an MHC class II antigen; (iv) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (v) optionally, at least one polyadenylation sequence.

[0020] In some aspects, the promoter controlled by the TETr comprises one or more TET operator (TETo) nucleic acid sequences, optionally wherein the one or more TETo nucleic acid sequences comprise the nucleotide sequence shown in SEQ ID NO: 60. In some aspects, one or more TETo nucleic acid sequences comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more TETo nucleic acid sequences, optionally wherein each of the TETo nucleic acid sequences comprises the nucleotide sequence shown in SEQ ID NO: 60. In some aspects, 2 or more TETo nucleic acid sequences are linked together. In some aspects, 2 or more TETo nucleic acid sequences are directly linked together. In some aspects, 2 or more TETo nucleic acid sequences are linked together using a linker sequence, wherein the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides, and optionally wherein the linker sequence comprises a linker nucleotide sequence shown in SEQ ID NO: 61. In some aspects, one or more TETo nucleic acid sequences are 5' of the RNA polymerase binding sequence of the promoter sequence. In some aspects, one or more TETo nucleic acid sequences are 3' of the RNA polymerase binding sequence of the promoter sequence. In some aspects, at least one promoter sequence comprises a CMV, SV40, EF-1, RSV, PGK, HSA, MCK or EBV promoter sequence. In some aspects, at least one promoter sequence is a CMV-derived promoter sequence, optionally wherein the CMV promoter sequence comprises a CMV promoter nucleotide sequence as shown in SEQ ID NO: 64. In some aspects, the CMV-derived promoter sequence is a minimal CMV promoter sequence, optionally wherein the minimal CMV promoter sequence comprises a minimal CMV promoter nucleotide sequence as shown in SEQ ID NO: 61.

[0021] In some aspects, the TETr-controlled promoter operably linked to at least one payload nucleic acid sequence comprises an ordered sequence described by the following formula, comprising from 5′ to 3′: (TL Y ) X-PN, wherein N comprises one of at least one payload nucleic acid sequence, optionally wherein each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, optionally wherein there is at least one change such that the encoded epitope sequence is different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, P, an RNA polymerase binding sequence operably linked to a promoter sequence of at least one of the at least one payload nucleic acid sequence, T comprises a TETo nucleic acid sequence comprising a nucleotide sequence as shown in SEQ ID NO: 60, and L comprises a linker sequence, wherein for each X, Y=0 or 1, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some aspects, the TETr-controlled promoter operably linked to at least one payload nucleic acid sequence comprises an ordered sequence described by the formula, comprising from 5′ to 3′: P-(TL Y ) X -N, wherein N comprises one of at least one payload nucleic acid sequence, optionally wherein each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, optionally there is at least one change that makes the encoded epitope sequence different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, P, an RNA polymerase binding sequence operably linked to the promoter sequence of at least one of the at least one payload nucleic acid sequence, T comprises a TETo nucleic acid sequence comprising the nucleotide sequence shown in SEQ ID NO: 60, and L comprises a linker sequence, wherein for each X, Y=0 or 1, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0022] In some aspects, the TETr-controlled promoter comprises: (1) a minimal CMV promoter sequence; (2) 7 TETo nucleic acid sequences, wherein each of the TETo nucleic acid sequences comprises the nucleotide sequence shown in SEQ ID NO: 60, and wherein each of the TETo nucleic acid sequences is linked together using a linker sequence, the 7 TETo nucleic acid sequences are 5′ of the minimal CMV promoter sequence, and optionally wherein the TETr-controlled promoter comprises the nucleotide sequence shown in SEQ ID NO: 61. In some aspects, the TETr-controlled promoter comprises: (1) a CMV promoter sequence; (2) 2 TETo nucleic acid sequences, wherein each of the TETo nucleic acid sequences comprises the nucleotide sequence shown in SEQ ID NO: 60, and wherein each of the TETo nucleic acid sequences is directly linked together, the 2 TETo nucleic acid sequences are 3′ of the CMV promoter sequence, and optionally wherein the TETr-controlled promoter comprises the nucleotide sequence shown in SEQ ID NO: 64.

[0023] In some aspects, the viral vector comprises a vector backbone, wherein the vector backbone comprises a chimpanzee adenoviral vector, optionally wherein the chimpanzee adenoviral vector is a ChAdV68 vector.

[0024] In some aspects, the cassette comprises an ordered sequence described by the following formula, comprising from 5' to 3': a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g -A h, wherein N comprises one of the at least one payload nucleic acid sequences, optionally wherein each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, optionally there is at least one change such that the encoded epitope sequence is different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, wherein c=1, P comprises at least one promoter sequence operably linked to at least one of the at least one payload nucleic acid sequences, wherein a=1, L5 comprises a 5′ linker sequence, wherein b=0 or 1, L3 comprises a 3′ linker sequence, wherein d=0 or 1, G5 comprises one of the at least one nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 56), wherein e=0 or 1, G3 comprises one of the at least one nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 56), wherein g=0 or 1, U comprises at least one universal MHC encoding sequence One of the nucleic acid sequences of a class II antigen, wherein f=1, A comprises at least one polyadenylation sequence, wherein h=0 or 1, X=2 to 400, wherein for each X, the corresponding N c is a payload nucleic acid sequence, optionally wherein for each X, the corresponding N c are different payload nucleic acid sequences, and Y = 0-2, where for each Y, the corresponding U f is a universal nucleic acid sequence encoding an MHC class II antigen, optionally wherein for each Y, the corresponding U f There are different universal nucleic acid sequences encoding MHC class II antigens.

[0025] In some aspects, the cassette further comprises at least one additional payload nucleic acid sequence not encoded in the ordered sequence. In some aspects, b=1, d=1, e=1, g=1, h=1, X=10, Y=2, P is a CMV-derived promoter sequence, each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, L5 encodes the native N-terminal amino acid sequence of the epitope, and wherein the 5′ linker sequence encodes a peptide of at least 3 amino acids in length, L3 encodes the native C-terminal amino acid sequence of the epitope, and wherein the 3′ linker sequence encodes a peptide of at least 3 amino acids in length, U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence, and the vector comprises a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence set forth in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO: 1; (b) an E4 gene having a partial deletion of the E4 gene sequence set forth in SEQ ID NO: 1, and which lacks at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO: 1; NO:1, wherein the partially deleted E4 gene is 3′ to the nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence as shown in SEQ ID NO:1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence as shown in SEQ ID NO:1, corresponding to an E3 deletion; and (c) nucleotides 35,643 to 36,518 of the sequence as shown in SEQ ID NO:1, and wherein the partially deleted E4 gene is 5′ to the nucleotides 35,643 to 36,518.

[0026] In some aspects, the vector is a chimpanzee adenoviral vector, optionally wherein the chimpanzee adenoviral vector is a ChAdV68 vector.

[0027] In some aspects, the partially deleted E4 gene comprises: A. the E4 gene sequence shown in SEQ ID NO: 1, and it lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1, B. the E4 gene sequence shown in SEQ ID NO: 1, and it lacks at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, C. the E4 gene sequence shown in SEQ ID NO: 1, and it lacks at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, D. the E4 gene sequence shown in SEQ ID NO: 1 NO:1, and it lacks at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO:1, E. E4 deletion comprising at least a partial deletion of E4Orf2, a complete deletion of E4Orf3 and at least a partial deletion of E4Orf4, F. E4 deletion comprising at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3 and at least a partial deletion of E4Orf4, G. E4 deletion comprising at least a partial deletion of E4Orf1, a complete deletion of E4Orf2 and at least a partial deletion of E4Orf3, or H. E4 deletion comprising at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf.3.

[0028] In some aspects, the vector comprises one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences are selected from the group consisting of: chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence shown in SEQ ID NO: 1. In some aspects, the adenoviral backbone or modified ChAdV68 sequence further comprises a functional deletion with respect to the adenoviral genome or with respect to the sequence set forth in SEQ ID NO: 1 in at least one gene selected from the group consisting of adenoviral E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4 and L5 genes, optionally wherein the adenoviral backbone or modified ChAdV68 sequence is completely deleted or functionally deleted of the following: (1) E1A and E1B; or (2) E1A, E1B and E3, with respect to the adenoviral genome or with respect to the sequence set forth in SEQ ID NO: 1, optionally wherein the E1 gene is functionally deleted by an E1 deletion of at least nucleotides 577 to 3403 with respect to the sequence set forth in SEQ ID NO: 1, and optionally wherein the E3 gene is functionally deleted by an E3 deletion of at least nucleotides 27,125 to 31,825 with respect to the sequence set forth in SEQ ID NO: 1.

[0029] In some aspects, the cassette is present and inserted into the El region, the E3 region, and / or any deleted AdV region in the vector that allows for incorporation of the cassette.

[0030] In some aspects, the vector is produced by one of a first generation, second generation, or helper-dependent adenoviral vector.

[0031] In some aspects, the modified ChAdV68 sequence comprises nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3′ of nucleotides 2 to 34,916. In some aspects, nucleotides 2 to 34,916 lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO: 1, corresponding to an E1 deletion. In some aspects, nucleotides 2 to 34,916 lack nucleotides 456-3014 with respect to the sequence set forth in SEQ ID NO: 1. In some aspects, nucleotides 2 to 34,916 lack nucleotides 27,125-31,825 with respect to the sequence set forth in SEQ ID NO: 1, corresponding to an E3 deletion. In some aspects, nucleotides 2 to 34,916 lack nucleotides 27,816-31,333 with respect to the sequence set forth in SEQ ID NO: 1. In some aspects, nucleotides 2 to 34,916 lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO: 1, corresponding to an E1 deletion, and lack nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO: 1, corresponding to an E3 deletion. In some aspects, nucleotides 2 to 34,916 further lack nucleotides 3957-10346, nucleotides 21787-23370, nucleotides 33486-36193, or a combination thereof with respect to the sequence set forth in SEQ ID NO: 1.

[0032] In some aspects, at least one of the at least one payload nucleic acid sequence encodes an antigen, wherein the antigen comprises: an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof. In some aspects, at least one of the at least one payload nucleic acid sequence encodes a polypeptide sequence capable of antigen processing into an epitope, optionally wherein the epitope is known or suspected to be presented on a cell surface by MHC class I, optionally wherein the cell surface is a tumor cell surface or an infected cell surface.

[0033] In some aspects, at least one of the at least one payload nucleic acid sequence encodes a polypeptide sequence or portion thereof that is presented by MHC class I and / or MHC class II on the surface of a cell, optionally wherein the cell surface is a tumor cell surface or an infected cell surface. In some aspects, the tumor cell is selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer and small cell lung cancer, or the infected cell is selected from the group consisting of pathogen-infected cells, virus-infected cells, bacteria-infected cells, fungus-infected cells and parasite-infected cells, optionally wherein the virus-infected cell is selected from the group consisting of HIV-infected cells, severe acute respiratory syndrome-associated coronavirus (SARS)-infected cells, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells, Ebola virus-infected cells, hepatitis B virus (HBV)-infected cells, influenza virus-infected cells and hepatitis C virus (HCV)-infected cells.

[0034] In some aspects, at least one of the at least one payload nucleic acid sequence encodes a polypeptide sequence or a portion thereof comprising an epitope capable of stimulating a B cell response, optionally wherein the polypeptide sequence or portion thereof comprises a full-length protein, protein domain, protein subunit, or antigenic fragment predicted or known to be capable of being bound by an antibody.

[0035] In some aspects, at least one of the at least one payload nucleic acid sequence encodes an infectious disease organism peptide selected from the group consisting of: a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide. In some aspects, at least one of the at least one payload nucleic acid sequence encodes an epitope with at least one change, the change causing the encoded epitope sequence to be different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some aspects, at least one of the at least one payload nucleic acid sequence encodes an MHC class I epitope or MHC class II epitope with at least one change, the change causing the encoded peptide sequence to be different from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, optionally wherein the encoded polypeptide sequence or a portion thereof has an increased binding affinity, increased binding stability, and / or an increased likelihood of presentation on its corresponding MHC allele relative to the corresponding wild-type nucleic acid sequence being translated. In some aspects, the at least one change includes a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splicing variant, a genomic rearrangement, or a splicing antigen produced by a proteasome.

[0036] In some aspects, at least one of the at least one payload nucleic acid sequence encodes a full-length protein, a protein domain, or a protein subunit. In some aspects, at least one of the at least one payload nucleic acid sequence encodes an antibody, a cytokine, a chimeric antigen receptor (CAR), a T cell receptor, and a genome editing system nuclease.

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

[0038] In some aspects, each of the at least one payload nucleic acid sequence is directly connected to each other. In some aspects, at least one of the at least one payload nucleic acid sequence is connected to a different payload nucleic acid sequence using a nucleic acid sequence encoding a joint. In some aspects, the joint connects two payload nucleic acid sequences encoding an MHC class I epitope or connects a first payload nucleic acid sequence encoding an MHC class I epitope to a second payload nucleic acid sequence encoding an MHC class II epitope or encoding an epitope sequence capable of stimulating a B cell response. In some aspects, the linker is selected from the group consisting of: (1) consecutive glycine residues of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (2) consecutive alanine residues of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length that is efficiently processed by the mammalian proteasome; and (6) one or more native sequences flanking the antigen derived from a homologous protein and of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length. In some aspects, the linker connects two payload nucleic acid sequences encoding MHC class II epitopes or connects a first payload nucleic acid sequence encoding an MHC class II epitope to a second payload nucleic acid sequence encoding an MHC class I epitope or encoding an epitope sequence capable of stimulating a B cell response. In some aspects, the linker comprises the sequence GPGPG (SEQ ID NO: 56).

[0039] In some aspects, at least one of the at least one payload nucleic acid sequence is operably or directly linked to an isolated or continuous sequence that enhances expression, stability, cellular transport, processing and presentation and / or immunogenicity of the at least one payload nucleic acid sequence, and optionally the expression, stability, cellular transport, processing and presentation and / or immunogenicity of a polypeptide encoded by the at least one payload nucleic acid sequence. In some aspects, the isolated or continuous sequence comprises at least one of the following: a ubiquitin sequence; a ubiquitin sequence modified to increase proteasome targeting, optionally wherein the ubiquitin sequence contains a substitution of Gly to Ala at position 76; an immunoglobulin signal sequence, optionally wherein the immunoglobulin signal sequence comprises IgK; a major histocompatibility class I sequence; a lysosomal associated membrane protein (LAMP)-1; a human dendritic cell lysosomal associated membrane protein; and a major histocompatibility class II sequence; optionally wherein the ubiquitin sequence modified to increase proteasome targeting is A76.

[0040] In some aspects, expression of each of the at least one payload nucleic acid sequence is driven by the at least one promoter.

[0041] In some aspects, the at least one payload nucleic acid sequence includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 payload nucleic acid sequences. In some aspects, the at least one payload nucleic acid sequence includes at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to 400 payload nucleic acid sequences. In some aspects, the at least one payload nucleic acid sequence includes at least 2-400 payload nucleic acid sequences, and wherein at least one of the at least one payload nucleic acid sequence encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response or a combination thereof. In some aspects, the at least one payload nucleic acid sequence includes at least 2-400 payload nucleic acid sequences, and wherein, when applied to the subject and translated, at least one of the at least one payload nucleic acid sequence encodes an antigen presented on an antigen presenting cell, producing an immune response targeting the antigen. In some aspects, the at least one payload nucleic acid sequence comprises at least 2-400 MHC class I and / or class II antigen encoding nucleic acid sequences, wherein, when administered to the subject and translated, at least one of the MHC class I or class II antigens is presented on an antigen presenting cell, generating an immune response targeting at least one of the antigens on the surface of the cell, and optionally wherein expression of each of the at least 2-400 MHC class I or class II antigen encoding nucleic acid sequences is driven by the at least one promoter.

[0042] In some aspects, each MHC class I epitope is independently between 8 and 35 amino acids in length, optionally 7-15, 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length. In some aspects, there is at least one universal nucleic acid sequence encoding an MHC class II antigen. In some aspects, there is at least one universal nucleic acid sequence encoding an MHC class II antigen, and comprises at least one universal nucleic acid sequence encoding an MHC class II antigen, the nucleic acid sequence comprising at least one change that causes the encoded peptide sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence. In some aspects, the at least one universal nucleic acid sequence encoding an MHC class II antigen is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some aspects, there is at least one universal nucleic acid sequence encoding an MHC class II antigen, and wherein the at least one universal sequence comprises at least one of tetanus toxoid and PADRE.

[0043] In some aspects, the at least one promoter sequence is a regulatable promoter, optionally wherein the regulatable promoter is a promoter controlled by a tetracycline (TET) repressor protein (TETr), optionally wherein the regulatable promoter includes multiple TET operator (TETo) sequences at 5' or 3' of the RNA polymerase binding sequence of the promoter. Multiple TET operator (TETo) sequences are 5' or 3' of RNA, and the at least one promoter sequence is constitutive. Multiple TET operator (TETo) sequences are 5' or 3' of RNA, and the at least one promoter sequence is CMV, SV40, EF-1, RSV, PGK, HSA, MCK or EBV promoter sequences.

[0044] In some aspects, the cassette further comprises at least one polyadenylation (polyA) sequence operably linked to at least one of the at least one payload nucleic acid sequence, optionally wherein the polyA sequence is located 3' to the at least one payload nucleic acid sequence. In some aspects, the polyA sequence comprises an SV40 or bovine growth hormone (BGH) polyA sequence.

[0045] In some aspects, the cassette further comprises at least one of the following: an intron sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a furin cleavage site, a nucleotide sequence encoding a TEV cleavage site, or a sequence in the 5′ or 3′ noncoding region known to enhance nuclear export, stability, or translation efficiency of an mRNA operably linked to at least one of the at least one payload nucleic acid sequence.

[0046] In some aspects, the cassette comprises a reporter gene including, but not limited to, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, or a luciferase variant.

[0047] In some aspects, the vector further comprises one or more payload nucleic acid sequences encoding at least one immunomodulator, optionally wherein the at least one immunomodulator inhibits immune checkpoint molecules. In some aspects, the immunomodulator is an anti-CTLA4 antibody or its antigen binding fragment, an anti-PD-1 antibody or its antigen binding fragment, an anti-PD-L1 antibody or its antigen binding fragment, an anti-4-1BB antibody or its antigen binding fragment, or an anti-OX-40 antibody or its antigen binding fragment. In some aspects, the antibody or its antigen binding fragment is a Fab fragment, a Fab' fragment, a single-chain Fv (scFv), a single-domain antibody (sdAb) (e.g., a camelid antibody domain) in a monospecific or multispecific form connected together, or a full-length single-chain antibody (e.g., a full-length IgG with heavy and light chains connected by a flexible linker). In some aspects, the heavy and light chain sequences of the antibody are continuous sequences separated by a self-cleavage sequence or an IRES sequence such as 2A, optionally wherein the self-cleavage sequence has a furin cleavage site sequence at 5' of the self-cleavage sequence; or the heavy and light chain sequences of the antibody are connected by a flexible linker, such as a continuous glycine residue. In some aspects, the immunomodulatory factor is a cytokine. In some aspects, the cytokine is at least one of IL-2, IL-7, IL-12, IL-15 or IL-21 or their respective variants.

[0048] In some aspects, at least one of the at least one payload nucleic acid sequence is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor cell, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing a peptide sequence for each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical probabilities that each of the antigens is presented on a cell surface, optionally a tumor cell surface, or an infected cell surface, by one or more of the MHC alleles, the set of numerical probabilities having been identified 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 probabilities to generate a set of selected antigens, the set of selected antigens being used to generate the at least one payload nucleic acid sequence.

[0049] In some aspects, each of the at least one payload nucleic acid sequence is selected by performing the following steps: (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor cell, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing a peptide sequence of each of the antigen sets; (b) inputting the peptide sequence of each antigen into a presentation model to generate a numerical likelihood set of each of the antigens presented by one or more of the MHC alleles on a cell surface, optionally a tumor cell surface, or an infected cell surface, the numerical likelihood set having been identified based at least on the received mass spectrometry data; and (c) selecting a subset of the antigen set based on the numerical likelihood set to generate a selected antigen set, the selected antigen set being used to generate each of the at least one payload nucleic acid sequence. In some aspects, the number of the selected antigen sets is 2-20. In some aspects, the presentation model represents the dependency between: (a) the presence of a pair of a specific one of the MHC alleles and a specific amino acid at a specific position of a peptide sequence; and (b) the probability of presenting the peptide sequence comprising the specific amino acid at the specific position on a cell surface, optionally on a tumor cell surface or on an infected cell surface by the specific one of the MHC alleles of the pair. In some aspects, selecting the selected antigen set includes selecting an antigen with an increased probability of presentation on the cell surface relative to an unselected antigen based on the presentation model. In some aspects, selecting the selected antigen set includes selecting an antigen with an increased probability of being able to induce a cell-specific immune response in the subject relative to an unselected antigen based on the presentation model. In some aspects, selecting the selected antigen set includes selecting an antigen with an increased probability of being able to be presented to a naive T cell by a professional antigen presenting cell (APC) relative to an unselected antigen based on the presentation model, optionally wherein the APC is a dendritic cell (DC). In some aspects, the selected antigen set is selected to include antigens with reduced likelihood of suppressed central or peripheral tolerance relative to unselected antigens based on the presentation model. In some aspects, the selected antigen set is selected to include antigens with reduced likelihood of inducing an autoimmune response to normal tissues in the subject relative to unselected antigens based on the presentation model. In some aspects, exon group or transcriptome nucleotide sequencing data is obtained by sequencing tumor cells or tissues, infected cells or infectious disease organisms. In some aspects, the sequencing is next generation sequencing (NGS) or any massively parallel sequencing method.

[0050] In some aspects, the box comprises a junction epitope sequence formed by the adjacent sequences in the box. In some aspects, at least one or each junction epitope sequence has an affinity to MHC greater than 500nM. In some aspects, each junction epitope sequence is non-self. In some aspects, the box does not encode non-therapeutic MHC Class I or Class II epitopes, wherein the non-therapeutic epitopes are predicted to be displayed on the MHC alleles of the experimenter. In some aspects, the predicted non-therapeutic MHC Class I or Class II epitope sequence is a junction epitope sequence formed by the adjacent sequences in the box. In some aspects, the prediction is based on the presentation possibility generated by inputting the sequence of the non-therapeutic epitope into the presentation model. In some aspects, the order of the at least one payload nucleic acid sequence in the box is determined by a series of steps, comprising: i. generating a set of candidate box sequences corresponding to different orders of the at least one payload nucleic acid sequence; ii. for each candidate box sequence, determining a presentation score based on the presentation of a non-therapeutic epitope in the candidate box sequence; and iii. selecting a candidate box sequence associated with a presentation score below a predetermined threshold as the box sequence.

[0051] In some aspects, each of the MHC class I and / or class II epitopes is predicted or verified to be presentable by at least one HLA allele present in at least 5% of the human population. In some aspects, each of the MHC class I and / or class II epitopes is predicted or verified to be presentable by at least one HLA allele, wherein each antigen / HLA pair has at least 0.01% antigen / HLA incidence in the human population. In some aspects, each of the MHC class I and / or class II epitopes is predicted or verified to be presentable by at least one HLA allele, wherein each antigen / HLA pair has at least 0.1% antigen / HLA incidence in the human population. In some aspects, relative to the native nucleic acid sequence directly extracted from the subject's tissue or sample, the at least one useful load nucleic acid sequence encoding the polypeptide is codon optimized.

[0052] Also disclosed herein is a pharmaceutical composition comprising any carrier described herein and a pharmaceutically acceptable carrier. In some aspects, the composition further comprises an adjuvant. In some aspects, the composition further comprises an immunomodulatory factor. In some aspects, the immunomodulatory factor is an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-4-1BB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof.

[0053] Also disclosed herein is an isolated nucleotide sequence comprising a cassette of any of the vectors described herein and a gene of sequence SEQ ID NO: 1, optionally wherein the gene is selected from the group consisting of chimpanzee adenovirus ITR, E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence shown in SEQ ID NO: 1, and optionally wherein the nucleotide sequence is a cDNA.

[0054] Also disclosed herein is an isolated cell comprising any of the isolated nucleotide sequences described herein, optionally wherein the cell is a CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6 or AE1-2a cell.

[0055] Also disclosed is a vector comprising any of the isolated nucleotide sequences described herein.

[0056] Also disclosed herein is a kit comprising any vector or composition described herein and instructions for use.

[0057] Also disclosed herein is a method for stimulating an immune response of a subject, the method comprising administering any carrier or composition described herein to the subject. In some aspects, the carrier or composition is administered intramuscularly (IM), intradermally (ID) or subcutaneously (SC). In some aspects, the method further comprises administering an immunomodulatory factor to the subject, optionally wherein the immunomodulatory factor is administered before, simultaneously or after the carrier or the pharmaceutical composition is administered. In some aspects, the immunomodulatory factor is an anti-CTLA4 antibody or its antigen binding fragment, an anti-PD-1 antibody or its antigen binding fragment, an anti-PD-L1 antibody or its antigen binding fragment, an anti-4-1BB antibody or its antigen binding fragment, or an anti-OX-40 antibody or its antigen binding fragment. In some aspects, the immunomodulatory factor is administered intravenously (IV), intramuscularly (IM), intradermally (ID) or subcutaneously (SC). In some aspects, the subcutaneous administration is close to the carrier or the composition application site or very close to one or more carriers or compositions draining lymph nodes.

