Heterologous prime-boost vaccine compositions and methods of use

Abstract

The present application describes a method of inducing an immune response in a subject, comprising administering a prime-boost vaccine, where the priming vaccine comprises DNA (e.g., ceDNA) encoding a first peptide, and the boosting vaccine comprises (i) ribonucleic acid (RNA), or (ii) a second peptide. Also provided is a vaccine regimen comprising a priming vaccine comprising DNA, where the DNA encodes the first peptide, and a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, where the RNA encodes the second peptide.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 319,505, filed March 14, 2022, the entire contents of which are expressly incorporated herein by reference. [Background technology]

[0002] background Antigen-specific memory CD8 + Generating large populations of T cells and inducing long-lasting immune memory are desirable goals for vaccine design against various animal and human diseases. Typically, more than one immunization is required for a vaccine to induce efficient protection, and effective vaccines often require more than one immunization in the form of a prime-boost regime. For example, for pediatric populations, up to five immunizations may be required, as is the case for the diphtheria, tetanus, and pertussis (DTP) vaccine, which is given three doses during the first six months of life, followed by a fourth dose in the second year of life, and a final boost between the ages of four and six. Still, some vaccines require additional boosts even in adults who have already received a full immunization series, e.g., tetanus-diphtheria vaccine, for which boosters are recommended every 10 years. Such additional administrations may be performed with the same vaccine (homologous boost) or with a different vaccine (heterologous boost). Homologous prime-boost immunizations, which utilize re-administration of the same immunizing agent, have been used since the early development of vaccines. Classical vaccination approaches rely on homologous prime-boost regimens, which have traditionally failed to elicit immune responses strong enough to address more challenging diseases. For example, while this method is typically effective in boosting humoral responses to antigens, it often fails to elicit increased numbers of CD8+ cells due to rapid clearance of the homologous booster by the primed immune system. +These vaccines are generally considered to be far less effective at generating T cells and, furthermore, fail to boost cellular immunity. One strategy to overcome this limitation has been the sequential administration of vaccines using different antigen delivery systems. This approach is called heterologous prime / boost.

[0003] While heterologous prime-boost has been reported to increase responses in certain situations, not all combinations demonstrate improved immunity, indicating the importance of determining which combinations are effective. Many factors, including antigen selection, vector type, delivery route, dose, adjuvant, boost regimen, vector injection sequence, and interval between different vaccinations, affect the outcome of prime-boost immunization approaches, making results difficult to predict. Finding vaccine combinations that induce broad, durable, and long-lasting immunity is important for conferring robust protection.

[0004] Recombinant AAV (rAAV) is perhaps the best-tested vector for gene transfer in humans, with hundreds of clinical trials demonstrating the safety of transduction. Adeno-associated viruses (AAV) belong to the Parvoviridae family and, more specifically, constitute the Dependoparvovirus genus. Vectors derived from AAV (i.e., rAVV or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons; (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic in humans; (iv) in contrast to wild-type AAV, they are able to integrate into the host cell genome; replication-deficient AAV vectors lack replication (rep) genes and generally persist episomally, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) in comparison to other vector systems, AAV vectors are generally considered to be relatively poor immunogens and therefore do not elicit significant immune responses (see (ii)), thus resulting in persistence of vector DNA and, potentially, long-term expression of therapeutic transgenes.

[0005] However, there are several major drawbacks to using AAV particles as gene delivery vectors. One major drawback associated with rAAV is its limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, the use of AAV vectors has been limited to protein-coding capacities of less than 150,000 Da. Particularly relevant to antibody delivery, AAV's packaging limitations present a significant challenge for efficient delivery of both the heavy and light chains that form the native antibody structure. A second drawback is that, as a result of the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy must be screened for the presence of neutralizing antibodies that eliminate the vector from patients. A third drawback is related to capsid immunogenicity, which prevents re-administration to patients who were not excluded from initial treatment. The patient's immune system can respond to the vector, effectively acting as a "booster" shot to stimulate the immune system to produce high titers of anti-AAV antibodies that would prevent future treatment. Pre-existing immunity can severely limit transduction efficiency. Some recent reports have raised concerns about immunogenicity in high-dose settings. Another significant drawback is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA before heterologous gene expression can occur.

[0006] Adenoviral vectors, which express unknown antigenic proteins, have been extensively tested for gene and cancer therapy and vaccines. Apart from their broad safety profile, the advantages of using adenoviral vectors are that they are relatively stable, easily reach high titers, and can infect multiple cell lines, which contributes to their efficacy. Recombinant adenoviral vectors are widely used today due to their high transduction efficiency and transgene expression. However, because a large proportion of the population has been exposed to adenovirus (Id), there is a possibility of pre-existing immunity to the vector. This proved detrimental in a human immunodeficiency virus (HIV-1) phase IIb vaccine trial, in which a vector-based vaccine provided favorable conditions for HIV-1 replication (Smaill, F. et al., Sci. Transl. Med. (2013) 5:205). There remains a need in the art for heterologous prime-boost regimens that provide robust immunogenicity without inducing anti-vector immunity. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Smaill,F.et al.,Sci.Transl.Med.(2013)5:205 Summary of the Invention

[0008] overview The present disclosure provides prime-boost compositions and methods, including a priming vaccine comprising a first peptide encoded by DNA and a booster vaccine comprising a second peptide. In some embodiments, the second peptide is encoded by mRNA. In some embodiments, the DNA may be in the form of, for example, a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. In some embodiments, the priming vaccine comprises plasmid DNA. In some embodiments, the priming vaccine comprises closed-end linear double-stranded DNA (ceDNA).

[0009] As demonstrated in the Examples herein, the ceDNA vaccine platform can be successfully used as a priming vaccine in a heterologous prime / boost regimen to elicit and enhance both humoral and cellular responses to the encoded model antigen. The results presented herein suggest that a heterologous prime / boost regimen can confer a synergistically stronger response and greater protection against an antigen than immunization with the same vaccine alone. It is a finding of the present disclosure that priming with a DNA priming platform, e.g., a ceDNA vector platform, and boosting with an mRNA- or peptide-based platform (heterologous prime / boost regimen) can improve immune responses. Heterologous prime / boost regimens using two immunologically distinct platforms described herein are advantageously engineered using DNA (e.g., ceDNA) as the priming vaccine, such that subsequent administrations (boosts) activate the immune system in a different manner that synergizes with the initial administration (prime). Furthermore, the prime / boost compositions and methods described herein can also enhance immune responses by increasing CD8 + Generate memory T cell responses.

[0010] Applications of the prime-boost compositions and methods described herein, in which the priming vaccine comprises DNA (e.g., minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimally immunologically defined gene expression (MIDGE) vector, viral vector or non-viral vector), in which the DNA encodes a first peptide and RNA encoding a second peptide, or the boosting vaccine comprising the second peptide, are useful for treating, preventing, or reducing the severity of a disease or disorder in a subject, being minimally invasive in delivery, repeatable and dose-dependent, having a rapid onset of therapeutic effect, and / or providing sustained expression of an antigen or immunogenic peptide.

[0011] Unlike conventional vaccines that are produced ex vivo and may induce undesirable cellular responses, the ceDNA vaccines used herein as prime vaccines are presented to cell lines in a more natural manner. By using ceDNA vectors to deliver antigen-encoding transgenes (e.g., nucleic acid sequences) to cells or tissues, adaptive immune responses are bypassed and desired antibody specificities are produced without the use of immunization or passive transfer. That is, the ceDNA vector enters cells via endocytosis, then escapes from the endosomal compartment and is transported to the nucleus. The transcriptionally active ceDNA episome results in the expression of the antigen, which can then be secreted from the cell into the circulation. The ceDNA vector may therefore enable continuous, sustained, and long-term delivery of an antibody (e.g., a therapeutic antibody described herein, or an antigen-binding fragment therein) administered via a single injection. This is particularly advantageous in the context of the described nucleic acid vaccine compositions, where DNA primed vaccines show a slower increase in expression and more sustained expression compared to mRNA vaccines, which may show more increased initial expression, but expression is not sustained and declines more rapidly.

[0012] According to some aspects, the present disclosure provides a method for inducing an immune response to a first peptide and a second peptide in a subject, comprising administering to the subject a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes the first peptide; and administering (i) ribonucleic acid (RNA), wherein the RNA encodes the second peptide, or (ii) a boosting vaccine comprising the second peptide to the subject, thereby inducing an immune response to the first peptide and the second peptide in the subject. According to some embodiments, the priming vaccine comprises DNA encoding the first peptide, and the boosting vaccine comprises RNA encoding the second peptide. According to some embodiments, the priming vaccine comprises DNA encoding the first peptide, and the boosting vaccine comprises the second peptide. According to further embodiments of any of the embodiments herein, the DNA comprises a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimally immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. According to further embodiments of any of the embodiments herein, the first and second peptides are derived from a bacterial, viral, fungal, or parasitic infectious agent. According to further embodiments of any of the embodiments herein, the first and second peptides are derived from the same pathogenic organism. According to further embodiments of any of the embodiments herein, the first and second peptides are the same in the priming vaccine and the boosting vaccine. According to further embodiments of any of the embodiments herein, at least one epitope of the first and second peptides is different in the priming vaccine and the boosting vaccine. In some embodiments, the DNA comprises a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between flanking inverted terminal repeats (ITRs), wherein the at least one nucleic acid sequence encodes a peptide.According to some embodiments, the first and / or second peptide is a tumor-associated antigen or is associated with an autoimmune condition. According to further embodiments of any of the aspects or embodiments herein, the first or second peptide is selected from one or more of those shown in Tables 1-8. According to further embodiments of any of the embodiments herein, the ceDNA vector further comprises a promoter sequence linked to the at least one nucleic acid sequence. According to further embodiments of any of the embodiments herein, the ceDNA vector comprises at least one polyA sequence. According to further embodiments of any of the embodiments herein, the ceDNA vector comprises a 5'UTR and / or an intron sequence. According to further embodiments of any of the embodiments herein, the ceDNA vector comprises a 3'UTR sequence. According to further embodiments of any of the embodiments herein, the ceDNA vector comprises an enhancer sequence. According to further embodiments of any of the embodiments herein, at least one of the ITRs comprises a functional terminal resolution site and a Rep binding site. According to further embodiments of any of the embodiments herein, at least one or both of the ITRs are from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV). According to further embodiments of any of the embodiments herein, the adjacent ITRs are symmetrical or asymmetrical with respect to each other. According to some embodiments, the adjacent ITRs are symmetrical or substantially symmetrical. According to some embodiments, the adjacent ITRs are asymmetrical. According to further embodiments of any of the embodiments herein, one of the adjacent ITRs is wild-type, or both of the adjacent ITRs are wild-type ITRs. According to further embodiments of any of the embodiments herein, the adjacent ITRs are derived from different viral serotypes. According to further embodiments of any of the embodiments herein, the adjacent ITRs are selected from any pair of viral serotypes shown in Table 8. According to further embodiments of any of the embodiments herein, one or both of the ITRs comprise a sequence selected from one or more of the sequences in Table 9.According to further embodiments of any of the embodiments herein, at least one of the flanking ITRs is modified from the wild-type AAV ITR sequence by a deletion, addition, or substitution that affects the overall three-dimensional structure of the ITR. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs is derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs is synthetic. According to further embodiments of any of the embodiments herein, one of the flanking ITRs is not a wild-type ITR, or both of the flanking ITRs are not wild-type ITRs. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs is modified by a deletion, insertion, and / or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D, and D'. According to some embodiments, the deletion, insertion, and / or substitution results in the deletion of all or a portion of the stem-loop structure formed by the A, A', B, B', C, or C' regions. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs are modified by deletion, insertion, and / or substitution, which results in the deletion of all or a portion of the stem-loop structure formed by the B and B' regions. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs are modified by deletion, insertion, and / or substitution, which results in the deletion of all or a portion of the stem-loop structure formed by the C and C' regions. According to further embodiments of any of the embodiments herein, one or both of the flanking ITRs are modified by deletion, insertion, and / or substitution, which results in the deletion of a portion of the stem-loop structure formed by the B and B' regions and / or a portion of the stem-loop structure formed by the C and C' regions.According to further embodiments of any of the embodiments herein, one or both of the adjacent ITRs comprise a single stem-loop structure in a region that, in a wild-type ITR, would include a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. According to further embodiments of any of the embodiments herein, one or both of the adjacent ITRs comprise a single stem and two loops in a region that, in a wild-type ITR, would include a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. According to further embodiments of any of the embodiments herein, one or both of the adjacent ITRs comprise a single stem and a single loop in a region that, in a wild-type ITR, would include a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions. According to further embodiments of any of the embodiments herein, both of the adjacent ITRs are modified in a manner that results in overall three-dimensional symmetry when the ITRs are inverted relative to each other. According to further embodiments of any of the embodiments herein, the DNA is delivered in a lipid nanoparticle (LNP). According to further embodiments of any of the embodiments herein, the RNA is delivered in a lipid nanoparticle (LNP). According to further embodiments of any of the embodiments herein, the RNA is messenger RNA (mRNA). According to further embodiments, the RNA comprises at least one nucleotide analog. According to further embodiments of any of the embodiments herein, the immune response is an antibody response. According to further embodiments of any of the embodiments herein, the immune response is a T cell response. According to other further embodiments, the immune response is a memory (CD8. +According to some aspects and embodiments described herein, the method comprises administering the boosting vaccine at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 1-2 weeks, at least about 2-3 weeks, at least about 3-4 weeks, at least about 4-5 weeks, at least about 5-6 weeks, at least about 6-7 weeks, at least about 7-8 weeks, at least about 8-9 weeks, at least about 9-10 weeks, at least about 10-11 weeks, at least about 11-12 weeks, at least about 12-13 weeks, at least about 13-14 weeks, at least about 14-15 weeks, or at least about 15-16 weeks after administration of the priming vaccine. According to other further embodiments of any of the embodiments herein, the method comprises administering the boosting vaccine at least 8 weeks after administering the priming vaccine. According to other embodiments of any of the embodiments herein, the method comprises administering the boosting vaccine about 8 weeks after administering the priming vaccine.According to some aspects and embodiments described herein, the interval between administration of the priming vaccine and administration of the boosting vaccine is at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, at least about 66 days, at least about 67 days At least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, or at least at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, At least about 66 days, at least about 67 days, at least about 68 days, at least about 69 days, at least about 70 days, at least about 71 days, at least about 72 days, at least about 73 days, at least about 74 days, at least about 75 days, at least about 76 days, at least about 77 days, at least about 78 days, at least about 79 days, at least about 80 days, at least about 81 days, at least about 82 days, at least about 83 days, at least about 84 days, at least about 85 days, at least about 86 days, at least about 87 days. , at least about 88 days, at least about 89 days, at least about 90 days, at least about 91 days, at least about 92 days, at least about 93 days, at least about 94 days, at least about 95 days, at least about 96 days, at least about 97 days, at least about 98 days, at least about 99 days, at least about 100 days, at least about 101 days, at least about 102 days, at least about 103 days, at least about 104 days, at least about 105 days, at least about 106 days, at least about 107 days, at least about 108 days, at least about 109 days, at least about 110 days, at least about 111 days, or at least about 112 days. According to further embodiments, the interval between administration of the priming vaccine and administration of the boosting vaccine is about 64 days. According to some aspects and embodiments described herein, the method comprises administering two or more doses of a boosting vaccine to the subject. According to some aspects and embodiments described herein, the method comprises administering each dose of the boosting vaccine at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 1-2 weeks, at least about 2-3 weeks, at least about 3-4 weeks, at least about 4-5 weeks, at least about 5-6 weeks, at least about 6-7 weeks, at least about 7-8 weeks, at least about 8-9 weeks, at least about 9-10 weeks, at least about 10-11 weeks, at least about 11-12 weeks, at least about 12-13 weeks, at least about 13-14 weeks, at least about 14-15 weeks, or at least about 15-16 weeks after administration of the previous vaccine. According to some embodiments of the aspects and embodiments described herein, the interval between the administration of each dose of the boosting vaccine and the administration of the previous vaccine is at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days,At least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days , at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, at least about 66 days , at least about 67 days, at least about 68 days, at least about 69 days, at least about 70 days, at least about 71 days, at least about 72 days, at least about 73 days, at least about 74 days, at least about 75 days, at least about 76 days, at least about 77 days, at least about 78 days, at least about 79 days, at least about 80 days, at least about 81 days, at least about 82 days, at least about 83 days, at least about 84 days, at least about 85 days, at least about 86 days, at least about 87 days, at least about 88 days, at least about 89 days, at least about 90 days days, at least about 91 days, at least about 92 days, at least about 93 days, at least about 94 days, at least about 95 days, at least about 96 days, at least about 97 days, at least about 98 days, at least about 99 days, at least about 100 days, at least about 101 days, at least about 102 days, at least about 103 days, at least about 104 days, at least about 105 days, at least about 106 days, at least about 107 days, at least about 108 days, at least about 109 days, at least about 110 days, at least about 111 days, or at least about 113 days. According to further embodiments of any of the embodiments herein, the subject is treated with a bacterial infection,

[0013] According to further embodiments of any of the embodiments herein, the subject has a viral infection, a parasitic infection, or a fungal infection. According to further embodiments of any of the embodiments herein, the subject has cancer. According to further embodiments of any of the embodiments herein, the subject has an autoimmune disease or disorder. According to further embodiments of any of the embodiments herein, one or more of the priming vaccine or boosting vaccine comprises a pharmaceutically acceptable carrier. According to some embodiments, at least one of the priming vaccine composition and the boosting vaccine composition further comprises an adjuvant. According to further embodiments of any of the embodiments herein, at least one of the priming vaccine and the boosting vaccine is administered by a route selected from intramuscular, intraperitoneal, buccal, inhalation, intranasal, intrathecal, intravenous, subcutaneous, intradermal, and intratumoral, or is administered into the interstitial space of a tissue.

[0013] According to some aspects, the present disclosure provides vaccine regimens comprising a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes a first peptide, followed by a boosting vaccine comprising (i) ribonucleic acid (RNA) encoding a second peptide, or (ii) a second peptide. According to some embodiments, the priming vaccine comprises an immunologically effective amount of DNA encoding the first peptide, and the boosting vaccine comprises an immunologically effective amount of RNA encoding the second peptide. According to some embodiments, the priming vaccine comprises an immunologically effective amount of DNA encoding the first peptide, and the boosting vaccine comprises an immunologically effective amount of the second peptide. According to further embodiments of any of the embodiments herein, the DNA comprises a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. According to further embodiments of any of the embodiments herein, the first peptide and the second peptide are derived from a bacterial, viral, fungal, or parasitic infectious agent. According to further embodiments of any of the embodiments herein, the first peptide and the second peptide are derived from the same pathogenic organism. According to further embodiments of any of the embodiments herein, the first peptide and the second peptide are the same in the priming vaccine and the boosting vaccine. According to further embodiments of any of the embodiments herein, at least one epitope of the first peptide and the second peptide is different in the priming vaccine and the boosting vaccine. According to some embodiments, the DNA comprises a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between adjacent inverted repeat terminal repeats (ITRs), wherein the at least one nucleic acid sequence encodes the first peptide. According to some embodiments, the first peptide and / or the second peptide is a tumor-associated antigen.According to some embodiments, the first peptide and / or the second peptide is associated with an autoimmune condition. According to further embodiments of any of the embodiments herein, the first peptide or the second peptide is selected from one or more of those set forth in Tables 1-8.

[0014] The disclosure also features a method of treating a subject with a bacterial, viral, parasitic, or fungal infection, comprising performing the method of any of the aspects or embodiments herein or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0015] The disclosure also features a method of treating a subject with cancer, comprising performing the method of any of the aspects or embodiments herein, or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0016] The disclosure also features a method of treating a subject with an autoimmune disease or disorder, comprising performing the method of any of the aspects or embodiments herein, or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0017] The disclosure also features a method of preventing a bacterial, viral, parasitic, or fungal infection in a subject, comprising practicing the method of any of the aspects or embodiments herein or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0018] The disclosure also features a method of preventing cancer in a subject, comprising performing the method of any of the aspects or embodiments herein, or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0019] The disclosure also features a method of preventing autoimmune disease in a subject, comprising practicing the method of any of the aspects or embodiments herein, or administering to the subject a vaccine regimen of any one of the aspects and embodiments herein.