[0058] In some aspects, the method further comprises administering a second vaccine composition to the subject. In some aspects, the second vaccine composition is administered before administering any vector or composition described herein. In some aspects, the second vaccine composition is administered after administering any vector or composition described herein. In some aspects, the second vaccine composition is identical to any vector or composition described herein. In some aspects, the second vaccine composition is different from any vector or composition described herein. In some aspects, the second vaccine composition comprises a self-amplifying RNA (samRNA) vector encoding at least one payload nucleic acid sequence. In some aspects, the at least one payload nucleic acid sequence encoded by the samRNA vector is identical to at least one of the at least one payload nucleic acid sequence of any one of the aforementioned vector claims.

[0059] Also disclosed herein is a method for making a vector as claimed in any of the preceding vector claims, the method comprising: obtaining a plasmid sequence comprising an adenovirus vector or a chimpanzee adenovirus vector; transfecting the plasmid sequence into one or more host cells; and isolating the vector from the one or more host cells. In some aspects, the separation comprises: dissolving the one or more host cells to obtain a cell lysate comprising the vector; and purifying the vector from the cell lysate and optionally from the culture medium for culturing the one or more host cells. In some aspects, the plasmid sequence is produced using one of the following: DNA recombination in bacterial cells or bacterial recombination or whole genome DNA synthesis or whole genome DNA synthesis with synthetic DNA amplification. In some aspects, 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 aspects, purifying the vector from the cell lysate involves one or more of chromatographic separation, centrifugation, virus precipitation, and filtration.

[0060] Also provided herein is a method for producing a virus, wherein the virus is produced using any of the vectors described herein. In some aspects, relative to the production of a virus produced using a vector without a partially deleted E4 gene, the production of the virus is increased using the vector comprising the partially deleted E4 gene. In some aspects, relative to the titer of the infectious unit of a virus produced using a vector without a partially deleted E4 gene, the titer of the infectious unit of the virus is increased using the vector comprising the partially deleted E4 gene. In some aspects, the increase in production is at least 1.5 times, at least 2 times, at least 2.5 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or at least 9 times relative to the production using a vector without the partially deleted E4 gene. In some aspects, the increase in production is at least 10 times, at least 18 times, at least 20 times, at least 25 times, or at least 27 times relative to the production using a vector without the partially deleted E4 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] These and other features, aspects and advantages of the present invention will be better understood with respect to the following description and accompanying drawings, in which:

[0062] Figure 1 The development of an in vitro T cell activation assay is illustrated. Schematic representation of an assay in which delivery of a vaccine cassette to antigen presenting cells results in expression, processing and MHC restricted presentation of a unique peptide antigen. Reporter T cells engineered to have a T cell receptor matching a specific peptide-MHC combination are activated, resulting in luciferase expression.

[0063] Figure 2A The evaluation of the linker sequence in the short box is described and five MHC class I restricted epitopes (epitopes 1 to 5) connected in series in the same position relative to each other are shown, followed by two universal MHC class II epitopes (MHC-II). Various iterations are produced using different linkers. In some cases, T cell epitopes are directly connected to each other. In other cases, the T cell epitopes are flanked on one or both sides of its native sequence. In other iterations, the T cell epitopes are connected by non-native sequences AAY, RR and DPP.

[0064] Figure 2B Evaluation of linker sequences in cassettes is illustrated and sequence information about the T cell epitopes embedded in the cassettes is shown. Figure 2B SEQ ID NOs 78-79, 82, 81, 80, 49 and 194 are disclosed respectively, in order of appearance.

[0065] Figure 3The evaluation of cell targeting sequences added to the model vaccine cassette is described. The targeting cassette extends the short cassette design with ubiquitin (Ub), signal peptide (SP) and / or transmembrane (TM) domains, features the five marker human T cell epitopes (epitopes 1 to 5) and two mouse T cell epitopes SIINFEKL (SII) (SEQ ID NO: 72) and SPSYAYHQF (A5) (SEQ ID NO: 72) immediately adjacent to the five marker human T cell epitopes, and SPSYAYHQF (A5) (SEQ ID NO: 72), and uses a non-natural linker AAY- or natural linker to flank the T cell epitopes on both sides (25-mer).

[0066] Figure 4 In vivo evaluation of linker sequences in short cassettes is illustrated. A) Experimental design for in vivo evaluation of vaccine cassettes using HLA-A2 transgenic mice.

[0067] Figure 5A The in vivo evaluation of the impact of epitope position in the 21-mer long cassette is described and shows that the design of the long cassette requires five marker class I epitopes (epitopes 1 to 5) contained in the 25-mer native sequence separated by additional well-known T-cell class I epitopes (epitopes 6 to 21) contained in the 25-mer native sequence (linker = native flanking sequence), and two universal class II epitopes (MHC-II0, where only the relative position of the class I epitopes varies.

[0068] Figure 5B In vivo evaluation of the effect of epitope position within a 21-mer long cassette is illustrated and sequence information on the T cell epitopes used is shown. Figure 5B SEQ ID NOs 78-79, 82, 81, 80, 195-197, 83 and 198-209 are disclosed, respectively, in order of appearance.

[0069] Fig. 6A The final cassette design for the preclinical IND-enabling study is illustrated and shows that the final cassette design contains 20 MHC I epitopes contained in the 25-mer native sequence (linker = native flanking sequence) and 2 universal MHC class II epitopes, wherein the 20 MHC I epitopes consist of 6 non-human primate (NHP) epitopes, 5 human epitopes, and 9 murine epitopes.

[0070] Figure 6B The final cassette design for preclinical IND enabling studies is illustrated and shows sequence information for all used T cell epitopes presented on MHC class I of non-human primate, mouse and human origin, as well as the sequences of 2 universal MHC class II epitopes PADRE and tetanus toxoid. Figure 6BSEQ ID NOs 112-117, 80-82, 78-79, 72-73, 142, 210, 146-148, 144-145, 49 and 47 are disclosed, respectively, in order of appearance.

[0071] Fig. 7A The production of ChAdV68.4WTnt.GFP virus after transfection is illustrated. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using a calcium phosphate protocol. Ten days after transfection, viral replication was observed and ChAdV68.4WTnt.GFP viral plaques were observed using light microscopy (40× magnification).

[0072] Figure 7B Demonstrated production of ChAdV68.4WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using a calcium phosphate protocol. Viral replication was observed 10 days after transfection and ChAdV68.4WTnt.GFP viral plaques were observed using fluorescence microscopy at 40× magnification.

[0073] Figure 7C Demonstrated production of ChAdV68.4WTnt.GFP virus after transfection. HEK293A cells were transfected with ChAdV68.4WTnt.GFP DNA using a calcium phosphate protocol. Viral replication was observed 10 days after transfection and ChAdV68.4WTnt.GFP viral plaques were observed using fluorescence microscopy at 100× magnification.

[0074] Fig. 8A The production of ChAdV68.5WTnt.GFP virus after transfection is illustrated. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the lipofectamine protocol. Viral replication (plaques) was observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. After 3 days, ChAdV68.5WTnt.GFP viral plaques were observed using light microscopy (40× magnification) and photographed.

[0075] Figure 8BThe production of ChAdV68.5WTnt.GFP virus after transfection is illustrated. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the lipofectamine protocol. Viral replication (plaques) was observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. After 3 days, ChAdV68.5WTnt.GFP viral plaques were observed at 40× magnification using fluorescence microscopy and photographed.

[0076] Figure 8C The production of ChAdV68.5WTnt.GFP virus after transfection is illustrated. HEK293A cells were transfected with ChAdV68.5WTnt.GFP DNA using the lipofectamine protocol. Viral replication (plaques) was observed 10 days after transfection. Lysates were prepared and used to reinfect 293A cells in T25 flasks. After 3 days, ChAdV68.5WTnt.GFP viral plaques were observed and photographed at 100× magnification using fluorescence microscopy.

[0077] Fig. 9 The viral particle manufacturing protocol is illustrated.

[0078] Fig.10 Alphavirus-derived VEE self-replicating RNA (srRNA) vectors are described.

[0079] Fig.11 In vivo reporter gene expression after inoculation of C57BL / 6J mice with VEE-luciferase srRNA is illustrated. Representative luciferase signal images after immunization of C57BL / 6J mice with VEE-luciferase srRNA at various time points (10 ug per mouse, bilateral intramuscular injection, MC3 encapsulation) are shown.

[0080] Fig. 12AThe T cell response measured 14 days after immunization with VEE srRNA formulated with MC3LNP in mice bearing B16-OVA tumors is illustrated. 10ug VEE-luciferase srRNA (control), VEE-UbAAY srRNA (Vax), VEE-luciferase srRNA and anti-CTLA-4 (aCTLA-4) or VEE-UbAAY srRNA and anti-CTLA-4 (Vax+aCTLA-4) were injected into C57BL / 6J mice bearing B16-OVA tumors. In addition, all mice were treated with anti-PD1 mAb starting from day 7. Each group consisted of 8 mice. 14 days after immunization, mice were sacrificed and spleens and lymph nodes were collected. SIINFEKL-specific T cells ("SIINFEKL" disclosed in SEQ ID NO: 72) responses were evaluated by IFN-γ ELISPOT and reported as spot-forming cells (SFC) per 106 splenocytes. Lines indicate median values.

[0081] Fig. 12B The T cell response measured 14 days after immunization with VEE srRNA formulated with MC3LNP in mice bearing B16-OVA tumors is illustrated. 10ug VEE-luciferase srRNA (control), VEE-UbAAY srRNA (Vax), VEE-luciferase srRNA and anti-CTLA-4 (aCTLA-4) or VEE-UbAAYsrRNA and anti-CTLA-4 (Vax+aCTLA-4) were injected into C57BL / 6J mice bearing B16-OVA tumors. In addition, all mice were treated with anti-PD1 mAb starting from the 7th day. Each group consisted of 8 mice. 14 days after immunization, mice were killed and spleens and lymph nodes were collected. SIINFEKL-specific T cells ("SIINFEKL" disclosed in SEQ ID NO: 72) responses were evaluated by MHCI-pentamer staining, and the percentage of pentamer-positive cells in CD8-positive cells was reported. The line represents the median.

[0082] Fig.13AThe antigen-specific T cell response after heterologous priming / boosting in mice with B16-OVA tumors is described. C57BL / 6J mice with B16-OVA tumors were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA (control) formulated with MC3 LNP or injected with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. The third group was treated with a combination of Ad5-GFP priming / VEE-luciferase srRNA boosting and anti-CTLA-4 (aCTLA-4), while the fourth group was treated with a combination of Ad5-UbAAY priming / VEE-UbAAY boosting and anti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated with anti-PD-1 mAb from the 21st day. T cell responses were measured by IFN-γ ELISPOT. Fourteen days after immunization with adenovirus, mice were sacrificed and spleens and lymph nodes were collected.

[0083] Fig. 13B The antigen-specific T cell response after heterologous priming / boosting in mice with B16-OVA tumors is described. C57BL / 6J mice with B16-OVA tumors were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA (control) formulated with MC3 LNP or injected with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. The third group was treated with a combination of Ad5-GFP priming / VEE-luciferase srRNA boosting and anti-CTLA-4 (aCTLA-4), while the fourth group was treated with a combination of Ad5-UbAAY priming / VEE-UbAAY boosting and anti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated with anti-PD-1 mAb from the 21st day. T cell responses were measured by IFN-γ ELISPOT. Fourteen days after immunization with adenovirus and 14 days after boosting with srRNA (day 28 after priming), mice were sacrificed and spleens and lymph nodes were collected.

[0084] Fig. 13CThe antigen-specific T cell response after heterologous primary / boost immunization in mice with B16-OVA tumors is described. C57BL / 6J mice with B16-OVA tumors were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA (control) formulated with MC3 LNP or injected with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. The third group was treated with a combination of Ad5-GFP primary / VEE-luciferase srRNA boost immunization and anti-CTLA-4 (aCTLA-4), while the fourth group was treated with a combination of Ad5-UbAAY primary / VEE-UbAAY boost immunization and anti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated with anti-PD-1 mAb from the 21st day. T cell responses were measured by MHC class I pentamer staining. Fourteen days after immunization with adenovirus, mice were sacrificed and spleens and lymph nodes were collected.

[0085] Fig.13D The antigen-specific T cell response after heterologous primary / boost immunization in mice with B16-OVA tumors is described. C57BL / 6J mice with B16-OVA tumors were injected with adenovirus expressing GFP (Ad5-GFP) and boosted with VEE-luciferase srRNA (control) formulated with MC3 LNP or injected with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. The third group was treated with a combination of Ad5-GFP primary / VEE-luciferase srRNA boost immunization and anti-CTLA-4 (aCTLA-4), while the fourth group was treated with a combination of Ad5-UbAAY primary / VEE-UbAAY boost immunization and anti-CTLA-4 (Vax+aCTLA-4). In addition, all mice were treated with anti-PD-1 mAb from the 21st day. T cell responses were measured by MHC class I pentamer staining. Fourteen days after immunization with adenovirus and 14 days after boosting with srRNA (day 28 after priming), mice were sacrificed and spleens and lymph nodes were collected.

[0086] Fig.14AAntigen-specific T cell responses after heterologous prime / boost immunization in mice bearing CT26 (Balb / c) tumors are described. Mice were immunized with Ad5-GFP and boosted with VEE-luciferase srRNA formulated with MC3 LNPs 15 days after adenovirus priming (control), or primed with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. A combination of Ad5-GFP / VEE-luciferase srRNA prime / boost immunization with anti-PD-1 (aPD1) was administered to an independent group, while a fourth group received a combination of Ad5-UbAAY / VEE-UbAAYsrRNA prime / boost immunization with anti-PD-1 mAb (Vax+aPD1). T cell responses to AH1 peptides were measured using IFN-γ ELISPOT. Twelve days after immunization with adenovirus, mice were sacrificed and spleens and lymph nodes were collected.

[0087] Fig. 14B Antigen-specific T cell responses after heterologous prime / boost immunization in mice bearing CT26 (Balb / c) tumors are described. Mice were immunized with Ad5-GFP and boosted with VEE-luciferase srRNA formulated with MC3 LNPs 15 days after adenovirus prime (control), or primed with Ad5-UbAAY and boosted with VEE-UbAAYsrRNA (Vax). The control group and Vax group were also treated with IgG control mAb. A combination of Ad5-GFP / VEE-luciferase srRNA prime / boost immunization with anti-PD-1 (aPD1) was administered to an independent group, while a fourth group received a combination of Ad5-UbAAY / VEE-UbAAYsrRNA prime / boost immunization with anti-PD-1 mAb (Vax+aPD1). T cell responses to AH1 peptide were measured using IFN-γELI SPOT. Twelve days after immunization with adenovirus and 6 days after boosting with srRNA (day 21 after priming), mice were sacrificed and spleens and lymph nodes were collected.

[0088] Fig.15 ChAdV68 elicits T cell responses against mouse tumor antigens in mice. Mice were immunized with ChAdV68.5WTnt.MAG25mer and T cell responses were measured against the MHC class I epitope SIINFEKL (SEQ ID NO: 72) (OVA) in C57BL / 6J female mice and against the MHC class I epitope AH1-A5 in Balb / c mice. The results of each 10 6 The mean number of spot forming cells (SFC) per splenocyte was calculated. Error bars indicate standard deviation.

[0089] Fig.16 The cellular immune response after a single immunization with ChAdV6, ChAdV+anti-PD-1, srRNA, srRNA+anti-PD-1, or anti-PD-1 alone in the CT26 tumor model is illustrated. Antigen-specific IFN-γ production in splenocytes from 6 mice in each group was measured using ELISpot. Results are presented as 10 6 The number of spot forming cells (SFC) per splenocyte. The median of each group is indicated by a horizontal line. P values ​​were determined using Dunnett's multiple comparison test; ***P<0.0001, **P<0.001, *P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

[0090] Fig.17 CD8 T cell responses after a single immunization with ChAdV6, ChAdV+anti-PD-1, srRNA, srRNA+anti-PD-1, or anti-PD-1 alone in the CT26 tumor model are illustrated. Antigen-specific IFN-γ production in CD8 T cells was measured using ICS and results are presented as the percentage of antigen-specific CD8 T cells to total CD8 T cells. The median of each group is indicated by a horizontal line. P values ​​were determined using Dunnett's multiple comparison test; ***P<0.0001, **P<0.001, *P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

[0091] Fig.18 Tumor growth after immunization with ChAdV / srRNA heterologous prime / boost, srRNA / ChAdV heterologous prime / boost, or srRNA / srRNA homologous prime / boost in the CT26 tumor model is illustrated. Comparisons with prime / boost with or without anti-PD1 during prime and boost are also shown. Tumor volume was measured twice a week and the average tumor volume for 21 days before the study was presented. There were 22-28 mice per group at the start of the study. Error bars represent the standard error of the mean (SEM). P values ​​were determined using Dunnett's test; ***P<0.0001, **P<0.001, *P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

[0092] Fig.19Survival after immunization with ChAdV / srRNA heterologous prime / boost, srRNA / ChAdV heterologous prime / boost, or srRNA / srRNA homologous prime / boost in the CT26 tumor model is illustrated. Comparisons with prime / boost with or without anti-PD1 during prime and boost are also shown. P values ​​were determined using the log-rank test; ***P<0.0001, **P<0.001, *P<0.01. ChAdV=ChAdV68.5WTnt.MAG25mer; srRNA=VEE-MAG25mer srRNA.

[0093] Fig. 20A The antigen-specific cellular immune responses measured using ELISpot are illustrated. Antigen-specific IFN-γ production against six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot against VEE-MAG25mer srRNA-LNP1 (30 μg) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after the initial immunization (6 macaques per group). The results are presented as stacked bar graphs for each epitope per 10 6 The mean spot forming cell (SFC) number of PBMCs was calculated. The values ​​for each animal were normalized to the level before bleeding (week 0).

[0094] Fig. 20B The antigen-specific cellular immune responses measured using ELISpot are illustrated. Antigen-specific IFN-γ production against six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot against VEE-MAG25mer srRNA-LNP1 (100 μg) 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after the initial immunization (6 macaques per group). The results are presented as stacked bar graphs for each epitope per 10 6 The mean spot forming cell (SFC) number of PBMCs was calculated. The values ​​for each animal were normalized to the level before bleeding (week 0).

[0095] Fig. 20C The antigen-specific cellular immune response measured using ELISpot is illustrated. Antigen-specific IFN-λ production against six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot for VEE-MAG25mer srRNA-LNP2 (100 μg) homologous prime / boost immunizations 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after initial immunization (6 macaques per group). The results are presented as stacked bar graphs for each epitope per 10 6The mean spot forming cell (SFC) number of PBMCs was calculated. The values ​​for each animal were normalized to the level before bleeding (week 0).

[0096] Fig.20D The antigen-specific cellular immune response measured using ELISpot is illustrated. Antigen-specific IFN-γ production against six different mamuA01-restricted epitopes was measured in PBMCs using ELISpot for ChAdV68.5WTnt.MAG25mer / VEE-MAG25mersrRNA heterologous prime / boost groups 1, 2, 3, 4, 5, 6, 8, 9, or 10 weeks after initial immunization (6 macaques per group). The results are presented as stacked bar graphs for each epitope per 10 6 The mean spot forming cell (SFC) number of PBMCs was calculated. The values ​​for each animal were normalized to the level before bleeding (week 0).

[0097] Fig.21 Antigen-specific cellular immune responses measured using ELISpot are shown. Antigen-specific IFN-γ production to six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot prior to immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after the initial immunization following immunization with a heterologous ChAdV68.5WTnt.MAG25mer / VEE-MAG25mer srRNA prime / boost regimen. Results are presented as stacked bar graphs for each epitope (6 macaques per group) per 10 6 The average spot forming cell (SFC) number of PBMCs was calculated.

[0098] Fig. 22 Antigen-specific cellular immune responses measured using ELISpot are shown. Antigen-specific IFN-γ production to six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot before immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after the initial immunization following immunization with a homologous prime / boost regimen of VEE-MAG25mer srRNA LNP2. Results are presented as stacked bar graphs for each epitope (6 macaques per group) per 10 6 The average spot forming cell (SFC) number of PBMCs was calculated.

[0099] Fig.23Antigen-specific cellular immune responses measured using ELISpot are shown. Antigen-specific IFN-λ production to six different mamu A01-restricted epitopes was measured in PBMCs using ELISpot before immunization and 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 weeks after the initial immunization following immunization with a homologous prime / boost regimen of VEE-MAG25mer srRNA LNP1. Results are presented as stacked bar graphs for each epitope (6 macaques per group) per 10 6 The average spot forming cell (SFC) number of PBMCs was calculated.

[0100] Fig.24A Example peptide spectra generated from the Promega dynamic range standard are shown. Fig.24A SEQ ID NO: 77 is disclosed.

[0101] Fig. 24B Example peptide spectra generated from the Promega dynamic range standard are shown.

[0102] Fig.25 The production capacity of eight selected ChAdV68-MAGs for rapid growth of plaques, assessed by IU titer, compared to non-purified pooled virus is shown. The numbers above the bars in the graph indicate the fold increase of the pooled virus at an MOI of 0.1 in controlled infections.

[0103] Fig.26 A schematic diagram of the E4 locus identified in clone 1A and the 727 bp deletion between E4orf2-E4orf4 is shown.

[0104] Fig. 27 Virus productivity of viruses plus and minus E4 deletion is shown. Numbers above the bars indicate fold improvement compared to non-E4 deleted viruses. Comparison of ChAdV68-MAG and ChAdV68-MAG-E4 viruses was performed in 3 independent cases. In each case, 400 mL production runs were performed with both viruses at an MOI of 1.0. Virus particle (VP) titers (left panel) and infectious unit (IU) titers (right panel) are shown.

[0105] Fig.28 Western blot analysis of MAG expression using rabbit anti-class II epitope antibodies in cells infected with ChAdV68.5WTnt.MAG25mer ("MAG") and ChAdV68-MAG-E4 deletion ("MAG-E4") viruses is shown. Samples were treated with and without the proteasome inhibitor MG-132 as indicated by plus and minus signs.

[0106] Fig.29 The general organization of model epitopes for various species with large antigenic cassettes of 30 (L), 40 (XL), or 50 (XXL) epitopes is illustrated.

[0107] Fig.30 Shown is the ChAd vector expression long cassette, as indicated by the above-mentioned western blot using the anti-class II (PADRE) antibody identified as a sequence common to all cassettes. HEK293 cells are infected with the ChAdV68 vector expressing large cassettes of different sizes (ChAdV68-50XXL, ChAdV68-40XL and ChAdV68-30L). The infection is set at an MOI of 0.2. After infection for twenty-four hours, the proteasome inhibitor MG132 is added to a group of infection wells (indicated by a plus sign). Another group of virus-treated wells are not treated with MG132 (indicated by a minus sign). Uninfected HEK293 cells (293F) are used as negative controls. After infection forty-eight hours, cell pellets are collected and analyzed by SDS / PAGE electrophoresis and immunoblotting using rabbit anti-class II PADRE antibodies. HRP anti-rabbit antibodies and ECL chemiluminescent substrates are used for detection.

[0108] Fig.31 CD8+ immune responses in mice immunized with ChAdV68 large cassette detected by ICS to AH1 (top) and SIINFEKL (SEQ ID NO: 72) (bottom) are shown. Data are presented as IFNg+ cells to model epitopes as a percentage of total CD8 cells.

[0109] Fig.32 CD8+ responses to LD-AH1+ (top) and Kb-SIINFEKL+ (bottom) ("SIINFEKL" disclosed as SEQ ID NO: 72) tetramers after ChAdV68 large cassette vaccination are shown. Data are presented as the percentage of total CD8 cells reactive to the model tetrameric peptide complex. *p<0.05, **p<0.01 by ANOVA using Tukey's test. All p values ​​are compared to the MAG 20-antigen cassette.

[0110] Fig.33 CD8+ immune responses in mice treated with the alphavirus large cassette detected by ICS for AH1 (top) and SIINFEKL (SEQ ID NO: 72) (bottom) are shown. Data are presented as IFNg+ cells as a percentage of total CD8 cells to the model epitope. *p<0.05, **p<0.01, **p<0.001 by ANOVA using Tukey's test. All p values ​​are compared to the MAG20-antigen cassette.

[0111] Fig.34 Vaccination strategies for evaluating the immunogenicity of vectors containing antigenic cassettes in rhesus macaques are illustrated. Triangles indicate ChAdV68 vaccination (1e12 vp / animal) at weeks 0 and 32. Circles indicate alphavirus vaccination at weeks 0, 4, 12, 20, 28, and 32. Squares indicate administration of anti-CTLA4 antibodies.

[0112] Fig.35 Shown is the time course of CD8+ anti-epitope responses in rhesus macaques dosed with chAd-MAG alone (Group 4). Mean SFC / 1e6 splenocytes are shown.

[0113] Fig.36 Shown is the time course of CD8+ anti-epitope responses in rhesus macaques dosed with IV delivered chAd-MAG plus anti-CTLA4 antibody (Ipilimumab) (Group 5). Mean SFC / 1e6 splenocytes are shown.

[0114] Fig.37 Shown is the time course of CD8+ anti-epitope responses in rhesus macaques dosed with SC delivered chAd-MAG plus anti-CTLA4 antibody (ipilimumab) (Group 6). Mean SFC / 1e6 splenocytes are shown.

[0115] Fig.38 The antigen-specific memory responses produced by the ChAdV68 / samRNA vaccine regimen measured by ELISpot are shown. The results are presented as individual dot plots, where each dot represents a single animal. The memory responses at baseline before immunization (left) and 18 months after primary immunization (right) are shown.

[0116] Fig.39 Memory cell phenotype of antigen-specific CD8+ T cells by flow cytometry using combined tetramer staining and CD45RA / CCR7 co-staining is shown.