[0020] According to further embodiments of any of the embodiments herein, the method comprises administering two or more doses of a boosting vaccine to the subject. According to further embodiments of any of the embodiments herein, the method comprises administering the boosting vaccine about 8 weeks after administering the priming vaccine. According to further embodiments of any of the embodiments herein, the method further comprises administering one or more additional therapeutic agents to the subject.

[0021] According to other aspects, the priming vaccine and the boosting vaccine are each formulated in a pharmaceutical composition. According to some embodiments, one or both of the priming vaccine and the boosting vaccine further comprise one or more additional therapeutic agents. According to other further embodiments, one or both of the priming vaccine and the boosting vaccine further comprise a lipid. According to some embodiments, the lipid is a lipid nanoparticle (LNP). According to further embodiments, one or both of the priming vaccine and the boosting vaccine are lyophilized.

[0022] The present disclosure also features a pharmaceutical composition comprising the vaccine regimen of any one of the aspects and embodiments herein. According to some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0023] The present disclosure also features a vaccine regimen according to any one of the aspects and embodiments herein, and a composition comprising a lipid. According to some embodiments, the lipid is a lipid nanoparticle (LNP). According to further embodiments of any of the embodiments herein, the composition is lyophilized.

[0024] In another aspect, the present disclosure provides a kit comprising the vaccine regimen of any one of the aspects and embodiments herein and instructions for use. In another aspect, the present disclosure provides a kit comprising one or both of the priming vaccine and boosting vaccine of any one of the aspects and embodiments herein and instructions for use. In some embodiments, the kit comprises a lipid.

[0025] These and other aspects of the disclosure are described in further detail below. [Brief explanation of the drawings]

[0026] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to illustrative embodiments thereof, which are depicted in the accompanying drawings. However, because the present disclosure may admit of other equally effective embodiments, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope.

[0027] [Figure 1] FIG. 1 is a graph depicting spike protein antibody titers determined on day 49 of the study described in Example 5.

[0028] [Figure 2] FIG. 2 is a graph depicting spike protein antibody titers determined on day 77 of the study described in Example 5.

[0029] [Figure 3] FIG. 3 is a graph depicting spike protein antibody titers determined on day 105 of the study described in Example 5.

[0030] [Figure 4] FIG. 4 is a graph depicting the percentage of CD8+ T cells in the population that were IFNγ+, IFNγ+ and CD107+, IFNγ+ and TNFα+, or IL4+ on day 77 of the assay.

[0031] [Figure 5] FIG. 5 is a graph depicting spike protein antibody titers determined on days 21 and 49 of the study described in Example 6.

[0032] [Figure 6] Figure 6 is a graph depicting the percentage of IFNγ+ antigen-specific memory CD8+ T cells in mouse splenocyte suspensions 8 weeks after immunization with mRNA, ceDNA, or plasmid encoding the COVID spike protein.

[0033] [Figure 7] FIG. 7 is a graph depicting the percentage of IFNγ+ antigen-specific memory CD8+ T cells in mice primed and boosted at either 4-, 6-, or 8-week intervals with ceDNA-ceDNA, mRNA-mRNA, or ceDNA-mRNA regimens.

[0034] [Figure 8] FIG. 8 is a graph depicting the percentage of IFNγ+ antigen-specific memory CD8+ T cells after heterologous prime-boost regimens of 0.3 μg mRNA-3 μg mRNA, 1 μg mRNA-3 μg mRNA, 3 μg mRNA-3 μg mRNA, 3 μg ceDNA-3 μg mRNA, and 10 μg ceDNA-3 μg mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description The present disclosure generally relates to the use of compositions and methods for inducing an immune response in a subject using a heterologous prime-boost immunization regimen. Included herein is a method of inducing an immune response in a subject against a first peptide and a second peptide, comprising administering to the subject a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes the first peptide; and administering to the subject a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, wherein the RNA encodes the second peptide, thereby inducing an immune response in the subject against the first peptide and the second peptide, which can be used prophylactically and / or therapeutically. In some embodiments, the compositions and methods disclosed herein can be used for producing a molecule of interest, e.g., a therapeutic polypeptide, in a subject.

[0036] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein, and as such may vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6 thEdition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, DM and Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4 thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN 1936113414);Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN 044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN 0124199542);Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN047150338X,9780471503385),Current Protocols in Protein Science(CPPS),John E.Coligan(ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, A.D.A. M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0037] As used herein, the term "immunization" or "active immunization" refers to the production of active immunity, meaning immunity resulting from naturally acquired infection or deliberate vaccination (artificial active immunity).

[0038] As used herein, the term "adjuvant" is meant to refer to an agent that, when used in combination with a particular immunogen in a formulation, enhances or otherwise alters or modifies the resulting immune response. Modification of an immune response includes enhancing or broadening the specificity of an immune response (e.g., either or both of an antibody response and a cellular immune response). Modification of an immune response can also mean reducing or suppressing a particular antigen-specific immune response.

[0039] As used herein, the term "antigen" refers to a molecule containing one or more epitopes (either linear, conformational, or both) that stimulate the host's immune system to generate a humoral and / or cellular antigen-specific response. This term is used interchangeably with the term "immunogen." B cell epitopes typically contain at least about five amino acids, but can be as small as three or four amino acids. T cell epitopes, such as CTL epitopes, contain at least about seven to nine amino acids, and helper T cell epitopes contain at least about 12 to 20 amino acids. Typically, epitopes contain between about seven and fifteen amino acids, such as 9, 10, 11, 12, 13, 14, or 15 amino acids. This term also encompasses polypeptides containing modifications, such as deletions, additions, and substitutions (generally conservative in nature), compared to the native sequence, so long as the protein retains its ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, for example, through mutations of hosts which produce the antigens.

[0040] The term "epitope," also referred to as antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) capable of being specifically bound by a binding agent, immunoglobulin, or T-cell receptor. Epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, i.e., composed of non-linear amino acids. An epitope recognized by an antibody or antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope may be formed by contributions from several amino acid residues that interact with the CDR of the antibody to generate specificity. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody specifically binds to an antigen if it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. For example, antibodies are said to "bind to the same epitope" if the antibodies cross-compete (one prevents the binding or modulatory effect of the other).

[0041] As used herein, the term "autoimmune disorder" generally refers to a condition in which a subject's immune system attacks the body's own cells, causing tissue destruction. Autoimmune disorders can be diagnosed using blood tests, cerebrospinal fluid analysis, electromyography (measuring muscle function), and magnetic resonance imaging of the brain, but antibody testing in the blood for autologous antibodies (or autoantibodies) is particularly useful. IgG class antibodies are usually associated with autoimmune diseases.

[0042] The terms "B lymphocyte" or "B cell" are used interchangeably to refer to a broad class of lymphocytes that are precursors of antibody-secreting cells expressing clonally diverse cell-surface immunoglobulin (Ig) receptors (BCRs) that recognize specific antigenic epitopes. Mammalian B cell development involves a series of steps that begin in primary lymphoid tissues (e.g., human fetal liver and fetal / adult bone marrow) followed by functional maturation in secondary lymphoid tissues (e.g., human lymph nodes and spleen). The functional / protective endpoint is antibody production by terminally differentiated plasma cells. Mature B cells can be activated by encounter with an antigen that expresses an epitope recognized by its cell-surface immunoglobulin (Ig). The activation process can be direct, dependent on cross-linking of membrane Ig molecules by antigen (cross-linking-dependent B cell activation), or indirect, occurring most efficiently in the context of close interaction with helper T cells (the "cognate help process"). (LeBien, TW&TF Tedder, B lymphocytes: how they develop and function.Blood(2008)112(5):1570-80).

[0043] As used herein, the term "cancer" refers to a disease in which abnormal cells divide uncontrollably and can invade other tissues. There are over 100 types of cancer. Most cancers are named for the organ or type of cell in which they begin, for example, cancer that begins in the colon is called colon cancer; cancer that begins in the melanocytes of the skin is called melanoma. Types of cancer can be grouped into broader categories. The major categories of cancer include: carcinoma (meaning cancer that begins in the skin or in tissues lining or covering the inside or outside of organs, and its subtypes, including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma); sarcoma (meaning cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue); leukemia (meaning cancer that begins in blood-forming tissues (e.g., bone marrow) and causes large numbers of abnormal blood cells to be produced, which then enter the bloodstream); lymphoma and myeloma (meaning cancer that begins in cells of the immune system); and central nervous system (CNS) cancer (meaning cancer that begins in the tissues of the brain and spinal cord). The term "myelodysplastic syndrome" refers to a type of cancer in which the bone marrow does not make enough healthy blood cells (white blood cells, red blood cells, and platelets) and there are abnormal cells in the blood and / or bone marrow. Myelodysplastic syndrome can lead to acute myeloid leukemia (AML). In certain embodiments, the cancer is selected from, but not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), anal cancer, bile duct cancer, bladder cancer, bone cancer, intestinal cancer, brain cancer, breast cancer, cancer of unknown primary origin, cancer that has spread to the bone, cancer that has spread to the brain, cancer that has spread to the liver, cancer that has spread to the lungs, carcinoid, cervical cancer, choriocarcinoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumor (GTT), hair follicle cancer, and the like. The cancer is selected from cancers including melanoma-like cell leukemia, head and neck cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma skin cancer, mesothelioma, male cancer, molar pregnancy, mouth and oropharyngeal cancer, myeloma, nasal and paranasal sinus cancer, nasopharyngeal cancer, non-Hodgkin's lymphoma (NHL), esophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectal cancer, salivary gland cancer, secondary cancers, skin cancer (non-melanoma), soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer, cancer of unknown primary, uterine cancer, vaginal cancer, and vulvar cancer.

[0044] As used herein, the term "cross-protection" is used to describe immunity against at least two subgroups, subtypes, strains, and / or variants of a virus, bacterium, parasite, or other pathogen, following a single inoculation with one of the subgroups, subtypes, strains, and / or variants.

[0045] As used herein, the term "cytokine" refers to small soluble protein substances secreted by cells that have a variety of effects on other cells. Cytokines mediate many important physiological functions, including growth, development, wound healing, and immune responses. They act by binding to their cell-specific receptors located in the cell membrane, which allows distinct signaling cascades to be initiated in the cell, which ultimately lead to biochemical and phenotypic changes in the target cell. Generally, cytokines act locally. They include type I cytokines, which include many of the interleukins and several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules, including TNFα and lymphotoxin; immunoglobulin superfamily members, including interleukin-1 (IL-1); and chemokines, a family of molecules that play critical roles in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on cells depending on the cellular context. Cytokines often regulate the expression of other cytokines, triggering cascades thereof.

[0046] As used herein, the term "detectable response" refers to any signal or response that can be detected in an assay, which may be performed with or without a detection reagent. Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection, or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrometry, ultraviolet spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and X-ray diffraction. Alternatively, a detectable response may be the result of an assay to measure one or more properties of a biological material, such as melting point, density, conductivity, surface acoustic wave, catalytic activity, or elemental composition. A "detection reagent" is any molecule that generates a detectable response that indicates the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences, and enzymes. To facilitate detection, a detection reagent may include a marker.

[0047] As used herein, the term "effector cell" refers to a cell that carries out the final response or function. The primary effector cells of the immune system are, for example, activated lymphocytes and phagocytes.

[0048] As used herein, the term "herd immunity" refers to the protection conferred on unvaccinated individuals in a population produced by a decline in the natural reservoir for other vaccinations and infections.

[0049] As used herein, the term "heterosubtypic immunity (HSI)" refers to immunity based on immune recognition of antigens conserved across all viral strains.

[0050] As used herein, the term "heterotypic" is used to refer to a different or unusual species or form (e.g., a different subgroup, subtype, strain, and / or variant of a virus, bacterium, parasite, or other pathogen).

[0051] As used herein, the term "homotypic" is used to refer to the same species or form, e.g., the same subgroup, subtype, strain, and / or variant of a virus, bacterium, parasite, or other pathogen.

[0052] As used herein, the terms "immune response" and "immune-mediated" are used interchangeably to refer to any functional manifestation of a subject's immune system against either a foreign antigen or a self-antigen, regardless of whether the outcome of these responses is beneficial or harmful to the subject. As used herein, the term "immunological response" to an antigen or composition is meant to refer to the development in a subject of a humoral and / or cellular immune response to an antigen present in the composition of interest. For purposes of this disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T lymphocytes and / or other white blood cells. One important aspect of cellular immunity includes antigen-specific responses by cytolytic T cells (CTLs). CTLs have specificity for peptide antigens that are presented in the context of proteins encoded by major histocompatibility complexes (MHC) and expressed on the surface of cells. CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity includes antigen-specific responses by helper T cells. Helper T cells act to help stimulate the function and focus the activity of nonspecific effector cells against cells that display peptide antigens in the context of MHC molecules on their surface. "Cellular immune responses" also involve CD4 + and CD8 +An immunological response refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other white blood cells, including those derived from T cells. Thus, an immunological response may include one or more of the following effects: the production of antibodies by B cells; and / or the activation of suppressor T cells and / or γδ T cells specifically directed against the antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity and / or mediate antibody complement or antibody-dependent cellular cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays well known in the art.

[0053] As used herein, the term "immunophenotype" or "immunotype" refers to the collective frequency of various immune cell populations and their functional responses (cell signaling and antibody responses) to stimuli. (See Kaczorowski, KJ et al. Proc. Nat. Acad. Sci. USA (2017)).

[0054] As used herein, the term "immune system" refers to the body's defense system against disease, including the innate immune system and the adaptive immune system. The innate immune system provides a nonspecific first line of defense against pathogens. It includes a physical barrier (e.g., skin) and both cellular (granulocytes, natural killer cells) and humoral (complement system) defense mechanisms. The response of the innate immune system is immediate, but unlike the adaptive immune system, it does not provide permanent immunity to pathogens. The adaptive immune response is the response of the vertebrate immune system to a specific antigen, which typically generates immunological memory.

[0055] As used herein, the term "immunodominant epitope" refers to an epitope to which the majority of antibodies are produced or to which the majority of T cells respond.

[0056] As used herein, the term "immunogenic amount" or "immunologically effective amount" refers to an amount of an active ingredient (e.g., an immunogenic peptide) sufficient to elicit either an antibody or T cell response, or both, and to have a beneficial effect, e.g., a prophylactic or therapeutic effect, on a subject.

[0057] The term "immunological repertoire" refers to the collection of transmembrane antigen receptor proteins located on the surface of T cells and B cells. (Benichou, J. et al. Immunology (2011) 135:183-191)) The combinatorial machinery involved in encoding receptors does so by reshuffling the genetic code, potentially generating over 10 distinct T cell receptors (TCRs) in humans (Venturi, Y. et al. Nat. Rev. Immunol. (2008) 8:231-8) and a much larger B cell repertoire. These sequences are sequentially transcribed and then translated into proteins that are displayed on the cell surface. The recombination process that rearranges gene segments to construct receptors is critical for the generation of an immune response, and the correct formation of the rearranged receptors is crucial for their future binding affinity to antigens.

[0058] A peptide, oligopeptide, polypeptide, protein, or polynucleotide encoding such a molecule is "immunogenic" and thus an immunogen within the context of the present disclosure if it is capable of inducing an immune response. In this disclosure, immunogenicity is more specifically defined as the ability to induce a CTL-mediated response. Thus, an immunogen is a molecule capable of inducing an immune response, and in this disclosure, a molecule capable of inducing a CTL response. An immunogen may have one or more isoforms, sequence variants, or splice variants that have equivalent biological and immunological activity and thus, for purposes of this disclosure, are also considered to be immunogenic equivalents of the original native polypeptide.

[0059] The term "priming" or "prime" refers to the administration of a vaccine (priming vaccine) or immunogenic composition that, when followed by a subsequent administration of the same or a different vaccine immunogenic composition, induces a higher level of immune response than that achieved by administration with a single vaccine or immunogenic composition. In some embodiments, the "priming vaccine" is a DNA priming vaccine. In some embodiments, the DNA priming vaccine may be in the form of, for example, a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. In some embodiments, the priming vaccine comprises closed-end linear double-stranded DNA (ceDNA). In some embodiments, the priming vaccine comprises plasmid DNA.

[0060] The term "boosting" or "boost" refers to the administration of a subsequent vaccine (a "boosting vaccine") or immunogenic composition after the administration of a priming vaccine or immunogenic composition, where the subsequent administration produces a higher level of immune response than the immune response to a single administration of the vaccine or immunogenic composition. The boosting vaccine can be the same or a different vaccine immunogenic composition as the priming vaccine or immunogenic composition.

[0061] The term "heterologous prime-boost" as used herein refers to a regimen that includes priming an immune response with an immunogenic peptide or antigen, and subsequently boosting the immune response with an immunogenic peptide or antigen delivered by a different molecule and / or vector.For example, the heterologous prime-boost regimen of the present invention includes priming with a ceDNA vector and boosting with an mRNA vector, and priming with a ceDNA vector and boosting with an immunogenic peptide.The heterologous prime-boost regimen of the present invention can also include, for example, priming with a plasmid DNA and boosting with an mRNA vector, and priming with a plasmid DNA and boosting with an immunogenic peptide.

[0062] The term "specifically binds," as used herein, refers to the ability of a polypeptide or polypeptide complex to recognize and bind a ligand in vitro or in vivo while not substantially recognizing or binding other molecules in the surrounding environment. In some embodiments, specific binding is at least about 1 x 10 6 The binding may be characterized by an equilibrium dissociation constant of M or less (e.g., a smaller equilibrium dissociation constant indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0063] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Example 1 of U.S. Patent No. 6,258,562 and Jonsson et al. (1993) Ann. Biol. Clin. 51:19; Jonsson et al. (1991) Biotechniques 11:620-627; Johnson et al. (1995) J. Mol. Recognit. 8:125; and Johnson et al. (1991) Anal. Biochem. 198:268.

[0064] As used herein, the terms "heterologous nucleic acid sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated, delivered, and expressed by a ceDNA vector disclosed herein. According to some embodiments, the term "heterologous nucleic acid" is meant to refer to a nucleic acid (or transgene) that is not present in, expressed by, or derived from a cell or subject with which it is contacted.

[0065] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acid comprising a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but not including capsid coding sequences, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0066] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of between about 5 and about 100 nucleotides, either single- or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides, also known as "oligomers" or "oligos," may be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded polynucleotides (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments.

[0067] The terms DNA and DNA molecule are used interchangeably herein and are meant to refer to DNA, which may be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups. DNA may be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), a Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. RNA may be in the form of small interfering RNA (siRNA), a Dicer substrate dsRNA, a short hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), an mRNA, a rRNA, a tRNA, a viral RNA (vRNA), or combinations thereof. According to preferred embodiments, the DNA of the priming vaccine includes minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimal immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0068] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0069] "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0070] As used herein, the term "nucleic acid construct" refers to either a single-stranded or double-stranded nucleic acid molecule, which is isolated from a native gene or modified to contain a segment of nucleic acid in a manner that would not otherwise occur in nature or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present disclosure. An "expression cassette" contains a DNA coding sequence operably linked to a promoter.

[0071] By "hybridizable" or "complementary" or "substantially complementary," it is meant that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under suitable in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). It is also known in the art that guanine (G) base pairs with uracil (U) for hybridization between two RNA molecules (e.g., dsRNA). For example, G / U base pairing, in the context of tRNA anticodon base pairing with codons in mRNA, is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, a guanine (G) of the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, if a G / U base pair can be made at a given nucleotide position of the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, that position is not considered non-complementary, but instead is considered complementary.

[0072] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0073] A DNA sequence that "encodes" a particular antigen or immunogenic peptide is a DNA nucleic acid sequence that is transcribed into a specific RNA and / or protein. The DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA-targeting RNA; also called "non-coding" RNA or "ncRNA").

[0074] As used herein, the term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence containing at least one minimal required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence ("RBS") (also referred to as an RBE (Rep binding element)) and terminal resolution site ("TRS") together constitute the "minimal required origin of replication," and thus, a TR contains at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs," respectively. In the context of viruses, ITRs mediate replication, viral packaging, integration, and proviral rescue. Unexpectedly, TRs that are not reverse complements across their entire length can still perform the traditional functions of ITRs; thus, the term "ITR" is used herein to refer to TRs in a ceDNA genome or ceDNA vector that are capable of mediating replication of the ceDNA vector. It will be understood by those skilled in the art that in complex ceDNA vector configurations, there may be more than two ITRs or asymmetric ITR pairs. The ITRs can be AAV or non-AAV ITRs, or can be derived from AAV or non-AAV ITRs. For example, the ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin that serves as the SV40 origin of replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Parvoviridae viruses consist of two subfamilies: the Parvovirinae subfamily infects vertebrates, and the Densovirinae subfamily infects invertebrates. Dependoparvoviruses include the adeno-associated viruses (AAV) family of viruses that are capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, canine, equine, and bovine species.For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a ceDNA vector will be referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette in a ceDNA vector will be referred to as the "3' ITR" or "right ITR."

[0075] "Wild-type ITR" or "WT-ITR" refers to a native ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may vary slightly from the standard native sequence due to degeneracy or drift in the genetic code; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from natural variations (e.g., replication errors) that occur during the production process.

[0076] As used herein, the term "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refers to a pair of WT-ITRs within a single ceDNA genome or ceDNA vector, both of which are wild-type ITRs with reverse-complementary sequences across their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from the standard native sequence, as long as the changes do not affect the sequence's characteristics and overall three-dimensional structure. In some embodiments, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (e.g., as measured using BLAST with default settings) and has a symmetrical three-dimensional spatial configuration relative to the other WT-ITR, such that their 3D structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in 3D space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (trs) that pairs with an appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0077] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation may result in a change in the three-dimensional spatial configuration (i.e., its 3D structure in geometric space) compared to the 3D spatial configuration of a WT-ITR of the same serotype, which may result in a change according to some or more of the A, C, C', B, and B' regions in the ITR.

[0078] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are not reverse complements across their entire length. As one non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial configuration relative to its cognate ITR, such that their 3D structures are different shapes in geometric space. Stated differently, an asymmetric ITR pair has a different overall geometric structure, i.e., they have a different configuration of their A, C-C', and B-B' loops in 3D space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to the cognate ITR). The difference in sequence between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. According to some embodiments, one ITR of an asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs that have different shapes in geometric space (i.e., different overall geometric structures). According to some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm, or a short B-B' arm, etc.) such that it has a different three-dimensional spatial configuration compared to its cognate asymmetric mod-ITR.

[0079] As used herein, the term "symmetric ITRs" refers to a pair of ITRs within a single ceDNA genome or ceDNA vector that are mutated or modified relative to the wild-type Dependvirus ITR sequence and are reverse complements across their entire length. Neither of the ITRs is the wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may have differences in sequence from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a ceDNA vector will be referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette in a ceDNA vector will be referred to as the "3' ITR" or "right ITR."

[0080] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs within a single ceDNA genome or ceDNA vector that both have reverse complementary sequences throughout their entire length. For example, a modified ITR may be considered substantially symmetric even if it has some nucleotide sequence deviations from its reverse complementary sequence, as long as the changes do not affect its properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (as measured using BLAST with default settings) and also has a symmetric three-dimensional spatial organization relative to its cognate modified ITR, such that their 3D structures are the same shape in geometric space. Stated differently, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops organized in 3D space. According to some embodiments, the ITRs from a mod-ITR pair may have different reverse-complementary nucleotide sequences, but still have the same symmetrical three-dimensional spatial organization (i.e., both ITRs have mutations that result in the same overall 3D shape). For example, one ITR (e.g., the 5' ITR) in a mod-ITR pair may be from one serotype and the other ITR (e.g., the 3' ITR) may be from a different serotype, but both may have the same corresponding mutation (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR from the different serotype will have a deletion at the corresponding position in the C' region), and the modified ITR pair will have the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in a modified ITR pair may be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and modifications along some ITRs are mirrored at corresponding positions in the cognate ITRs from the different serotypes. According to some embodiments, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the nucleotide sequence differences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in 3D space.Non-limiting examples of mod-ITRs include those that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference mod-ITR, as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and have symmetrical three-dimensional spatial configurations such that their 3D structures are the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in 3D space; for example, if the modified ITR in a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, then the cognate mod-ITR will have a corresponding deletion of the C-C' loop and a similar 3D structure of the remaining A and B-B' loops that are the same shape in geometric space as its cognate mod-ITR.

[0081] As used herein, "internal ribosome entry site" (IRES) is meant to refer to a nucleotide sequence (>500 nucleotides) that allows translation initiation in the middle of an mRNA sequence (Kirn, J.I.T. et al., 2011. PLoS One 6(4):8556; the contents of which are incorporated herein by reference in their entirety). The use of an IRES sequence ensures co-expression of genes preceding and following the IRES, although the sequence following the IRES may be transcribed and translated at a lower level than the sequence preceding the IRES sequence.

[0082] As used herein, "2A peptide" is meant to refer to a small self-cleaving peptide derived from a virus, such as foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Zosea asignavirus (T2A), or equine rhinitis A virus (E2A). The 2A designation specifically refers to a region of the picornavirus polyprotein where a glycyl-prolyl bond at the O-terminus of the 2A peptide leads to ribosomal skipping (Kim, J. I. T. et al. 2011. PLoS One 6(4); the contents of which are incorporated herein by reference in their entirety). This skipping results in cleavage between the 2A peptide and the peptide immediately downstream.

[0083] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked by A and C. The same is true for the sequence AxBxC. Thus, a flanking sequence may precede or follow the flanking sequence, but need not be contiguous with or immediately adjacent to the flanking sequence. In some embodiments, the term "flanking" refers to the terminal repeats at each end of a linear double-stranded ceDNA vector.

[0084] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. According to some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0085] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. According to some embodiments, the ceDNA spacer region maintains two functional elements at a desired distance for optimal functionality. According to some embodiments, the ceDNA spacer region provides or adds to the genetic stability of the ceDNA genome, for example, within a plasmid or baculovirus. According to some embodiments, the ceDNA spacer region facilitates easy genetic manipulation of the ceDNA genome by providing a convenient location for cloning sites and the like. For example, in certain aspects, oligonucleotide "polylinkers" containing several restriction endonuclease sites, or non-open reading frame sequences designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in the ceDNA genome to separate cis-acting elements, e.g., inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between the polyadenylation signal sequence and the 3' terminal degradation site.

[0086] As used herein, the terms "Rep binding site," "Rep binding element," "RBE," and "RBS" are used interchangeably and refer to a binding site for a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding by the Rep protein, allows the Rep protein to perform its site-specific endonuclease activity on a sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'. Any known RBS sequence can be used in embodiments of the present disclosure, including other known AAV RBS sequences and other naturally occurring, known, or synthetic RBS sequences. Without being bound by theory, it is believed that the nuclease domain of the Rep protein binds to the double-stranded nucleic acid sequence GCTC, and thus two known AAV Rep proteins directly bind to and stably assemble on the double-stranded oligonucleotide 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3'. Alternatively, a soluble aggregate conformation (i.e., an undefined number of interrelated Rep proteins) dissociates and binds to an oligonucleotide containing the Rep binding site. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interactions with the nitrogenous bases provide sequence specificity, whereas the interactions with the phosphodiester backbone are non- or low-sequence specific and stabilize the protein-DNA complex.

[0087] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine, generating a 3' OH residue that serves as a substrate for DNA elongation via cellular DNA polymerases, such as DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may participate in a synchronous ligation reaction. In some embodiments, the TRS minimally contains non-base-paired thymidines. In some embodiments, the nicking efficiency of the TRS may be controlled, at least in part, by its intramolecular distance from the RBS. If the acceptor substrate is a complementary ITR, then the resulting product is an intramolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' specified in AAV2. Any known TRS sequence can be used in embodiments of the present disclosure, including other known AAV TRS sequences as well as other naturally occurring, known, or synthetic TRS sequences, such as AGTT, GGTTGG, AGTTGG, AGTTGA, and other motifs, such as RRTTRR.

[0088] As used herein, the term "ceDNA" refers to capsid-free, closed-end, linear, double-stranded (ds) DNA for non-viral gene transfer, synthesis, or other methods. A detailed description of ceDNA is provided in International Application PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Specific methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in International Application PCT / US18 / 49996, filed September 7, 2018, and Example 1 of International Application PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for producing synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference. As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector containing at least one terminal palindrome. According to some embodiments, the ceDNA contains two covalently closed ends.

[0089] As used herein, the term "ceDNA plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0090] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome containing the ceDNA genome as an intermolecular duplex that can be propagated in E. coli as a plasmid and thus act as a shuttle vector for baculovirus.

[0091] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0092] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) that have been infected with a ceDNA-baculovirus.

[0093] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector with at least one covalently closed end, at least a portion of the vector having an intramolecular double-stranded structure.

[0094] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferases cause cells to catalyze light-producing reactions, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0095] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, e.g., as a reporter polypeptide, or more appropriately, as a cell-killing polypeptide, e.g., a toxin or drug that sensitizes a cell to killing with, or lack thereof, a selected agent. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins can include, but are not limited to, restriction endonucleases that target host cell DNA sequences (whether genomic or on extrachromosomal elements), proteases that degrade polypeptide targets necessary for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. According to some embodiments, expression of an effector protein controlled by a synthetic biological circuit described herein can participate as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the responsiveness of the biological circuit system.

[0096] Transcriptional regulators refer to transcriptional activators and repressors that activate or repress transcription of a transgene (e.g., a nucleic acid encoding an antibody or antigen-binding fragment thereof described herein). A promoter is a region of nucleic acid that initiates transcription of a particular gene. Transcriptional activators typically bind near a transcriptional promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to a transcriptional promoter and sterically hinder transcription initiation by RNA polymerase. Other transcriptional regulators can act as either activators or repressors, depending on where they bind and on cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine zipper proteins.

[0097] As used herein, a "repressor protein" or "inducer protein" is a protein that binds to a regulatory sequence element and represses or activates, respectively, transcription of a sequence operatively linked to the regulatory sequence element. Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input or environmental input. Preferred proteins described herein are modular in form, for example, containing separable DNA-binding and input-binding or response elements or domains.

[0098] As used herein, "carrier" includes any and all solvents, dispersion media, excipients, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0099] As used herein, an "input agent responsive domain" is a domain of a transcription factor that binds to or otherwise responds to a condition or input agent in a manner that renders the linked DNA-binding fusion domain responsive to the presence of that condition or input. According to some embodiments, the presence of the condition or input results in a conformational change in the input agent responsive domain, or in the protein to which it is fused, that modifies the transcriptional regulatory activity of the transcription factor.

[0100] The term "in vivo" refers to an assay or process that occurs in or within an organism, such as a multicellular animal. According to some of the aspects described herein, a method or use can be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" particularly refers to methods and uses that are carried out using living cells with intact membranes that are outside the body of a multicellular animal or plant, such as explants, cultured cells including primary cells and cell lines, transformed cell lines, and extracted tissues or cells including blood cells, among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and can refer to the introduction of programmable synthetic biology circuits in a non-cellular system, such as cell-free medium, or in a cellular system, such as a cell extract.

[0101] The term "promoter," as used herein, refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. According to some embodiments of the aspects described herein, a promoter can drive the expression of transcription factors that regulate its own expression. Within the promoter sequence, transcription initiation sites and protein binding domains involved in the binding of RNA polymerase are found. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, may be used to drive the expression of transgenes in the ceDNA vectors disclosed herein. A promoter sequence may be bounded at its 3' end by a transcription start site and may extend upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at levels detectable above background. According to some embodiments, the promoters of the present disclosure are liver-specific promoters.

[0102] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 10 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream of the start site of the gene they regulate or downstream of the start site of the gene. Enhancers can be located within intron regions or in exon regions of unrelated genes.

[0103] A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The terms "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to the nucleic acid sequence it regulates so as to control transcription initiation and / or expression of that sequence. A "reverse promoter," as used herein, refers to a promoter in which the nucleic acid sequence is in the reverse orientation, so that what was the coding strand is now the non-coding strand, or vice versa. Reverse promoter sequences can be used in various embodiments to regulate the state of a switch. Also, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0104] A promoter can be one that is naturally associated with a gene or sequence, such as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons of a given gene or sequence. Such a promoter can be referred to as "endogenous." Similarly, in some embodiments, an enhancer can be one that is naturally associated with a nucleic acid sequence and located either downstream or upstream of that sequence.

[0105] In some embodiments, a coding nucleic acid segment is placed under the control of a "recombinant promoter" or "heterologous promoter," both of which refer to a promoter that is not normally associated with a coding nucleic acid sequence to which it is operably linked in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell; and synthetic promoters or enhancers that are not "native," i.e., contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in the context of the synthetic biology circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, and the like, may also be used.

[0106] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducing agent. An "inducer" or "inducing agent," as defined herein, can be an endogenous or, usually exogenous, compound or protein administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be an inducer protein expressed by another component or module), which can itself be under the control or inducible promoter. According to some embodiments, an inducible promoter is induced in the absence of a particular agent, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter; and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0107] The terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, and the like, that provide for and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide, or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0108] As used herein, the term "open reading frame (ORF)" refers to a sequence of several nucleotide triplets that can be translated into a peptide or protein. An open reading frame preferably includes an initiation codon, i.e., a combination of three subsequent nucleotides that usually encodes the amino acid methionine (ATG), at its 5' end, and a subsequent region that usually exhibits a length that is a multiple of three nucleotides. The ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). Typically, this is the only stop codon in the open reading frame. Thus, an open reading frame in the context of the present disclosure is a nucleotide sequence consisting of a number of nucleotides divisible by three, preferably beginning with an initiation codon (e.g., ATG) and preferably ending with a stop codon (e.g., TAA, TGA, or TAG). An open reading frame may be isolated, or it may be incorporated into a longer nucleic acid sequence, for example, into a ceDNA vector described herein.

[0109] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. An "expression cassette" comprises a DNA sequence operably linked to a promoter or other regulatory sequence sufficient to direct transcription of a transgene in a ceDNA vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0110] As used herein, the term "subject" refers to a human or animal to whom treatment, including prophylactic treatment, using a ceDNA vector according to the present disclosure is provided. As used herein, the term "subject" includes humans and other animals. Typically, a subject is a human. For example, a subject may be an adult, teenager, child (ages 2-14), infant (birth up to age 2), or newborn (up to 2 months). In certain embodiments, a subject is up to 4 months of age or up to 6 months of age. In some embodiments, an adult is about 65 years of age or older, or about 60 years of age or older. In some embodiments, a subject is a pregnant woman or a woman who intends to become pregnant. In other embodiments, a subject is not a human, e.g., a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, a subject may be a pet, such as a dog or cat.

[0111] As used herein, the term "host cell" includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or ceDNA expression vector of the present disclosure. By way of non-limiting example, host cells include isolated primary cells, pluripotent stem cells, CD34 + Host cells can be human cells, induced pluripotent stem cells, or any of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, host cells can be in situ or in vivo cells in a tissue, organ, or organism.

[0112] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced, by a process involving the hand of man, into a biological system, such as a cell or organism in which it is not normally found, and in which it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced, by a process involving the hand of man, into a biological system, such as a cell or organism in which it is found in relatively low abundance, and in which it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0113] The term "sequence identity" refers to the relatedness between two nucleotide sequences. For purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0114] As used herein, the term "homology" or "homologous" is defined as the percentage of nucleotide residues that are identical to the nucleotide residues in the corresponding sequence on the target chromosome after aligning the sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage. Alignment for purposes of determining the percent nucleotide sequence homology can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of a homologous arm is considered to be "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., a genomic sequence) of the host cell.

[0115] The term "heterologous," as used herein, refers to a nucleotide sequence or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence that encodes a chimeric polypeptide. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleic acid sequence that encodes a fusion variant polypeptide. Alternatively, the term "heterologous" may refer to a nucleic acid sequence that does not naturally occur in a cell or subject.

[0116] A "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," can be attached so as to bring about replication of the attached segment in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be of viral or non-viral origin and / or final form, however, for purposes of this disclosure, "vector," as that term is used herein, generally refers to a ceDNA vector. The term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replication and can transfer genetic sequences into a cell. According to some embodiments, a vector can be an expression vector or a recombinant vector.

[0117] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thus enabling it to be maintained in two organisms, for example, in human cells for expression and in prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins, and, where appropriate, secreting proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a (DNA) nucleic acid sequence that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0118] By "recombinant vector" is meant a vector containing a heterologous nucleic acid sequence, or "transgene," capable of being expressed in vivo. It should be understood that the vectors described herein, in some embodiments, can be combined with other appropriate compositions and treatments. In some embodiments, the vector is episomal. Use of an appropriate episomal vector provides a means of maintaining a nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0119] As used herein, the terms "administration," "administering," and variants thereof refer to the introduction of a composition or agent (e.g., a ceDNA described herein) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. Introduction of a composition or agent into a subject is by any suitable route, including oral, intrapulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be by any suitable route. The appropriate route of administration enables the composition or agent to perform its intended function. For example, if the appropriate route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0120] As used herein, the administration of a composition "following" the administration of a composition indicates that a time interval has elapsed between the administration of the first composition and the administration of the second composition, regardless of whether the first and second compositions are the same or different.

[0121] As used herein, the term "infection" refers to the initial invasion of a pathogen into a host; and to a state in which the pathogen is established in or on the cells or tissues of the host; such a state does not necessarily constitute or lead to disease.

[0122] As used herein, the term "biological sample" refers to any type of material of biological origin isolated from a subject, including, for example, DNA, RNA, lipids, carbohydrates, and proteins. The term "biological sample" includes tissues, cells, and biological fluids isolated from a subject. Biological samples include, for example, but are not limited to, whole blood, plasma, serum, semen, saliva, tears, urine, fecal material, sweat, oral cavity, skin, cerebrospinal fluid, bone marrow, bile, hair, muscle biopsy, organ tissue, or other materials of biological origin known to those of skill in the art. Biological samples can be obtained from subjects for diagnostic or research purposes, or can be obtained from healthy subjects as controls or for basic research. As used herein, the term "dose" refers to the amount of a substance (e.g., ceDNA described herein) ingested or administered to a subject at one time.

[0123] The term "dose," as used herein, refers to the administration of a substance (e.g., a ceDNA described herein) to achieve a therapeutic purpose (e.g., treatment).

[0124] The term "combination" in the phrase "a first agent in combination with a second agent" includes simultaneous administration of the first agent and the second agent, which may, for example, be dissolved or mixed in the same pharmaceutically acceptable carrier, or administration of the first agent followed by the second agent, or administration of the second agent followed by the first agent. The present disclosure therefore includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

[0125] The term "combined" in the phrase "combined therapeutic treatment" includes administering an agent in the presence of a second agent. Combination therapeutic treatment methods include methods in which a first, second, third, or additional agent is co-administered. Combination therapeutic treatment methods also include methods in which a first or additional agent is administered in the presence of a second or additional agent, where the second or additional agent may, for example, have been previously administered. Combination therapeutic treatment methods may be performed in stages by different actors. For example, one actor may administer a first agent to a subject, and a second actor may administer a second agent to a subject, and the administering steps may be performed simultaneously, nearly simultaneously, or at separate times, so long as the first agent (and additional agent) is in the presence of the second agent (and additional agent) after administration. The actor and subject may be the same entity (e.g., a human).

[0126] The term "combination therapy," as used herein, refers to the administration of two or more therapeutic agents, e.g., an antigen, or immunogenic protein, as described herein, and another drug. The other drug may be administered simultaneously with, before, or after the administration of the antigen, or immunogenic protein, as described herein.

[0127] As used herein, the terms "nucleic acid therapeutic," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any therapeutic modality that uses nucleic acids as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or nonviral synthetic DNA vector, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, Doggybone™ DNA vector, minimal immunologically defined gene expression (MIDGE) vector, nonviral ministring DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA"). According to some embodiments, the therapeutic nucleic acid is ceDNA.

[0128] As used herein, the term "therapeutic effect" refers to the outcome of treatment, which is deemed to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of disease symptoms. A therapeutic effect can also include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease symptoms.

[0129] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro tests and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is within the ability of one of ordinary skill in the art to adjust the dose to achieve maximum efficacy based on the methods described above and other well-known methods. The following summarizes general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference.

[0130] Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired degree of therapeutic efficacy with minimal, acceptable adverse effects. In situations where the plasma concentration of a drug is measured and may be related to the therapeutic window, additional guidance for dosage modifications can be obtained.