[0117] Fig.40 Shown is the distribution of memory cell types within the sum of the four Mamu-A*01 tetramer+CD8+ T cell populations at study month 18. Memory cells were characterized as follows: CD 45RA+CCR7+=naive, CD45RA+CCR7-=effector (Teff), CD45RA-CCR7+=central memory (Tcm), CD45RA-CCR7-=effector memory (Tem).

[0118] Fig.41The frequency of CD8+ T cells recognizing the CT26 tumor antigen AH1 in CT26 tumor-bearing mice is shown. P values ​​were determined using one-way ANOVA with Tukey's multiple comparison test; **P<0.001, *P<0.05. ChAdV=ChAdV68.5WTnt.MAG25mer; aCTLA4=anti-CTLA4 antibody, clone 9D9.

[0119] Fig.42A Shown is the CD8+ immune response of IFN-γ production assessed by ICS after stimulation with AH1 (a dominant epitope of the murine leukemia virus envelope protein gp70) in Balb / c mice treated with ChAdV68-MAG and ChAdV68-E4δ-MAG vectors. Balb / c mice were immunized by bilateral injection of 50uL of virus into the quadriceps femoris (100uL total, 50uL / leg).

[0120] Fig.42B Shown are T cell responses for IFN-λ production assessed by ELISpot after stimulation with 6 different rhesus macaque Mamu-A*01 class I epitopes at week 2 in rhesus macaques immunized with ChAdV68-CMV-MAG (left) and ChAdV68-E4d-CMT-MAG (right), and in both cases anti-CTLA4 antibody (ipilimumab) was administered.

[0121] Fig.42C Shown are T cell responses for IFN-γ production assessed by ELISpot following stimulation with six different rhesus macaque Mamu-A*01 class I epitopes over a time course in rhesus macaques immunized with ChAdV68-CMV-MAG (left) and ChAdV68-E4d-CMT-MAG (right), and in both cases anti-CTLA4 antibody (ipilimumab) was administered.

[0122] Fig.43 The general strategy of a tetracycline-controlled viral production system using an example of an antigen-encoding vaccine is illustrated.

[0123] Fig.44A A schematic diagram showing the arrangement of the "TETo" reaction zone with respect to the promoter and cassette to be expressed is presented.

[0124] Fig.44B A schematic diagram showing the arrangement of the "CMT" reaction zone with respect to the promoter and cassette to be expressed is presented.

[0125] Fig.45AShown is TETr-mediated regulation of GFP expressed from a ChAdV68 vector with a TETo sequence. GFP is significantly reduced in 293F cells expressing TETr (clone 17, right) relative to the parental 293F cell line (left). Cells were infected with ChAdV68-TETo-GFP at an MOI of 1, and GFP was evaluated 24 hours later by fluorescence microscopy under a 10X objective.

[0126] Fig.45B TETr-mediated regulation of SEAP expressed from a ChAdV68 vector with a CMT sequence is shown. SEAP is significantly reduced in 293F cells expressing TETr (clone 17, second column from the left) relative to the parental 293F cell line (left column). Background signal was established using a ChAdV68 vector expressing a control expression cassette (right two columns). 293F cells were infected at an MOI of 0.3, and culture media were collected 24 hours later for SEAP determination (Phospha-LightTM System (Applied Biosystems), using a chemiluminescent substrate to detect secreted alkaline phosphatase), which was operated according to the manufacturer's description.

[0127] Fig.46 The virus production of ChAdV68-Teto-MAG vector in 293F TETr repressor system (clone 17) is shown relative to the production in the parental 293F system. The experiment was performed in triplicate (operations 1-3). In each experiment, 400mL 293F cells were infected with an MOI of about 3 and incubated for 48-72 hours before collection. The virus was purified by two discontinuous CsCl ultracentrifugation steps and dialyzed into storage buffer. Virus particles were measured by absorbance at 260nm. Virus particles (VP; upper figure) and infection unit (IU; lower figure) titers are shown.

[0128] Fig.47A The overall production capacity of Tet-regulated viruses ("TETo-MAG") in the 293F TETr line (clone 17) is shown relative to non-regulated viruses ("MAG") with the same cassette in the normal 293F cell line. Data from multiple 400 mL production runs followed by centrifugation are shown. The numbers above the graph represent the fold increase of Tet-regulated viruses.

[0129] Fig.47B Virus production of ChAdV68-CT-TSNA, ChAdV68-TETo-TSNA, ChAdV68-CMT-TSNA, and ChAdV68-E4d-CMT-TSNA viruses relative to ChAdV68-CMV-TSNA is shown.

[0130] Fig.47C Virus production of model antigen cassettes 50XXL and M2.2 using adenoviral vectors with CMT-responsive regions in a tTS-expressing cell line is shown.

[0131] Fig.48 Antigen-specific T cell responses after vaccination with regulated vectors versus unregulated vectors are shown. Antigen-specific IFN-γ production in CD8 T cells was measured using ICS, and the results are presented as the percentage of antigen-specific CD8 T cells to total CD8 T cells. Horizontal lines represent the median of each group. Vaccines were administered with 1 x 10 10 Balb / c mice were immunized with ChAdV68 vaccine expressing the model antigen cassette of VP. Spleens were collected 12 days after vaccination and single cell suspensions were prepared. DETAILED DESCRIPTION

[0132] I. Definitions

[0133] In general, the terms used in the claims and this specification are intended to be interpreted as having the ordinary meaning as understood by one of ordinary skill in the art. For greater clarity, certain terms are defined below. In the event of a conflict between the ordinary meaning and the provided definition, the provided definition will apply.

[0134] As used herein, the term "antigen" is a substance that induces an immune response. An antigen may be a novel antigen. An antigen may be a "shared antigen," which is an antigen found among a specific population, such as a specific population of cancer patients or infected subjects. An antigen may be associated with or derived from an infectious disease organism.

[0135] As used herein, the term "neoantigen" is an antigen with at least one change that makes it different from the corresponding wild-type antigen, for example, via a mutation in a tumor cell or a post-translational modification specific to a tumor cell. A neoantigen may include a polypeptide sequence or a nucleic acid sequence. Mutations may include frameshift or non-frameshift insertions and deletions, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that produce a neoORF. Mutations may also include splice variants. Post-translational modifications specific to tumor cells may include abnormal phosphorylation. Post-translational modifications specific to tumor cells may also include spliced ​​antigens produced by the proteasome. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. October 21, 2016; 354(6310): 354-358. Subjects may be identified for administration using various diagnostic methods (e.g., patient selection methods described further below).

[0136] As used herein, the term "tumor antigen" is an antigen that is present in tumor cells or tissues of a subject but not in corresponding normal cells or tissues of the subject, or is derived from a polypeptide that is known or found to have altered expression in tumor cells or cancer tissues compared to normal cells or tissues.

[0137] As used herein, the term "antigen-based vaccine" is a vaccine composition based on one or more antigens (e.g., multiple antigens). The vaccine can be a nucleotide-based (e.g., virus-based, RNA-based, or DNA-based) vaccine, a protein-based (e.g., peptide-based) vaccine, or a combination thereof.

[0138] As used herein, the term "candidate antigen" is a mutation or other aberration that produces a sequence that may represent an antigen.

[0139] As used herein, the term "coding region" is the portion of a gene that encodes a protein.

[0140] As used herein, the term "coding mutation" is a mutation that occurs in a coding region.

[0141] As used herein, the term "ORF" means open reading frame.

[0142] As used herein, the term "NEO-ORF" is a tumor-specific ORF resulting from mutation or other aberrations (eg, splicing).

[0143] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another amino acid.

[0144] As used herein, the term "nonsense mutation" is a substitution of an amino acid that results in a stop codon or a mutation that results in the removal of a typical start codon.

[0145] As used herein, the term "frameshift mutation" is a mutation that causes a change in the protein framework.

[0146] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0147] As used herein, in the context of two or more nucleic acids or polypeptide sequences, the term percentage "identity" refers to two or more sequences or subsequences having a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to the skilled person) or by visual inspection. Depending on the application, the "identity" percentage may be present in a region of the sequence being compared, such as a functional domain, or in the full length of the two sequences to be compared.

[0148] Regarding sequence comparison, usually a sequence serves as a reference sequence compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the specified program parameters. Alternatively, sequence similarity or dissimilarity can be established by combining the presence or absence of specific nucleotides, or for translated sequences, at the amino acid at the selected sequence position (e.g., sequence motif).

[0149] Optimal alignment of compared sequences can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); 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.).

[0150] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is 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.

[0151] As used herein, the term "no stop or read-through" is a mutation that results in the removal of the natural stop codon.

[0152] As used herein, the term "epitope" is the specific portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0153] As used herein, the term "immunogenicity" is the ability to elicit an immune response, for example, via T cells, B cells, or both.

[0154] As used herein, the terms "HLA binding affinity", "MHC binding affinity" mean the affinity of binding between a specific antigen and a specific MHC allele.

[0155] As used herein, the term "bait" is a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.

[0156] As used herein, the term "variant" is a difference between a subject's nucleic acid and a reference human genome used as a control.

[0157] As used herein, the term "variant call" is an algorithmic determination of the presence of a variant, typically based on sequencing.

[0158] As used herein, the term "polymorphism" is a germline variant, ie, a variant found in all DNA-bearing cells of an individual.

[0159] As used herein, the term "somatic variant" is a variant arising in non-germline cells of an individual.

[0160] As used herein, the term "allele" is a form of a gene or a form of a gene sequence or a form of a protein.

[0161] As used herein, the term "HLA type" is the complement of HLA gene alleles.

[0162] As used herein, the term "nonsense-mediated decay" or "NMD" is the degradation of mRNA by cells due to premature stop codons.

[0163] As used herein, the term "trunk mutation" is a mutation that originates early in tumor development and is present in the majority of tumor cells.

[0164] As used herein, the term "subclonal mutation" is a mutation that originates late in tumor development and is present only in a subset of tumor cells.

[0165] As used herein, the term "exome" is a subset of a genome that encodes proteins. An exome may be the collective exons of a genome.

[0166] As used herein, the term "logistic regression" is a regression model for binary data from statistics in which the logistic of the probability that the dependent variable is equal to 1 is modeled as a linear function of the dependent variable.

[0167] As used herein, the term "neural network" is a machine learning model for classification or regression that consists of multiple layers of linear transformations followed by element-wise nonlinearities that are typically trained via stochastic gradient descent and back-propagation.

[0168] As used herein, the term "proteome" is the collection of all proteins expressed and / or translated by a cell, a population of cells, or an individual.

[0169] As used herein, the term "peptide group" is the collection of all peptides presented by MHC-I or MHC-II on the cell surface. The peptide group can refer to the characteristics of a cell or a collection of cells (e.g., a tumor peptidome, meaning the union of the peptide groups of all cells constituting a tumor).

[0170] As used herein, the term "ELISPOT" means enzyme-linked immunosorbent spot assay, which is a common method used to monitor immune responses in humans and animals.

[0171] As used herein, the term "dextran peptide multimer" is a dextran-based peptide-MHC multimer used for antigen-specific T cell staining in flow cytometry.

[0172] As used herein, the term "tolerance or immune tolerance" is a state of immune unresponsiveness to one or more antigens (eg, self-antigens).

[0173] As used herein, the term "central tolerance" is tolerance incurred in the thymus by deleting autoreactive T cell clones or by promoting the differentiation of autoreactive T cell clones into immunosuppressive regulatory T cells (Tregs).

[0174] As used herein, the term "peripheral tolerance" is tolerance incurred in the periphery by down-regulating or not activating autoreactive T cells undergoing central tolerance or promoting differentiation of these T cells into Tregs.

[0175] The term "sample" may include single or multiple cells or cell fragments or aliquots of body fluids obtained from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage samples, scraping, surgical incision or intervention, or other means known in the art.

[0176] The term "subject" encompasses human or non-human, whether in vivo, ex vivo or in vitro, male or female cells, tissues or organisms. The term subject includes mammals including humans.

[0177] The term "mammal" encompasses humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0178] The term "clinical factor" refers to a measure of a subject's condition, such as disease activity or severity. "Clinical factors" encompass all markers of a subject's health status, including non-sample markers, and / or other features of the subject, such as, but not limited to, age and gender. A clinical factor can be a score, value, or set of values ​​obtained by evaluating a sample (or sample population) or subject from a subject under certain conditions. Clinical factors can also be predicted by markers and / or other parameters (such as gene expression surrogates). Clinical factors can include tumor type, tumor subtype, and smoking history.

[0179] The term "derived from" refers to sequences extracted directly from a subject tissue or sample (e.g., a tumor, a cell, an infected cell, or an infectious disease organism), such as via RT-PCR; or sequence data obtained by sequencing a subject tissue or sample and then synthesizing a nucleic acid sequence using the sequencing data, such as via various synthetic or PCR-based methods known in the art. "Derived from" may include nucleic acid sequence variants, such as codon-optimized nucleic acid sequence variants, which encode the same polypeptide sequence as the corresponding native nucleic acid sequence (e.g., the corresponding native infectious disease organism nucleic acid sequence). "Derived from" may also include variants encoding modified polypeptide sequences, such as infectious disease organism polypeptide sequences having one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to the native polypeptide sequence (e.g., the native infectious disease organism polypeptide sequence). For example, the modified polypeptide sequence may have one or more missense mutations (e.g., engineered mutations) relative to the native polypeptide sequence.

[0180] The term "alphavirus" refers to a member of the Togaviridae family and is a positive-sense single-stranded RNA virus. Alphaviruses are generally classified as Old World, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest virus, or New World, such as eastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equine encephalitis and its derivative strain TC-83. Alphaviruses are generally self-replicating RNA viruses.

[0181] The term "alphavirus backbone" refers to the minimal sequence of an alphavirus that allows the viral genome to replicate itself. The minimal sequence may include conserved sequences for nonstructural protein-mediated amplification, nonstructural protein 1 (nsP1) gene, nsP2 gene, nsP3 gene, nsP4 gene and polyA sequence, as well as sequences for subgenomic viral RNA expression, including 26S promoter elements.

[0182] The term "sequence for nonstructural protein-mediated amplification" includes alphavirus conserved sequence elements (CSEs) well known to those skilled in the art. CSEs include (but are not limited to) alphavirus 5'UTR, 51-ntCSE, 24-ntCSE or other 26S subgenomic promoter sequences, 19-ntCSE and alphavirus 3'UTR.

[0183] The term "RNA polymerase" includes polymerases that catalyze the production of RNA polynucleotides from a DNA template. RNA polymerases include, but are not limited to, polymerases derived from bacteriophages, including T3, T7, and SP6.

[0184] 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 some cases 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 dilinoleyl methyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules.

[0185] The term "lipid nanoparticle" or "LNP" includes a vesicle-like structure formed around an aqueous interior using a lipid-containing membrane, also referred to as a liposome. Lipid nanoparticles include lipid-based compositions with a solid lipid core stabilized by a surfactant. The core lipid can be a mixture of fatty acids, acylglycerols, waxes, and these surfactants. Biomembrane lipids, such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol), can be used as stabilizers. Lipid nanoparticles can be formed using different lipid molecules of a defined ratio, including, but not limited to, one or more cationic lipids, anionic lipids, or neutral lipids of a defined ratio. Lipid nanoparticles can encapsulate molecules in an outer membrane shell, and can subsequently contact target cells to deliver the encapsulated molecules to the host cell cytosol. Lipid nanoparticles can be modified or functionalized with non-lipid molecules, including on their surface. Lipid nanoparticles can be monolayer (monolayer) or multilayer (multilayer). Lipid nanoparticles can be complexed with nucleic acids. Monolayer lipid nanoparticles can be complexed with nucleic acids, wherein the nucleic acids are in the aqueous interior. Multilamellar lipid nanoparticles can be complexed with nucleic acids, with the nucleic acids being in the aqueous interior, or formed or entrapped therein.

[0186] 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.

[0187] It must be noted 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.

[0188] Unless the context specifically states or is otherwise apparent, as used herein, the term "about" should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. Approximately can be understood to be 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 the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term about.

[0189] Any term not directly defined herein should be understood to have a meaning generally associated with that understood in the technical field of the present invention. Certain terms are discussed herein to provide practitioners with additional guidance on the compositions, devices, methods, etc., describing aspects of the present invention and how to make or use them. It should be understood that the same thing can be represented in more than one way. Therefore, alternative wordings and synonyms can be used for any one or more of the terms discussed herein. It does not matter whether the terms are described or discussed herein. Some synonyms or replaceable methods, materials, etc. are provided. Unless explicitly stated, the narration of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents. The use of examples including term examples is for illustrative purposes only and does not limit the scope and meaning of aspects of the present invention herein.

[0190] All references, issued patents, and patent applications cited within the text of this specification are hereby incorporated by reference in their entirety for all purposes.

[0191] II. Methods for Identifying Antigens

[0192] Methods for identifying antigens (e.g., antigens derived from a tumor or infectious disease organism) include identifying antigens that are likely to be presented on a cell surface (e.g., presented on a tumor cell, an infected cell, or an immune cell (including professional antigen presenting cells, such as dendritic cells) via MHC), and / or that are likely to be immunogenic. For example, one such method may include the following steps: obtaining at least one of the following from a tumor, an infected cell, or an infectious disease organism: exome, transcriptome, or whole genome nucleotide sequencing and / or expression data, wherein the nucleotide sequencing data and / or expression data is used to obtain data representing a peptide sequence of each of a set of antigens (e.g., antigens derived from a tumor or infectious disease organism); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical probabilities that each of the antigens is presented on a cell surface of a subject, such as a tumor cell or an infected cell, by one or more MHC alleles, the set of numerical probabilities having been identified based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical probabilities to generate a set of selected antigens.

[0193] In one example of a tumor vaccine and adaptable to an infectious disease vaccine, the presentation model may include a statistical regression or machine learning (e.g., deep learning) model trained on a reference data set (also referred to as a training data set), the reference data set including a corresponding set of markers, wherein the reference data set is obtained from each of a plurality of different subjects, wherein optionally some subjects may have a tumor, and wherein the reference data set includes at least one of the following: data representing exome nucleic acid sequences from tumor tissue, data representing exome nucleic acid sequences from normal tissue, data representing transcriptome nucleic acid sequences from tumor tissue, data representing proteome sequences from tumor tissue, data representing MHC peptide group sequences from tumor tissue, and data representing MHC peptide group sequences from normal tissue. The reference data may further include mass spectrometry data, sequencing data, RNA sequencing data, expression profile data, and proteomics data of monoallelic cell lines engineered to express predetermined MHC alleles subsequently exposed to synthetic proteins, normal and tumor human cell lines, and fresh and frozen primary samples, as well as T cell assays (e.g., ELISPOT). In certain aspects, the reference data set includes each form of reference data.

[0194] The presentation model may comprise a feature set derived at least in part from the reference data set, and wherein the feature set comprises at least one of an allele-dependent feature and an allele-independent feature. In certain aspects, each feature is included.

[0195] The method for identifying antigens also includes generating an output for constructing a personalized vaccine by identifying one or more antigens that may be presented on the surface of a subject's cells (e.g., tumor cells or infected cells). As an example of a tumor vaccine and adaptable to an infectious disease vaccine, one such method may include the following steps: obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing and / or expression data from tumor cells and normal cells of the subject, wherein the nucleotide sequencing and / or expression data is used to obtain a peptide sequence representing each of a set of antigens identified by comparing the nucleotide sequencing and / or expression data from tumor cells with the nucleotide sequencing and / or expression data from normal cells (e.g., in the case of a new antigen, wherein the peptide sequence of each new antigen comprises at least one change that makes it different from the corresponding wild-type peptide sequence, or in the case of an antigen without a mutation, wherein the peptide is derived from a known or found to be expressed in a tumor cell or tissue compared to a normal cell or tissue). any polypeptide with altered expression in cancer tissue), data of peptide sequences identified from normal cells of the subject; encoding the peptide sequence of each of the antigens in a corresponding numerical vector, each numerical vector including information about a plurality of amino acids constituting the peptide sequence and a set of positions of the amino acids in the peptide sequence; using a computer processor to input the numerical vector into a deep learning presentation model to generate a set of presentation probabilities for the antigen set, each presentation probability in the set representing the probability that the corresponding antigen is presented on the surface of the tumor cells of the subject by one or more class II MHC alleles, a deep learning presentation model; selecting a subset of the antigen set based on the set of presentation probabilities to generate a selected antigen set; and generating an output for constructing a personalized cancer vaccine based on the selected antigen set.

[0196] Specific methods for identifying antigens (including neoantigens) are known to those of skill in the art, such as the methods described in more detail in International Patent Application Publications WO / 2017 / 106638, WO / 2018 / 195357, and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0197] Disclosed herein is a method of treating a subject having a tumor, the method comprising the steps of performing any of the antigen identification methods described herein, and further comprising obtaining a tumor vaccine comprising a selected set of antigens, and administering the tumor vaccine to the subject.

[0198] The methods disclosed herein may also include identifying one or more T cells that have antigen specificity for at least one of the antigens in the subset. In some embodiments, identification includes co-culturing one or more T cells with one or more antigens in the subset under conditions of amplifying one or more antigen-specific T cells. In other embodiments, identification includes contacting one or more T cells with a tetramer containing one or more antigens in the subset under conditions that allow binding between T cells and tetramers. In even other embodiments, the methods disclosed herein may also include identifying one or more T cell receptors (TCRs) of one or more identified T cells. In certain embodiments, identifying one or more T cell receptors includes sequencing the T cell receptor sequences of one or more identified T cells. The methods disclosed herein may also include genetically engineering multiple T cells to express at least one of the one or more identified T cell receptors; culturing the multiple T cells under conditions of amplifying the multiple T cells; and injecting the amplified T cells into the subject. In some embodiments, genetically engineering multiple T cells to express at least one of the one or more identified T cell receptors includes cloning the T cell receptor sequences of one or more identified T cells into an expression vector; and transfecting each of the multiple 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 infusing the expanded T cells into the subject.

[0199] Also disclosed herein is an isolated T cell having antigen specificity for at least one selected antigen in the subset.

[0200] Also disclosed herein is a method for making a tumor vaccine, the method comprising the following steps: obtaining at least one of the following from a tumor cell of a subject: exome, transcriptome or whole genome tumor nucleotide sequencing and / or expression data, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequence of each of a set of antigens (e.g., in the case of new antigens, wherein the peptide sequence of each new antigen comprises at least one change that makes it different from the corresponding wild-type peptide sequence, or in the case of antigens without mutations, wherein the peptide is derived from any polypeptide known or found to have altered expression in tumor cells or cancer tissues compared to normal cells or tissues); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical probabilities that each of the antigens is presented by one or more MHC alleles on the tumor cell surface of the tumor cell of the subject, the set of numerical probabilities having been identified based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical probabilities to generate a set of selected antigens; and preparing or having prepared a tumor vaccine comprising the set of selected antigens.

[0201] Also disclosed herein is a tumor vaccine comprising a selected set of antigens selected by performing a method comprising the following steps: obtaining at least one of the following from a tumor cell of a subject: exome, transcriptome or whole genome tumor nucleotide sequencing and / or expression data, wherein the tumor nucleotide sequencing and / or expression data is used to obtain data representing the peptide sequence of each of the set of antigens, and wherein the peptide sequence of each antigen (e.g., in the case of a neoantigen, wherein the peptide sequence of each neoantigen comprises at least one change that makes it different from the corresponding wild-type peptide sequence, or in the case of an antigen without a mutation, wherein the peptide is derived from any polypeptide known or found to have altered expression in a tumor cell or cancer tissue compared to a normal cell or tissue); inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical probabilities that each of the antigens is presented by one or more MHC alleles on the tumor cell surface of the tumor cell of the subject, the set of numerical probabilities having been identified based at least on the received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical probabilities to generate a set of selected antigens; and preparing or having prepared a tumor vaccine comprising the set of selected antigens.

[0202] Tumor vaccines may include one or more of nucleic acid sequences, polypeptide sequences, RNA, DNA, cells, plasmids, or vectors.

[0203] Tumor vaccines may include one or more antigens presented on the surface of tumor cells.

[0204] Tumor vaccines may include one or more antigens that are immunogenic in a subject.

[0205] Tumor vaccines may not include one or more antigens that induce an autoimmune response against normal tissue in a subject.

[0206] Tumor vaccines may include an adjuvant.

[0207] Tumor vaccines may include excipients.

[0208] The methods disclosed herein may also include selecting antigens having an increased probability of being presented on the surface of tumor cells relative to unselected antigens based on the presentation model.

[0209] The methods disclosed herein may also include selecting an antigen that has an increased likelihood of being able to induce a tumor-specific immune response in a subject relative to an unselected antigen based on the presentation model.

[0210] The methods disclosed herein may further comprise selecting an antigen having an increased probability of being presented to naive T cells by a professional antigen presenting cell (APC) relative to an unselected antigen based on the presentation model, optionally wherein the APC is a dendritic cell (DC).

[0211] The methods disclosed herein may also include selecting antigens that have a reduced likelihood of being inhibited via central or peripheral tolerance relative to unselected antigens based on the presentation model.

[0212] The methods disclosed herein may also include selecting an antigen that has a reduced likelihood of being able to induce an autoimmune response to normal tissue in the subject relative to an unselected antigen based on the presentation model.

[0213] Exome or transcriptome nucleotide sequencing and / or expression data can be obtained by sequencing tumor tissue.

[0214] The sequencing can be next generation sequencing (NGS) or any massively parallel sequencing method.