[0131] As used herein, "viral infection" is meant to refer to the invasion and proliferation of a virus in the body of a subject.

[0132] As used herein, the term "treatment" is meant to refer to either (i) prevention of infection or reinfection, as in traditional vaccines; (ii) reduction or elimination of symptoms; and (iii) substantial or complete elimination of the pathogen in question. Treatment can be effected prophylactically (before infection) or therapeutically (after infection). Treating can further refer to achieving one or more of: (a) reducing the severity of the disorder; (b) limiting the worsening of symptoms characteristic of the disorder being treated; (c) limiting the recurrence of the disorder in patients who previously had the disorder; and (d) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0133] Beneficial or desirable clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who would be susceptible to the disease, disorder, or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0134] As used herein, the term "vaccinated" is meant to refer to treatment with a vaccine.

[0135] As used herein, the term "vaccination" is meant to refer to treatment with a vaccine.

[0136] As used herein, the term "vaccine" is meant to refer to a formulation that is in a form that can be administered to a vertebrate and induces a protective immune response sufficient to induce immunity and / or prevent and / or ameliorate infection and / or reduce at least one symptom of infection and / or enhance the efficacy of another dose of the formulation. Typically, vaccines comprise a conventional saline or buffered aqueous solution medium in which a composition of the present disclosure is suspended or dissolved. In this form, the composition of the present disclosure can be conveniently used to prevent, ameliorate, or otherwise treat viral infection. Upon introduction into a host, the vaccine can elicit an immune response, including, but not limited to, the production of antibodies and / or cytokines and / or the activation of cytotoxic T cells, antigen-presenting cells, helper T cells, dendritic cells, and / or other cellular responses.

[0137] As used herein, the term "vaccine therapy" is meant to refer to a type of therapy that uses a substance or group of substances to stimulate the immune system to destroy tumors or infectious organisms.

[0138] Those "in need of treatment" include mammals, such as humans, who already have the disease or disorder, infection, or cancer.

[0139] As used herein, the terms "increase," "enhance," "elevate" (and similar terms) generally refer to the act of increasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to natural, expected, or average conditions, or compared to a control condition.

[0140] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of decreasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to natural, expected, or average conditions, or compared to control conditions.

[0141] As used herein, "control" is meant to refer to a reference standard. According to some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with a disease or disorder, infection, or cancer. In still other embodiments, the control is a historical control or standard reference value or range of values ​​(such as a previously tested control sample or a group of samples representing baseline or normal values). The difference between the test sample and the control can be an increase or, conversely, a decrease. The difference can be a qualitative difference or a quantitative difference, e.g., a statistically significant difference. In some embodiments, the difference is an increase or decrease of at least about 5%, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% compared to the control.

[0142] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the method or composition, but are open to the inclusion of unspecified elements, whether essential or not.

[0143] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment. The use of "comprises" indicates inclusion rather than limitation.

[0144] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of an embodiment.

[0145] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0146] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%. The present disclosure is further illustrated by the following examples, but the scope of the present disclosure should not be limited thereto.

[0147] Groupings of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member can be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification herein includes the modified group and, in this way, is deemed to satisfy the written description of all Markush groups used in the appended claims.

[0148] Other terms are defined herein within the description of various aspects of the disclosure.

[0149] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples thereof, the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, where necessary, to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made in protein structure without affecting biological or chemical activity in kind or amount. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0150] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Furthermore, while advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages, to fall within the scope of the present disclosure.

[0151] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It is to be understood that the present disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0152] II. Cells of the immune system There are many cellular interactions, including in the immune system, that occur through specific receptor-ligand pairs that signal in both directions, such that each cell is instructed based on the temporal and spatial distribution of those signals.

[0153] Mouse models are highly useful in discovering immunoregulatory pathways, however, the clinical utility of these pathways does not always translate from inbred mouse strains to outbred human populations because outbred human populations may have individuals that rely on individual immunoregulatory pathways to varying degrees.

[0154] Cells of the immune system include lymphocytes, monocytes / macrophages, dendritic cells, the closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. In addition, a series of specialized epithelial and stromal cells provide the anatomical environment in which immunity occurs, often by secreting key factors that regulate growth and / or gene activation in cells of the immune system, which also play a direct role in the inductive and effector phases of the response. (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0155] Cells of the immune system are found in peripheral organized tissues, such as the spleen, lymph nodes, Peyer's patches of the intestine, and tonsils. Lymphocytes are also found in central lymphoid organs, the thymus, and bone marrow, where they undergo developmental processes that prepare them to mediate the myriad responses of the mature immune system. A substantial portion of lymphocytes and macrophages comprise the recirculating pool of cells found in the blood and lymph, providing a means for delivering immunocompetent cells to sites of need and enabling locally generated immunity to be generalized. (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0156] The term "lymphocyte" refers to small white blood cells formed in lymphoid tissues throughout the body and, in normal adults, constitute approximately 22–28% of the total number of circulating white blood cells, which play a major role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to responding to a limited set of structurally related antigens through the recombination of their genetic material (e.g., to create T cell receptors and B cell receptors). This commitment exists before the immune system's initial contact with a given antigen and is expressed by the presence of receptors specific for determinants (epitopes) on the lymphocyte's surface membrane. Each lymphocyte has a unique population of receptors, all of which have identical binding sites. One set, or clone, of lymphocytes differs from another clone in the structure of its receptor binding region and thus in the epitopes it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors but also in their function. (Paul, WE, “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0157] Two broad classes of lymphocytes are recognized: B lymphocytes (B cells), which are the precursors of antibody-secreting cells, and T lymphocytes (T cells).

[0158] B lymphocytes B lymphocytes originate from hematopoietic cells in the bone marrow. Mature B cells can be activated by antigens expressing epitopes recognized by their cell surface. The activation process can be direct, dependent on cross-linking of membrane Ig molecules by antigen (cross-linking-dependent B cell activation), or indirect, via interaction with helper T cells, a process referred to as cognate help. In many physiological situations, receptor cross-linking stimuli and cognate receptors act synergistically to produce a more vigorous B cell response (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0159] Cross-linking-dependent B cell activation requires that the antigen express multiple copies of an epitope complementary to the binding site of the cell surface receptor because each B cell expresses an Ig molecule with an identical variable region. Such a requirement is fulfilled by other antigens with repeated epitopes, such as the capsular polysaccharides of microbial or viral envelope proteins. Cross-linking-dependent B cell activation is the primary protective immune response mounted against these microorganisms (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0160] Cognate help enables B cells to mount responses to antigens that cannot cross-link receptors, while at the same time providing costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linking events. Cognate help is dependent on the binding of antigen by the B cell's membrane immunoglobulin (Ig), endocytosis of the antigen, and its fragmentation into peptides within the endosomal / lysosomal compartment of the cell. Portions of the resulting peptides are loaded into grooves of a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules. The resulting class II / peptide complexes are expressed on the cell surface and bind to CD4 + Acts as a ligand for the antigen-specific receptor of a set of T cells termed T cells. CD4 + T cells bear receptors on their surface specific for the class II / peptide complex of B cells. B cell activation not only depends on the binding of a T cell through its T cell receptor (TCR), but this interaction also allows an activating ligand (CD40 ligand) on the T cell to bind to its receptor (CD40) on the B cell, signaling B cell activation. T helper cells also secrete several cytokines that regulate the growth and differentiation of stimulated B cells by binding to cytokine receptors on B cells (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0161] During cognate recruitment for antibody production, CD40 ligand binds activated CD4 +Transiently expressed on T helper cells, it binds to CD40 on antigen-specific B cells, thereby transducing a second costimulatory signal. The latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of antigen-encountered germline B cells. Overexpression of CD40 ligand on both B and T cells has been implicated in pathogenic autoantibody production in human SLE patients (Desai-Mehta, A. et al., "Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest. Vol. 97(9), 2063-2073, (1996)).

[0162] T lymphocytes T lymphocytes, derived from precursors in hematopoietic tissues, undergo differentiation in the thymus and then seed peripheral lymphoid tissues and the recirculating pool of lymphocytes. T lymphocytes, or T cells, mediate a wide range of immune functions. These include the ability to help B cells develop into antibody-producing cells, augment the microbe-killing activity of monocytes / macrophages, inhibit specific types of immune responses, directly kill target cells, and mobilize inflammatory responses. These effects depend on T cell expression of specific cell surface molecules and secretion of cytokines (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0163] T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulins are B cell receptors that bind to individual epitopes on soluble molecules or particle surfaces. B cell receptors recognize epitopes expressed on the surface of natural molecules. While antibodies and B cell receptors evolved to bind to and defend against microorganisms in extracellular fluids, T cells recognize antigens on the surface of other cells and mediate their function by interacting with and modifying the behavior of these antigen-presenting cells (APCs). In peripheral lymphoid organs, there are three main types of APCs that can activate T cells: dendritic cells, macrophages, and B cells. The most potent of these are dendritic cells, whose sole function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, intestine, and airways. When they encounter invading microorganisms at these sites, they endocytose the pathogens and their products and transport them via the lymph to local lymph nodes or gut-associated lymphoid organs. Encountering a pathogen induces dendritic cells to mature from antigen-capturing cells into APCs capable of activating T cells. APCs display three types of protein molecules on their surface that are responsible for activating T cells to become effector cells: (1) MHC proteins, which present foreign antigens to the T cell receptor; (2) costimulatory proteins, which bind to complementary receptors on the surface of T cells; and (3) cell-cell adhesion molecules, which allow T cells to bind to APCs long enough to be activated ("Chapter 24: The adaptive immune system," Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, (2002)).

[0164] T cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T cells express a T cell receptor (TCR) consisting of an α chain and a β chain. A small group of T cells expresses receptors made up of γ chains and δ chains. Among α / β T cells, there are two sub-lineages: those that express the core receptor molecule CD4 (CD4+ T cells) and those expressing CD8 (CD8 + T cells). These cells differ in how they recognize antigens and in their effector and regulatory functions.

[0165] CD4 + T cells are the primary regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell surface molecules, such as CD40 ligand, whose expression is induced upon T cell activation, and on the wide range of cytokines that they secrete upon activation.

[0166] T cells also mediate important effector functions, which are determined in part by the pattern of cytokines they secrete, which can be directly toxic to target cells and can mobilize powerful inflammatory mechanisms.

[0167] Also, T cells, especially CD8 + T cells can develop into cytotoxic T lymphocytes (CTLs) that can efficiently lyse target cells expressing the antigen recognized by the CTL (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0168] T cell receptors (TCRs) recognize complexes consisting of peptides derived by proteolysis of antigens bound to specialized grooves of class II or class I MHC proteins. CD4 + T cells recognize only peptide / class II complexes, whereas CD8 +T cells recognize peptide / class I complexes (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0169] Ligands for TCRs (i.e., peptide / MHC protein complexes) are generated within APCs. Generally, class II MHC molecules bind peptides derived from proteins internalized by APCs through the process of endocytosis. These peptide-bearing class II molecules are then expressed on the surface of cells, where they bind to CD4 receptors associated with TCRs capable of recognizing the expressed cell surface complexes. + available for binding by CD4 T cells. + T cells are specialized to react with antigens derived from extracellular sources (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0170] In contrast, class I MHC molecules are loaded primarily with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are generated from cytoplasmic proteins by proteolysis via the proteosome and translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound to class I MHC molecules and transported to the cell surface, where they are targeted to CD8 cells expressing the appropriate receptor. + It can be recognized by T cells, which allows it to be recognized by T cell lines, especially CD8 +T cells are given the ability to detect cells that express proteins that are different from, or produced in much greater amounts than, those in the rest of the organism (e.g., viral antigens) or mutant antigens (e.g., active oncogene products), but these proteins in their intact form may not be expressed on the cell surface or secreted (Paul, W.E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W.E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0171] T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells.

[0172] Helper T cells Helper T cells are T cells that stimulate B cells to generate antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes appear only once or at limited times, rendering them unable or inefficiently cross-linkable with B cell membrane immunoglobulins (Ig). B cells bind antigens through their membrane Ig, and the complexes undergo endocytosis. Within endosomal and lysosomal compartments, the antigens are fragmented into peptides by proteolytic enzymes, and one or more of the resulting peptides are packaged into class II MHC molecules, which translocate through this vesicular compartment. The resulting peptide / class II MHC complexes are then exported to the B cell surface membrane. T cells with receptors specific for the peptide / class II molecule complex recognize this complex on the B cell surface. (Paul, WE, “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia (1999)).

[0173] B cell activation depends on both the engagement of a T cell through its TCR and the interaction of T cell CD40 ligand (CD40L) with CD40 on the B cell. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of interaction with APCs that express both the T cell's TCR and the cognate antigen recognized by CD80 or CD86. CD80 / CD86 are generally expressed by activated B cells, but not by resting B cells, allowing helper interactions involving activated B cells and T cells to lead to efficient antibody production. In many cases, however, the initial induction of CD40L on T cells is dependent on their recognition of antigens on the surface of APCs, such as dendritic cells, that constitutively express CD80 / 86. Such activated helper T cells can then efficiently interact with and help B cells. Cross-linking of membrane Ig on B cells, even if inefficient, can synergize with CD40L / CD40 interactions to result in intense B cell activation. Subsequent events in the B cell response, including proliferation of expressed Ig classes, Ig secretion, and class switching, are either dependent on or enhanced by the action of T cell-derived cytokines (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0174] CD4 + T cells tend to differentiate into cells that primarily secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that primarily produce IL-2, IFN-γ, and lymphotoxin (TH1 cells). TH2 cells are highly effective in helping B cells develop into antibody-producing cells, whereas TH1 cells are effective inducers of cellular immune responses, including enhancing the microbe-killing activity of monocytes and macrophages and resulting increased efficiency in lysing microbes in intracellular vesicular compartments. The CD4+ phenotype associated with TH2 cells+ Although T cells (i.e., IL-4, IL-5, IL-6, and IL-10) are efficient helper cells, TH1 cells also have the ability to be helpers (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0175] T cell involvement in the induction of cellular immunity T cells can also act to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon gamma (IFN-γ) produced by helper T cells enhances several mechanisms by which mononuclear phagocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of tumor necrosis factor (TNF) production. TH1 cells are effective in enhancing microbial killing because they produce IFN-γ. In contrast, two of the major cytokines produced by TH2 cells, IL-4 and IL-10, block these activities (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0176] Regulatory T (Treg) cells Immune homeostasis is maintained by a controlled balance between initiation and down-regulation of immune responses. Both apoptosis and T cell anergy (a tolerance mechanism in which T cells are essentially functionally inactivated after antigen encounter (Schwartz, RH, "T cell anergy", Annu. Rev. Immunol., Vol. 21:305-334 (2003)) contribute to the down-regulation of immune responses. A third mechanism is the activation of suppressor cells or regulatory CD4 +It is provided by the active suppression of activated T cells by T (Treg) cells (reviewed in Kronenberg, M. et al., "Regulation of immunity by self-reactive T cells", Nature, Vol. 435:598-604 (2005)). The IL-2 receptor alpha (IL-2Rα) chain (CD4 + CD25 + ) constitutively expressing CD4 + Tregs are a natural T cell subset that are anergic and suppressive (Taams, L. et al., "Human anergic / suppressive CD4 + CD25 + CD4 + CD25 + Depletion of Tregs leads to systemic autoimmune disease in mice. Furthermore, transfer of these Tregs prevents the development of autoimmune disease. + CD25 + Tregs, like their murine counterparts, are generated in the thymus and are characterized by their ability to suppress the proliferation of responder T cells through a cell-cell contact-dependent mechanism, their inability to produce IL-2, and an anergic phenotype in vitro. + CD25 + T cells are classified as suppressor (CD25 high ) cells and non-inhibitory (CD25 low ) cells. FOXP3, a member of the forkhead family of transcription factors, mediates the differentiation of mouse and human CD4 + CD25 + It has been shown to be expressed in Tregs and CD4 + CD25 +It appears to be the master gene controlling Treg development (Battaglia, M. et al., "Rapamycin promotes expansion of functional CD4 + CD25 + Foxp3 + regulator T cells of both healthy subjects and type 1 diabetic patients”, J. Immunol., Vol. 177:8338-8347, (2006)).

[0177] cytotoxic T lymphocytes CD8 recognizes peptides from proteins produced within target cells + T cells have cytotoxic properties in that they lead to the lysis of target cells. The mechanism of CTL-induced lysis involves the production by CTLs of perforin, a molecule that can insert into the membrane of target cells and promote their lysis. Perforin-mediated lysis is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many activated CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTLs with fas on the surface of target cells initiates apoptosis in the target cells, leading to their death. CTL-mediated lysis appears to be the primary mechanism for the destruction of virus-infected cells.

[0178] Lymphocyte activation The terms "activation" or "lymphocyte activation" refer to the stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells, resulting in the synthesis of RNA, protein, and DNA and the production of lymphokines; this is followed by the proliferation and differentiation of various effector and memory cells. T cell activation is dependent on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events driven by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases, which lead to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include phospholipase Cγ1, a set of adaptor proteins that link the TCR to the ras pathway; its tyrosine phosphorylation increases its catalytic activity, engaging the inositol phospholipid metabolic pathway and leading to an increase in intracellular free calcium concentration and the activation of protein kinase C, as well as a series of other enzymes that control cell growth and differentiation. Full T cell responsiveness requires, in addition to receptor engagement, additional cell-delivered costimulatory activity, such as engagement of CD28 on the T cell by CD80 and / or CD86 on the APC.

[0179] T memory cells After pathogen recognition and eradication through the adaptive immune response, the majority of T cells (90-95%) undergo apoptosis, and the remaining cells form a pool of memory T cells: designated central memory T cells (TCM), effector memory T cells (TEM), and resident memory T cells (TRM) (Clark, RA, "Resident memory T cells in human health and disease", Sci. Transl. Med., 7, 269rv1, (2015)). CD45RA is expressed on naive T cells and both CD4 and CD8 T cells on effector cells. After antigen experience, central memory T cells and effector memory T cells gain CD45RO expression and lose CD45RA expression. Thus, either CD45RA or CD45RO is commonly used to distinguish naive from memory populations. CCR7 and CD62L are two other markers that can be used to distinguish central memory T cells from effector memory T cells. Naive and central memory cells express CCR7 and CD62L to migrate to secondary lymphoid organs. Thus, naive T cells express CD45RA + CD45RO - CCR7 + CD62L + and central memory T cells are CD45RA - CD45RO + CCR7 + CD62L + and effector memory T cells express CD45RA - CD45RO + CCR7 - CD62L - is.

[0180] Compared to standard T cells, these memory T cells are long-lived with distinct phenotypes, such as the expression of specific surface markers, the rapid production of distinct cytokine profiles, the capacity for direct effector cell function, and unique homing distribution patterns. Memory T cells respond rapidly upon re-exposure to their respective antigens to eliminate re-infection of the attacker and thereby rapidly restore balance to the immune system. Increasing evidence demonstrates that autoimmune memory T cells hinder most attempts to treat or cure autoimmune diseases (Clark, RA, "Resident memory T cells in human health and disease", Sci. Transl. Med., Vol. 7, 269rv1, (2015)).

[0181] III. Expression of peptides from DNA vectors Provided herein are methods for inducing an immune response to a first peptide and a second peptide in a subject, comprising administering to the subject a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes the first peptide; and administering to the subject a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, wherein the RNA encodes the second peptide, thereby inducing an immune response to the first peptide and the second peptide in the subject.

[0182] Also provided is a vaccine regimen comprising: a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes a first peptide; and a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, wherein the RNA encodes the second peptide.

[0183] In some embodiments, the priming vaccine comprises DNA in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. In some embodiments, the priming vaccine comprises DNA in the form of a plasmid. In some embodiments, the priming vaccine comprises DNA in the form of ceDNA.

[0184] In some embodiments, the priming vaccine comprises DNA in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector, and the boosting vaccine comprises RNA (e.g., mRNA).

[0185] In some embodiments, the priming vaccine comprises DNA in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector, and the boosting vaccine comprises a peptide.

[0186] DNA plasmids In some embodiments, the priming vaccine comprises DNA in the form of a DNA plasmid containing a nucleic acid sequence encoding a selected antigen against which an immune response is desired, wherein the selected antigen is under the control of regulatory sequences that direct its expression in mammalian or vertebrate cells.