[0215] The set of numerical probabilities can be further identified by at least one MHC-allele interaction feature, the feature comprising at least one of the following: the predicted affinity of the MHC allele for binding to the antigen-encoding peptide; the predicted stability of the antigen-encoding peptide-MHC complex; the sequence and length of the antigen-encoding peptide; the probability of presentation of an antigen-encoding peptide with a similar sequence in cells from other individuals expressing the particular MHC allele, as assessed by mass spectrometry proteomics or other means; the expression level of the particular MHC allele in the subject in question (e.g., as measured by RNA-seq or mass spectrometry); the overall probability of sequence independence of new antigen-encoding peptides presented by a particular MHC allele in other different subjects expressing the particular MHC allele; the overall probability of sequence independence of new antigen-encoding peptides presented by MHC alleles in the same family of molecules (e.g., HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DR, HLA-DP) in other different subjects.

[0216] The set of numerical likelihoods is further identified by at least MHC-allele non-interactive features, including at least one of the following: C-terminal and N-terminal sequences flanking the neoantigen encoding peptide within its source protein sequence; the presence of a protease cleavage motif in the neoantigen encoding peptide, optionally weighted according to the expression of the corresponding protease in tumor cells (as measured by RNA-seq or mass spectrometry); the turnover rate of the source protein as measured in an appropriate cell type; the length of the source protein, optionally taking into account the specific splice variants ("isoforms") that are most highly expressed in tumor cells, as measured by RNA-seq or proteomic mass spectrometry as measured, or as predicted from annotations of germline or somatic splicing mutations detected in DNA or RNA sequence data; expression levels of proteasomes, immunoproteasomes, thymic proteasomes or other proteases in tumor cells (which can be measured by RNA-seq, proteomic mass spectrometry or immunohistochemistry); expression of source genes encoding new antigen peptides (e.g., as measured by RNA-seq or mass spectrometry); typical tissue-specific expression of source genes encoding new antigen peptides during various stages of the cell cycle; a comprehensive catalog of features of source proteins and / or their domains, such as in, for example, uniProt or PDB available at http: / / www.rcsb.org / pdb / home / home.do; features describing the domain properties of the source protein containing the peptide, such as: secondary or tertiary structure (e.g., alpha helices versus beta sheets); alternative splicing; the probability that the peptide will be presented by the source protein encoding the neoantigen in question in other different subjects; the probability that the peptide will not be detected or is overrepresented by mass spectrometry due to technical bias; the expression of various gene modules / pathways that provide information about the state of tumor cells, stroma, or tumor infiltrating lymphocytes (TILs) as measured by RNASeq (which does not require the source protein containing the peptide); the copy number of the source gene encoding the neoantigen in tumor cells; the probability of peptide binding to TAP or the measured or predicted binding affinity of the peptide to TAP; the expression level of TAP in tumor cells (which can be measured by RNA-seq, proteomic mass spectrometry, immunohistochemistry);The presence or absence of tumor mutations, including but not limited to: known cancer driver genes (e.g., EGFR, KRAS, ALK, RET, ROS1, TP53, CDKN2A, CDKN2B, NTRK1, NTRK2, NTRK3), and genes encoding proteins involved in the antigen presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C, TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, Driver mutations in any of the genes encoding components of the proteasome or immunoproteasome). Peptides whose presentation depends on components of the antigen presentation machinery that have undergone loss-of-function mutations in the tumor have a reduced probability of presentation; the presence or absence of functional germline polymorphisms, including but not limited to: in genes encoding proteins involved in the antigen presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C, TAP-1, TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB, HLA-DO, HLA-DOA, HLA-DOB, HLA-DP, HLA-DPA1, HLA -DPB1, HLA-DQ, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 or any of the genes encoding proteasome or immunoproteasome components); tumor type (e.g., NSCLC, melanoma); clinical tumor subtype (e.g., squamous lung cancer vs. non-squamous); smoking history; typical expression of the source gene of the peptide in the relevant tumor type or clinical subtype, optionally stratified by driver mutations.;

[0217] The at least one alteration may be a frameshift or non-frameshift indel, a missense or nonsense substitution, a splice site alteration, a genomic rearrangement or gene fusion, or any genomic or expression alteration that produces a neoORF.

[0218] The tumor cells may be selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer and small cell lung cancer.

[0219] The methods disclosed herein may further comprise obtaining a tumor vaccine comprising the selected set of neoantigens or a subset thereof, optionally further comprising administering the tumor vaccine to a subject.

[0220] At least one of the neoantigens in the selected neoantigen set may include at least one of the following when in polypeptide form: a binding affinity to MHC of less than 1000 nM with an IC50 value, a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids for MHC class I polypeptides, and 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 that promotes proteasomal cleavage within or near the polypeptide in the parent protein sequence, and the presence of a sequence motif that promotes TAP transport. For MHC class II, there are sequence motifs located within or near the peptide that promote cleavage by HLA binding catalyzed by extracellular or lysosomal proteases (eg, cathepsins) or HLA-DM.

[0221] Disclosed herein is a method for identifying one or more neoantigens that may be presented on a tumor cell surface of a tumor cell, the method comprising performing the following steps: receiving mass spectrometry data comprising data associated with a plurality of isolated peptides eluted from a major histocompatibility complex (MHC) derived from a plurality of fresh or frozen tumor samples; obtaining a training data set by at least identifying a set of training peptide sequences present in the tumor samples and presented on one or more MHC alleles associated with each training peptide sequence; obtaining a set of training protein sequences based on the training peptide sequences; and training a set of numerical parameters of a presentation model using the training protein sequences and the training peptide sequences, the presentation model providing a plurality of numerical probabilities that a peptide sequence from a tumor cell is presented by one or more MHC alleles on the tumor cell surface.

[0222] The presentation model can represent the dependency between: the presence of a pair of a specific one of the MHC alleles and a specific amino acid at a specific position in a peptide sequence; and the likelihood that such a peptide sequence comprising the specific amino acid at the specific position will be presented on the surface of a tumor cell by the specific one of the MHC alleles of the pair.

[0223] The methods disclosed herein may further comprise selecting a subset of neoantigens, wherein the subset of neoantigens are selected due to an increased likelihood of each being presented on the surface of a tumor cell relative to one or more different tumor neoantigens.

[0224] The methods disclosed herein may further comprise selecting a subset of neoantigens, wherein the subset of neoantigens are selected due to an increased likelihood that each of the neoantigens is capable of eliciting a tumor-specific immune response in a subject relative to one or more different tumor neoantigens.

[0225] The methods disclosed herein may further comprise selecting a subset of neoantigens, wherein the subset of neoantigens is selected for an increased likelihood that each can be presented on a naive T cell by a professional antigen presenting cell (APC) relative to one or more different tumor neoantigens, optionally wherein the APC is a dendritic cell (DC).

[0226] The methods disclosed herein may further comprise selecting a subset of neoantigens, wherein the subset of neoantigens are selected due to a reduced likelihood of each being inhibited via central or peripheral resistance relative to one or more different tumor neoantigens.

[0227] The methods disclosed herein may also include selecting a subset of neoantigens, wherein the subset of neoantigens is selected because of a reduced likelihood that each of the neoantigens is capable of inducing an autoimmune response against normal tissue in a subject relative to one or more different tumor neoantigens.

[0228] The methods disclosed herein may further comprise selecting a subset of neoantigens, wherein the subset of neoantigens are selected due to a reduced likelihood that each will be differentially post-translationally modified in a tumor cell relative to an APC, optionally wherein the APC is a dendritic cell (DC).

[0229] Unless otherwise indicated, the practice of the methods herein will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, ed., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd edition. (Plenum Press) Volumes A and B (1992).

[0230] III. Identification of Tumor-Specific Mutations in Neoantigens

[0231] Also disclosed herein is a method for identifying certain mutations (e.g., variants or alleles present in cancer cells). Specifically, these mutations may be present in the genome, transcriptome, proteome, or exome of cancer cells in subjects with cancer, rather than in normal tissues of the subject. Specific methods for identifying neoantigens (including shared neoantigens) specific to tumors are known to those skilled in the art, such as U.S. Patent No. 10,055,540, U.S. Application Publication No. US20200010849A1, and International Patent Application Publication WO / 2018 / 195357 and WO / 2018 / 208856, which are each incorporated herein by reference in their entirety for all purposes.

[0232] If a genetic mutation in a tumor causes a change in the amino acid sequence of a protein that is unique to the tumor, it is considered to be useful for immune targeting of the tumor. Useful mutations include: (1) non-synonymous mutations, resulting in different amino acids in the protein; (2) read-through mutations, in which the stop codon is modified or deleted, resulting in the translation of a longer protein with a novel tumor-specific sequence at the C-terminus; (3) splice site mutations, resulting in the inclusion of introns in the mature mRNA and thus resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements, resulting in a chimeric protein with a tumor-specific sequence at the junction of two proteins (i.e., gene fusion); (5) frameshift mutations or deletions, resulting in a new open reading frame with a novel tumor-specific protein sequence. Mutations may also include one or more of non-frameshift insertions and deletions, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that produce neoORFs.

[0233] Peptides or mutant polypeptides with mutations resulting from, for example, splice site, frameshift, read-through or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA or protein in tumor and normal cells.

[0234] Mutations may also include previously identified tumor-specific mutations. Known tumor mutations can be found in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.

[0235] A variety of methods can be used to detect the presence of specific mutations or alleles in the DNA or RNA of an individual. Advances in the art have provided accurate, easy and cheap large-scale SNP genotyping. For example, several techniques have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrophosphate sequencing, oligonucleotide specific ligation, TaqMan system and various DNA "chip" technologies, such as Affymetrix SNP chips. These methods utilize target gene regions that are usually amplified by PCR. There are still other methods, based on the production of small signal molecules by invasive cutting, followed by mass spectrometry or immobilized padlock probes and rolling circle amplification. Several methods known in the art for detecting specific mutations are summarized below.

[0236] Detection means based on PCR can include multiple markers of multiple amplification simultaneously. For example, it is well known in the art to select PCR primers to produce PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, different markers can be amplified by primers that are differentially labeled and can therefore be detected by differences. Of course, detection means based on hybridization allow differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiple analysis of multiple markers.

[0237] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. For example, single base polymorphisms can be detected by using specialized exonuclease-resistant nucleotides, as disclosed in, for example, Mundy, CR (U.S. Patent No. 4,656,127). According to the method, a primer complementary to the allele sequence immediately adjacent to the 3′ of the polymorphic site is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to the specific exonuclease-resistant nucleotide derivative present, the derivative will be incorporated into the end of the hybridization primer. Such incorporation makes the primer resistant to exonucleases, thereby allowing its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer is already resistant to exonucleases reveals that the nucleotides present in the polymorphic site of the target molecule are complementary to the nucleotide derivatives used in the reaction. The advantage of this method is that it does not require the determination of a large amount of unrelated sequence data.

[0238] Solution-based methods can be used to determine the identity of the nucleotide at the polymorphic site. Cohen, D. et al. (French Patent 2,650,840; PCT Application No. WO91 / 02087). For example, in the Mundy method of U.S. Patent No. 4,656,127, a primer complementary to the allele sequence immediately 3' to the polymorphic site is used. The method uses a labeled dideoxynucleotide derivative to determine the identity of the nucleotide at the site, which will be incorporated at the end of the primer if it is complementary to the nucleotide at the polymorphic site.

[0239] An alternative method, called genetic locus analysis or GBA, is described by Goelet, P. et al. (PCT Application No. 92 / 15712). The method of Goelet, P. et al. uses a mixture of labeled terminators and primers complementary to the 3' sequence of the polymorphic site. The incorporated labeled terminator is thus determined by and complementary to the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Application No. WO91 / 02087), the method of Goelet, P. et al. can be a heterogeneous assay in which either the primers or the target molecule are immobilized on a solid phase.

[0240] Several primer-directed nucleotide incorporation procedures for determining polymorphic sites in DNA have been described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C. et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they utilize the incorporation of labeled deoxynucleotides to distinguish bases at polymorphic sites. In this format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms occurring in a run of the same nucleotide can produce a signal proportional to the length of the run (Syvanen, A.-C. et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0241] Many schemes directly obtain sequence information in parallel from millions of individual DNA or RNA molecules. Real-time single-molecule synthesis sequencing technology relies on the detection of fluorescent nucleotides because they are incorporated into the nascent chain of DNA complementary to the template being sequenced. In one method, oligonucleotides of 30-50 bases in length are covalently anchored to a glass cover slide at the 5′ end. These anchor chains perform two functions. First, if the template is configured to have a capture tail complementary to the oligonucleotide bound to the surface, it serves as a capture site for the target template chain. They also serve as primers for primer extension guided by templates, forming the basis for sequence reading. Capture primers serve as fixed sites so that multiple cycles of synthesis, detection and chemical cutting dye-joints are used to remove dyes for sequence determination. Each cycle consists of adding polymerase / labeled nucleotide mixtures, washing, imaging and dye cutting. In an alternative method, polymerase is modified with fluorescent donor molecules and fixed on a slide, and each nucleotide is color-coded with an acceptor fluorescent moiety connected to γ-phosphate. The system detects the interaction between a fluorescently labeled polymerase and fluorescently modified nucleotides as the nucleotides are incorporated into the de novo chain.Other sequencing-by-synthesis technologies also exist.

[0242] Any suitable synthetic sequencing platform can be used to identify mutations. As mentioned above, four major synthetic sequencing platforms are currently available: the genome sequencer from Roche / 454Life Sciences, the 1G analyzer from Illumina / Solexa, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. The synthetic sequencing platform has also been described by Pacific BioSciences and VisiGen Biotechnologies. In some embodiments, a plurality of nucleic acid molecules sequenced are bound to a support (e.g., a solid support). In order to fix the nucleic acid on the support, a capture sequence / universal priming site can be added at the 3′ and / or 5′ end of the template. Nucleic acid can be bound to the support by hybridizing the capture sequence with a complementary sequence covalently attached to the support. The capture sequence (also referred to as a universal capture sequence) is a nucleic acid sequence complementary to the sequence attached to the support, which can double as a universal primer.

[0243] As an alternative to a capture sequence, one member of a coupling pair (e.g., antibody / antigen, receptor / ligand, or an avidin-biotin pair as described, for example, in U.S. Patent Application No. 2006 / 0252077) can be attached to each fragment for capture on a surface coated with the corresponding second member of the coupling pair.

[0244] After capture, the sequence can be analyzed, for example, by single molecule detection / sequencing, for example, as described in the Examples and U.S. Patent No. 7,283,337, including template-dependent synthesis sequencing. In synthesis sequencing, surface-bound molecules are exposed to multiple labeled nucleotide triphosphates in the presence of a polymerase. The sequence of the template is determined by the order of the labeled nucleotides incorporated into the 3' end of the growing chain. This can be performed in real time or in a step-and-repeat mode. For real-time analysis, different optical labels can be incorporated into each nucleotide and multiple lasers can be used to stimulate the incorporated nucleotides.

[0245] Sequencing can also include other massively parallel sequencing or next generation sequencing (NGS) technologies and platforms. Additional examples of massively parallel sequencing technologies and platforms are Illumina HiSeq or MiSeq, Thermo PGM or Proton, Pac Bio RSII or Sequel, Qiagen's Gene Reader and Oxford Nanopore MinION. Other similar current massively parallel sequencing technologies, as well as the progeny of these technologies, can be used.

[0246] Any cell type or tissue can be used to obtain nucleic acid samples for the methods described herein. For example, DNA or RNA samples can be obtained from a tumor or a body fluid, such as blood or saliva obtained by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on a dry sample (e.g., hair or skin). In addition, a sample can be obtained from a tumor for sequencing and another sample can be obtained from a normal tissue for sequencing, wherein the normal tissue has the same tissue type as the tumor. A sample can be obtained from a tumor for sequencing and another sample can be obtained from a normal tissue for sequencing, wherein the normal tissue has a different tissue type relative to the tumor.

[0247] The tumor may include one or more of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.

[0248] Alternatively, protein mass spectrometry can be used to identify or verify the presence of mutant peptides that bind to MHC proteins on tumor cells.Peptides can be acid eluted from tumor cells or from tumor immunoprecipitated HLA molecules and then identified using mass spectrometry.

[0249] IV. Antigens

[0250] Antigens may include nucleotides or polypeptides. For example, an antigen may be an RNA sequence encoding a polypeptide sequence. Antigens that can be used in vaccines may therefore include nucleic acid sequences or polypeptide sequences.

[0251] Disclosed herein are isolated peptides comprising tumor-specific mutations identified by the methods disclosed herein, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods disclosed herein. A new antigenic peptide can be described in the context of its coding sequence, wherein the new antigen includes a nucleic acid sequence (e.g., DNA or RNA) encoding a related polypeptide sequence.

[0252] Also disclosed herein are peptides derived from any polypeptide known or found to have altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, such as any polypeptide known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides for which antigenic peptides can be obtained can be found, for example, in the COSMIC database. COSMIC curates comprehensive information on somatic mutations in human cancers. The peptides contain tumor-specific mutations.

[0253] The modified adenoviral vectors and other constructs described herein can be used to deliver antigens from any organism, including toxins or other byproducts thereof, to prevent and / or treat infection or other adverse reactions associated with the organism or its byproducts.

[0254] Antigens that can be incorporated into vaccines (e.g., encoded in a box) include immunogens that can be used to immunize humans or non-human animals against viruses, such as pathogenic viruses that infect humans and non-human vertebrates. Antigens can be selected from a variety of virus families. Examples of ideal virus families for which immune responses are expected include the Picornaviridae family, including the rhinovirus genus, which causes about 50% of common cold cases; the Enterovirus genus, including poliovirus, coxsackievirus, echovirus, and human enterovirus, such as hepatitis A virus; and the Foot-and-Mouth Disease virus genus, which mainly causes foot-and-mouth disease in non-human animals. In the viruses of the Picornaviridae family, target antigens include VP1, VP2, VP3, VP4, and VPG. Another virus family includes the Calicivirus family, which covers the Norwalk virus group, which is an important pathogen of epidemic gastroenteritis. Another ideal virus family for targeting antigens to induce immune responses in humans and non-human animals is the togavirus family, which includes the alphavirus genus, including Sindbis virus, Ross River virus, and Eastern and Western Venezuelan equine encephalitis viruses; and the rubivirus genus, including rubella virus. Flaviviridae includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Other target antigens can be generated from the hepatitis C or coronavirus family, which includes many non-human viruses, such as infectious bronchitis virus (poultry), porcine transmissible gastroenteritis virus (pigs), porcine hemagglutinating encephalomyelitis virus (pigs), feline infectious peritonitis virus (cats), feline enteric coronavirus (cats), canine coronavirus (dogs), and human respiratory coronaviruses, which may cause the common cold and / or non-A, B, or C hepatitis. In the coronavirus family, target antigens include E1 (also known as M or matrix protein), E2 (also known as S or spike protein), E3 (also known as HE or hemagglutinin-elterose) glycoprotein (not present in all coronaviruses), or N (nucleocapsid). Other antigens can also target the rhabdovirus family, including the vesicular virus genus (e.g., vesicular stomatitis virus) and the lyssavirus genus (e.g., rabies). In the rhabdovirus family, suitable antigens can be derived from the G protein or the N protein. The filoviridae family, including hemorrhagic fever viruses such as Marburg virus and Ebola virus, can be a suitable source of antigens. The paramyxovirus family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubella virus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, morbilliviruses, which include measles and canine distemper, and pneumoviruses, including respiratory syncytial virus (e.g., sugar (G) protein and fusion (F) protein, sequences of which can be obtained from GenBank).Influenza viruses are classified as orthomyxoviruses and may be suitable sources of antigens (e.g., HA protein, N1 protein). The bunyavirus family includes the genus bunyavirus (California encephalitis, La Crosse), phlebovirus (Rift Valley fever), hantavirus (puremala is a hemorrhagic fever virus), nairovirus (Nairobi sheep disease), and various unspecified bugaviruses. The rhinovirus family provides a source of antigens for LCM and Lassa fever viruses. The reovirus family includes the genus reovirus, rotavirus (can cause acute gastroenteritis in children), orbivirus, and cultivirus (Colorado tick fever, Lebombo (human), equine organic encephalopathy, blue tongue). The retrovirus family includes the Enterovirinae, covering human and veterinary diseases such as feline leukemia virus, HTLVI and HTLVII, lentiviruses (including human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus and spumavirinal subfamily (spumavirinal)). Among lentiviruses, many suitable antigens have been described and can be easily selected. Examples of suitable HIV and SIV antigens include, but are not limited to, gag, pol, Vif, Vpx, VPR, Env, Tat, Nef and Rev proteins, and various fragments thereof. For example, a suitable fragment of the Env protein may include any of its subunits, such as gp120, gp160, gp41 or a smaller fragment thereof, such as at least about 8 amino acids in length. Similarly, a fragment of the tat protein may be selected. [See U.S. Patent No. 5,891,994 and U.S. Patent No. 6,193,981]. See also the HIV and SIV proteins described in DH Barouch et al., J. Virol., 75(5):2462-2467 (March 2001) and RR Amara et al., Science, 292:69-74 (April 6, 2001). In another example, HIV and / or SIV immunogenic proteins or peptides can be used to form fusion proteins or other immunogenic molecules. See, for example, the HIV-1 Tat and / or Nef fusion proteins and immunization protocols described in WO01 / 54719 published on August 2, 2001 and WO99 / 16884 published on April 8, 1999. The present invention is not limited to the HIV and / or SIV immunogenic proteins or peptides described herein. In addition, various modifications of the proteins have been described or can be readily made by those skilled in the art. See, for example, the modified gag proteins described in U.S. Pat. No. 5,972,596. Furthermore, any desired HIV and / or SIV immunogens may be delivered alone or in combination.These combinations may include expression from a single vector or from multiple vectors. The papovavirus family includes polyomavirus subfamily (BKU and JCU viruses) and papillomavirus subfamily (associated with the malignant progression of cancer or papilloma). The adenovirus family includes viruses that cause respiratory diseases and / or enteritis (EX, AD7, ARD, OB). Parvoviridae feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus and porcine parvovirus. The herpesvirus family includes alpha herpesvirus subfamily, which covers herpes simplex virus (HSVI, HSVII), varicella virus (pseudorabies, varicella zoster); and beta herpesvirus subfamily, which includes cytomegalovirus (human CMV), murine cytomegalovirus; and gamma herpesvirus subfamily, which includes lymphocytic virus, EBV (Burkitts lymphoma), infectious rhinotracheitis, Marek's disease (Marek's disease) virus and rhinovirus. The poxvirus family includes the chordopoxvirinae, which encompasses the orthopoxvirus (Variola (Smallpox) and cowpox (Vaccinia (Cowpox)), parapoxvirus, fowlpox virus, capripoxvirus, rabbitpox virus, swinepox virus, and entomopoxyirinae subfamilies. The hepacivirus family includes hepatitis B virus. An unclassified virus that may be a suitable source of antigens is hepatitis D virus. Other viral sources may include avian infectious bursal disease virus and porcine respiratory and reproductive syndrome virus. The alphavirus family includes equine arteritis virus and various encephalitis viruses.

[0255] Antigens that can be incorporated into vaccines (e.g., encoded in a cassette) also include immunogens that can be used to immunize humans or non-human animals against pathogens, including bacteria, fungi, parasitic microorganisms, or multicellular parasites that infect humans and non-human vertebrates. Examples of bacterial pathogens include pathogenic Gram-positive cocci, including pneumococci; Staphylococci; and Streptococci. Pathogenic Gram-negative cocci include meningococci; gonococci. Pathogenic enteric gram-negative bacilli include Enterobacteriaceae; pseudomonas, acinetobacteria, and eikenella; melioidosis; salmonella; shigella; haemophilus (Haemophilus influenzae, Haemophilus solnnus); moraxella; H. ducreyi (which causes chancroid); brucella; Franisella tularensis (which causes tularemia); yersinia (pasteurella); Streptobacillus moniliformis; and spirillum. Gram-positive bacilli include Listeria monocytogenes; Erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); Donovan's disease (inguinal granuloma); and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism; other Clostridia; tuberculosis; leprosy; and other mycobacteria.Examples of specific bacterial species are, but are not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Moraxella catarrhalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Salmonella typhi, Salmonella typhimurium, Salmonella cholera, and Salmonella typhimurium. choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae pleuropneumoniae and Mycoplasma gallisepticum.Pathogenic spirochetal diseases include syphilis; treponematoses: yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by highly pathogenic bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis (Cryptococcus), blastomycosis (Blastomyces), histoplasmosis (Histoplasma), and coccidioidomycosis (Coccidiodes); candidiasis (Candida), aspergillosis (Aspergillis), and mucormycosis; sporotrichosis; paracoccidioidomycosis, petriellidiosis, torculosis, mycetoma, and chromomycosis; and dermatophytosis. Rickettsial infections include typhus, Rocky Mountain spotted fever, Q fever, and Rickettsialpox. Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infection. Pathogenic eukaryotic organisms encompass pathogenic protozoa and helminths, and infections resulting therefrom include: amoebiasis; malaria; leishmaniasis (e.g., caused by Leishmania major); trypanosomiasis; toxoplasmosis (e.g., caused by Toxoplasma gondii); Pneumocystis carinii; Trichans; Toxoplasma; babesiosis; giardiasis (e.g., caused by Giardia); trichinosis (e.g., caused by Trichomonas); filariasis; schistosomiasis (e.g., caused by Schistosoma); nematodes; trematodes or flukes; and tapeworm (tapeworm) infections. Other parasitic infections can be caused by Ascaris, Trichuris, Cryptosporidium, and Pneumocystis carinii.

[0256] Also disclosed herein are peptides derived from any polypeptide associated with an infectious disease organism, an infection in a subject, or an infected cell of a subject. Antigens may be derived from nucleic acid sequences or polypeptide sequences of infectious disease organisms. The polypeptide sequences of infectious disease organisms include, but are not limited to, pathogenic peptides, viral peptides, bacterial peptides, fungal peptides, and / or parasite peptides. Infectious disease organisms include, but are not limited to, severe acute respiratory syndrome-related coronaviruses (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola virus, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), and tuberculosis.

[0257] Antigens predicted to be presented on the cell surface of cells (e.g., tumor cells, infected cells, or immune cells, including professional antigen presenting cells, such as dendritic cells) may be selected. Antigens predicted to be immunogenic may be selected.

[0258] One or more polypeptides encoded by the antigen nucleic acid sequence may include at least one of the following: an IC50 value of less than 1000 nM binding affinity to MHC, a length of 8-15, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids for MHC class I peptides, a sequence motif promoting proteasome cleavage in or near the peptide, and a sequence motif promoting TAP transport. For MHC class II peptides 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 in length, a sequence motif promoting HLA binding by extracellular or lysosomal proteases (e.g., cathepsin) cleavage or HLA-DM catalysis in or near the peptide.

[0259] One or more antigens may be presented on the surface of a tumor. One or more antigens may be presented on the surface of an infected cell.

[0260] One or more antigens may be immunogenic in a subject with a tumor, for example, capable of inducing a T cell response or a B cell response in the subject. One or more antigens may be immunogenic in a subject with or suspected of having an infection, for example, capable of causing a T cell response or a B cell response in the subject. One or more antigens may be immunogenic in a subject at risk of infection, for example, capable of causing a T cell response or a B cell response in the subject, which provides immune protection (i.e., immunity) against infection, for example, stimulating the production of memory T cells, memory B cells, or infection-specific antibodies.

[0261] One or more antigens may be capable of eliciting a B cell response, such as the production of antibodies that recognize the one or more antigens. Antibodies may recognize linear polypeptide sequences or recognize secondary and tertiary structures. Thus, B cell antigens may include linear polypeptide sequences or polypeptides with secondary and tertiary structures, including but not limited to full-length proteins, protein subunits, protein domains, or any polypeptide sequence known or predicted to have secondary and tertiary structures.

[0262] In the context of vaccine production, one or more antigens that induce an autoimmune response in a subject may be disregarded for the subject.

[0263] The size of at least one antigenic peptide molecule (e.g., epitope sequence) can include, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid molecule residues, and any range derivable therein. In a specific embodiment, the antigenic peptide molecule is equal to or less than 50 amino acids.

[0264] Antigenic peptides and polypeptides may be: 15 residues or less in length and typically consist of between about 8 and about 11 residues, particularly 9 or 10 residues, for MHC class I; 6-30 residues (inclusive) for MHC class II.

[0265] If desired, longer peptides can be designed in several ways. In one case, when the presentation probability of the peptide on the HLA allele is predicted or known, the longer peptide can consist of either: (1) individually presented peptides with 2-5 amino acid extensions toward the N-terminus and C-terminus of each corresponding gene product; (2) concatenation of some or all of the presented peptides with the respective extension sequences. In another case, when sequencing reveals long (>10 residues) neoepitope sequences present in the tumor (e.g., due to frameshifting, read-through, or intron inclusion that generates novel peptide sequences), the longer peptide will consist of: (3) an entire stretch of novel tumor-specific or infectious disease-specific amino acids, thus bypassing the need to select shorter peptides for strongest HLA presentation based on calculations or in vitro testing. In both cases, the use of longer peptides allows for endogenous processing by patient cells and can result in more efficient antigen presentation and induction of T cell responses.

[0266] Antigenic peptides and polypeptides can be presented on HLA proteins. In some aspects, antigenic peptides and polypeptides are presented on HLA proteins with greater affinity than wild-type peptides. In some aspects, the IC50 of the antigenic peptide or polypeptide can be at least less than 5000nM, at least less than 1000nM, at least less than 500nM, at least less than 250nM, at least less than 200nM, at least less than 150nM, at least less than 100nM, at least less than 50nM or less.

[0267] In some aspects, the antigenic peptides and polypeptides do not induce an autoimmune response and / or induce immune tolerance when administered to a subject.

[0268] Compositions comprising at least two or more antigenic peptides are also provided. In some embodiments, the composition contains at least two different peptides. At least two different peptides may be derived from the same polypeptide. Different polypeptides mean that the peptides vary according to length, amino acid sequence or both. Peptides may be derived from any polypeptide known or found to contain tumor-specific mutations, or peptides may be derived from any polypeptides with altered expression in tumor cells or cancerous tissues compared to normal cells or tissues, such as any polypeptides known or found to be abnormally expressed in tumor cells or cancerous tissues compared to normal cells or tissues. Suitable polypeptides for obtaining antigenic peptides may be found, for example, in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database. COSMIC curates comprehensive information on somatic mutations in human cancers. AACRGENIE summarizes and links clinical-grade cancer genome data to clinical outcomes from tens of thousands of cancer patients. In some aspects, tumor-specific mutations are driver mutations for specific cancer types. The peptides can be derived from any polypeptide known or suspected to be associated with an infectious disease organism, or from any polypeptide known or found to have altered expression in infected cells compared to normal cells or tissues (e.g., infectious disease polynucleotides or polypeptides, including infectious disease polynucleotides or polypeptides whose expression is restricted to host cells).

[0269] Antigenic peptides and polypeptides with desired activities or properties can be modified to provide certain desired attributes, such as improved pharmacological characteristics, while increasing or at least retaining the biological activity of substantially all unmodified peptides in binding to desired MHC molecules and activating appropriate T cells. For example, antigenic peptides and polypeptides can be subjected to various changes, such as conservative or non-conservative substitutions, wherein such changes can provide certain advantages in their use, such as improved MHC binding, stability or presentation. Conservative substitution means replacing an amino acid residue with another amino acid residue that is biologically and / or chemically similar (e.g., a hydrophobic residue replaces another amino acid residue, or a polar residue replaces another amino acid residue). Substitutions include the following 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 probed using D-amino acids. Such modifications can be made using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany and Merrifield, The Peptides, Gross and Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2nd ed. (1984).

[0270] Peptides and polypeptides modified with various amino acid mimetics or unnatural amino acids can be particularly useful in improving the in vivo stability of peptides and polypeptides. Stability can be determined in a variety of ways. For example, peptidases and various biological media (such as human plasma and serum) have been used to test stability. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11: 291-302 (1986). The half-life of peptides can be conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows. Pooled human serum (AB type, 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, a small amount of the reaction solution is removed and added to a 6% trichloroacetic acid or ethanol aqueous solution. The turbid reaction sample is cooled (4° C.) for 15 minutes, and then the precipitated serum protein is spun. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatographic conditions.

[0271] Peptides and polypeptides can be modified to provide the desired attributes in addition to improved serum half-life. For example, the ability of peptides to induce CTL activity can be enhanced by being connected to a sequence containing at least one epitope that can induce T helper cell reactions. Immunogenic peptide / T helper cell conjugates can be connected by spacer molecules. Spacers are usually composed of relatively small neutral molecules (such as amino acids or amino acid mimetics), which are basically uncharged under physiological conditions. Spacers are usually selected from other neutral spacers such as Ala, Gly or non-polar amino acids or neutral polar amino acids. It should be understood that the optional spacer does not need to be composed of the same residues, and therefore can be a hetero-oligomer or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the peptide can be connected to the T helper peptide without a spacer.

[0272] The antigenic peptide can be linked to the T helper peptide directly or via a spacer at the amino or carboxyl terminus of the peptide. The amino terminus of the antigenic peptide or T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

[0273] Proteins or peptides can be made by any technique known to those skilled in the art, including expressing proteins, polypeptides or peptides via 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 ordinary skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information located at the website of the National Institutes of Health. The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or known to those of ordinary skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides and peptides are known to those of ordinary skill in the art.

[0274] On the other hand, antigens include nucleic acids (e.g., polynucleotides) encoding antigenic peptides or portions thereof. Polynucleotides may be, for example, DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), single-stranded and / or double-stranded, or native or stable forms of polynucleotides, such as polynucleotides with a thiophosphate backbone, or combinations thereof, and they may or may not contain introns. On the other hand, an expression vector capable of expressing a polypeptide or portion thereof is provided. Expression vectors of different cell types are well known in the art and can be selected without excessive experimentation. In general, DNA is inserted into an expression vector (e.g., a plasmid) with appropriate orientation and expressed in the correct reading frame. If desired, DNA may be connected to appropriate transcription and translation regulatory control nucleic acid sequences recognized by the desired host, but such controls may generally be used in expression vectors. The vector is then introduced into the host via standard techniques. Guidance may be found in, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0275] V. Delivery Compositions

[0276] Also disclosed herein is an immunogenic composition, such as a vaccine composition, capable of eliciting a specific immune response (e.g., a tumor-specific immune response or an infectious disease organism-specific immune response). Vaccine compositions typically include one or more antigens selected, for example, using the methods described herein. Vaccine compositions may also be referred to as vaccines.

[0277] The vaccine may contain 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 include post-translational modifications. The vaccine may contain 1 to 100 or more 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 or more different nucleic acid sequences; 6, 7, 8, 9, 10, 11, 12, 13, or 14 different nucleic acid sequences; or 12, 13, or 14 different nucleic acid sequences. The vaccine may contain 1 to 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, or 14 different antigen sequences.

[0278] The vaccine may 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 , 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. Antigen encoding nucleic acid sequence can refer to the antigen coding portion of an antigen " box ". The feature of box is described in more detail below.

[0279] The vaccine may contain 1 to 30 different epitope encoding nucleic acid sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 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 sequence encoding an epitope may refer to the sequence of a single epitope sequence.

[0280] The vaccine may contain at least two repeats of an epitope encoding nucleic acid sequence. As used herein, "repeat sequence" refers to two or more iterations of the same nucleic acid epitope encoding nucleic acid sequence (including optional 5' linker sequences and / or optional 3' linker sequences described herein) within an antigen encoding nucleic acid sequence. In one example, the antigen encoding nucleic acid sequence portion of the box encodes at least two repeats of an epitope encoding nucleic acid sequence. In another non-limiting example, the antigen encoding nucleic acid sequence portion of the box encodes more than one different epitope, and at least one different epitope is encoded by at least two repeats of the nucleic acid sequence encoding the different epitopes (i.e., at least two different epitope encoding nucleic acid sequences). In an illustrative non-limiting example, the antigen encoding nucleic acid sequence encodes epitopes A, B, and C encoded by the epitope encoding nucleic acid sequence, i.e., epitope encoding sequence A (E A ), epitope encoding sequence B (E B ) and epitope encoding sequence C(E C ), and an exemplary antigen encoding nucleic acid sequence having at least one repeating sequence of a different epitope is illustrated by the following formula, but is not limited thereto:

[0281] - A repeating sequence of a different epitope (repeating sequence of epitope A):

[0282] E A -E B -E C -E A ;or

[0283] E A -E A -E B -E C

[0284] - Repeating sequences of multiple different epitopes (repeating sequences of epitopes A, B and C):

[0285] E A -E B -E C -E A -E B -E C ;or

[0286] E A -E A -E B -E B -E C -E C

[0287] - Multiple repeats of multiple different epitopes (repeats of epitopes A, B and C):

[0288] E A -EB -E C -E A -E B -E C -E A -E B -E C ;or

[0289] E A -E A -E A -E B -E B -E B -E C -E C -E C

[0290] The above examples are not limiting, and the antigen encoding nucleic acid sequence having at least one repeating sequence of different epitopes can encode each of the different epitopes in any order or frequency. For example, the order and frequency can be a random arrangement of different epitopes, for example, in an example having epitopes A, B and C, the formula is E A -E B -E C -E C -E A -E B -E A -E C -E A -E C -E C -E B .

[0291] Also provided herein is an antigen encoding cassette having at least one antigen encoding nucleic acid sequence described by the following formula from 5′ to 3′:

[0292] (E x -(E N n ) y ) z

[0293] wherein E represents a nucleic acid sequence comprising at least one of at least one different epitope encoding nucleic acid sequence,

[0294] n represents the number of independent different epitope encoding nucleic acid sequences and is any integer including 0,

[0295] E N represents a nucleic acid sequence comprising an independent different epitope encoding nucleic acid sequence for each corresponding n,

[0296] For each iteration of z: for each n, x=0 or 1, y=0 or 1, and at least one of x or y=1, and

[0297] z = 2 or greater, wherein the antigen encoding nucleic acid sequence comprises at least two iterations of E, given E N or a combination thereof.

[0298] Each E or E N Can independently comprise any epitope encoding nucleic acid sequence described herein. For example, each E or E N Can independently contain the formula (L5 b -N c -L3 d ) from 5' to 3' of the nucleic acid sequence, wherein N contains each E or E N Related different epitope encoding nucleic acid sequences, 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, for example see the "VA box" section.

[0299] The repeats of the epitope encoding nucleic acid sequence (including the optional 5' linker sequence and / or the optional 3' linker sequence) can be directly linearly linked to each other (e.g., as shown above in E A -E A -...). Repeats of the epitope encoding nucleic acid sequence may be separated by one or more other nucleotide sequences. In general, repeats of the epitope encoding nucleic acid sequence may be separated by nucleic acid sequences of any size suitable for use in the compositions described herein. In one example, repeats of the epitope encoding nucleic acid sequence may be separated by separate different epitope encoding nucleic acid sequences (e.g., E shown above). A -EB-E C -E A ...). In the example where the repeat sequences are separated by a single independent different epitope encoding nucleic acid sequence and each epitope encoding nucleic acid sequence (including the optional 5' linker sequence and / or the optional 3' linker sequence) encodes a peptide of 25 amino acids in length, the repeat sequences may be separated by 75 nucleotides, for example in the example where the repeat sequences are separated by E A -E B -E A ...in the antigen encoding nucleic acid represented by E AIn one illustrative example, an antigen encoding nucleic acid having the sequence VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 74) encodes a 25-mer antigen repeating sequence Trp1 (VINTEMFVTAPDNLGYMYEVQWPGQ (SEQ ID NO: 75)) and Trp2 (TQPQIANCSVYDFFVWLHYYSVRDT (SEQ ID NO: 76)), the repeating sequence of Trp1 being separated by a 25-mer Trp2 and thus the repeating sequence of the Trp1 epitope encoding nucleic acid sequence being separated by a 75-nucleotide Trp2 epitope encoding nucleic acid sequence. In examples where the repeat sequences are separated by 2, 3, 4, 5, 6, 7, 8 or 9 separate different epitope encoding nucleic acid sequences and each epitope encoding nucleic acid sequence (including an optional 5' linker sequence and / or an optional 3' linker sequence) encodes a peptide of 25 amino acids in length, the repeat sequences may be separated by 150, 225, 300, 375, 450, 525, 600 or 675 nucleotides, respectively.

[0300] In one embodiment, different peptides and / or polypeptides or nucleic acid sequences encoding the same are selected such that the peptides and / or polypeptides are capable of binding to different MHC molecules (e.g., different MHC class I molecules and / or different MHC class II molecules). In some aspects, a vaccine composition comprises coding sequences for peptides and / or polypeptides capable of associating with the most frequently occurring MHC class I molecules and / or different MHC class II molecules. Thus, a vaccine composition may comprise different fragments capable of associating with at least 2 preferred, at least 3 preferred, or at least 4 preferred MHC class I molecules and / or different MHC class II molecules.

[0301] The vaccine composition is capable of eliciting a specific cytotoxic T cell response and / or a specific helper T cell response.

[0302] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are given below. The composition may be associated with a carrier, such as a protein or an antigen presenting cell, such as a dendritic cell (DC) capable of presenting the peptide to a T cell.

[0303] An adjuvant is any substance that is mixed into a vaccine composition to increase or otherwise modify the immune response to an antigen. The carrier may be a scaffold structure, such as a polypeptide or polysaccharide that is capable of associating with the antigen. Optionally, the adjuvant is covalently or non-covalently conjugated.

[0304] The ability of an adjuvant to increase the immune response to an antigen is usually manifested as a significant or substantial increase in an immune-mediated reaction or a decrease in disease symptoms. For example, enhancement of humoral immunity is typically manifested as a significant increase in the titer of antibodies produced against the antigen, and enhancement of T cell activity is typically manifested as an increase in cell proliferation or cellular cytotoxicity or cytokine secretion. An adjuvant can also alter the immune response, for example by changing a predominantly humoral or Th response to a predominantly cellular or Th response.

[0305] 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, ISPatch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide 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 traps, R848, β-glucan, Pam3Cys, Aquila's saponin-derived QS21 stimulator (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other specialty adjuvants, such as Ribi's Detox.Quil or Superfos. Adjuvants such as Freund's incomplete or GM-CSF are useful. Several immunoadjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11). Cytokines may also be used. Several cytokines have been directly linked to: affecting the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen presenting cells of T-lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, which is expressly incorporated herein by reference in its entirety), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6): 414-418).

[0306] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting.Other TLR binding molecules may also be used, such as TLR 7, TLR 8 and / or TLR9 that bind RNA.

[0307] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly (I: C) (e.g., poly i: C12U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives can be readily determined by those skilled in the art without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0308] The vaccine composition may contain more than one different adjuvant. In addition, the therapeutic composition may contain any adjuvant substance, including any one of the above or a combination thereof. It is also contemplated that the vaccine and adjuvant may be administered together or separately in any appropriate order.

[0309] The carrier (or excipient) may exist independently of the adjuvant. The function of the carrier may, for example, be to increase the molecular weight of a particular mutant to increase activity or immunogenicity, impart stability, increase biological activity, or increase serum half-life. In addition, the carrier may assist in presenting 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. The carrier protein may be, but is 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 use in human immunization, the carrier is generally a physiologically acceptable carrier that is acceptable to humans and safe. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be a dextran, such as agarose.

[0310] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules rather than the complete foreign antigen itself. The MHC molecules themselves are located on the cell surface of antigen presenting cells. Therefore, if there is a trimeric complex of peptide antigen, MHC molecules and APC, it is possible to activate CTL. Accordingly, if not only the peptide is used to activate CTL, but also if APC with corresponding MHC molecules is added in addition, the immune response can be enhanced. Therefore, in some embodiments, the vaccine composition additionally contains at least one antigen presenting cell.

[0311] Antigens may also be included in vaccine platforms based on viral vectors, such as vaccinia, fowlpox, self-replicating alphaviruses, marabavirus, 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 recombinant lentiviruses of any generation, designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Development by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880). Depending on the packaging capacity of the above-mentioned viral vector-based vaccine platform, this method can deliver one or more nucleic acid sequences encoding one or more antigenic peptides.The sequence may be flanked by non-mutated sequences, may be separated by a linker, or may be preceded by one or more sequences that target a subcellular compartment (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 cancerantigens 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 eliciting a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods that can be used in immunization protocols are described, e.g., in U.S. Pat. No. 4,722,848. Another vector is Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)). Based on the description herein, various other vaccine vectors that can be used for therapeutic administration or immunization of antigens, such as Salmonella typhi vectors, etc., will be apparent to those skilled in the art.

[0312] Also disclosed herein is an adenoviral vector delivery composition capable of delivering one or more payload nucleic acid sequences. The payload nucleic acid sequence may be any nucleic acid sequence desired to be delivered to the target cell. Typically, a payload is a nucleic acid sequence connected to a promoter to drive the expression of a nucleic acid sequence. The payload nucleic acid sequence may encode a polypeptide (i.e., a nucleic acid sequence that can be transcribed and translated into a protein). Typically, a payload nucleic acid sequence encoding a peptide may encode any protein desired to be expressed in a cell. Examples of proteins include, but are not limited to, antigens (e.g., MHC I class epitopes, MHC II class epitopes, or epitopes that can stimulate B cell responses), antibodies, cytokines, chimeric antigen receptors (CARs), T cell receptors, or genome editing system components (e.g., nucleases used in genome editing systems). Genome editing systems include, but are not limited to, CRISPR systems, zinc finger systems, meganuclease systems, or TALEN systems. The payload nucleic acid sequence may be non-coding (i.e., a nucleic acid sequence that can be transcribed but not translated into a protein). Typically, a non-coding payload nucleic acid sequence may be any non-coding polynucleotide desired to be expressed in a cell. Examples of non-coding polynucleotides include, but are not limited to, RNA interference (RNAi) polynucleotides (e.g., antisense oligonucleotides, shRNA, siRNA, miRNA, etc.) or genome editing system polynucleotides (e.g., guide RNA [gRNA], single guide RNA [sgRNA], trans-activating CRISPR [tracrRNA] and / or CRISPR RNA [crRNA]). The payload nucleic acid sequence may encode two or more (e.g., 2, 3, 4, 5 or more) different polypeptides (e.g., two or more different epitope sequences linked together) or contain two or more different non-coding sequence nucleic acid sequences (e.g., two or more different RNAi polynucleotides). The payload nucleic acid sequence may have a combination of a polypeptide encoding nucleic acid sequence and a non-coding nucleic acid sequence.

[0313] VA1 Box

[0314] In view of the teachings provided herein, it is within the skill of the art to select one or more antigens, clone and construct "boxes" and insert them into viral vectors. The box may have one or more payload nucleic acid sequences, such as boxes containing multiple payload nucleic acid sequences, each independently operably connected to a separate promoter and / or connected together using other polycistronic systems, such as 2A ribosomal skipping sequence elements (e.g., E2A, P2A, F2A or T2A sequences) or internal ribosome entry site (IRES) sequence elements. In a box containing more than one payload nucleic acid sequence, each payload nucleic acid sequence may contain one or more payloads, such as, each payload nucleic acid sequence may encode two or more polypeptides or contain two or more non-coding nucleic acid sequences. The box may have a combination of a polypeptide encoding nucleic acid sequence and a non-coding nucleic acid sequence.

[0315] The cassette may be an antigen cassette. "Antigen cassette" means a combination of a selected antigen or multiple antigens and other regulatory elements necessary for transcribing the antigen and expressing the transcription product. The antigen cassette may include one or more antigens. The selected one or more antigens may refer to different epitope sequences, for example, the antigen encoding nucleic acid sequence in the cassette may encode an epitope encoding nucleic acid sequence (or multiple epitope encoding nucleic acid sequences) so that the epitope is transcribed and expressed.

[0316] The effective load nucleic acid sequence or multiple effective load nucleic acid sequences can be operably connected to the regulatory components in a manner that allows transcription. Such components include conventional regulatory elements that can drive antigen expression in cells transfected with viral vectors. Therefore, the box can also contain a selected promoter, which is connected to one or more effective load nucleic acid sequences and positioned within the selected viral sequence of the recombinant vector with other optional regulatory elements.

[0317] Useful promoters may be constitutive promoters or regulated (e.g., inducible) promoters, which will be able to control the amount of payload nucleic acid sequence to be expressed, and generally the amount of peptide (e.g., antigen) in the case of a payload nucleic acid sequence. For example, a desirable promoter is the promoter of the cytomegalovirus immediate early promoter / enhancer [see, e.g., Boshart et al., Cell, 41:521-530 (1985)]. Another desirable promoter includes the Rous sarcoma virus LTR promoter / enhancer. Another promoter / enhancer sequence is the chicken cytoplasmic β-actin promoter [TA Kost et al., Nucl. Acids Res., 11 (23): 8287 (1983)]. Other suitable or desirable promoters may be selected by those skilled in the art, such as CMV, SV40, EF-1, RSV, PGK, HSA, MCK or EBV promoter sequences.

[0318] The cassette may also include nucleic acid sequences heterologous to the viral vector sequence, including sequences that signal efficient polyadenylation (poly(A), poly-A or pA) of the transcript and introns with functional splice donor and acceptor sites. A common poly-A sequence employed in an exemplary vector of the invention is a poly-A sequence derived from the papovavirus SV-40. The poly-A sequence may generally be inserted into the cassette after the payload nucleic acid sequence and before the viral vector sequence. Common intron sequences may also be derived from SV-40 and are referred to as SV-40T intron sequences. The cassette may also contain such introns, located between the promoter / enhancer sequence and the payload nucleic acid sequence. The selection of these and other common vector elements is conventional [see, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual.", 2nd edition, Cold Spring Harbor Laboratory, New York (1989) and references cited therein] and many such sequences are available from commercial and industrial sources and Genbank.

[0319] The box can have one or more payload nucleic acid sequences. For example, a given box 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 payload nucleic acid sequences. The payload nucleic acid sequences can be directly connected to each other. The payload nucleic acid sequences can also be connected to each other using a joint.

[0320] The box can have one or more payload nucleic acid sequences encoding polypeptides. For example, a given box 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 payload nucleic acid sequences encoding polypeptides. The box can have one or more payload nucleic acid sequences, wherein each payload nucleic acid sequence encodes a different polypeptide. The box can have one or more payload nucleic acid sequences, wherein each payload nucleic acid sequence encodes one or more polypeptides. The box can have one or more payload nucleic acid sequences, wherein one or more payload nucleic acid sequences encode one or more polypeptides. The polypeptides encoded by the payload nucleic acid sequences can be in any orientation relative to each other, including N to C or C to N.

[0321] The antigen cassette may have one or more antigens. For example, a given cassette may 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 connected to each other. The antigens may also be connected to each other using a linker. The antigens may be in any orientation relative to each other, including N to C or C to N.

[0322] As stated above, the cassette can be positioned in any selected deletion site in the viral vector, such as the site of the E1 gene region deletion or the E3 gene region deletion, as well as other sites that may be selected.