[0187] The components of the plasmid itself are known in the art.

[0188] Non-viral plasmid vectors useful in the present invention include isolated and purified DNA sequences that encode a selected antigen, such as those described herein. The DNA molecule may be derived from a viral or non-viral, e.g., bacterial, species engineered to encode an exogenous or heterologous nucleic acid sequence. Such plasmids or vectors may contain sequences from viruses or phages. A variety of non-viral vectors are known in the art and may include, but are not limited to, plasmids, bacterial vectors, bacteriophage vectors, "naked" DNA, and DNA condensed with cationic lipids or polymers.

[0189] Examples of bacterial vectors include, but are not limited to, sequences derived from Bacillus Calmette-Guerin (BCG), Salmonella, Rhodophyta, Escherichia coli, and Listeria, among others. Suitable plasmid vectors include, for example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101, pAC105, pVA51, pKH47, pUB110, pMB9, pBR325, Col El, pSC101, pBR313, pML21, RSF2124, pCR1, RP4, pBAD18, and pBR328.

[0190] Examples of suitable inducible Escherichia coli expression vectors include pTrc (Amann et al., 1988 Gene, 69:301-315), arabinose expression vectors (e.g., pBAD18, Guzman et al., 1995 J. Bacteriol., 177:4121-4130), and pETIId (Studier et al., 1990 Methods in Enzymology, 85:60-89).

[0191] The promoter and other regulatory sequences that drive expression of the antigen in the desired mammalian or vertebrate host may similarly be selected from an extensive list of promoters known to be useful for that purpose. A variety of such promoters are disclosed below. Exemplary promoters include, but are not limited to, the human cytomegalovirus (HCMV) promoter / enhancer (e.g., those described in U.S. Pat. Nos. 5,168,062 and 5,385,839), as well as the SCMV promoter enhancer.

[0192] Additional regulatory sequences for inclusion in a nucleic acid sequence, molecule, or vector include, but are not limited to, enhancer sequences, polyadenylation sequences, splice donor and splice acceptor sequences, sites for transcription initiation and termination positioned at the beginning and end, respectively, of the polypeptide to be translated, ribosome binding sites for translation in the transcribed region, epitope tags, nuclear localization sequences, IRES elements, Goldberg-Hogness "TATA" elements, restriction enzyme cleavage sites, selectable markers, and the like. Enhancer sequences include, for example, the 72 bp tandem repeat of SV40 DNA or retroviral long terminal repeats or LTRs, and are used to increase transcription efficiency.

[0193] These other components useful in DNA plasmids, including, for example, origins of replication, polyadenylation sequences (e.g., BGH polyA, SV40 polyA), drug resistance markers (e.g., kanamycin resistance), and the like, may also be selected from among sequences well known in the art.

[0194] Selection of promoters and other common vector elements is conventional, and many such sequences are available for designing plasmids useful in the present invention. See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1989), and references cited therein, e.g., pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989). All components of the plasmids can be readily selected by one of skill in the art from materials known in the art and available from the pharmaceutical industry.

[0195] Examples of suitable DNA plasmid constructs that can be used in the priming vaccines described herein are detailed in the following patent publications: International Patent Publication Nos. WO98 / 17799, WO99 / 43839, and WO98 / 17799; and U.S. Patent Nos. 5,593,972; 5,817,637; 5,830,876; and 5,891,505, which are incorporated herein by reference in their entireties.

[0196] ceDNA vector According to some embodiments, the technology described herein is generally directed to the expression and / or production of antigens in cells from one or more non-viral DNA vectors, e.g., the ceDNA vectors described herein. ceDNA vectors for antigen expression are described herein in the section entitled "ceDNA Vectors Generally." As previously discussed, a distinct advantage of ceDNA vectors over traditional AAV vectors, and even lentiviral vectors, is that there are no size constraints on the nucleic acid sequence or sequences encoding peptides (e.g., antigens). Based on the disclosure provided herein, one skilled in the art will understand that a multitude of peptide antigens can be used to produce an almost limitless variety of ceDNA vectors, once armed with the teachings provided herein.

[0197] In some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises a pair of ITRs (e.g., symmetric or asymmetric as described herein), between which is located a nucleic acid encoding the ITR pair, antigen, or immunogenic peptide as described herein, and operably linked to a promoter or regulatory sequence. A distinct advantage of ceDNA vectors for expression of antigens or immunogenic peptides over traditional AAV vectors, and even lentiviral vectors, is that there are no size constraints on the nucleic acid sequence encoding the desired antigen or immunogenic peptide.

[0198] As will be understood, the ceDNA vector technology described herein can be adapted to any level of complexity or used in a modular fashion, where expression of different components of the ceDNA vector can be controlled in an independent manner. The following embodiments are specifically contemplated herein and can be adapted by those of skill in the art as desired.

[0199] In some aspects, the present disclosure provides one or more ceDNA vectors comprising one or more nucleic acid sequences encoding antigens. In some embodiments, the one or more nucleic acid sequences encode one or more peptides (e.g., antigens) from various pathogens, including, for example, bacterial, viral, fungal, and parasitic infectious agents. In some embodiments, the one or more nucleic acid sequences encode one or more peptides (e.g., antigens) that are cancer or cancer-associated antigens. In some embodiments, the antigen or immunogenic peptide is a tumor antigen. In some embodiments, the one or more nucleic acid sequences encode one or more peptides (e.g., antigens) associated with an autoimmune condition, such as rheumatoid arthritis (RA) or multiple sclerosis (MS). In some embodiments, the antigen is an antigen associated with an autoimmune disorder or condition, such as an autoimmune disease induced by an infectious agent, or with an infectious disease or pathogen.

[0200] Cancer or tumor-associated antigens In some embodiments, the ceDNA comprises nucleic acid sequences encoding cancer or tumor-associated antigens. In some embodiments, the ceDNA comprises nucleic acid sequences encoding one or more antigens selected from the Cancer Antigen Peptide Database, publicly available at caped.icp.ucl.ac.be / about. This database includes, for each identified antigen, the peptide sequence and its location in the protein sequence.

[0201] According to some embodiments, the ceDNA comprises a nucleic acid sequence encoding a tumor-associated antigen selected from one or more of the antigens set forth in Table 1 below. [Table 1]

[0202] Recent analyses of The Cancer Genome Atlas (TCGA) datasets have implicated neoantigen load in the tumor genomic environment in tumor immunity and driving T cell responses (Brown et al., Genome Res. 2014 May;24(5):743-50, 2014) and identified somatic mutations associated with immune infiltration (Rutledge et al., Clin Cancer Res. 2013 Sep 15;19(18):4951-60, 2013). Rooney et al. (2015 Jan 15;160(1-2):48-61) uncovered known and novel mutations that enable tumors to resist immune attack, suggesting that neoantigens and viruses likely drive cytolytic activity.

[0203] In some embodiments, the antigen is a neoantigen identified from cancer cells in a subject. In some embodiments, the neoantigen is a shared neoantigen. Methods for identifying neoantigens are known in the art and are described, for example, in U.S. Patent No. 10,055,540, which is incorporated herein by reference in its entirety. Neoantigenic polypeptides and shared neoantigenic polypeptides are described, for example, in PCT / US2016 / 033452, U.S. Publication No. 20180055922, Schumacher and Hacohen et al. (Curr Opin Immunol. 2016 Aug;41:98-103), Gubin, MM et al. (Nature. 2014 Nov 27;515(7528):577-81), Schumacher and Schreiber, Science. 2015 Apr 3;348(6230):69-74), Ott PA., et al., Nature. 2017 Jul 13;547(7662):217-221, all of which are incorporated herein by reference in their entireties.

[0204] Thus, in some embodiments, the antigen is a neoantigen polypeptide. In some embodiments, the antigen is a neoantigen polypeptide listed in The Comprehensive Tumor-Specific Neoantigen Database (TSNAdb v1.0), available at biopharm.zju.edu.cn / tsnadb, and described in Wu et al., Genomics Proteomics Bioinformatics 16 (2018) 276-282. In some embodiments, the antigen is a neoantigen polypeptide listed in U.S. Patent No. 10,055,540, incorporated herein by reference in its entirety.

[0205] autoimmune disease antigens In some embodiments, the antigen is associated with an autoimmune disease. In some embodiments, the ceDNA comprises a nucleic acid sequence encoding one or more antigens selected from those in Table 2 below. [Table 2-1] [Table 2-2]

[0206] In some embodiments, the autoimmune disease is induced by an infectious agent. In some embodiments, the present disclosure provides ceDNA as described herein that includes a nucleic acid sequence encoding one or more peptides (e.g., antigens) for treating an autoimmune disease or disorder associated with or induced by an infectious agent. Exemplary autoimmune diseases or disorders associated with or induced by an infectious agent are provided in Table 3. [Table 3]

[0207] infectious disease In some embodiments, the present disclosure provides ceDNAs as described herein that include a nucleic acid sequence encoding one or more peptides (e.g., antigens) for treating an infectious disease. In some embodiments, the antigen is an antigen of a pathogen or infectious agent (wherein pathogen and infectious agent are used interchangeably herein), such as a viral pathogen, a bacterial pathogen, a fungal pathogen, or a parasitic pathogen.

[0208] In some embodiments, the antigen is a viral antigen. In some embodiments, the present disclosure provides a ceDNA as described herein, comprising a nucleic acid sequence encoding one or more viral antigens.

[0209] Viral infections include adenovirus, coxsackievirus, hepatitis A virus, poliovirus, Epstein-Barr virus, herpes simplex type 1, herpes simplex type 2, human cytomegalovirus, human herpesvirus type 8, varicella-zoster virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, papillomavirus, rabies virus, and rubella virus. Other viral targets include Paramyxoviridae (e.g., Pneumovirus, Morbillivirus, Metapneumovirus, Respirovirus, or Rubulavirus), Adenoviridae (e.g., Adenovirus), Arenaviridae (e.g., Arenaviruses, such as Lymphocytic Choriomeningitis virus), Arteriviridae (e.g., Porcine Respiratory and Reproductive Syndrome virus or Equine Arteritis virus), Bunyaviridae (e.g., Phlebovirus or Hantavirus), Caliciviridae (e.g., Norwalk virus), Coronaviridae (e.g., Coronavirus or Torovirus), Filoviridae (e.g., Ebola-like virus), Flaviviridae (e.g., Hepacivirus or Flavivirus), Herpesvirus Family (e.g., Mycoviridae, Varicellovirus, Cytomegalovirus, Roseolovirus, or Lymphocryptovirus), Orthomyxoviridae (e.g., Influenza virus or Thogotovirus), Parvoviridae (e.g., Parvovirus), Picornaviridae (e.g., Enterovirus or Hepatovirus), Poxviridae (e.g., Orthopoxvirus, Avipoxvirus, or Leporipoxvirus), Retroviridae (e.g., Lentivirus or Spumavirus), Reoviridae (e.g., Rotavirus), Rhabdoviridae (e.g., Lyssavirus, Novirhabdovirus, or Vesiculovirus), and Togaviridae (e.g., Alphavirus or Rubivirus). Specific examples of these viruses include human respiratory coronavirus, influenza viruses A-C, hepatitis viruses A-G, and herpes simplex viruses 1-9.

[0210] Exemplary viral pathogens are shown in Table 4 below. [Table 4-1] [Table 4-2]

[0211] According to some embodiments, the present disclosure provides ceDNA as described herein, which comprises a nucleic acid sequence encoding one or more peptides (e.g., antigens) for treating COVID-19. According to some embodiments, the nucleic acid encodes the SARS-CoV-2 spike protein.

[0212] The spike protein contains an S1 subunit that promotes the binding of coronaviruses to cell surface proteins. Thus, the S1 subunit of the spike protein controls which cells are infected by coronaviruses. The spike protein also contains an S2 subunit, which is a transmembrane subunit that promotes viral and cellular membrane fusion.

[0213] The complete genome of the severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 is shown under GenBank accession number MN908947.3. The amino acid sequence of the wild-type spike glycoprotein (S) is shown below as SEQ ID NO:

[0214] [ka]

[0215] According to some embodiments, the peptide is a stabilized pre-fusion SARS-CoV-2 spike protein (SARS-CoV-2 S(2P)).

[0216] In some embodiments, the peptide is a bacterial antigen. In some embodiments, the present disclosure provides a ceDNA as described herein, comprising a nucleic acid sequence encoding one or more bacterial antigens.

[0217] Bacterial infections include, but are not limited to, mycobacteria, rickettsia, mycoplasma, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella, Vibrio cholerae, Streptococcus, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynebacterium diphtheriae, Clostridium spp., enterotoxigenic Escherichia coli, Bacillus anthracis, rickettsia, Bartonella henselae, Bartonella quintana, Coxiella burnetii, Chlamydia, Mycobacterium leprae, Salmonella; Shige Including: Yersinia enterocolitica; Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis; Listeria monocytogenes; Mycoplasma spp.; Pseudomonas fluorescens; Vibrio cholerae; Haemophilus influenzae; Bacillus anthracis; Treponema pallidum; Leptospira; Borrelia; Corynebacterium diphtheriae; Francisella; Brucella melitensis; Campylobacter jejuni; Enterobacter; Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

[0218] Exemplary bacterial infections are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

[0219] In some embodiments, the antigen is a fungal antigen or an immunogenic peptide. In some embodiments, the present disclosure provides a ceDNA as described herein, comprising a nucleic acid sequence encoding one or more fungal antigens.

[0220] Exemplary fungal infections are shown in Table 6 below. [Table 6-1] [Table 6-2]

[0221] In some embodiments, the peptide is a parasitic antigen. In some embodiments, the present disclosure provides a ceDNA as described herein, comprising a nucleic acid sequence encoding one or more fungal antigens.

[0222] Exemplary parasitic infections are shown in Table 7 below. [Table 7-1] [Table 7-2] [Table 7-3]

[0223] Other diseases and disorders are contemplated for treatment with the ceDNA vectors of the present disclosure. Examples include, but are not limited to, cardiovascular diseases and immune disorders.

[0224] For example, it is well within the ability of one of ordinary skill in the art to take a known and / or publicly available protein sequence of an antigen and reverse engineer a cDNA sequence to encode such a protein.

[0225] IV. ceDNA Vectors for Use in Antigen Production Embodiments of the present disclosure are based on methods and compositions including priming vaccines comprising deoxyribonucleic acid (DNA), wherein the DNA is a closed-end linear double-stranded (ceDNA) vector capable of expressing a peptide. As described herein, peptides (e.g., antigens) may be selected from a variety of pathogens, including, for example, bacterial, viral, fungal, and parasitic infectious agents, or cancer or cancer-associated antigens, or the like. Still other targets may include autoimmune conditions, such as rheumatoid arthritis (RA) or multiple sclerosis (MS).

[0226] In some embodiments, the transgene is a nucleic acid sequence encoding an antigen. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, e.g., expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vector has covalently closed ends and is thus resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for 1 hour at 37°C.

[0227] Generally, a ceDNA vector for expression of a peptide (e.g., an antigen) disclosed herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. The ITR sequences are selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetrically modified ITR); (ii) two modified ITRs, where the mod-ITR pair has a different three-dimensional spatial configuration relative to each other (e.g., an asymmetrically modified ITR); (iii) a symmetric or substantially symmetric WT-WT ITR pair, where each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair, where each mod-ITR has the same three-dimensional spatial configuration.

[0228] Included herein are methods and compositions comprising ceDNA vectors for the production of peptides (e.g., antigens), which may further comprise a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system. Non-limiting exemplary liposomal nanoparticle systems encompassed for use are disclosed herein. According to some embodiments, the present disclosure provides lipid nanoparticles comprising ceDNA and an ionizable lipid. For example, lipid nanoparticle formulations made and loaded with ceDNA vectors obtained by the present process are disclosed in International Application PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein by reference.

[0229] The ceDNA vector disclosed herein does not have the packaging constraints imposed by the limited space within the viral capsid.In contrast to the enclosed AAV genome, the ceDNA vector represents a viable eukaryotic alternative to the prokaryotic production of plasmid DNA vectors.This allows the insertion of control elements, such as the regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0230] 1A-1E of International Publication No. WO / 2019 / 051255, incorporated herein by reference in its entirety, show schematic diagrams of the corresponding sequences of non-limiting exemplary ceDNA vectors or ceDNA plasmids for the expression of peptides (e.g., antigens). ceDNA vectors for the expression of peptides (e.g., antigens) are capsid-free and can be obtained from a plasmid encoding, in this order: a first ITR, an expression cassette containing a transgene, and a second ITR. The expression cassette may include one or more regulatory sequences that enable and / or control expression of the transgene, for example, where the expression cassette can include, in this order: one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA).

[0231] The expression cassette may also contain an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir-regulatable elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments, the ITRs can act as promoters for the transgene. In some embodiments, the ceDNA vectors contain additional components for regulating the expression of the transgene, such as a regulatory switch to control and regulate the expression of the peptide (e.g., antigen), and, if desired, a kill switch to enable controlled cell death of cells containing the ceDNA vector.

[0232] An expression cassette can comprise more than 4000 nucleotides, more than 5000 nucleotides, more than 10,000 nucleotides, or more than 20,000 nucleotides, or more than 30,000 nucleotides, or more than 40,000 nucleotides, or more than 50,000 nucleotides, or any range between about 4000-10,000 nucleotides, 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, an expression cassette can comprise a transgene in the length range of 500-50,000 nucleotides. In some embodiments, an expression cassette can comprise a transgene in the length range of 500-75,000 nucleotides. In some embodiments, an expression cassette can comprise a transgene in the length range of 500-10,000 nucleotides. In some embodiments, an expression cassette can comprise a transgene in the length range of 1000-10,000 nucleotides. In some embodiments, the expression cassette can contain a transgene ranging in length from 500 to 5,000 nucleotides. The ceDNA vector does not have the size limitation of encapsidated AAV vectors, thus enabling delivery of large-sized expression cassettes to provide efficient transgene expression. In some embodiments, the ceDNA vector lacks prokaryote-specific methylation.

[0233] The sequences provided in the expression cassettes, expression constructs, of the ceDNA vectors for expression of the peptides (e.g., antigens) described herein can be codon-optimized for the target host cell. As used herein, the term "codon-optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of a vertebrate of interest, e.g., a mouse or human, by replacing at least one, two, or a significant number of codons in the native sequence (e.g., a prokaryotic sequence) with codons more frequently or most frequently used in the genes of that vertebrate. Different species exhibit specific biases for particular codons for particular amino acids. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's GENE FORGE® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another publicly available database. According to some embodiments, the nucleic acid is optimized for human expression.

[0234] The transgene expressed by the ceDNA vector for expression of the peptides (e.g., antigens) disclosed herein encodes the antigen. There are many structural features of ceDNA vectors that distinguish them from plasmid-based expression vectors. ceDNA vectors can have one or more of the following characteristics: lack of original (i.e., uninserted) bacterial DNA; lack of a prokaryotic origin of replication; self-contained, i.e., they do not require sequences other than the two ITRs and contain Rep binding sites and terminal resolution sites (RBS and TRS); exogenous sequences between the ITRs; the presence of ITR sequences that form hairpins; and the absence of bacterial-type DNA methylation or indeed any other methylation that would be considered abnormal by a mammalian host. Generally, it is preferred that the vector does not contain any prokaryotic DNA; however, it is contemplated that some prokaryotic DNA may be inserted as an exogenous sequence, as a non-limiting example in the promoter or enhancer region. Another important feature that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, while plasmids are always double-stranded DNA.

[0235] The ceDNA vectors for expression of peptides (e.g., antigens) produced by the methods provided herein preferably have a linear and contiguous structure, rather than a discontinuous structure, as determined by restriction enzyme digestion assays (see, e.g., Figure 4D of International Publication No. WO / 2019 / 051255, incorporated herein by reference in its entirety). Linear and contiguous structures are believed to be more stable from attack by cellular endonucleases and less likely to recombine and cause mutagenesis. Thus, ceDNA vectors in linear and contiguous structures are preferred embodiments. Continuous, linear, single-stranded intramolecular double-stranded ceDNA vectors can have covalently linked ends without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally distinct from plasmids (including the ceDNA plasmids described herein), which are circular double-stranded nucleic acid molecules of bacterial origin. While the complementary strands of a plasmid can be separated after denaturation to produce two nucleic acid molecules, in contrast, a ceDNA vector, while having complementary strands, is a single DNA molecule and therefore remains a single molecule even when denatured. According to some embodiments, the ceDNA vectors described herein, unlike plasmids, can be produced without prokaryotic-type DNA base methylation. Thus, ceDNA vectors and ceDNA plasmids differ in terms of their structure (particularly linear vs. circular), in terms of the methods used to produce and purify these different entities (see below), and in terms of their DNA methylation, which is prokaryotic for ceDNA-plasmids and eukaryotic for ceDNA vectors.