[0323] A cassette may be described using the following formula to describe the ordered sequence of each element, from 5' to 3':

[0324] (P a -(L5 b -N c -L3 d ) X ) Z -(P2 h -(G5 e -U f ) Y ) W -G3 g

[0325] Wherein P and P2 comprise promoter nucleic acid sequences, N comprises different epitope encoding nucleic acid sequences, L5 comprises a 5' linker sequence, L3 comprises a 3' linker sequence, G5 comprises a nucleic acid sequence encoding an amino acid linker, G3 comprises one of at least one nucleic acid sequence encoding an amino acid linker, U comprises an MHC class II antigen encoding nucleic acid sequence, wherein for each X, the corresponding Nc is an epitope encoding nucleic acid sequence, wherein for each Y, the corresponding Uf is an antigen encoding nucleic acid sequence. The compositions and ordered sequences can be further defined by selecting the number of elements present, for example, wherein a=0 or 1, wherein b=0 or 1, wherein c=1, wherein d=0 or 1, wherein e=0 or 1, wherein f=1, wherein g=0 or 1, wherein h=0 or 1, X=1 to 400, Y=0, 1, 2, 3, 4 or 5, Z=1 to 400, and W=0, 1, 2, 3, 4 or 5.

[0326] In one example, the elements present include where a=0, b=1, d=1, e=1, g=1, h=0, X=10, Y=2, Z=1, and W=1, which describes where no other promoter is present (i.e., only the promoter nucleic acid sequence provided by the RNA alphavirus backbone is present), 20 MHC class I epitopes are present, a 5' linker is present for each N, a 3' linker is present for each N, 2 MHC class II epitopes are present, a linker is present to connect the two MHC class II epitopes, a linker is present to connect the 5' ends of the two MHC class II epitopes to the 3' linker of the final MHC class I epitope, and a linker is present to connect the 3' ends of the two MHC class II epitopes to the RNA alphavirus backbone. Examples of connecting the 3' end of the cassette to the RNA alphavirus backbone include direct connection to a 3'UTR element (e.g., 3'19-ntCSE) provided by the RNA alphavirus backbone. Examples of linking the 5' end of the cassette to the RNA alphavirus backbone include direct linking to the 26S promoter sequence, the alphavirus 5'UTR, a 51-nt CSE, or a 24-nt CSE.

[0327] Other examples include: wherein a=1, describing wherein a promoter is present in addition to the promoter nucleic acid sequence provided by the RNA alphavirus backbone; wherein a=1 and Z is greater than 1, wherein multiple promoters are present in addition to the promoter nucleic acid sequence provided by the RNA alphavirus backbone, each of which drives the expression of one or more different MHC class I epitope encoding nucleic acid sequences; wherein h=1, describing wherein a separate promoter is present to drive the expression of the MHC class II antigen encoding nucleic acid sequence; and wherein g=0, describing wherein the MHC class II antigen encoding nucleic acid sequence (if present) is directly linked to the RNA alphavirus backbone.

[0328] Other examples include where each MHC class I epitope present may have a 5' linker, a 3' linker, neither, or both. In examples where more than one MHC class I epitope is present in the same cassette, some MHC class I epitopes may have both a 5' linker and a 3' linker, while others may have a 5' linker, a 3' linker, or neither. In other examples where more than one MHC class I epitope is present in the same cassette, some MHC class I epitopes may have a 5' linker or a 3' linker, while others may have a 5' linker, a 3' linker, or neither.

[0329] In instances where more than one MHC class II epitope is present in the same cassette, some of the MHC class II epitopes may have both a 5′ linker and a 3′ linker, while others may have a 5′ linker, a 3′ linker, or neither. In other instances where more than one MHC class II epitope is present in the same cassette, some of the MHC class II epitopes may have a 5′ linker or a 3′ linker, while others may have a 5′ linker, a 3′ linker, or neither.

[0330] The promoter nucleic acid sequence P and / or P2 may be identical to the promoter nucleic acid sequence provided by the RNA alphavirus backbone. For example, the promoter sequences Pn and P2 provided by the RNA alphavirus backbone may each comprise a 26S subgenomic promoter. The promoter nucleic acid sequence P and / or P2 may be different from the promoter nucleic acid sequence provided by the RNA alphavirus backbone, and may be different from each other.

[0331] 5' linker L5 can be a native sequence or a non-native sequence. Non-native sequences include, but are not limited to, AAY, RR, and DPP. 3' linker L3 can also be a native sequence or a non-native sequence. In addition, L5 and L3 can both be native sequences, both be non-native sequences, or one can be native and the other can be non-native. For each X, the amino acid linker can 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, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 200, 201, 202, 203, 204, 205 The amino acid linker can 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 for each X.

[0332] For each Y, the amino acid linker G5 can 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 , 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 amino acids in length. The amino acid linker can 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 long for each Y.

[0333] The amino acid linker G3 can 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, 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 amino acids in length. G3 can 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 long.

[0334] For each X, each N may encode an MHC class I epitope of 7-15 amino acids in length. For each X, each N may also encode an MHC class I epitope of 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 of 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.

[0335] The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less. The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less, and encodes 2 different epitope encoding nucleic acid sequences. The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less, and encodes at least 2 different epitope encoding nucleic acid sequences. The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less, and encodes 3 different epitope encoding nucleic acid sequences. The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less, and encodes at least 3 different epitope encoding nucleic acid sequences. The box of encoding the effective load nucleic acid sequence can be 700 nucleotides or less, and includes 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens.

[0336] The length of the box of the encoding payload nucleic acid sequence can be between 375-700 nucleotides. The length of the box of the encoding payload nucleic acid sequence can be between 375-700 nucleotides, and encodes 2 different epitope encoding nucleic acid sequences. The box length of the encoding payload nucleic acid sequence can be between 375-700 nucleotides, and encodes at least 2 different epitope encoding nucleic acid sequences. The length of the box of the encoding payload nucleic acid sequence can be between 375-700 nucleotides, and encodes 3 different epitope encoding nucleic acid sequences. The length of the box of the encoding payload nucleic acid sequence is between 375-700 nucleotides, and encodes at least 3 different epitope encoding nucleic acid sequences. The box length of the encoding payload nucleic acid sequence can be between 375-700 nucleotides, and includes 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens.

[0337] The length of the box encoding the effective load nucleic acid sequence can be 600, 500, 400, 300, 200 or 100 nucleotides or less. The length of the box encoding the effective load nucleic acid sequence can be 600, 500, 400, 300, 200 or 100 nucleotides or less, and encodes 2 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence can be 600, 500, 400, 300, 200 or 100 nucleotides or less, and encodes at least 2 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence can be 600, 500, 400, 300, 200 or 100 nucleotides or less, and encodes 3 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence can be 600, 500, 400, 300, 200 or 100 nucleotides or less, and encodes at least 3 different epitope encoding nucleic acid sequences. The cassette encoding the payload nucleic acid sequence may be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens.

[0338] The length of the box encoding the effective load nucleic acid sequence may be 375-600, 375-500, or 375-400 nucleotides. The length of the box encoding the effective load nucleic acid sequence may be 375-600, 375-500, or 375-400 nucleotides, and encodes 2 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence may be 375-600, 375-500, or 375-400 nucleotides, and encodes at least 2 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence may be 375-600, 375-500, or 375-400 nucleotides, and encodes 3 different epitope encoding nucleic acid sequences. The length of the box encoding the effective load nucleic acid sequence may be 375-600, 375-500, or 375-400 nucleotides, and encodes at least 3 different epitope encoding nucleic acid sequences. The cassette encoding the payload nucleic acid sequence can be 375-600, 375-500, or 375-400 nucleotides in length and include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens.

[0339] VA2 TET promoter system

[0340] Also disclosed herein is a viral vector comprising a cassette having at least one payload sequence operably linked to a regulatable promoter, the regulatable promoter being a TET promoter system, such as a TET-On system or a TET-Off system. Without wishing to be bound by theory, the TET promoter system can be used to minimize the transcription of payload nucleic acids encoded in the cassette (e.g., antigens encoded in a vaccine cassette) during viral production. The TET promoter system may include a promoter controlled by a tetracycline (TET) repressor protein (TETr). Therefore, also disclosed herein is a viral vector comprising a cassette having at least one payload sequence operably linked to a promoter controlled by a tetracycline (TET) repressor protein (TETr). The TETr sequence (tTS) may include an amino acid sequence encoded by a nucleotide sequence shown in SEQ ID NO: 63 and / or by SEQ ID NO: 62. The promoter controlled by TETr may include a 19bpTET operator (TETo) sequence TCCCTATCAGTGATAGAGA (SEQ ID NO: 60). The promoter controlled by TETr may include 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more TETo nucleic acid sequences. There are 2 or more TETo nucleic acid sequences in the promoter controlled by TETr, and the TETo sequences can be connected together. There are 2 or more TETo nucleic acid sequences in the promoter controlled by TETr, and the TETo sequences can be directly connected together. There are 2 or more TETo nucleic acid sequences in the promoter controlled by TETr, and the TETo sequences can be connected together using a joint sequence, such as a joint sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides. In one example, the joint sequence has a joint nucleotide sequence shown in SEQ ID NO: 61. Generally, the promoter controlled by TETr can use any desired promoter sequence, such as SV40, EF-1, RSV, PGK, HSA, MCK or EBV promoter sequences. The promoter controlled by TETr can use a CMV promoter sequence. The promoter controlled by TETr can use the minimal CMV promoter sequence. The TETo sequence can be upstream (5') of the promoter sequence region, where RNA polymerase binds. In an illustrative example, 7 TETo sequences are upstream (5') of the promoter sequence. The promoter controlled by TETr that is operably connected to at least one payload nucleic acid sequence and has a TETo sequence upstream of the promoter sequence region can have an ordered sequence described by the following formula, from 5' to 3':

[0341] (TL Y ) X -PN

[0342] Wherein N is a payload nucleic acid sequence, P is an RNA polymerase binding sequence of a promoter sequence operably linked to the payload nucleic acid sequence, T is a TETo nucleic acid sequence comprising a nucleotide sequence as shown in SEQ ID NO: 60, and L is a linker sequence, wherein for each X, Y=0 or 1, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In an illustrative example, X=7 and Y=1 for each X describes that 7 TETo sequences are upstream (5′) of the promoter sequence and each TETo sequence is separated by a linker.

[0343] The TETo sequence may be downstream (3') of the promoter sequence region where RNA polymerase binds. In another illustrative example, two TETo sequences are downstream (3') of the promoter sequence. A TETr-controlled promoter operably linked to at least one payload nucleic acid sequence and having a TETo sequence downstream of the promoter sequence region may have an ordered sequence described by the following formula, from 5' to 3':

[0344] P-(TL Y ) X -N

[0345] Wherein N is a payload nucleic acid sequence, P is an RNA polymerase binding sequence of a promoter sequence operably linked to the payload nucleic acid sequence, T is a TETo nucleic acid sequence comprising a nucleotide sequence as shown in SEQ ID NO: 60, and L is a linker sequence, wherein for each X, Y=0 or 1, and wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In an illustrative example, X=2 and Y=1 for each X describes that 2 TETo sequences are downstream (3′) of the promoter sequence and each TETo sequence is separated by a linker.

[0346] The virus production of the vector with the promoter controlled by TETr can use any virus production cell line engineered to express the TETr sequence (tTS), such as the 293 cell line or its derivative (such as 293F cell line) engineered to express tTS. The virus production of the vector with the promoter controlled by TETr in the cell expressing tTS can improve virus production. The virus production of the vector with the promoter controlled by TETr in the cell expressing tTS can improve virus infectivity, which is defined as the virus particles (VP) per infection unit (IU). The virus production of the vector with the promoter controlled by TETr in the cell expressing tTS can improve virus yield and / or virus infectivity, relative to the production in non-tTS expressing cells, improve at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times. Virus production of a vector with a TETr-controlled promoter in cells expressing tTS can increase virus yield and / or virus infectivity by at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold relative to production in non-tTS-expressing cells. Virus production of a vector with a TETr-controlled promoter in cells expressing tTS can increase virus yield and / or virus infectivity by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to production of a vector without a TETr-controlled promoter. Viral production of a vector with a TETr-controlled promoter in cells expressing tTS can increase viral yield and / or viral infectivity by at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold relative to production of a vector without a TETr-controlled promoter.

[0347] VB immune checkpoint

[0348] The vectors described herein, such as the C68 vectors described herein or the alphavirus vectors described herein, may include a nucleic acid encoding at least one antigen and the same or separate vectors may include a nucleic acid encoding at least one immunomodulatory factor (e.g., an antibody such as scFv) that binds and blocks the activity of an immune checkpoint molecule. The vector may include a cassette and one or more nucleic acid molecules encoding a checkpoint inhibitor.

[0349] Illustrative immune checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to one or more of the following and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160 and CGEN-15049. Illustrative immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), Ipilimumab, MK-3475 (PD-1 blocker), Nivolumab (Nivolumab) (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody) and Yervoy / Ipilimumab (anti-CTLA-4 checkpoint inhibitor). Antibody coding sequences can be engineered into a vector such as C68 using common skills in the art. Exemplary methods are described in Fang et al., Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005 May;23(5):584-90. Epub 2005 Apr 17; which is incorporated herein by reference for all purposes.

[0350] Additional considerations for VC vaccine design and manufacture

[0351] VC1. Identification of peptide sets covering all tumor subclones

[0352] Trunk peptides, meaning those peptides presented by all or most tumor subclones, may be preferentially included in the vaccine. 53Optionally, if there are no Torso peptides predicted to be presented with high probability and to be immunogenic, or if the number of Torso peptides predicted to be presented with high probability and to be immunogenic is small enough that additional non-Torso peptides can be included in the vaccine, then other peptides can be prioritized by estimating the number and identity of tumor subclones and selecting peptides to maximize the number of tumor subclones covered by the vaccine. 54 .

[0353] VC2. Antigen prioritization

[0354] After applying all of the above antigen filters, there may still be more candidate antigens available for vaccine inclusion than the vaccine technology can support. In addition, uncertainty about various aspects of antigen analysis can be retained, and there may be compromises between different characteristics of candidate vaccine antigens. Therefore, an integrated multidimensional model can be considered to replace the predetermined filters in each step of the selection process, placing the candidate antigens in a space with at least the following axes and optimizing the selection using an integrated approach.

[0355] 1. Risk of autoimmunity or tolerance (germline risk) (lower risk of autoimmunity is usually preferred)

[0356] 2. Probability of sequencing artifacts (lower probability of artifacts is usually preferred)

[0357] 3. Probability of immunogenicity (a higher probability of immunogenicity is usually preferred)

[0358] 4. Probability of presentation (a higher probability of presentation is usually preferred)

[0359] 5. Gene expression (higher expression is usually preferred)

[0360] 6. HLA gene coverage (a larger number of HLA molecules involved in antigen presentation can reduce the probability of tumors escaping immune attack through downregulation or mutation of HLA molecules)

[0361] 7. Coverage of HLA categories (covering both HLA-I and HLA-II increases the probability of treatment response and decreases the probability of tumor escape)

[0362] In addition, optionally, if the antigen is predicted to be presented by an HLA allele that is lost or inactivated in all or a portion of the patient's tumor or infected cells, the priority of the antigen can be removed from vaccination (e.g., the antigen is excluded). HLA allele loss can occur by somatic mutation, loss of heterozygosity, or homozygous loss of the locus. Methods for detecting somatic mutations of HLA alleles are well known in the art, for example (Shukla et al., 2015). Methods for detecting somatic LOH and homozygous loss (including HLA loci) are also well described. (Carter et al., 2012; McGranahan et al., 2017; Van Loo et al., 2010). If the mass spectrometry data indicates that the predicted antigen is not presented by the predicted HLA allele, the priority of the antigen can also be removed.

[0363] VD alphavirus

[0364] VD1. Alphavirus Biology

[0365] Alphavirus is a member of the Togaviridae family, and is a positive single-stranded RNA virus. Members are usually classified as the Old World, such as Sindbis, Ross River, Mayaro, Chikungunya and Semliki Forest virus, or the New World, such as Eastern Equine Encephalitis, Aola, Fort Morgan or Venezuelan Equine Encephalitis and its derivative virus strain TC-83 (Strauss Microbrial Review 1994). Natural alphavirus genome is usually about 12kb long, wherein the first two-thirds contain genes encoding non-structural proteins (nsP), and the non-structural proteins form an RNA replication complex for viral genome self-replication, and the last third contains a subgenomic expression cassette (Frolov RNA 2001) encoding the structural proteins produced by virions.

[0366] The model life cycle of alphaviruses involves several different steps (Strauss Microbrial Review 1994, Jose Future Microbiol 2009). After the virus attaches to the host cell, the virion fuses with the membrane within the endocytic compartment, resulting in the eventual release of the genomic RNA into the cytosol. The genomic RNA, which is oriented in the positive strand and contains a 5' methylguanylate cap and a 3' polyA tail, is translated to produce the nonstructural proteins nsP1-4 that form the replication complex. Early in the infection, the positive strand is then replicated by the complex into a negative strand template. In the current model, the replication complex is further processed as the infection progresses, so that the resulting processed complex is converted to transcribe the negative strand into a full-length positive strand genomic RNA and a 26S subgenomic positive strand RNA containing structural genes. Several conserved sequence elements (CSEs) of alphaviruses have been identified as potentially playing a role in various steps of RNA replication, including: the complementary sequence of the 5′ UTR in positive-strand RNA replication of negative-strand templates, the 51-nt CSE in negative-strand synthetic replication of genomic templates, the 24-nt CSE in the junction region between nsP and 26S RNA in subgenomic RNA transcription of negative strands, and the 3′ 19-nt CSE in negative-strand synthesis of positive-strand templates.

[0367] After replication of the various RNA species, viral particles are then usually assembled in the natural life cycle of the virus. The 26S RNA is translated and the resulting protein is further processed to produce structural proteins, which include capsid proteins, glycoproteins E1 and E2, and two small polypeptides E3 and 6K (Strauss 1994). Encapsidation of the viral RNA occurs, and the capsid proteins are usually specific only for the packaged genomic RNA, and then the virions assemble and bud on the membrane surface.

[0368] VD2. Alphavirus as a delivery vehicle

[0369] Alphaviruses (including alphavirus sequences, features, 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 previously been engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages, particularly in vaccine settings where heterologous antigen expression may be desired. Due to the ability to self-replicate in the host cytosol, alphavirus vectors are generally able to produce high copy numbers of expression cassettes within the cell, resulting in high levels of heterologous antigen production. In addition, the vectors are generally transient, resulting in improved biosafety and reduced induction of immune tolerance to the vectors. The public also generally lacks pre-existing immunity to alphavirus vectors compared to other standard viral vectors (e.g., human adenoviruses). Alphavirus-based vectors also generally result in cytotoxic responses to infected cells. To some extent, cytotoxicity can be important in a vaccine setting for properly eliciting an immune response to the expressed heterologous antigen. However, the degree of cytotoxicity required can be a balancing act, and thus several attenuated alphaviruses have been developed, including the TC-83 strain of VEE. Thus, examples of antigen expression vectors described herein can utilize an alphavirus backbone that allows for high levels of antigen expression, elicits a robust immune response to the antigen, does not elicit an immune response to the vector itself, and can be used in a safe manner. In addition, the antigen expression cassette can be designed to elicit different levels of immune response by optimizing the alphavirus sequences used by the vector, including but not limited to sequences derived from VEE or its attenuated derivative TC-83.

[0370] Several expression vector design strategies have been engineered using alphavirus sequences (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 heterologous gene (Frolov 1993). Thus, in addition to the native nonstructural and structural proteins, additional subgenomic RNA is produced that expresses the heterologous protein. In this system, all elements for the production of infectious virions are present, and therefore repeated rounds of infection with the expression vector in non-infected cells may occur.

[0371] Another expression vector design utilizes a helper virus system (Pushko 1997). In this strategy, structural proteins are replaced by heterologous genes. Therefore, after the self-replication of viral RNA mediated by still intact non-structural genes, the 26S subgenomic RNA provides the expression of heterologous proteins. Traditionally, the additional vectors expressing structural proteins are then supplied in trans, for example, by co-transfection of cell lines, to produce infectious viruses. The system is described in detail in USPN 8,093,021, which is incorporated herein by reference as a whole for all purposes. The helper vector system provides the benefit of limiting the possibility of forming infectious particles, thereby improving biosafety. In addition, the helper vector system reduces the total vector length, potentially improving replication and expression efficiency. Therefore, the example of the antigen expression vector described herein can utilize the alphavirus backbone replaced by the box in which the structural protein is replaced, and the resulting vector reduces the biosafety problem, while promoting effective expression due to the overall expression vector size reduction.

[0372] VD3. In vitro alphavirus production

[0373] Alphavirus delivery vectors are generally positive RNA polynucleotides. A convenient technique for producing RNA well known in the art is in vitro transcription IVT. In this technique, a DNA template of the desired vector is first produced by techniques well known to those skilled in the art, including standard molecular biology techniques such as cloning, restriction digestion, ligation, gene synthesis, and polymerase chain reaction (PCR). The DNA template contains an RNA polymerase promoter at the 5' end of the sequence desired to be transcribed into RNA. Promoters include, but are not limited to, bacteriophage polymerase promoters, such as T3, T7, or SP6. The DNA template is then incubated with an appropriate RNA polymerase, a buffer, and nucleotides (NTPs). The resulting RNA polynucleotide may optionally be further modified, including, but not limited to, adding a 5' cap structure, such as 7-methylguanosine or related structures, and optionally modifying the 3' end to include a polyadenylic acid (polyA) tail. The RNA may then be purified using techniques well known in the art, such as phenol-chloroform extraction.

[0374] VD4. Delivery via lipid nanoparticles

[0375] An 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 (e.g., certain human adenovirus systems), or in the form of immunity to the vector that develops after vaccine administration. The latter is an important consideration if multiple administrations of the same vaccine are to be performed (e.g., separate priming and boosting doses), or if different cassettes are delivered using the same vaccine vector system.

[0376] In the case of alphavirus vectors, the standard delivery method is the helper virus system discussed previously, which provides the capsid, E1 and E2 proteins in trans to produce infectious viral particles. However, it is important to note that the E1 and E2 proteins are often the primary targets of neutralizing antibodies (Strauss 1994). Therefore, if neutralizing antibodies target infectious particles, the efficacy of using alphavirus vectors to deliver the target antigen to target cells may be reduced.

[0377] An alternative to viral particle-mediated gene delivery is to use nanomaterials to deliver expression vectors (Riley 2017). Importantly, nanomaterial vehicles can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials may include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials. Lipids may be cationic, anionic, or neutral. The material may be synthetic or naturally derived and in some cases biodegradable. Lipids may include fats, cholesterol, phospholipids, lipid conjugates, including, but not limited to, polyethylene glycol (PEG) conjugates (pegylated lipids), waxes, oils, glycerides, and fat-soluble vitamins.

[0378] Lipid nanoparticles (LNPs) are attractive delivery systems because the amphiphilicity of lipids enables the formation of membranes and vesicle-like structures (Riley 2017). In general, these vesicles deliver expression vectors by absorbing into the membranes of target cells and releasing nucleic acids into the cytosol. In addition, LNPs can be further modified or functionalized to help target specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions generally include a limited mixture of cationic, neutral, anionic and amphoteric lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that help additional parts to attach. Lipid compositions can affect overall LNP size and stability. In one example, the lipid composition includes dilinoleyl methyl-4-dimethylaminobutyrate (MC3) and MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols or neutral lipids.

[0379] Nucleic acid vectors (e.g., expression vectors) directly exposed to serum may have several undesirable results, including nucleic acid degradation by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Therefore, encapsulation of alphavirus vectors can be used to avoid degradation while also avoiding potential off-target effects. In some instances, alphavirus vectors are completely encapsulated in a delivery vehicle, such as in the aqueous interior of LNP. Alphavirus vectors can be encapsulated in LNP by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation performed on a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-joint devices or flow focusing devices. In one example, a desired lipid formulation (e.g., a composition containing MC3 or MC3-like) is provided to a droplet generation device in parallel with an alphavirus delivery vector and other desired agents, so that the delivery vector and the desired agent are completely encapsulated in the LNP interior based on MC3 or MC3-like. In one example, the droplet generation device can control the size range and size distribution of the LNP produced. For example, the size of the LNP can range from 1 to 1000 nanometers in diameter, such as 1, 10, 50, 100, 500, or 1000 nanometers.After droplet generation, the delivery vehicle encapsulating the expression vector can be further processed or modified to make it ready for administration.

[0380] VE Chimpanzee Adenovirus (ChAd)

[0381] VE1. Viral delivery using chimpanzee adenovirus

[0382] Vaccine compositions for delivery of one or more antigens (e.g., via a cassette encoding one or more antigens or neoantigens) can be produced by providing chimpanzee-derived adenovirus nucleic acid sequences, a variety of novel vectors, and cell lines expressing chimpanzee adenovirus genes. The nucleic acid sequence of chimpanzee C68 adenovirus (also referred to herein as ChAdV68) can be used in vaccine compositions for antigen delivery (see SEQ ID NO: 1). The use of vectors derived from C68 adenovirus is further described in detail in USPN 6,083,716, which is incorporated herein by reference in its entirety for all purposes.

[0383] In another aspect, provided herein is a recombinant adenovirus (e.g., C68) comprising a DNA sequence of a chimpanzee adenovirus and a cassette operatively connected to a regulatory sequence directing its expression. The recombinant virus is capable of infecting mammalian cells, preferably human cells, and is capable of expressing the cassette payload in the cells. In such a vector, the native chimpanzee E1 gene and / or the E3 gene and / or the E4 gene may be deleted. The cassette may be inserted into any of these gene deletion sites. The cassette may include an antigen for which an immune response elicited is desired.

[0384] In another aspect, provided herein is a mammalian cell infected with a chimpanzee adenovirus (eg, C68).

[0385] In another aspect, a novel mammalian cell line is provided that expresses a chimpanzee adenovirus gene (eg, from C68) or a functional fragment thereof.

[0386] In another aspect, provided herein is a method for delivering a cassette into a mammalian cell, comprising the steps of introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, that has been engineered to express the cassette.

[0387] Another aspect provides a method for eliciting an immune response in a mammalian host to treat cancer. The method may include administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, comprising a cassette encoding one or more antigens from a tumor targeted by the immune response.

[0388] Another aspect provides a method for eliciting an immune response in a mammalian host to treat or prevent a disease, such as an infectious disease, in a subject. The method may include the step of administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, comprising an antigenic cassette encoding one or more antigens, such as from an infectious disease targeted by the immune response.

[0389] Also disclosed is a non-simian mammalian cell expressing a chimpanzee adenovirus gene obtained from the sequence of SEQ ID NO: 1. The gene may 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.

[0390] Also disclosed is a nucleic acid molecule comprising a chimpanzee adenovirus DNA sequence 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 aspects, the nucleic acid molecule comprises SEQ ID NO: 1. In some aspects, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, lacking 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.

[0391] Also disclosed is a vector comprising a chimpanzee adenovirus DNA sequence obtained from SEQ ID NO: 1 and a cassette operably linked to one or more regulatory sequences that direct expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenovirus DNA sequence comprises at least the cis elements necessary for replication and virion encapsidation, the cis elements flanking the cassette and regulatory sequences. In some aspects, the chimpanzee adenovirus DNA sequence comprises genes selected from the group consisting of: E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 gene sequences of SEQ ID NO: 1. In some aspects, the vector may lack the E1A and / or E1B genes.

[0392] Also disclosed herein are host cells transfected with the vectors disclosed herein, such as C68 vectors engineered to express the cassette. Also disclosed herein are human cells expressing a selected gene introduced therein via introduction of the vectors disclosed herein into the cells.

[0393] Also disclosed herein is an adenoviral vector comprising: 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 may comprise an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence set forth in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 may comprise an at least partial deletion of nucleotides 34,916 to 34,942 of the sequence set forth in SEQ ID NO: 1, an at least partial deletion of nucleotides 34,952 to 35,305 of the sequence set forth in SEQ ID NO: 1, and an E4 deletion of at least partial deletion of nucleotides 35,302 to 35,642 of the sequence set forth in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 may comprise a deletion of E4 of at least nucleotides 34,980 to 36,516 of the sequence set forth in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 may comprise a deletion of E4 of at least nucleotides 34,979 to 35,642 of the sequence set forth in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 may comprise an E4 deletion of at least partial deletion of E4Orf2, complete deletion of E4Orf3, and at least partial deletion of E4Orf4 of the sequence set forth in SEQ ID NO: 1. The partially deleted E4 may comprise an E4 deletion of at least partial deletion of E4Orf2, at least partial deletion of E4Orf3, and at least partial deletion of E4Orf4. The partially deleted E4 may comprise an E4 deletion of at least partial deletion of E4Orf1, complete deletion of E4Orf2, and at least partial deletion of E4Orf3. The partially deleted E4 may include an E4 deletion in which E4Orf2 is at least partially deleted and E4Orf3 is at least partially deleted. 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 near the start site of E4Orf1. The partially deleted E4 may be an E4 deletion near the start site of E4Orf2. The partially deleted E4 may be an E4 deletion near the start site of E4Orf3. The partially deleted E4 may be an E4 deletion near the start site of E4Orf4.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 nucleotides or less. The E4 deletion may be at least 1550 nucleotides or less.

[0394] The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305 of the sequence shown in SEQ ID NO: 1, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, and lacks at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1. The partially deleted E4 gene may be the E4 gene sequence shown in SEQ ID NO: 1, and lacks at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1. The adenoviral vector with a partially deleted E4 gene may have a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence. The adenoviral vector with a partially deleted E4 gene may have one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO: 1. The adenoviral vector with a partially deleted E4 gene may have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3′ to nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO: 1, corresponding to an E3 deletion. The adenoviral vector with a partially deleted E4 gene may have nucleotides 35,643 to 36,518 of the sequence set forth in SEQ ID NO: 1, and wherein the partially deleted E4 gene is 5′ to nucleotides 35,643 to 36,518.An adenoviral vector having a partially deleted E4 gene may have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO:1, wherein the partially deleted E4 gene is 3′ of nucleotides 2 to 34,916, which additionally lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion, and lack nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion. An adenoviral vector having a partially deleted E4 gene may have nucleotides 2 to 34,916 of the sequence set forth in SEQ ID NO:1, wherein the partially deleted E4 gene is 3′ of nucleotides 2 to 34,916, and nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence set forth in SEQ ID NO:1, corresponding to an E1 deletion, and lack nucleotides 27,125 to 31,825 of the sequence set forth in SEQ ID NO:1, corresponding to an E3 deletion, and have nucleotides 35,643 to 36,518 of the sequence set forth in SEQ ID NO:1, and wherein the partially deleted E4 gene is 5′ of nucleotides 35,643 to 36,518.

[0395] The partially deleted E4 gene may be an E4 gene sequence as shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1, nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3′ of nucleotides 2 to 34,916, and nucleotides 2 to 34,916 further lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and lacks nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion, and has nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, and wherein the partially deleted E4 gene is 5′ of nucleotides 35,643 to 36,518.

[0396] Adenoviral vectors with a partially deleted E4 gene may have

[0397] Also disclosed herein is a method for delivering a cassette to a mammalian cell, the method comprising introducing into the cell an effective amount of a vector disclosed herein, such as a C68 vector engineered to express a cassette.

[0398] Also disclosed herein is a method for production, comprising introducing the vector disclosed herein into mammalian cells, culturing the cells under suitable conditions and producing the antigen.

[0399] VE2. Complementation cell line expressing E1

[0400] To generate a recombinant chimpanzee adenovirus (Ad) that lacks 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 complementing or packaging cell line). For example, to generate a replication-defective chimpanzee adenovirus vector, a cell line expressing the E1 gene product of a human or chimpanzee adenovirus can be used; such a cell line can include HEK293 or a variant thereof. The protocol for generating a cell line expressing the chimpanzee E1 gene product (Examples 3 and 4 of USPN 6,083,716) can be followed to generate a cell line expressing any selected chimpanzee adenovirus gene.

[0401] The AAV enhancement assay can be used to identify cell lines that express chimpanzee adenovirus El. This assay can be used to identify El function in cell lines prepared by using, for example, El genes from other uncharacterized adenoviruses from other species. The assay is described in Example 4B of USPN 6,083,716.

[0402] The selected chimpanzee adenovirus gene (e.g., E1) can be used for expression in the selected parental cell line under the transcriptional control of a promoter. Inducible or constitutive promoters can be used for this purpose. Among the inducible promoters are the sheep metallothionein promoter, which can be induced by zinc, or the mouse mammary tumor virus (MMTV) promoter, which can be induced by glucocorticoids, particularly dexamethasone. Other inducible promoters, such as those identified in International Patent Application WO 95 / 13392, which is incorporated herein by reference, can also be used to generate packaging cell lines. Constitutive promoters that control the expression of chimpanzee adenovirus genes can also be used.

[0403] Parent cells can be selected to produce novel cell lines expressing any desired C68 gene. Such parent cell lines can be, but are not limited to, HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72], and WI-38 [CCL 75] 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.

[0404] Cell lines expressing E1 can be used to generate recombinant chimpanzee adenovirus E1-deficient vectors. Cell lines expressing one or more other chimpanzee adenovirus gene products constructed using essentially the same procedures are used to generate recombinant chimpanzee adenovirus vectors that are deficient in the genes encoding those products. In addition, cell lines expressing other human AdE1 gene products can also be used to generate chimpanzee recombinant Ad.

[0405] VE3. Recombinant viral particles as vectors

[0406] Compositions disclosed herein may include viral vectors that deliver at least one antigen to cells. Such vectors include chimpanzee adenovirus DNA sequences (e.g., C68) and cassettes that are operably connected to regulatory sequences that direct cassette expression. C68 vectors are capable of expressing cassettes in infected mammalian cells. C68 vectors may functionally delete one or more viral genes. The cassettes include at least one antigen under the control of one or more regulatory sequences (e.g., promoters). Optional helper viruses and / or packaging cell lines may supply any necessary products of the adenovirus genes deleted to chimpanzee virus vectors.

[0407] The term "functional loss" means removing or otherwise changing (e.g., by mutation or modification) a sufficient amount of a gene region so that the gene region is no longer able to produce a functional product expressed by one or more genes. Mutations or modifications that may result in functional loss include, but are not limited to, nonsense mutations, such as the introduction of premature stop codons and the removal of typical and atypical start codons, mutations that change mRNA splicing or other transcriptional processing, or a combination thereof. If desired, the entire gene region may be removed.

[0408] Modifications of the nucleic acid sequences forming the vectors disclosed herein, including sequence deletions, insertions, and other mutations, can be made using standard molecular biology techniques and are within the scope of the present invention.

[0409] VE4. Construction of viral plasmid vector

[0410] Chimpanzee adenovirus C68 vectors useful in the present invention include recombinant defective adenoviruses, i.e., chimpanzee adenovirus sequences that are functionally deleted in the E1a or E1b gene and optionally carry other mutations (e.g., temperature-sensitive mutations or deletions in other genes). It is expected that these chimpanzee sequences can also be used to form hybrid vectors from other adenovirus and / or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses are described in the open literature [see, e.g., Kozarsky I and II cited above, and references cited therein, U.S. Pat. No. 5,240,846].

[0411] In constructing useful chimpanzee adenovirus C68 vectors that can be used to deliver cassettes to human (or other mammalian) cells, a series of adenovirus nucleic acid sequences can be used for the vectors. Vectors containing minimal chimpanzee C68 adenovirus sequences can be used in conjunction with a helper virus to produce infectious recombinant viral particles. The helper virus provides the essential gene products required for viral infectivity and propagation of minimal chimpanzee adenovirus vectors. When only one or more selected deletions of chimpanzee adenovirus genes are produced in an otherwise functional viral vector, the deleted gene products can be supplied during viral vector production by propagating the virus in a selected packaging cell line that provides the deleted gene function in trans.

[0412] VE5. Recombinant minimal adenovirus

[0413] The minimal chimpanzee Ad C68 virus is a virus particle that contains only the adenoviral cis elements necessary for replication and virion encapsidation. That is, the vector contains the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences of the adenovirus (which serve as replication origins) and the native 5' packaging / enhancer domain (which contains the sequences necessary for packaging the linear Ad genome and the enhancer element of the E1 promoter). See, for example, the techniques for preparing "minimal" human Ad vectors described in International Patent Application WO96 / 13597 and incorporated herein by reference.

[0414] VE6. Other defective adenoviruses

[0415] The recombinant replication-defective adenovirus may also contain more than the minimal chimpanzee adenovirus sequence. These other Ad vectors may be characterized by the deletion of various portions of the viral gene regions and by the formation of infectious viral particles, optionally using a helper virus and / or a packaging cell line.

[0416] For example, a suitable vector can be formed by deleting all or a sufficient portion of the C68 adenovirus immediate early gene E1a and delayed early gene E1b, thereby eliminating their normal biological functions. The replication-defective E1-deleted virus is able to replicate and produce infectious virus when grown on a complementing cell line transformed with a chimpanzee adenovirus containing functional adenoviral E1a and E1b genes that provide the corresponding trans gene products. Based on the homology of known adenoviral sequences, it is expected that the resulting recombinant chimpanzee adenovirus will be able to infect many cell types and express antigens, just like human recombinant E1-deleted adenoviruses in the art, but will not be able to replicate in most cells that do not carry chimpanzee E1 region DNA unless the cells are infected at an extremely high infection multiplicity.

[0417] As another example, all or a portion of the C68 adenovirus delayed early gene E3 can be eliminated from the chimpanzee adenovirus sequences that form part of the recombinant virus.

[0418] A chimpanzee adenovirus C68 vector can also be constructed with a deletion of the E4 gene. Another vector can contain a deletion in the delayed early gene E2a.

[0419] Deletions may also be obtained in any of the late genes L1 to L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions in the intermediate genes IX and IVa2 may be useful for some purposes. Other deletions may be obtained in other structural or nonstructural adenovirus genes.

[0420] The above deletions can be used alone, i.e., the adenovirus sequence can contain only the E1 deletion. Alternatively, deletions of complete genes or portions thereof that effectively destroy or reduce their biological activity can be used in any combination. For example, in an exemplary vector, the adenovirus C68 sequence can delete the E1 gene and the E4 gene, or delete the E1, E2a, and E3 genes, or delete the E1 and E3 genes, or delete the E1, E2a, and E4 genes with or without the deletion of E3, and so on. As discussed above, such deletions can be used in combination with other mutations (e.g., temperature-sensitive mutations) to achieve the desired results.

[0421] The cassette containing the antigen is optionally inserted into any of the deleted regions of the chimpanzee C68 Ad virus. Alternatively, if desired, the cassette can be inserted into an existing gene region to disrupt the function of that region.

[0422] VE7.Helper virus

[0423] Depending on the chimpanzee adenovirus gene content of the viral vector used to carry the cassette, a helper adenovirus or a non-replicating viral fragment may be used to provide sufficient chimpanzee adenovirus gene sequences to produce infectious recombinant viral particles containing the cassette.

[0424] 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-defective and contain a variety of adenoviral genes in addition to the sequences described above. The helper virus may be used in combination with the cell lines expressing E1 described herein.

[0425] For C68, the "helper" virus can be a fragment formed by cutting the C-terminus of the C68 genome with SspI, which removes about 1300 bp from the left end of the virus. This sheared virus is then co-transfected with plasmid DNA into a cell line expressing E1, thereby forming a recombinant virus by homologous recombination with the C68 sequence in the plasmid.

[0426] The helper virus can also form a polycation conjugate, as described in Wu et al., J. Biol. Chem., 264: 16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299: 49 (April 1, 1994). The helper virus may optionally contain a reporter gene. Many such reporter genes are known in the art. Unlike the cassette on the adenoviral vector, the presence of the reporter gene on the helper virus allows the Ad vector and the helper virus to be monitored independently. This second reporter gene is used to separate the resulting recombinant virus from the helper virus after purification.

[0427] VE8. Assembly of viral particles and infection of cell lines

[0428] The assembly of the selected DNA sequence, cassette and other vector elements of the adenovirus into various intermediate plasmids and shuttle vectors, and the use of plasmids and vectors to make recombinant virus particles can all be achieved using conventional techniques. Such techniques include conventional cloning techniques of cDNA, in vitro recombination techniques (e.g., Gibson assembly), the use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method for providing the desired nucleic acid sequence. Standard transfection and co-transfection techniques are used, such as CaPO4 precipitation techniques or liposome-mediated transfection methods, such as lipofectamine. Other conventional methods used include homologous recombination of viral genomes, plaques of viruses in agar overlays, methods for measuring signal generation, and the like.

[0429] For example, after the desired cassette-containing viral vector is constructed and assembled, the vector can be transfected into a packaging cell line in the presence of a helper virus. Homologous recombination occurs between the helper sequence and the vector sequence, which allows the adenovirus-antigen sequence in the vector to be replicated and packaged into the virion capsid, thereby producing a recombinant viral vector particle.

[0430] The resulting recombinant chimpanzee C68 adenovirus can be used to transfer the cassette into cells of choice.In in vivo experiments using recombinant virus grown in packaging cell lines, the El deleted recombinant chimpanzee adenovirus demonstrated utility in transferring the cassette into non-chimpanzee (preferably human) cells.

[0431] VE9. Uses of recombinant viral vectors

[0432] The resulting recombinant chimpanzee C68 adenovirus containing the cassette (prepared by cooperating an adenoviral vector with a helper virus or an adenoviral vector with a packaging cell line, as described above) thus provides an effective gene transfer vehicle for delivering antigens to a subject in vivo or in vitro.

[0433] The above recombinant vectors are administered to humans according to the disclosed methods of gene therapy. The chimpanzee virus vector carrying the cassette can be administered to the patient, preferably suspended in a biocompatible solution or a 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 to those skilled in the art as pharmaceutically acceptable carriers can be used for this purpose.

[0434] Chimpanzee adenoviral vectors are administered in amounts sufficient to transduce human cells and provide sufficient levels of antigen transfer and expression, thereby providing therapeutic benefit without excessive adverse effects or with medically acceptable physiological effects, which can be determined by those skilled in the art of medicine. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parenteral routes of administration. Routes of administration may be combined if desired.

[0435] The dosage of the viral vector will depend primarily on factors such as the condition being treated, the patient's age, weight and health, and may therefore vary among patients. The dosage will be adjusted to balance the therapeutic benefit with any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is employed. Antigen expression levels may be monitored to determine the frequency of dosage administration.

[0436] The recombinant replication-deficient adenovirus can be administered in a "pharmaceutically effective amount", i.e., an amount of the recombinant adenovirus that effectively transfects the desired cells in the route of administration and provides sufficient expression levels of the selected genes to provide a vaccine benefit (i.e., some measurable level of protective immunity). The C68 vector containing the cassette can be co-administered with an adjuvant. The adjuvant can be separate from the vector (e.g., alum) or encoded within the vector, particularly when the adjuvant is a protein. Adjuvants are well known in the art.

[0437] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral, and other parenteral routes of administration. If desired, the routes of administration may be combined or adjusted, depending on the immunogen or disease. For example, in rabies prevention, subcutaneous, intratracheal, and intranasal routes are preferred. The route of administration will depend primarily on the nature of the disease being treated.

[0438] The level of immunity to the antigen can be monitored to determine whether a booster is needed. For example, after assessing the antibody titer in the serum, an optional booster immunization may be needed.

[0439] VI. Treatment and Manufacturing Methods

[0440] Also provided is a method of inducing a tumor-specific immune response in a subject, vaccinating against a tumor, treating and / or ameliorating a symptom of cancer in a subject by administering one or more antigens (e.g., a plurality of antigens identified using the methods disclosed herein) to the subject.

[0441] Also provided is a method of inducing an infectious disease organism-specific immune response in a subject, vaccinating against an infectious disease organism, treating and / or ameliorating symptoms of infection associated with an infectious disease organism in a subject by administering one or more antigens (e.g., a plurality of antigens identified using the methods disclosed herein) to the subject.

[0442] In some aspects, the subject has been diagnosed with cancer or is at risk of developing cancer. The subject may be a human, dog, cat, horse, or any animal that requires a tumor-specific immune response. The tumor may be any solid tumor, such as a breast tumor, an ovarian tumor, a prostate tumor, a lung tumor, a kidney tumor, a stomach tumor, a colon tumor, a testicular tumor, a head and neck tumor, a pancreatic tumor, a brain tumor, a melanoma, and other tissue and organ tumors, and a blood tumor, such as a lymphoma and a leukemia, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0443] In some aspects, the subject has been diagnosed as having an infection or being at risk for an infection (e.g., age, geographic / travel, and / or work-related increased risk or susceptibility to infection, or at risk for seasonal and / or novel disease infection).

[0444] The antigen may be administered in an amount sufficient to induce a CTL response. The antigen may be administered in an amount sufficient to induce a T cell response. The antigen may be administered in an amount sufficient to induce a B cell response.

[0445] Antigens can be administered alone or in combination with other therapeutic agents. Therapeutic agents are, for example, chemotherapeutic agents, radiation, or immunotherapy. Any suitable therapeutic treatment can be administered for a particular cancer. Therapeutic agents can include those targeting infectious disease organisms, such as antiviral agents or antibiotic agents.

[0446] In addition, the subject may be further administered an anti-immunosuppressant / immunostimulant, such as a checkpoint inhibitor. For example, an anti-CTLA antibody or anti-PD-1 or anti-PD-L1 may be further administered to the subject. Blocking CTLA-4 or PD-L1 by an antibody may enhance the immune response to cancer cells in the patient. In particular, CTLA-4 blockade has been shown to be effective when following a vaccination regimen.

[0447] The optimal amount and optimal dosing regimen of each antigen included in the vaccine composition can be determined. For example, the antigen or its variant can be prepared for intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, intramuscular (im) injection. Injection methods include sc, id, ip, im and iv. DNA or RNA injection methods include id, im, sc, ip and iv. Other methods of administration of the vaccine composition are known to those skilled in the art.

[0448] The vaccine can be compiled so that the selection, number and / or amount of antigens present in the composition is tissue, cancer, infectious disease and / or patient specific. For example, the exact selection of peptides can be guided by the expression pattern of the parent protein in a given tissue or by the mutation or disease state of the patient. The selection can depend on the specific type of cancer, the specific infectious disease, the disease state, the vaccination goal (e.g., preventive or targeting an ongoing disease), the earlier treatment regimen, the patient's immune status, and of course the patient's HLA haplotype. In addition, the vaccine can contain individualized components according to the personal needs of a particular patient. Embodiments include changing the selection of antigens according to the expression of the antigens in a particular patient or adjusting secondary treatments after the first round or treatment regimen.

[0449] Patients can be identified to administer antigen vaccines via various diagnostic methods (e.g., patient selection methods further described below). Patient selection may involve identifying mutations in one or more genes, or expression patterns. Patient selection may involve identifying infectious diseases of ongoing infection. Patient selection may involve identifying the risk of infection of infectious diseases. In some cases, patient selection involves identifying the haplotype of the patient. Various patient selection methods can be performed in parallel, such as sequencing diagnosis can identify mutations and haplotypes of patients. Various patient selection methods can be performed in sequence, such as a diagnostic test identification mutation and another diagnostic test identification patient's haplotype, and wherein each test can be the same (e.g., both are high-throughput sequencing) or different (e.g., one is high-throughput sequencing and the other is Sanger sequencing) diagnostic method.

[0450] For compositions to be used as cancer or infectious disease vaccines, antigens with similar normal self-peptides that are expressed in large amounts in normal tissues can be avoided or present in low amounts in the compositions described herein. On the other hand, if a patient's tumor or infected cells are known to express a large amount of a certain antigen, the corresponding pharmaceutical composition for treating such cancer or infection can be present in high amounts and / or can include more than one antigen specific for such a particular antigen or such an antigenic pathway.

[0451] Compositions comprising antigens can be administered to individuals suffering from cancer or infection. In therapeutic applications, compositions are administered to patients in an amount sufficient to induce effective CTL responses to tumor antigens or infectious disease organism antigens and to cure or at least partially contain symptoms and / or complications. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose". The effective amount for this purpose will depend on, for example, the composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. It should be remembered that compositions are generally useful for serious disease states, i.e. life-threatening or potentially life-threatening situations, especially when cancer has metastasized. In such cases, in view of the minimization of foreign matter and the relative nontoxicity of antigens, the treating physician may and may feel the need to administer substantial excesses of these compositions.

[0452] For therapeutic use, administration may be initiated at the time of detection or surgical removal of a tumor or at the time of detection or treatment of an infection. Booster doses may follow until symptoms are at least substantially abated and for a period of time thereafter.

[0453] Pharmaceutical compositions (e.g., vaccine compositions) for therapeutic treatment are intended for parenteral, topical, nasal, oral or topical administration. Pharmaceutical compositions can be administered parenterally, such as intravenously, subcutaneously, intradermally or intramuscularly. The composition can be administered at the site of surgical resection to induce a local immune response against a tumor. The composition can be administered to target specific infected tissues and / or cells (e.g., antigen presenting cells) of a subject. Disclosed herein are compositions for parenteral administration, comprising a solution in which an antigen and a vaccine composition are dissolved or suspended in an acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional well-known sterilization techniques or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0454] Antigens can also be administered via liposomes, which target specific cell tissues, such as lymphoid tissue. Liposomes can also be used to increase half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. In these preparations, the antigen to be delivered is incorporated as part of the liposome alone or with molecules that are bound to receptors that are ubiquitous in, for example, lymphocytes (e.g., monoclonal antibodies that are bound to CD45 antigens) or with other therapeutic or immunogenic compositions. Therefore, liposomes filled with the desired antigen can be guided to the site of lymphocytes, where the liposomes then deliver the selected therapeutic / immunogenic composition. Liposomes can be formed by standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols (e.g., cholesterol). The selection of lipids is generally guided by considering, for example, liposome size, acid instability and stability of liposomes in the bloodstream. A variety of methods can be used to prepare liposomes as described, for example, 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.

[0455] For targeting immune cells, the ligand to be incorporated into the liposomes may include, for example, antibodies or fragments thereof that are specific for cell surface determinants of the desired immune system cells. The liposomal suspension may be administered intravenously, topically, topically, etc., at a dose that varies, inter alia, depending on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0456] For therapeutic or immunization purposes, nucleic acids encoding peptides and optionally one or more peptides described herein can also be administered to patients. A variety of methods are conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly in the form of "naked DNA". This method is described in, for example, Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patents Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described in, for example, U.S. Patent No. 5,204,253. Particles containing only DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. Methods for delivering nucleic acid sequences can include viral vectors, mRNA vectors, and DNA vectors in the presence or absence of electroporation.

[0457] Nucleic acids can also be delivered in combination with cationic compounds (e.g., cationic lipids). Lipid-mediated gene delivery methods are described in, for example, 9618372WOAWO 96 / 18372; 9324640WOAWO 93 / 24640; Mannino and Gould-Fogerite, BioTechniques 6 (7): 682-691 (1988); U.S. Pat. No. 5,279,833; Rose U.S. Pat. No. 5,279,833; 9106309WOAWO 91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

[0458] Antigens may also be included in vaccine platforms based on viral vectors, such as vaccinia, fowlpox, self-replicating alphaviruses, marabavirus, 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 recombinant lentiviruses of any generation, designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880). Depending on the packaging capacity of the above-mentioned viral vector-based vaccine platform, this method can deliver one or more nucleic acid sequences encoding one or more antigenic peptides.The sequence may be flanked by non-mutated sequences, may be separated by a linker, or may be preceded by one or more sequences targeting subcellular 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 cancerantigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20(13): 3401-10). After introduction into the host, the infected cells express the antigen, thereby eliciting a host immune (e.g., CTL) response against the peptide. Vaccinia vectors and methods that can be used in immunization protocols are described, e.g., in U.S. Pat. No. 4,722,848. Another vector is Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)). Based on the description herein, a variety of other vaccine vectors that can be used for therapeutic administration or immunization of antigens, such as Salmonella typhi vectors, etc., will be apparent to those skilled in the art.