[0236] There are several differences in using ceDNA vectors for expression of peptides (e.g., antigens) from plasmid-based expression vectors, including, but not limited to: 1) plasmids contain bacterial DNA sequences and are subject to prokaryote-specific methylation, e.g., 6-methyladenosine and 5-methylcytosine methylation, whereas capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryote-specific methylation; consequently, capsid-free AAV vectors are less likely to induce inflammatory and immune responses compared to plasmids; 2) circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, i.e., ITRs, which can be designed to confer resistance to nucleases and be targeted for delivery to the nucleus.

[0237] inverted terminal repeat (ITR) As disclosed herein, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises a transgene or nucleic acid sequence positioned between two inverted terminal repeat (ITR) sequences, where the ITR sequences can be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. The ceDNA vectors disclosed herein can comprise ITR sequences selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetrically modified ITR); (ii) two modified ITRs, wherein the mod-ITR pair has a different three-dimensional spatial configuration relative to each other (e.g., an asymmetrically modified ITR); or (iii) a symmetric or substantially symmetric WT-WT ITR pair, wherein each WT-ITR has the same three-dimensional spatial configuration; or (iv) a symmetric or substantially symmetric modified ITR pair, wherein each mod-ITR has the same three-dimensional spatial configuration, wherein the methods of the present disclosure may further include a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system.

[0238] In some embodiments, the ITR sequences can be from viruses of the Parvoviridae family, which includes two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. The Parvovirinae subfamily (also referred to as parvoviruses) includes the genus Dependovirus, whose members, under most conditions, require co-infection with a helper virus, such as an adenovirus or a herpesvirus, for productive infection. The Dependovirus genus includes adeno-associated viruses (AAVs), which typically infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0239] While the ITRs exemplified herein and in the Examples herein are AAV2 WT-ITRs, one skilled in the art can use ITRs from any known parvovirus, e.g., dependovirus, such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes; e.g., NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITRs, or ITRs from any synthetic AAV, as described above. In some embodiments, the AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are from B19 parvovirus (GenBank Accession No. NC 000883), minute virus of mice (MVM) (GenBank Accession No. NC 001510), goose parvovirus (GenBank Accession No. NC 001701), or snake parvovirus 1 (GenBank Accession No. NC 006148). In some embodiments, the 5'WT-ITR can be from one serotype and the 3'WT-ITR can be from a different serotype, as discussed herein.

[0240] Those skilled in the art recognize that ITR sequences have the consensus structure of a double-stranded Holliday junction, which is typically a T- or Y-shaped hairpin structure (see, e.g., Figures 2A and 3A of International Publication No. WO / 2019 / 051255, incorporated herein by reference in its entirety), in which each WT-ITR is formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded D sequence (where the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, e.g., structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6). Grimm et al., J. Virology, 2006;80(1);426-439; Yan et al., J. Virology, 2005;364-379; Duan et al., Virology 1999;261;8-14. Based on the exemplary AAV2 ITR sequences provided herein, one of skill in the art can readily determine the WT-ITR sequence from any AAV serotype for use in a ceDNA vector or ceDNA plasmid. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1 to AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006;80(1);426-439, which shows the % identity of the left ITR of AAV2 to left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0241] Symmetric ITR pair According to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) described herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other—i.e., the ceDNA vector can comprise ITR sequences that have a symmetrical three-dimensional spatial organization, and their structures are the same shape in geometric space or have the same A, C-C', and B-B' loops in 3D space. In such embodiments, the symmetric or substantially symmetric ITR pair can be a modified ITR (e.g., a mod-ITR) that is not a wild-type ITR. A mod-ITR pair can have one or more modifications from the wild-type ITR and have the same sequence that is the reverse complement (reverse orientation) of each other. In alternative embodiments, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair has different sequences but can have corresponding or identical symmetrical three-dimensional shapes.

[0242] (i) Wild-type ITR According to some embodiments, the symmetric or substantially symmetric ITRs are wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs from the same AAV serotype. That is, according to some embodiments, one WT-ITR can be from one AAV serotype and the other WT-ITR can be from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, i.e., they can have one or more conservative nucleotide modifications while still retaining a symmetric three-dimensional spatial organization.

[0243] Thus, as disclosed herein, a ceDNA vector comprises a transgene or nucleic acid sequence positioned between two adjacent wild-type inverted terminal repeat (WT-ITR) sequences, which are reverse complements (inverted) of each other, or alternatively, are substantially symmetrical with respect to each other - i.e., the WT-ITR pair has a symmetrical three-dimensional spatial configuration. According to some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) comprises a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: __) and a functional terminal resolution site (TRS; e.g., 5'-AGTT-3', SEQ ID NO: __).

[0244] According to some aspects, a ceDNA vector for expression of a peptide (e.g., an antigen) can be obtained from a vector polynucleotide encoding a nucleic acid operatively positioned between two wild-type inverted terminal repeats (WT-ITRs) (e.g., AAV WT-ITRs). That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs from the same AAV serotype. That is, according to some embodiments, one WT-ITR can be from one AAV serotype and the other WT-ITR can be from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they can have one or more conservative nucleotide modifications while still retaining a symmetrical three-dimensional spatial configuration. According to some embodiments, the 5'WT-ITR is from one AAV serotype and the 3'WT-ITR is from the same or a different AAV serotype. According to some embodiments, the 5'WT-ITR and the 3'WT-ITR are mirror images of each other, i.e., they are symmetrical. According to some embodiments, the 5'WT-ITR and the 3'WT-ITR are from the same AAV serotype.

[0245] WT ITRs are well known. In some embodiments, the two ITRs are from the same AAV2 serotype. In certain embodiments, WT ITRs from other serotypes can be used. There are many homologous serotypes, such as AAV2, AAV4, AAV6, and AAV8. In some embodiments, closely homologous ITRs (e.g., ITRs with similar loop structures) can be used. In other embodiments, more diverse AAV WT ITRs, such as AAV2 and AAV5, can be used, and in still other embodiments, substantially WT ITRs can be used—i.e., they have the basic loop structure of the WT but have some conservative nucleotide changes that do not alter or affect the properties. When WT ITRs from the same viral serotype are used, one or more regulatory sequences may also be used. In certain embodiments, the regulatory sequence is a regulatory switch that allows for modulation of the activity of the ceDNA, for example, the expression of the encoded antigen or immunogenic peptide.

[0246] According to some embodiments, one aspect of the technology described herein relates to a ceDNA vector for expression of a peptide (e.g., an antigen), wherein the ceDNA vector comprises at least one nucleic acid sequence encoding, for example, an HC and / or LC, operably positioned between two wild-type inverted terminal repeats (WT-ITRs), wherein the WT-ITRs can be from the same serotype, different serotypes, or substantially symmetric with respect to each other (i.e., have a symmetric three-dimensional spatial organization, such that their structures are the same shape in geometric space or have the same A, C-C', and B-B' loops in 3D space). According to some embodiments, the symmetric WT-ITRs comprise functional terminal resolution sites and Rep binding sites. According to some embodiments, the nucleic acid sequence encodes a transgene, wherein the vector is not in a viral capsid.

[0247] In some embodiments, the WT-ITRs are identical but are reverse complements of each other. For example, the sequence AACG in the 5' ITR may be CGTT (i.e., the reverse complement) in the 3' ITR at the corresponding site. In some examples, the 5' WT-ITR sense strand comprises the sequence ATCGATCG, and the corresponding 3' WT-ITR sense strand comprises CGATCGAT (i.e., the reverse complement of ATCGATCG). In some embodiments, the WT-ITR ceDNA further comprises a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), e.g., a Rep binding site.

[0248] Exemplary WT-ITR sequences for use in ceDNA vectors for expression of peptides (e.g., antigens) containing WT-ITRs are shown in Table 8 herein, which shows pairs of WT-ITRs (5'WT-ITR and 3'WT-ITR).

[0249] As an illustrative example, the present disclosure provides a ceDNA vector for expression of a peptide (e.g., an antigen) comprising a promoter operably linked to a transgene (e.g., a nucleic acid sequence), with or without a regulatory switch, wherein the ceDNA lacks a capsid protein and is (a) produced from a ceDNA plasmid encoding WT-ITRs (see, e.g., Figures 1F-1G of International Publication No. WO / 2019 / 051255, incorporated herein by reference in its entirety), wherein each WT-ITR has the same number of intramolecular double-stranded base pairs in its hairpin secondary configuration (preferably excluding the loss of any AAA or TTT terminal loops in this configuration compared to these reference sequences), and (b) identified as a ceDNA using the assay for identifying ceDNA by agarose gel electrophoresis under native and denaturing conditions in Example 1.

[0250] In some embodiments, adjacent WT-ITRs are substantially symmetrical to one another. In this embodiment, the 5' WT-ITR can be from one AAV serotype and the 3' WT-ITR can be from a different AAV serotype, such that the WT-ITRs are not identical reverse complements. For example, the 5' WT-ITR can be from AAV2 and the 3' WT-ITR can be from a different serotype (e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). In some embodiments, the WT ITRs can be selected from two different parvoviruses selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. In some embodiments, such a combination of WT ITRs is a combination of WT ITRs from AAV2 and AAV6. In some embodiments, substantially symmetric WT ITRs are at least 90% identical, at least 95% identical, at least 96%...97%...98%...99%...99.5% identical, and all points in between, when one is inverted relative to the other ITR, and have the same symmetric three-dimensional spatial configuration. According to some embodiments, a WT-ITR pair is substantially symmetric because they have a symmetric three-dimensional spatial organization, e.g., the same 3D organization of the A, C-C', B-B', and D arms. According to some embodiments, a substantially symmetric WT-ITR pair is inverted relative to the other and is at least 95% identical to each other, at least 96%...97%...98%...99%...99.5% and all points in between, wherein one WT-ITR retains a Rep binding site (RBS) and terminal resolution site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60).According to some embodiments, a substantially symmetric WT-ITR pair is inverted relative to each other and is at least 95% identical to each other, at least 96%...97%...98%...99%...99.5% and all points in between, where one WT-ITR retains a Rep binding site (RBS) and terminal resolution site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: __), and also a variable palindromic sequence that allows for hairpin secondary structure formation. Homology can be determined by standard means known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN with default settings, etc.

[0251] In some embodiments, the structural element of an ITR can be any structural element involved in the functional interaction of an ITR with a large Rep protein (e.g., Rep78 or Rep68). In certain embodiments, the structural element provides selectivity for the interaction of an ITR with a large Rep protein, i.e., at least partially determines whether a Rep protein will functionally interact with the ITR. In other embodiments, the structural element physically interacts with a large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleic acid sequence of the ITR, the spacing between two or more elements, or any combination of the above. In some embodiments, the structural element is selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arm, Rep binding site (RBE) and RBE' (i.e., complementary RBE sequences), and terminal resolution site (trs).

[0252] By way of example only, Table 8 shows exemplary WT-ITR combinations.

[0253] Table 8: Exemplary combinations of WT-ITRs from the same or different serotypes, or different parvoviruses. The order shown does not indicate the ITR positions, for example, "AAV1, AAV2" demonstrates that the ceDNA can comprise a WT-AAV1 ITR at the 5' position and a WT-AAV2 ITR at the 3' position, or vice versa, a WT-AAV2 ITR at the 5' position and a WT-AAV1 ITR at the 3' position. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AA) V8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12); AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes (e.g., NCBI:NC 002077;NC 001401;NC001729;NC001829;NC006152;NC 006260;NC 006261), ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), canine, equine, and ovine AAV), ITRs from B19 parvovirus (GenBank accession number: NC 000883), minute virus from mice (MVM) (GenBank accession number NC 001510); goose: goose parvovirus (GenBank accession number NC 001701); snake: snake parvovirus 1 (GenBank accession number NC 006148). [Table 8-1] [Table 8-2] [Table 8-3]

[0254] By way of example only, Table 9 shows the sequences of exemplary WT-ITRs from some different AAV serotypes. [Table 9]

[0255] According to some embodiments, the nucleic acid sequence of the WT-ITR sequence can be modified (e.g., by modifying 1, 2, 3, 4, or 5 or more nucleotides, or any range thereof), whereby the modifications are substitutions for complementary nucleotides, e.g., G for C and vice versa, T for A and vice versa.

[0256] In certain embodiments of the present disclosure, a ceDNA vector for expression of a peptide (e.g., an antigen) does not have a WT-ITR consisting of a nucleic acid sequence selected from any of the following: SEQ ID NOs: 1, 2, 5-14. In alternative embodiments of the present disclosure, a ceDNA vector has a WT-ITR comprising a nucleic acid sequence selected from any of the following: SEQ ID NOs: 1, 2, 5-14, where the adjacent ITR is also WT, and the ceDNA vector comprises a regulatory switch, e.g., as disclosed herein and in International Application PCT / US18 / 49996 (see, e.g., Table 11 of PCT / US18 / 49996, incorporated herein by reference in its entirety). According to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises a regulatory switch as disclosed herein and a selected WT-ITR having a nucleic acid sequence selected from any of the group consisting of SEQ ID NOs: 1, 2, 5-14.

[0257] The ceDNA vectors for expression of peptides (e.g., antigens) described herein can comprise a WT-ITR structure that retains functional RBE, trs, and RBE' portions. Figures 2A and 2B illustrate one possible mechanism for the operation of the trs portion within the wild-type ITR portion of a ceDNA vector, using the wild-type ITR for illustrative purposes. According to some embodiments, a ceDNA vector for expression of a peptide (e.g., antigen) comprises one or more functional WT-ITR polynucleotide sequences that include a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: __) for AAV2) and a terminal resolution site (TRS; 5'-AGTT (SEQ ID NO: __)). According to some embodiments, at least one WT-ITR is functional. In alternative embodiments, when a ceDNA vector for expression of a peptide (e.g., antigen) comprises two WT-ITRs that are substantially symmetrical to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional.

[0258] Common modified ITRs (mod-ITRs) for ceDNA vectors containing asymmetric or symmetric ITR pairs As discussed herein, ceDNA vectors for expression of peptides (e.g., antigens) can comprise a symmetric ITR pair or an asymmetric ITR pair. In both instances, one or both ITRs can be modified ITRs; the difference is that in the first instance (i.e., symmetric mod-ITRs), the mod-ITRs have the same three-dimensional spatial organization (i.e., have the same A-A', C-C', and B-B' arm arrangement), whereas in the second instance (i.e., asymmetric mod-ITRs), the mod-ITRs have a different three-dimensional spatial organization (i.e., have a different arrangement of the A-A', C-C', and B-B' arms).

[0259] In some embodiments, modified ITRs are ITRs that are modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., an AAV ITR). In some embodiments, at least one of the ITRs in the ceDNA vector comprises a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2) and a functional terminal resolution site (TRS; e.g., 5'-AGTT-3'). In some embodiments, at least one of the ITRs is a non-functional ITR. In some embodiments, each of the different or modified ITRs is not a wild-type ITR from a different serotype.

[0260] Specific modifications and mutations in ITRs are described in detail herein; however, in the context of an ITR, "modification," or "mutation," or "modification," it indicates that nucleotides have been inserted, deleted, and / or substituted relative to the wild-type sequence, reference sequence, or original ITR sequence. A modified or mutated ITR can be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be engineered if at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man so that it differs from the aspect that exists in nature.

[0261] According to some embodiments, mod-ITRs may be synthetic. According to some embodiments, synthetic ITRs are based on ITR sequences from more than one AAV serotype. In other embodiments, synthetic ITRs do not contain AAV-based sequences. In yet other embodiments, synthetic ITRs preserve the ITR structure described above, but have only a portion or none of the AAV source sequence. According to some aspects, synthetic ITRs may preferentially interact with wild-type Rep or Rep of a particular serotype, or, in some instances, are not recognized by wild-type Rep and are only recognized by mutant Rep.

[0262] Those skilled in the art can determine the corresponding sequences in other serotypes by known means. For example, determine whether the changes are in the A, A', B, B', C, C', or D regions and determine the corresponding region in another serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used in their default settings to determine the corresponding sequences. The present disclosure further provides populations and multiple ceDNA vectors containing mod-ITRs from combinations of different AAV serotypes. That is, one mod-ITR can be from one AAV serotype and the other mod-ITR can be from a different serotype. While not wishing to be bound by theory, according to some embodiments, one ITR can be from or based on the AAV2 ITR sequence, and the other ITR of the ceDNA vector can be from or based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0263] Any parvovirus ITR can be used as an ITR or as a base ITR for modification. Preferably, the parvovirus is a dependovirus. More preferably, it is an AAV. The serotype selected can be based on the serotype's tissue tropism. AAV2 has broad tissue tropism, AAV1 preferentially targets neuromuscular and skeletal muscle, and AAV5 preferentially targets nerves, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. According to some embodiments, the modified ITR is based on the AAV2 ITR.

[0264] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleic acid sequence of the structural element can be modified compared to the wild-type sequence of the ITR. According to some embodiments, structural elements of the ITR (e.g., the A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from a different parvovirus. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITRs can be AAV2 ITRs, with the A or A' arms or RBE replaced with structural elements from AAV5. In another example, the ITRs can be AAV5 ITRs, with the C or C' arms, RBE, and tr replaced with structural elements from AAV2. In another example, the AAV ITRs can be AAV5 ITRs, with the B and B' arms replaced with the AAV2 ITR B and B' arms.

[0265] By way of example only, Table 10 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X represents a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. According to some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the C and / or C' and / or B and / or B' regions retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. For example, if the modification results in a single-arm ITR (e.g., a single C-C' arm or a single B-B' arm), or a modified C-B' arm or C'-B arm, or a two-arm ITR with at least one cleavable arm (e.g., a cleavable C-C' arm and / or a cleavable B-B' arm), or at least one arm of a two-arm ITR (wherein one arm can be cleaved), three consecutive T nucleotides (i.e., TTT) are retained in at least one terminal loop. According to some embodiments, the cleavable C-C' arm and / or the cleavable B-B' arm have three consecutive T nucleotides (i.e., TTT) in the terminal loop. [Table 10]

[0266] According to some embodiments, mod-ITRs for use in ceDNA vectors for expression of peptides (e.g., antigens) comprise an asymmetric ITR pair or a symmetric mod-ITR pair as disclosed herein and can include any one of the combinations of modifications shown in Table 10, as well as a modification of at least one nucleotide in any one or more of the regions selected from between A' and C, between C' and C', between C' and B, between B and B', and between B' and A. According to some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the C or C' or B or B' region still maintains the terminal loop of the stem-loop. According to some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' maintains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and / or substitution) between C and C' and / or B and B' maintains three consecutive A nucleotides (i.e., AAA) in at least one terminal loop. According to some embodiments, modified ITRs for use herein can include any one of the combinations of modifications shown in Table 10, and also a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any one or more of the regions selected from the following: A', A, and / or D. For example, according to some embodiments, modified ITRs for use herein can include any one of the combinations of modifications shown in Table 10, and also a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A region. According to some embodiments, modified ITRs for use herein can include any one of the combinations of modifications shown in Table 10, and also a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A' region.In some embodiments, modified ITRs for use herein can include any one of the combinations of modifications shown in Table 10, and also a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A and / or A' region. In some embodiments, modified ITRs for use herein can include any one of the combinations of modifications shown in Table 10, and also a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0267] In some embodiments, the nucleotide sequence of a structural element can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any range thereof) to produce a modified structural element. In some embodiments, specific modifications to the ITRs are exemplified herein (e.g., SEQ ID NOS: 3, 4, 15-47, 101-116, or 165-187), or shown in Figures 7A-7B of International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018 (e.g., SEQ ID NOS: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 in PCT / US2018 / 064242). In some embodiments, the ITRs can be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein). In other embodiments, the ITR can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to one of the modified ITRs of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or the RBE-containing sections of the A-A' arm and the C-C' arm and the B-B' arm of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or to those set forth in Tables 2-9 of International Patent Application No. PCT / US18 / 49996 (i.e., SEQ ID NOs: 110-112, 115-190, 200-468), which are incorporated herein by reference in their entireties.

[0268] In some embodiments, the modified ITR can include, for example, the removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or alternatively, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs forming the stem of a loop, so long as a final loop capping the stem (e.g., a single arm) is still present (see, e.g., ITR-21 in Figure 7A of PCT / US2018 / 064242, filed December 6, 2018). In some embodiments, the modified ITR can include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. According to some embodiments, the modified ITR can include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm (see, e.g., ITR-1 in Figure 3B or ITR-45 in Figure 7A of International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018). According to some embodiments, the modified ITR can include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removal is contemplated; for example, 6 base pairs can be removed in the C-C' arm and 2 base pairs in the B-B' arm. As an illustrative example, Figure 3B shows an exemplary modified ITR having at least 7 base pairs deleted from each of the C and C' portions, a substitution of a nucleotide in the loop between the C and C' regions, and at least one base pair deletion from each of the B and B' regions, such that the modified ITR comprises two arms, where at least one arm (e.g., C-C') is truncated. According to some embodiments, the modified ITR also comprises at least one base pair deletion from each of the B and B' regions, such that the B-B' arm is also truncated relative to the WT ITR.

[0269] In some embodiments, the modified ITRs may have between 1 and 50 (e.g., 1, 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, or 50) nucleotide deletions relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITRs may have between 1 and 30 nucleotide deletions relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITRs may have between 2 and 20 nucleotide deletions relative to the full-length wild-type ITR sequence.

[0270] In some embodiments, the modified ITRs do not contain any nucleotide deletions in the RBE-containing portions of the A or A' regions so as not to interfere with DNA replication (e.g., binding to the RBE by Rep proteins or nicking at the terminal resolution site). In some embodiments, modified ITRs encompassed for use herein have one or more deletions in the B, B', C, and / or C regions described herein.

[0271] According to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprising a symmetric or asymmetric ITR pair comprises a regulatory switch as disclosed herein and at least one selected modified ITR having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187.

[0272] In another embodiment, the structure of the structural element can be modified. For example, the structural element is a change in stem height and / or the number of nucleotides in the loop. For example, the stem height can be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. According to some embodiments, the stem height can be about 5 nucleotides to about 9 nucleotides and functionally interact with Rep. In another embodiment, the stem height can be about 7 nucleotides and functionally interact with Rep. In another example, the loop can have 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0273] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites in an RBE or extended RBE can be increased or decreased. In some examples, an RBE or extended RBE can contain 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any range thereof. Each GAGY binding site can independently be the exact GAGY sequence or a sequence similar to GAGY, so long as the sequence is sufficient to bind to the Rep protein.

[0274] In another embodiment, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be altered (e.g., increased or decreased) to alter functional interactions with a large Rep protein. For example, the spacing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range therein.

[0275] The ceDNA vectors for expression of peptides (e.g., antigens) described herein can include ITR structures that are modified with respect to the wild-type AAV2 ITR structure disclosed herein, but still retain functional RBE, trs, and RBE' portions. Figures 2A and 2B illustrate one possible mechanism for the operation of the trs portion within the wild-type ITR portion of a ceDNA vector for expression of an antigen or immunogenic peptide. According to some embodiments, a ceDNA vector for expression of a peptide (e.g., antigen) includes one or more functional ITR polynucleotide sequences that include a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTCGCTC-3', AAV2) and a terminal resolution site (TRS; 5'-AGTT). According to some embodiments, at least one ITR (wt or modified ITR) is functional. In alternative embodiments, when a ceDNA vector for expression of a peptide (e.g., antigen) includes two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0276] According to some embodiments, the modified ITRs (e.g., left or right ITRs) of the ceDNA vectors for expression of peptides (e.g., antigens) described herein have modifications within the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs having modifications in the loop arm, cleavage arm, or spacer are listed in Table 2 (i.e., SEQ ID NOS: 135-190, 200-233); Table 3 (e.g., SEQ ID NOS: 234-263); Table 4 (e.g., SEQ ID NOS: 264-293); Table 5 (e.g., SEQ ID NOS: 294-318 herein); Table 6 (e.g., SEQ ID NOS: 319-468; and Tables 7-9 (e.g., SEQ ID NOS: 101-110, 111-112, 115-134) or Table 10A or 10B (e.g., SEQ ID NOS: 9, 100, 469-483, 484-499) of International Patent Application No. PCT / US18 / 49996, which are incorporated herein by reference in their entireties.

[0277] According to some embodiments, modified ITRs for use in ceDNA vectors for expression of peptides (e.g., antigens) comprising asymmetric ITR pairs or symmetric mod-ITR pairs are selected from any one or combination of those set forth in Tables 2, 3, 4, 5, 6, 7, 8, 9, and 10A-10B of International Patent Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0278] Additional exemplary modified ITRs for use in ceDNA vectors for expression of peptides (e.g., antigens) comprising asymmetric or symmetric mod-ITR pairs in each of the above classes are provided in Tables 11A and 11B. The predicted secondary structures of the right-modified ITRs in Table 11A are shown in Figure 7A of International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, and the predicted secondary structures of the left-modified ITRs in Table 11B are shown in Figure 7B of International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, which are incorporated herein by reference in their entireties.

[0279] Tables 11A and 11B list the sequence numbers of exemplary right- and left-modified ITRs. [Table 11A-1] [Table 11A-2] [Table 11B-1] [Table 11B-2]

[0280] According to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises, in a 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleic acid sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other, i.e., they have different 3D spatial arrangements. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, where the first ITR can be a mutated or modified ITR and the second ITR can be a wild-type ITR. According to some embodiments, both the first ITR and the second ITR are mod-ITRs, but have different sequences or have different modifications, and thus are not the same modified ITR but have different 3D spatial arrangements. In other words, a ceDNA vector with asymmetric ITRs comprises ITRs in which any changes according to one ITR relative to the WT-ITR are not reflected in the other ITR; or alternatively, the asymmetric ITRs can have a modified asymmetric ITR pair and have different sequences and different three-dimensional shapes relative to each other. Exemplary asymmetric ITRs in ceDNA vectors for expression of peptides (e.g., antigens) and for use in generating ceDNA plasmids are shown in Tables 11A and 11B.

[0281] In an alternative embodiment, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises two symmetric mod-ITRs, i.e., both ITRs have the same sequence but are reverse complements (inverted) of each other. According to some embodiments, a symmetric mod-ITR pair comprises at least one or any combination of deletions, insertions, or substitutions relative to the wild-type ITR sequence from the same AAV serotype. The additions, deletions, or substitutions in the symmetric ITRs are identical but reverse complementary to each other. For example, an insertion of three nucleotides in the C region of the 5' ITR would be reflected in the insertion of three reverse complement nucleotides in the corresponding section in the C' region of the 3' ITR. For illustrative purposes only, if the addition is AACG in the 5' ITR, the addition is CGTT in the 3' ITR at the corresponding site. For example, if the 5' ITR sense strand is ATCGATCG, with the addition of AACG between the G and A, resulting in the sequence ATCGAACGATCG. The corresponding 3' ITR sense strand is CGATCGAT (the reverse complement of ATCGATCG) with the addition of CGTT (i.e., the reverse complement of AACG) between the T and C to yield the sequence CGATCGTTCGAT (the reverse complement of ATCGAACGATCG).

[0282] In alternative embodiments, the modified ITR pair is substantially symmetrical as defined herein—i.e., the modified ITR pair can have different sequences but have corresponding or the same symmetrical three-dimensional shapes. For example, one modified ITR can be from one serotype and the other modified ITR can be from a different serotype, but they have the same mutation (e.g., nucleotide insertion, deletion, or substitution) in the same region. In other words, for illustrative purposes only, the 5'-modified ITR can be from AAV2 and have a deletion in the C region, and the 3'-modified ITR can be from AAV5 and have a corresponding deletion in the C' region, provided that the 5'-modified ITR and the 3'-modified ITR have the same or symmetrical three-dimensional spatial configuration, and are encompassed for use herein as a modified ITR pair.

[0283] According to some embodiments, a substantially symmetric mod-ITR pair has the same A, C-C', and B-B' loops in 3D space; for example, if a modified ITR in a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, then the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar 3D structure of the remaining A and B-B' loops in the same shape in the geometric space of the cognate mod-ITR. By way of example only, substantially symmetric ITRs can have a symmetric spatial organization, such that their structures are the same shape in geometric space. This can occur, for example, when a GC pair is modified to, for example, a CG pair, or vice versa, or when an AT pair is modified to, for example, a TA pair, or vice versa. Thus, using the above illustrative examples of a modified 5' ITR as ATCGAACGATCG and a modified 3' ITR as CGATCGTTCGAT (i.e., the reverse complement of ATCGAACGATCG), these modified ITRs may be modified, for example, by modifying the 5' ITR as ATCGAAC C It may still be symmetrical if it had the sequence ATCG, but where the G in the addition is modified to a C, resulting in a substantially symmetric 3' ITR having the sequence CGATCGTTCGAT without the corresponding modification of the T in the addition to a. According to some embodiments, such modified ITR pairs are substantially symmetrical because the modified ITR pair has symmetric stereochemistry.

[0284] Table 12 shows exemplary symmetrically modified ITR pairs (i.e., left-modified ITRs and symmetrically right-modified ITRs) for use in ceDNA vectors for expression of antigens or immunogenic peptides. The bold (red) portions of the sequences identify the partial ITR sequences (i.e., the sequences of the A-A', C-C', and B-B' loops) and are shown in Figures 31A-46B. These exemplary modified ITRs include an RBE of GCGCGCTCGCTCGCTC-3', a spacer of ACTGAGGC, the spacer complement, and an RBE' (i.e., the complement to the RBE) of GAGCGAGCGAGCGCGCGC. [Table 12-1] [Table 12-2] [Table 12-3]

[0285] According to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprising an asymmetric ITR pair can include an ITR with a modification corresponding to any of the modifications in the ITR sequences or ITR subsequences set forth in any one or more of Tables 11A-11B herein, or the sequences set forth in Figures 7A-7B of International Patent Application PCT / US2018 / 064242, filed December 6, 2018, which is incorporated herein by reference in its entirety, or the sequences disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A-10B of International Patent Application PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety.

[0286] Exemplary ceDNA Vectors As described above, the present disclosure relates to recombinant ceDNA expression vectors and ceDNA vectors encoding peptides (e.g., antigens) comprising any one of the above-described asymmetric ITR pairs, symmetric ITR pairs, or substantially symmetric ITR pairs. In certain embodiments, the present disclosure relates to recombinant ceDNA vectors for expression of peptides (e.g., antigens) having flanking ITR sequences and a transgene, wherein the ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein, and the ceDNA further comprises a nucleic acid sequence of interest (e.g., an expression cassette comprising the nucleic acid of a transgene) positioned between the flanking ITRs, wherein the nucleic acid molecule lacks a viral capsid protein-encoding sequence.

[0287] A ceDNA expression vector for expression of a peptide (e.g., an antigen) may be any ceDNA vector that can be conveniently subjected to recombinant DNA procedures and contains a nucleic acid sequence described herein, provided that at least one ITR is modified. The ceDNA vector for expression of a peptide (e.g., an antigen) of the present disclosure is compatible with the host cell into which the ceDNA vector is introduced. In certain embodiments, the ceDNA vector may be linear. In certain embodiments, the ceDNA vector may exist as an extrachromosomal entity. In certain embodiments, the ceDNA vector of the present disclosure may contain elements that allow for integration of the donor sequence into the genome of the host cell. As used herein, "transgene," "nucleic acid sequence," and "heterologous nucleic acid sequence" are synonymous and encode a peptide (e.g., an antigen) described herein.

[0288]

[0013] Referring now to Figures 1A-1G of International Publication No. WO / 2019 / 051255, which is incorporated herein by reference in its entirety, there are shown schematic diagrams of the functional components of two non-limiting plasmids useful in generating ceDNA vectors for the expression of peptides (e.g., antigens). Figures 1A, 1B, 1D, and 1F show the constructs of ceDNA vectors or the corresponding sequences of ceDNA plasmids for the expression of antigens or immunogenic peptides. The ceDNA vectors are capsid-free and can be derived from a plasmid encoding, in that order, a first ITR, an expressible transgene cassette, and a second ITR, where the first ITR sequence and the second ITR sequence are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. ceDNA vectors for expression of peptides (e.g., antigens) are capsid-free and can be derived from a plasmid encoding a first ITR, an expressible transgene (protein or nucleic acid), and a second ITR, in that order, where the first ITR sequence and the second ITR sequence are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. According to some embodiments, the expressible transgene cassette optionally includes an enhancer / promoter, one or more homology arms, a donor sequence, a post-transcriptional regulatory element (e.g., WPRE, e.g., SEQ ID NO: 67), and a polyadenylation and termination signal (e.g., BGH polyA, e.g., SEQ ID NO: 68).

[0289] Figure 5 of International Publication No. WO / 2019 / 051255, which is incorporated herein by reference in its entirety, is a gel confirming the production of ceDNA from multiple plasmid constructs using the methods described in the Examples. The ceDNA is confirmed by the characteristic banding pattern in the gel, as discussed with respect to Figure 4A of International Publication No. WO / 2019 / 051255, which is incorporated herein by reference in its entirety.

[0290] Regulatory elements The ceDNA vectors for expression of peptides (e.g., antigens) described herein, comprising asymmetric or symmetric ITR pairs as defined herein, can further comprise specific combinations of cis-regulatory elements, including but not limited to promoters, riboswitches, insulators, mir-regulatable elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers.

[0291] According to some embodiments, the sequences of the various cis-regulatory elements can be selected from any of those disclosed in International Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0292] In embodiments, the second nucleic acid sequence comprises a regulatory sequence and a nucleic acid sequence encoding a nuclease. In certain embodiments, a gene regulatory sequence is operably linked to the nucleic acid sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling expression of the nuclease in the host cell. In certain embodiments, the regulatory sequence comprises a suitable promoter sequence capable of directing transcription of a gene operably linked to the promoter sequence, such as a nucleic acid sequence encoding a nuclease of the present disclosure. In certain embodiments, the second nucleic acid sequence comprises an intron sequence linked to the 5' end of the nucleic acid sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the effectiveness of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, wherein the second nucleic acid sequence comprises an intron sequence upstream of the nucleic acid sequence encoding the nuclease, wherein the intron comprises one or more nuclease cleavage sites, and wherein the promoter is operably linked to the nucleic acid sequence encoding the nuclease.

[0293] Suitable promoters can be derived from viruses, and thus may be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6, e.g., SEQ ID NO: 80) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31 (17)), the human H1 promoter (H1) (e.g., SEQ ID NO: 81 or SEQ ID NO: 155), the CAG promoter, the human alpha 1-antitypsin (HAAT) promoter (e.g., SEQ ID NO: 82), and the like. In certain embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites, hi certain embodiments, the DNA containing the nuclease cleavage sites is exogenous to the promoter DNA.

[0294] In some embodiments, the promoter may also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein.

[0295] According to some embodiments, the promoter is a tissue-specific promoter. According to further embodiments, the tissue-specific promoter is a liver-specific promoter. In some embodiments, the antigen or immunogenic protein is targeted to and / or produced in the liver by a liver-specific promoter.

[0296] Any liver-specific promoter known in the art is contemplated for use in the present disclosure. In some embodiments, the liver-specific promoter is selected from, but not limited to, human alpha 1-antitypsin (HAAT), natural or synthetic. In some embodiments, delivery to the liver can be achieved by using the endogenous ApoE-specific targeting of the composition comprising the ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0297] Non-limiting examples of suitable promoters for use in accordance with the present disclosure include, but are not limited to, any of the following: CAG promoter, EF1a promoter, IE2 promoter and rat EF1-alpha promoter, mEF1 promoter, or 1E1 promoter fragment.

[0298] According to some embodiments, the promoter can be selected from any promoter sequence disclosed in International Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0299] Polyadenylation sequence: A sequence encoding a polyadenylation sequence can be included in a ceDNA vector for expression of a peptide (e.g., an antigen) to stabilize the mRNA expressed from the ceDNA vector and aid in nuclear transport and translation. In some embodiments, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) contains at least one, at least two, at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0300] The expression cassette can include any polyadenylation sequence or variation thereof known in the art. Some expression cassettes also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE sequence can be used in combination with an SV40 pA or heterologous polyA signal. The polyA sequence is positioned 3' to the transgene encoding the antigen or immunogenic peptide.

[0301] According to some embodiments, the polyadenylation sequence can be selected from any of the polyadenylation sequences disclosed in International Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0302] The expression cassette can also include post-transcriptional elements to increase transgene expression. In some embodiments, the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression. Other post-transcriptional processing elements can be used, such as the post-transcriptional elements from herpes simplex virus or the thymidine kinase gene of hepatitis B virus (HBV).

[0303] According to some embodiments, the post-transcriptional regulatory element can be selected from any of the post-transcriptional regulatory element sequences disclosed in International Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0304] In some embodiments, the nucleic acid sequence or sequences encoding the antigen or immunogenic protein can also encode a secretory sequence, which targets the protein to the Golgi apparatus and endoplasmic reticulum so that it can be folded into the correct conformation by chaperone molecules as it passes through the ER and exits the cell. Exemplary secretory sequences include, but are not limited to, VH-02 and VK-A26) and Ig K It contains a signal sequence, as well as a Gluc secretion signal that allows the tagged protein to be secreted out of the cytoplasm, and a TMD-ST secretion sequence that targets the tagged protein to the Golgi apparatus.

[0305] According to some embodiments, the secretory sequence can be selected from any of the secretory sequences disclosed in International Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0306] nuclear localization sequence In some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) comprises one or more nuclear localization sequences (NLSs), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the one or more NLSs are located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and / or one or more NLSs at the carboxy terminus). When two or more NLSs are present, each can be selected independently of the others, such that a single NLS is present in two or more copies and / or in combination with one or more other NLSs present in some or more copies.

[0307] In some embodiments, the NLS may be selected from any of the NLSs disclosed in International Patent Application No. PCT / US2021 / 023891, filed March 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0308] V. Methods for the production of ceDNA vectors General production Certain methods for producing ceDNA vectors for expression of peptides (e.g., antigens) comprising asymmetric or symmetric ITR pairs as defined herein are described in Section IV of International Application PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety. According to some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein can be produced using insect cells as described herein. In alternative embodiments, ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein can be produced synthetically in a cell-free manner, and according to some embodiments, as disclosed in International Application PCT / US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety.

[0309] As described herein, according to some embodiments, a ceDNA vector for expression of a peptide (e.g., an antigen) can be obtained, for example, by a process comprising the following steps: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., a ceDNA plasmid, a ceDNA bacmid, and / or a ceDNA baculovirus) lacking a viral capsid-encoding sequence in the presence of Rep proteins under conditions effective and for a sufficient time to induce production of the ceDNA vector in the host cells (the host cells do not contain a viral capsid-encoding sequence); and b) recovering and isolating the ceDNA vector from the host cells. The presence of Rep proteins induces replication of the vector polynucleotide with modified ITRs, resulting in the production of the ceDNA vector in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Thus, there are no size limitations, such as those naturally imposed by AAV or other virus-based vectors.

[0310] The presence of a ceDNA vector isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0311] In yet another aspect, the present disclosure provides for the use of host cell lines that have stably integrated a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, as described, for example, in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. When the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line can have a stably integrated polynucleotide vector template, and a second vector, e.g., a herpes virus, can be used to introduce Rep proteins into the cells, allowing for removal and amplification of the ceDNA in the presence of Rep and a helper virus.

[0312] According to some embodiments, the host cells used to generate ceDNA vectors for expression of peptides (e.g., antigens) described herein are insect cells, and baculovirus is used to deliver both the polynucleotide encoding the Rep protein and the non-viral DNA vector polynucleotide expression construct template for the ceDNA, e.g., as described in Example 1. According to some embodiments, the host cells are engineered to express the Rep protein.