[0459] The means of administering nucleic acids uses a minigene construct encoding one or more epitopes. To generate a DNA sequence (minigene) encoding a selected CTL epitope for expression in human cells, the amino acid sequence of the epitope is reverse translated. The human codon usage table is used to guide the codon selection for each amino acid. These epitope encoding DNA sequences are directly adjacent to each other, resulting in a continuous polypeptide sequence. To optimize expression and / or immunogenicity, other elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. In addition, MHC presentation of CTL epitopes can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitope. The minigene sequence is converted to DNA by assembling oligonucleotides encoding the positive and negative strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified, and ligated under appropriate conditions using well-known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene encoding the CTL epitope polypeptide can then be cloned into the desired expression vector.

[0460] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate buffered saline (PBS). A variety of methods have been described, and new techniques can be used. As noted above, nucleic acids are preferably formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds (collectively referred to as protection, interaction, non-condensation (PINC)) can also be complexed with purified plasmid DNA to affect variables such as stability, intramuscular dispersion or migration to specific organs or cell types.

[0461] Also disclosed is a method for making a vaccine, comprising the steps of performing the methods disclosed herein; and manufacturing a vaccine comprising a plurality of antigens or a subset of the plurality of antigens. Also disclosed is a method for making an adenoviral vector, comprising the steps of performing the methods disclosed herein; and producing an adenoviral vector comprising a box. For example, a method for making an adenoviral vector using a TET promoter system, such as a TETr-controlled promoter system described herein, is disclosed. Viral production using a TETr-controlled promoter system may include a. providing a viral vector comprising a box, the box comprising: (i) at least one payload nucleic acid sequence, and (ii) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, wherein the at least one promoter is a promoter controlled by a tetracycline (TET) repressor protein (TETr), b. providing cells engineered to express TETr proteins; and c. contacting the viral vector with the cells under conditions sufficient to produce the virus.

[0462] Antigens disclosed herein can be manufactured using methods known in the art. For example, methods for preparing antigens disclosed herein or vectors (e.g., vectors comprising at least one sequence encoding one or more antigens) may include culturing host cells under conditions suitable for expressing the antigen or vector, wherein the host cell comprises at least one polynucleotide encoding the antigen or vector, and purifying the antigen or vector. Standard purification methods include chromatographic techniques, electrophoresis, immunization, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0463] The host cell may include Chinese hamster ovary (CHO) cells, NS0 cells, yeast or HEK293 cells. The host cell may be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding an antigen or a vector disclosed herein, optionally wherein the isolated polynucleotide further comprises a promoter sequence operably linked to at least one nucleic acid sequence encoding an antigen or a vector. In certain embodiments, the isolated polynucleotide may be a cDNA.

[0464] VII. Antigen Use and Administration

[0465] Vaccination regimens can be used to administer one or more antigens to a subject. Prime vaccines and booster vaccines can be used to administer to a subject. Prime vaccines 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 booster vaccines 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 includes a box containing an antigen. The box may include about 20 antigens, which are separated by spacers (e.g., the natural sequence that usually surrounds each antigen) or other non-natural spacer sequences (e.g., AAY). The box may also include MHCII antigens, such as tetanus toxoid antigens and PADRE antigens, which can be considered as universal class II antigens. The box may also include a targeting sequence, such as a ubiquitin targeting sequence. In addition, each vaccine dose may be administered to a subject in combination with a checkpoint inhibitor (CPI) (e.g., simultaneously, before, or after). CPIs may include those that inhibit CTLA4, PD1, and / or PDL1, such as antibodies or antigen-binding portions thereof. Such antibodies may include tremelimumab or durvalumab.

[0466] The priming vaccine may be injected (e.g., intramuscularly) into the subject. Bilateral injections per dose may be used. For example, one or more injections of ChAdV68 (C68) may be used (e.g., a total dose of 1×10 12virus particles); one or more self-replicating RNA (srRNA) injections of a low vaccine dose selected from 0.001 to 1 ug RNA, particularly 0.1 or 1 ug can be used; or one or more srRNA injections of a high vaccine dose selected from 1 to 100 ug RNA, particularly 10 or 100 ug can be used.

[0467] A booster vaccine (booster vaccine) may be injected (e.g., intramuscularly) after the primary vaccination. The booster vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the primary vaccination, such as every 4 and / or 8 weeks. Bilateral injections per dose may be used. For example, one or more ChAdV68 (C68) injections (e.g., a total dose of 1×10 12 virus particles); one or more self-replicating RNA (srRNA) injections of a low vaccine dose selected from 0.001 to 1 ug RNA, particularly 0.1 or 1 ug can be used; or one or more srRNA injections of a high vaccine dose selected from 1 to 100 ug RNA, particularly 10 or 100 ug can be used.

[0468] Anti-CTLA-4 (e.g., tremelimumab) may also be administered to the subject. For example, anti-CTLA4 may be administered subcutaneously near the site of intramuscular vaccine injection (ChAdV68 priming or srRNA low dose) to ensure drainage to the same lymph node. Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4. The target anti-CTLA-4 (tremelimumab) subcutaneous dose is typically 70-75 mg (particularly 75 mg), with a dose range of, for example, 1-100 mg or 5-420 mg.

[0469] In some cases, anti-PD-L1 antibodies such as durvalumab (MEDI 4736) may be used. Durvalumab is a selective, high-affinity human IgG1 mAb that blocks PD-L1 binding to PD-1 and CD80. Durvalumab is generally administered iv at 20 mg / kg every 4 weeks.

[0470] Immune monitoring can be performed before, during and / or after vaccine administration. Such monitoring can inform safety and efficacy, among other parameters.

[0471] In order to perform immune monitoring, PBMCs are usually used. PBMCs can be separated before and after the primary vaccination (e.g., 4 and 8 weeks). PBMCs can be collected only before booster vaccinations and after each booster vaccination (e.g., 4 and 8 weeks).

[0472] T cell response can be assessed as part of an immune monitoring scheme. For example, the ability of the vaccine composition 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., stimulating the initial immune response in an initial subject) or boosting an immune response (e.g., stimulating an immune response in a subject with a pre-existing immune response to an antigen, such as a booster vaccine that enhances the immune response). T cell responses can be measured using one or more methods known in the art, such as ELISpot, intracellular cytokine staining, cytokine secretion and cell surface capture, T cell proliferation, MHC polymer staining or by cytotoxicity assay. T cell responses for epitopes encoded in vaccines can be monitored from PBMC by measuring the induction of cytokines (e.g., IFN-γ) using ELISpot assays. Specific CD4 or CD8 T cell responses for epitopes encoded in vaccines can be monitored from PBMC by using flow cytometry to measure the induction of intracellular or extracellular captured cytokines (e.g., IFN-γ). Specific CD4 or CD8 T cell responses for epitopes encoded in the vaccine can be monitored from PBMC by measuring T cell populations expressing T cell receptors specific for epitopes / MHC class I complexes using MHC multimer staining. Specific CD4 or CD8 T cell responses for epitopes encoded in the vaccine can be monitored from PBMC by measuring the ex vivo expansion of T cell populations after 3H-thymidine, bromodeoxyuridine and carboxyfluorescein-diacetate-succinimidyl ester (CFSE) are incorporated. The antigen recognition ability and lytic activity of T cells derived from PBMCs specifically for epitopes encoded in the vaccine can be functionally assessed by chromium release assays or alternative colorimetric cytotoxicity assays.

[0473] B cell responses can be measured using one or more methods known in the art, such as assays for determining B cell differentiation (e.g., differentiation into plasma cells), B cell or plasma cell proliferation, B cell or plasma cell activation (e.g., upregulation of co-stimulatory markers such as CD80 or CD86), antibody class switching, and / or antibody production (e.g., ELISA).

[0474] VIII. Antigen Identification

[0475] VIII.A. Identification of Antigen Candidates

[0476] Research methods for NGS analysis of tumor and normal exomes and transcriptomes have been described and applied to the antigen identification space. 6,14,15It is contemplated that certain optimizations for greater sensitivity and specificity for antigen identification in a clinical setting. These optimizations can be divided into two areas, those optimizations associated with laboratory methods and those optimizations associated with NGS data analysis. The described research methods may also be applied to identifying antigens in other situations, such as identifying antigens from infectious disease organisms, infection of subjects, or identification of infected cells of subjects. Optimization examples are known to those skilled in the art, such as those described in more detail in the following methods: U.S. Patent No. 10,055,540, U.S. Application Publication No. US20200010849A1, and International Patent Application Publications WO / 2018 / 195357 and WO / 2018 / 208856, each incorporated herein by reference in its entirety for all purposes.

[0477] VIII.B. Isolation and Detection of HLA Peptides

[0478] After lysis and solubilization of tissue samples, isolation of HLA-peptide molecules was performed using the classical immunoprecipitation (IP) approach (55-58). The clarified lysate was used for HLA-specific IP.

[0479] Immunoprecipitation is performed using antibodies coupled to beads, where the antibodies are specific for HLA molecules. For pan-class I HLA immunoprecipitation, a pan-class I CR antibody is used; for class II HLA-DR, an HLA-DR antibody is used. During an overnight incubation, the antibodies are covalently attached to NHS-agarose beads. After covalent attachment, the beads are washed and aliquoted for IP. (59, 60) Immunoprecipitation can also be performed with antibodies that are not covalently attached to the beads. This is usually done using agarose or magnetic beads coated with protein A and / or protein G, which fix the antibodies to the column. Listed below are some antibodies that can be used to selectively enrich for MHC / peptide complexes.

[0480] Antibody Name Specificity W6 / 32 Class I HLA-A, B, C L243 Class II - HLA-DR Tu36 Class II - HLA-DR LN3 Class II - HLA-DR Tu39 Class II - HLA-DR, DP, DQ

[0481] The clarified tissue lysate is added to the antibody beads for immunoprecipitation. After immunoprecipitation, the beads are removed from the lysate and the lysate is stored for additional experiments, including additional IP. The IP beads are washed to remove non-specific binding and standard techniques are used to elute the HLA / peptide complex from the beads. Molecular weight spin columns or C18 fractionation are used to remove protein components from the peptides. The resulting peptides are dried by SpeedVac evaporation and stored at -20°C before MS analysis in some cases.

[0482] The dried peptide is reconstructed in the HPLC buffer suitable for reverse phase chromatography and loaded on a C-18 microcapillary HPLC column to gradient elution in a Fusion Lumos mass spectrometer (Thermo). The MS1 spectrum of peptide mass / charge (m / z) is collected with high resolution in an Orbitrap detector, followed by MS2 low-resolution scans after HCD fragmentation of the selected ion in an ion trap detector. In addition, the MS2 spectrum can be obtained using CID or ETD fragmentation methods or any combination of the three techniques to achieve a larger amino acid coverage of the peptide. The MS2 spectrum can also be measured with high-resolution mass accuracy in an Orbitrap detector.

[0483] Protein database searches were performed on MS2 spectra from each analysis using Comet (61, 62), and peptide identifications were scored using Percolator (63-65). Additional sequencing was performed using PEAKS studio (Bioinformatics Solutions Inc.), and other search engines or sequencing methods, including spectral matching and de novo sequencing (97), may be used.

[0484] VIII.B.1. Studies of MS detection limits supporting comprehensive HLA peptide sequencing.

[0485] Using the peptide YVYVADVAAK (SEQ ID NO: 77), the detection limit was determined using different amounts of the peptide loaded on the LC column. The amounts of peptide tested were 1 pmol, 100 fmol, 10 fmol, 1 fmol and 100 amol. (Table 1) The results are shown in Fig.24A and Fig. 24B These results indicate that the lowest limit of detection (LoD) is in the attomole range (10 -18 ), the dynamic range spans five orders of magnitude, and the signal-to-noise ratio is sufficient to detect chromatin in the low femtomolar range (10 -15 ) sequencing.

[0486] Table 1

[0487]

[0488]

[0489] IX. Presentation Model

[0490] Presentation models can be used to identify the possibility of peptide presentation in patients. Various presentation models are known to those skilled in the art, such as those described in more detail in U.S. Patent No. 10,055,540, U.S. Application Publication No. US20200010849A1 and No. US20110293637, and International Patent Application Publication WO / 2018 / 195357, WO / 2018 / 208856, and WO2016187508, the documents are each incorporated herein by reference in their entirety for all purposes.

[0491] X. Training Module

[0492] The training module can be used to construct one or more presentation models based on the training data set, and the model generates the possibility that a peptide sequence will be presented by the MHC allele associated with the peptide sequence. Various training modules are known to those skilled in the art, such as the presentation models described in more detail in U.S. Patent No. 10,055,540, U.S. Application Publication No. US20200010849A1, and International Patent Application Publication WO / 2018 / 195357 and WO / 2018 / 208856, which are incorporated herein by reference as a whole for all purposes. The training module can construct a presentation model on an independent allele basis to predict the presentation possibility of a peptide. The training module can also construct a presentation model in a multi-allelic environment in which there are two or more MHC alleles to predict the presentation possibility of a peptide.

[0493] XI. Prediction Module

[0494] The prediction module can be used to receive sequence data and select candidate antigens in the sequence data using a presentation model. Specifically, the sequence data can be a DNA sequence, RNA sequence and / or protein sequence extracted from the patient's tumor tissue cells, infected cell patients or infectious disease organisms themselves. The prediction module can be compared with the sequence data extracted from the patient's normal tissue cells to identify the part containing one or more mutations, thereby identifying a new antigen in a mutant peptide sequence. The prediction module can be, for example, by comparing the sequence data extracted from the patient's normal tissue cells with the sequence data extracted from the patient's infected cells to identify the part containing one or more infectious disease organism-related antigens, thereby identifying candidate antigens, which are pathogen-derived peptides, virus-derived peptides, bacteria-derived peptides, fungus-derived peptides and parasite-derived peptides. The prediction module can be compared with the sequence data extracted from the patient's normal tissue cells to identify the candidate antigens that are inappropriately expressed, thereby identifying the candidate antigens that have a change in expression compared to normal cells or tissues in tumor cells or cancerous tissues. The prediction module can identify expressed candidate antigens (e.g., identifying polynucleotides and / or polypeptides expressed specifically for infectious diseases) by comparing sequence data extracted from normal tissue cells of the patient with sequence data extracted from infected tissue cells of the patient, thereby identifying candidate antigens expressed in infected cells or infected tissues compared to normal cells or tissues.

[0495] The presentation module can apply one or more presentation models to the processed peptide sequence to estimate the presentation probability of the peptide sequence. Specifically, the prediction module can select one or more candidate antigen peptide sequences that may be presented on tumor HLA molecules or infected cell HLA molecules by applying the presentation model to the candidate antigen. In one implementation, the presentation module selects candidate antigen sequences whose estimated presentation probability exceeds a predetermined threshold. In another implementation, the presentation model selects N candidate antigen sequences with the highest estimated presentation probability (where N is generally the maximum number of epitopes that can be delivered in the vaccine). A vaccine comprising a candidate antigen selected for a given patient can be injected into the patient to induce an immune response.

[0496] XI.B. Box Design Module

[0497] XI.B.1 Overview

[0498] The cassette design module can generate a vaccine cassette sequence based on the candidate peptide selected for injection into the patient. Various cassette design modules are known to those skilled in the art, such as those described in more detail in U.S. Patent No. 10,055,540, U.S. Application Publication No. US20200010849A1, and International Patent Application Publications WO / 2018 / 195357 and WO / 2018 / 208856, each of which is incorporated herein by reference in its entirety for all purposes.

[0499] Can produce therapeutic epitope set based on selected peptides associated with presentation probability exceeding predetermined threshold value determined by prediction module, wherein said presentation probability is determined by presentation model.However, it should be appreciated that in other embodiments, can be based on any one or more (alone or in combination) in a variety of methods, for example, based on binding affinity for HLA class I or class II allele of patient or predicted binding affinity, for HLA class I or class II allele of patient or predicted binding stability, random sampling etc. produce the set of therapeutic epitope.

[0500] The therapeutic epitope may correspond to a peptide of its own choice. In addition to the selected peptide, the therapeutic epitope may also include C-terminal and / or N-terminal flanking sequences. The N-terminal and C-terminal flanking sequences may be native N-terminal and C-terminal flanking sequences of the therapeutic vaccine epitope in the context of its source protein. The therapeutic epitope may represent an epitope of fixed length. The therapeutic epitope may represent an epitope of variable length, wherein the length of the epitope may vary according to, for example, the length of the C- flanking sequence or the N- flanking sequence. For example, the C-terminal flanking sequence and the N-terminal flanking sequence may each have a variable length of 2-5 residues, thereby generating 16 possible epitope selections.

[0501] The cassette design module can also generate a cassette sequence by considering the presentation of a junction epitope that spans the junction between a pair of therapeutic epitopes in the cassette. A junction epitope is a novel non-self but unrelated epitope sequence generated in the cassette as a result of the process of concatenating a therapeutic epitope and a linker sequence in the cassette. The novel sequence of a junction epitope is different from the therapeutic epitope itself of the cassette.

[0502] The cassette design module can generate a cassette sequence that reduces the likelihood of presentation of a junction epitope in a patient. Specifically, when the cassette is injected into a patient, the junction epitope is likely to be presented by the patient's class I HLA or class II HLA alleles and stimulate a CD8 or CD4 T cell response, respectively. Since T cell responses to junction epitopes have no therapeutic benefit and may attenuate the immune response to the selected therapeutic epitope in the cassette due to antigenic competition, such responses are often undesirable. 76

[0503] The cassette design module can iterate over one or more candidate cassettes and determine cassette sequences for which a presentation score of a junction epitope associated with the cassette sequence is below a numerical threshold. The junction epitope presentation score is a quantity associated with the likelihood of presentation of a junction epitope in the cassette, and a higher junction epitope presentation score value indicates a higher likelihood that the junction epitope of the cassette will be presented by class I HLA or class II HLA, or both.

[0504] In one embodiment, the cassette design module may determine the cassette sequence among the candidate cassette sequences that is associated with the lowest junction epitope presentation score.

[0505] The cassette design module may iterate over one or more candidate cassette sequences, determine a junction epitope presentation score for the candidate cassettes, and identify an optimal cassette sequence associated with a junction epitope presentation score below a threshold.

[0506] The cassette design module can further examine the one or more candidate cassette sequences to identify whet...

Claims

1. An adenoviral vector, comprising: an adenoviral backbone comprising one or more genes or regulatory sequences of the adenoviral genome, and wherein the adenoviral backbone comprises a partially deleted E4 gene with respect to the adenoviral genome, wherein the partially deleted E4 gene comprises a deleted or partially deleted E4orf2 region and a deleted or partially deleted E4orf3 region, and optionally a deleted or partially deleted E4orf4 region, and Optionally, the adenoviral vector further comprises a cassette comprising: (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises: -MHC class I epitopes, -MHC class II epitopes, - epitopes capable of stimulating a B cell response, or - combinations thereof, and Optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ adapter sequence and / or a 3′ adapter sequence, and optionally wherein; (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, (3) optionally, at least one universal nucleic acid sequence encoding an MHC class II antigen; (4) optionally, at least one linker sequence encoding GPGPG (SEQ ID NO: 56); and (5) Optionally, at least one polyadenylation sequence.

2. A chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; and (b) one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence shown in SEQ ID NO: 1; and Optionally, wherein the chimpanzee adenoviral vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

3. A chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; (b) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3' of nucleotides 2 to 34,916, and optionally nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion; and (c) nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, and wherein the partially deleted E4 gene is 5' to nucleotides 35,643 to 36,518, and Optionally, wherein the chimpanzee adenoviral vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.

4. A chimpanzee adenovirus vector, comprising: a. A modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (i) an E4 gene having a partial deletion of the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; (ii) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3' to said nucleotides 2 to 34,916, and optionally said nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of said sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of said sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion; and (iii) and nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, and wherein the partially deleted E4 gene is 5' of nucleotides 35,643 to 36,518, and; b. CMV-derived promoter sequence; c. SV40 polyadenylation signal nucleotide sequence; and d. A cassette comprising at least one payload nucleic acid sequence encoding: - at least one MHC class I epitope, optionally wherein the at least one MHC class I epitope comprises at least 2 different MHC class I epitopes, the at least 2 different MHC class I epitopes being linearly linked to each other and each optionally comprising: (A) at least one alteration that causes the encoded peptide sequence to differ from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, wherein the different MHC I epitope is 7-15 amino acids in length, (B) an N-terminal linker comprising a native N-terminal amino acid sequence of at least 3 amino acids in length of said different MHC I epitope, (C) a C-terminal linker comprising a native C-terminal acid sequence of at least 3 amino acids in length of the different MHC I epitope, or (D) any combination thereof, - at least one MHC class II epitope, optionally wherein said at least one MHC class II epitope comprises at least 2 different MHC class II epitopes, - at least one epitope capable of stimulating a B cell response, or - combinations thereof, and wherein the cassette is inserted into the deleted region of ChAdV68 and the CMV-derived promoter sequence is operably linked to the cassette.

5. The vector of any one of claims 1 to 4, wherein the cassette comprises an ordered sequence described by the following formula, from 5' to 3' comprising: P a -(L5 b -N c -L3 d ) X -(G5 e -U f ) Y -G3 g -A h in, N comprises one of the at least one payload nucleic acid sequence, optionally wherein each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, optionally with at least one change such that the encoded epitope sequence differs from the corresponding peptide sequence encoded by the wild-type nucleic acid sequence, wherein c=1, P comprises at least one promoter sequence operably linked to at least one of the at least one payload nucleic acid sequence, wherein a=1, L5 contains a 5' linker sequence, where b = 0 or 1, L3 contains a 3' linker sequence, where d = 0 or 1, G5 comprises at least one of the nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 56), wherein e=0 or 1, G3 comprises at least one of the nucleic acid sequences encoding a GPGPG amino acid linker (SEQ ID NO: 56), wherein g = 0 or 1, U comprises at least one of the common nucleic acid sequences encoding MHC class II antigens, wherein f=1, A comprises at least one polyadenylation sequence, where h = 0 or 1, X = 2 to 400, where for each X, the corresponding N c is a payload nucleic acid sequence, optionally wherein for each X, the corresponding N c are different payload nucleic acid sequences, and Y = 0-2, where for each Y, the corresponding U f is a universal nucleic acid sequence encoding an MHC class II antigen, optionally wherein for each Y, the corresponding Uf is a different universal nucleic acid sequence encoding an MHC class II antigen.

6. The vector of claim 5, wherein the cassette further comprises at least one additional payload nucleic acid sequence not encoded in the ordered sequence.

7. The vector according to claim 5 or 6, wherein: b=1, d=1, e=1, g=1, h=1, X=10, Y=2, P is a CMV-derived promoter sequence, Each N encodes an MHC class I epitope, an MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof, L5 encodes the native N-terminal amino acid sequence of the epitope, and wherein the 5' linker sequence encodes a peptide of at least 3 amino acids in length, L3 encodes the native C-terminal amino acid sequence of the epitope, and wherein the 3' linker sequence encodes a peptide of at least 3 amino acids in length, U is each of a PADRE class II sequence and a tetanus toxoid MHC class II sequence, and The vector comprises a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises: (a) an E4 gene having a partial deletion of the E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1; (b) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO: 1, wherein the partially deleted E4 gene is 3' of nucleotides 2 to 34,916, and optionally nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1, corresponding to an E1 deletion, and / or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, corresponding to an E3 deletion; and (c) nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO: 1, and wherein the partially deleted E4 gene is 5' to nucleotides 35,643 to 36,518.

8. The vector of any one of the preceding claims except claims 2-4 or 7, wherein the vector is a chimpanzee adenoviral vector, optionally wherein the chimpanzee adenoviral vector is a ChAdV68 vector.

9. The vector of any one of the preceding claims except claims 2-4 or 7, wherein the partially deleted E4 gene comprises: A. The E4 gene sequence shown in SEQ ID NO: 1, which lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO: 1, B. The E4 gene sequence shown in SEQ ID NO: 1, and lacking at least nucleotides 34,916 to 34,942, nucleotides 34,952 to 35,305, and nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, C. The E4 gene sequence shown in SEQ ID NO: 1, and which lacks at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, D. The E4 gene sequence shown in SEQ ID NO: 1, and which lacks at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO: 1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence shown in SEQ ID NO: 1, E. an E4 deletion comprising at least a partial deletion of E4Orf2, a complete deletion of E4Orf3 and at least a partial deletion of E4Orf4, F. an E4 deletion comprising at least a partial deletion of E4Orf2, at least a partial deletion of E4Orf3 and at least a partial deletion of E4Orf4, G. an E4 deletion comprising at least a partial deletion of E4Orf1, a complete deletion of E4Orf2 and at least a partial deletion of E4Orf3, or H. E4 deletion comprising at least a partial deletion of E4Orf2 and at least a partial deletion of E4Orf3.

10. The vector of any of the preceding claims except claims 2-4 or 7, wherein the vector comprises one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO: 1, optionally wherein the one or more genes or regulatory sequences are selected from the group consisting of: chimpanzee adenovirus inverted terminal repeats (ITRs), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of the sequence shown in SEQ ID NO: 1.

Images