[0313] The ceDNA vector is then recovered and isolated from the host cells. The time for recovering and collecting the ceDNA vector described herein from the cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the recovery time can be selected in terms of cell viability, cell morphology, cell growth, etc. In some embodiments, the cells are grown and recovered after baculovirus infection for a sufficient time to produce the ceDNA vector, but before the majority of the cells begin to die due to baculovirus toxicity. The DNA vector can be isolated using a plasmid purification kit, such as a Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. Generally, any nucleic acid purification method can be employed.

[0314] The DNA vector can be purified by any means known to those skilled in the art for purifying DNA. In some embodiments, the ceDNA vector is purified as a DNA molecule. In other embodiments, the ceDNA vector is purified as an exosome or microparticle.

[0315] The presence of a ceDNA vector for expression of a peptide (e.g., an antigen) can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0316] According to some embodiments, the ceDNA is produced synthetically in a cell-free environment.

[0317] ceDNA Plasmid A ceDNA plasmid is a plasmid used for subsequent production of a ceDNA vector for expression of a peptide (e.g., an antigen) described herein. According to some embodiments, a ceDNA plasmid can be constructed using known techniques to provide at least the following as operably linked components in the direction of transcription: (1) a modified 5' ITR sequence; (2) an expression cassette including cis-regulatory elements, such as a promoter, an inducible promoter, a regulatory switch, an enhancer, and the like; and (3) a modified 3' ITR sequence, where the 3' ITR sequence is symmetrical to the 5' ITR sequence. According to some embodiments, the expression cassette flanked by ITRs includes cloning sites for introducing exogenous sequences. The expression cassette replaces the rep-coding and cap-coding regions of the AAV genome.

[0318] According to some aspects, ceDNA vectors for expression of peptides (e.g., antigens) are derived from plasmids, referred to herein as "ceDNA plasmids," that encode, in that order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutated or modified AAV ITR, wherein the ceDNA plasmid lacks an AAV capsid protein coding sequence. In alternative embodiments, a ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette comprising a transgene, and a second (or 3') modified AAV ITR, wherein the ceDNA-plasmid lacks an AAV capsid protein coding sequence, wherein the 5' and 3' ITRs are symmetrical with respect to each other. In an alternative embodiment, the ceDNA plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein the ceDNA plasmid lacks the AAV capsid protein coding sequence, and wherein the 5' and 3' modified ITRs have the same modification (i.e., they are reverse complementary or symmetrical to each other).

[0319] In further embodiments, the ceDNA plasmid system lacks viral capsid protein coding sequences (i.e., it lacks the AAV capsid genes, but also the capsid genes of other viruses). Also, in certain embodiments, the ceDNA plasmid also lacks the AAV Rep protein coding sequences. Thus, in preferred embodiments, the ceDNA plasmid lacks functional AAV cap and AAV rep genes (GG-3') for AAV2, plus the variable palindrome sequence that allows hairpin formation.

[0320] The ceDNA plasmids of the present disclosure can be generated using the native nucleic acid sequence of the genome of any AAV serotype known in the art. In some embodiments, the ceDNA plasmid backbone is derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome. See, e.g., NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261; Kotin and Smith, The Springer Index of Viruses (available at URLs maintained by Springer) at the address oesys.springer.de / viruses / database / mkchapter.asp?virID=42.04. (Note—references to URLs or databases refer to the contents of the URL or database as of the effective filing date of this application.) In certain embodiments, the ceDNA plasmid backbone is derived from the AAV2 genome. In another specific embodiment, the ceDNA plasmid backbone is a synthetic backbone engineered to include the 5' and 3' ITRs derived from one of these AAV genomes.

[0321] The ceDNA plasmid can optionally include a selectable or selectable marker for use in establishing a ceDNA vector-producing cell line. According to some embodiments, the selectable marker can be inserted downstream (i.e., 3') of the 3' ITR sequence. In other embodiments, the selectable marker can be inserted upstream (i.e., 5') of the 5' ITR sequence. Suitable selectable markers include, for example, those that confer drug resistance. Selectable markers can be, for example, the blasticidin S resistance gene, kanamycin, geneticin, and the like. In a preferred embodiment, the drug selectable marker is the blasticidin S resistance gene.

[0322] Exemplary ceDNA (e.g., rAAV0) vectors for expression of peptides (e.g., antigens) are produced from rAAV plasmids. Methods for producing rAAV vectors include (a) providing host cells with the rAAV plasmid described above, wherein both the host cell and the plasmid lack a capsid protein-encoding gene, (b) culturing the host cells under conditions that allow for the production of a ceDNA genome, and (c) harvesting the cells and isolating the produced AAV genome from the cells.

[0323] Exemplary Methods for Producing ceDNA Vectors from ceDNA Plasmids Also provided herein are methods for generating capsid-free ceDNA vectors for expression of peptides (e.g., antigens), notably methods with sufficiently high yields to provide sufficient vector for in vivo experiments.

[0324] According to some embodiments, a method for producing a ceDNA vector for expression of a peptide (e.g., an antigen) includes (1) introducing a nucleic acid construct containing an expression cassette and two symmetric ITR sequences into host cells (e.g., Sf9 cells), (2) optionally establishing a clonal cell line, for example, by using a selectable marker present on the plasmid, (3) introducing a Rep-encoding gene into the insect cells (either by transfection or infection with a baculovirus carrying the gene), and (4) recovering the cells and purifying the ceDNA vector. The nucleic acid construct containing the expression cassette and two ITR sequences described above for producing a ceDNA vector can be in the form of a ceDNA plasmid, or a bacmid or baculovirus generated using the ceDNA plasmid, as described below. The nucleic acid construct can be introduced into the host cell by transfection, viral transduction, stable integration, or other methods known in the art.

[0325] cell line Host cell lines for use in producing ceDNA vectors for expression of peptides (e.g., antigens) can include insect cell lines derived from Spodoptera frugiperda, such as Sf9 and Sf21, or Trichoplusia nii cells, or other eukaryotic cell lines, including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to those skilled in the art can also be used, such as HEK293, Huh-7, HeLa, HepG2, Hep1A, 911, CHO, COS, MeWo, NIH3T3, A549, HT1180, monocytes, and mature and immature dendritic cells. Host cell lines can be transfected for stable expression of ceDNA plasmids for high-yield ceDNA vector production.

[0326] The CeDNA-plasmid can be introduced into Sf9 cells by transient transfection using reagents known in the art (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation). Alternatively, stable Sf9 cell lines can be established that have stably integrated the ceDNA-plasmid into their genome. Such stable cell lines can be established by incorporating a selection marker into the ceDNA plasmid, as described above. If the ceDNA plasmid used to transfect the cell line contains a selection marker, such as an antibiotic, cells transfected with the ceDNA plasmid and that have integrated the ceDNA plasmid DNA into their genome can be selected by adding the antibiotic to the cell growth medium. Resistant clones of cells can then be isolated and propagated by single-cell dilution or colony transfer techniques.

[0327] Isolation and purification of ceDNA vectors Exemplary processes for obtaining and isolating ceDNA vectors are described in Figures 4A-4E of International Publication No. WO / 2019 / 051255, which is incorporated herein by reference in its entirety. ceDNA vectors for expression of peptides (e.g., antigens) used as priming vaccines in the prime-boost compositions and methods described herein can be obtained from producer cells expressing AAV Rep proteins and further transformed with a ceDNA plasmid, ceDNA bacmid, or ceDNA baculovirus. Plasmids useful for producing ceDNA vectors include plasmids encoding peptides (e.g., antigens) or plasmids encoding one or more REP proteins.

[0328] According to some embodiments, the polynucleotide encodes an AAV Rep protein (Rep 78 or 68) that is delivered to a producer cell in a plasmid (Rep plasmid), a bacmid (Rep-bacmid), or a baculovirus (Rep-baculovirus). The Rep plasmid, Rep-bacmid, and Rep-baculovirus can be generated by the methods described above.

[0329] Described herein are methods for producing ceDNA vectors for expression of peptides (e.g., antigens) used as priming vaccines in the prime-boost compositions and methods described herein. The expression constructs used to generate ceDNA vectors for expression of peptides (e.g., antigens) described herein can be plasmids (e.g., ceDNA plasmids), bacmids (e.g., ceDNA-bacmids), and / or baculoviruses (e.g., ceDNA-baculoviruses). By way of example only, ceDNA vectors can be generated from cells co-infected with ceDNA-baculovirus and Rep-baculovirus. The Rep protein produced from the Rep-baculovirus can replicate the ceDNA-baculovirus to generate the ceDNA vector. Alternatively, ceDNA vectors for expression of peptides (e.g., antigens) can be generated from cells stably transfected with a construct containing a sequence encoding the AAV Rep protein (Rep78 / 52) delivered in a Rep plasmid, Rep-bacmid, or Rep-baculovirus. The CeDNA-baculovirus can be transiently transfected into cells and replicated by the Rep protein to produce the ceDNA vector.

[0330] A bacmid (e.g., a ceDNA bacmid) can be transfected into permissive insect cells, such as Sf9, Sf21, Tni (Trichoplusia ni) cells, or High Five cells, to generate a ceDNA-baculovirus, which is a recombinant baculovirus containing sequences including the symmetric ITRs and an expression cassette. The ceDNA-baculovirus can be reinfected into insect cells to generate the next generation of recombinant baculovirus. Optionally, the process can be repeated one or more times to produce larger quantities of recombinant baculovirus.

[0331] The time for harvesting and collecting ceDNA vectors for expression of peptides (e.g., antigens) described herein from cells can be selected and optimized to achieve high-yield production of ceDNA vectors. For example, the harvest time can be selected in consideration of cell viability, cell morphology, cell growth, etc. Typically, cells can be harvested after a sufficient time after baculovirus infection to produce ceDNA vectors (e.g., ceDNA vectors), but before most of the cells begin to die due to viral toxicity. ceDNA vectors can be isolated from Sf9 cells using a plasmid purification kit, such as the Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation can also be adapted for ceDNA vectors. Generally, any nucleic acid purification method known in the art, as well as commercially available DNA extraction kits, can be employed.

[0332] Alternatively, purification can be carried out by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As one non-limiting example, the process can be carried out by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acids, followed by elution (e.g., with a 1.2 M NaCl solution), and performing further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE). The capsid-free AAV vector is then recovered, for example, by precipitation.

[0333] In some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) can also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargoes via membrane microvesicle shedding (Cocucci et al., 2009; EP 10306226.1). Such vesicles include microvesicles (also referred to as microparticles) and exosomes (also referred to as nanovesicles), both of which contain proteins and RNA as cargo. Vesicles are generated by direct budding of the cell membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with the cell membrane. Thus, ceDNA vector-containing microvesicles and / or exosomes can be isolated from cells transduced with ceDNA plasmids or from bacmids or baculoviruses produced with ceDNA plasmids.

[0334] Microvesicles can be isolated by subjecting culture medium to filtration or ultracentrifugation at 20,000 x g and exosomes at 100,000 x g. The optimal duration of ultracentrifugation can be determined experimentally and depends on the specific cell type from which vesicles are isolated. Preferably, the culture medium is first removed by low-speed centrifugation (e.g., 2000 x g for 5-20 minutes) and then subjected to spin concentration, for example, using an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other microvesicle and exosome purification methods include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. During purification, vesicles are washed, for example, with phosphate-buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by containing on them membrane proteins that are recognized by specific receptors on each cell type (see also EP 10306226).

[0335] Another aspect of the present disclosure herein relates to a method for purifying ceDNA vectors from host cell lines that have stably integrated the ceDNA construct into their genome. According to some embodiments, the ceDNA vectors are purified as DNA molecules. In other embodiments, the ceDNA vectors are purified as exosomes or microparticles.

[0336] Figure 5 of International Application PCT / US18 / 49996 shows a gel confirming the production of ceDNA from several ceDNA-plasmid constructs.

[0337] VI. Administration Methods for heterologous prime-boost immunization It has been reported that multiple dose immunizations are often more effective than single dose immunizations for treatment or disease prevention. + Generating T cells is generally considered a desirable goal in vaccine design for various animal and human diseases because this number strongly correlates with host immunization and protection. One approach to generating large numbers of these cells is to use prime-boost immunization, which relies on restimulation of antigen-specific immune cells after primary memory formation. In such a process, a "priming" composition is administered to the subject initially, followed by one or more subsequent "boost" compositions.

[0338] The present disclosure further contemplates multiple administrations of one of the compositions (a priming vaccine) followed by multiple administrations of the other composition (a boost). In one embodiment, the priming composition is administered to the subject at least one or more times prior to administration of the boosting composition. The boosting composition is then subsequently administered to the subject at least one or more times. Boosting the immune response by a vaccine is widely believed to result in the generation of more effector cells required to mediate protection against pathogens upon infection.

[0339] According to aspects of the present disclosure, the methods described herein employ heterologous prime-boost immunization, or administration of an antigen or immunogenic peptide using two different modalities or platforms. According to embodiments, such approaches advantageously elicit improved immune responses in subjects. According to some embodiments, the improved immune responses resulting from the described heterologous prime-boost immunizations include improved memory responses, including, but not limited to, CD8 + These include a higher magnitude of T cell responses, a broader range of T cell epitopes recognized by the immune system, and an increase in T cell polyfunctionality. +The higher magnitude of the T cell response can be an increase of at least 20%, or at least 25%, or at least 30%, or at least 50%, or at least 75%, or at least 100%, or at least 150%, or at least 200%, or at least 250%, or at least 300% relative to a single dose administration or relative to a homologous prime-boost regimen. According to some embodiments, the heterologous prime-boost strategies described herein can result in a synergistic enhancement of the immune response when the ceDNA platform is used as the priming vaccine. According to further embodiments, the synergistic enhancement of the immune response is seen in increased numbers of antigen-specific T cells, the length of the immune memory response, and the magnitude of the immune memory response.

[0340] According to some embodiments, the present disclosure provides a method for inducing an immune response to a first peptide and a second peptide in a subject, comprising administering to the subject a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes the first peptide; and administering to the subject a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, wherein the RNA encodes the second peptide, thereby inducing an immune response to the first peptide and the second peptide in the subject. Also provided is a vaccine regimen comprising a priming vaccine comprising deoxyribonucleic acid (DNA), wherein the DNA encodes the first peptide; and a boosting vaccine comprising (i) ribonucleic acid (RNA), or (ii) a second peptide, wherein the RNA encodes the second peptide. According to some embodiments, the first and second peptides are derived from a bacterial, viral, fungal, or parasitic infectious agent. According to some embodiments, the first and second peptides are from the same pathogenic organism. In some embodiments, the first and second peptides are the same in the priming vaccine and the boosting vaccine. In some embodiments, at least one of the epitopes of the first and second peptides is different in the priming vaccine and the boosting vaccine.

[0341] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising a ceDNA vector encoding the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, wherein the boosting vaccine does not comprise a ceDNA vector.

[0342] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising a ceDNA vector encoding the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, wherein the boosting vaccine comprises the second peptide.

[0343] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising a ceDNA vector encoding the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, wherein the boosting vaccine comprises ribonucleic acid (RNA) encoding the second peptide.

[0344] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising a ceDNA vector encoding the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, the boosting vaccine comprising a ceDNA vector encoding the second peptide.

[0345] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising ribonucleic acid (RNA) encoding the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, wherein the boosting vaccine comprises a ceDNA vector encoding the second peptide.

[0346] Some embodiments disclosed herein relate to a method for inducing an immune response to a first peptide and a second peptide in a subject, the method comprising administering to the subject at least one dose of a priming vaccine comprising the first peptide; and subsequently administering to the subject at least one dose of a boosting vaccine, wherein the boosting vaccine comprises a ceDNA vector encoding the second peptide.

[0347] In some embodiments, the first peptide and the second peptide are identical to each other. In some embodiments, the amino acid sequences of the first peptide and the second peptide are homologous to each other. In some embodiments, the amino acid sequence of the first peptide exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the second peptide. In some embodiments, the first and second immunogenic peptides comprise at least one cross-reactive antigenic determinant. In some embodiments, the first and second immunogenic peptides or antigens induce substantially the same immune response in a subject.

[0348] In some embodiments, the priming composition is administered to the subject in a single dose. In some embodiments, the priming composition is administered to the subject in multiple doses. In some embodiments, the boosting composition is administered to the subject in a single dose. In some embodiments, the boosting composition is administered to the subject in multiple doses.

[0349] In some embodiments, the priming composition and / or boosting composition is administered to a subject over at least 2, at least 3, at least 4, at least 5, or at least 10 consecutive doses, or any number of doses in between. In some embodiments, the priming composition and / or boosting composition is administered to a subject over at least 10, at least 12, at least 14, at least 16, or at least 20 consecutive doses, or any number of doses in between.

[0350] In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to a subject about 1 week apart, or 2, 3, 4, 5, 6, 7, or 8 weeks apart, or 1-2, or 2-4, or 3-4 weeks apart. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to a subject about 4 weeks apart. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to a subject about 6 weeks apart. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to a subject about 8 weeks apart. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to a subject about 10 weeks apart.

[0351] In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 28 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 35 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 42 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 49 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 56 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 63 days. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of about 70 days or more. In some embodiments of the methods disclosed herein, at least one dose of the priming composition and the boosting composition is administered to the subject at intervals of between 28 and 56 days.

[0352] Those skilled in the art will further understand that for any particular subject, specific dosage regimens can be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. For example, the dose can be adjusted based on the clinical effects of the administered composition, such as toxic effects and / or clinical laboratory values. The dosage regimen can be adjusted to provide the optimum desired effect. For example, as discussed above, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It will be understood that determining appropriate dosages and regimens for administering the compositions disclosed herein is well within the skill of those skilled in the art once provided with the teachings disclosed herein.

[0353] Thus, based on the disclosure provided herein, those skilled in the art will understand that doses and administration regimens will be adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated dose can be readily established, and the effective amount that provides a subject with a detectable therapeutic benefit can also be determined, as can the time requirements for administering each agent to provide a patient with a detectable therapeutic benefit. Thus, while specific doses and administration regimens are exemplified herein, these examples in no way limit the doses and administration regimens that can be provided to patients when practicing the present disclosure.

[0354] Administration of the priming and boosting compositions disclosed herein can be by any method that allows delivery of the composition to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), topical administration, and rectal administration. Infusions can be administered by drip, continuous infusion, infusion pump, metered pump, depot formulation, or any other suitable means. In some embodiments, at least one dose of the priming composition is administered intramuscularly to a subject. In some embodiments, at least one dose of the boosting composition is administered intramuscularly to a subject. In some embodiments disclosed herein, the methods of the present disclosure further comprise one or more subsequent boost administrations. In some embodiments, the methods of the present disclosure further comprise at least 2, at least 3, at least 4, at least 5, or at least 10 consecutive boost administrations, or any number therebetween. In some embodiments, the subsequent boost administrations are administered at gradually increasing dosages over time. In some embodiments, the subsequent boost administrations are administered at gradually decreasing dosages over time.

[0355] VII. Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided, comprising a ceDNA vector for expression of a peptide (e.g., an antigen) used as a priming vaccine in the prime-boost compositions and methods described herein, and a pharmaceutically acceptable carrier or diluent.

[0356] The ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein can be incorporated into pharmaceutical compositions suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises a ceDNA vector disclosed herein and a pharmaceutically acceptable carrier.

[0357] Pharmaceutical formulations disclosed herein include liquids that can be administered directly, such as aqueous solutions, and lyophilized powders that can be reconstituted into a solution by adding a diluent prior to administration. In certain embodiments, formulations containing the ceDNA vectors disclosed herein can be formulated as lyophilizates using suitable excipients, with or without at least one additional therapeutic agent. Lyophilization can be performed using a standard lyophilization cycle on commercially available lyophilizers (e.g., VirTis Lab Scale lyophilizers).

[0358] Pharmaceutical compositions for therapeutic purposes typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of ceDNA vector compound in an appropriate buffer with one or a combination of the ingredients listed above, as required, followed by filtered sterilization.

[0359] In certain embodiments, formulations for parenteral administration may be stored in lyophilized form or in a solution. hi certain embodiments, parenteral formulations generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0360] According to some embodiments, the methods provided herein include delivering one or more ceDNA vectors to host cells for expression of peptides (e.g., antigens) used as priming vaccines in the prime-boost compositions and methods described herein. Also provided herein are cells produced by such methods and organisms (e.g., animals, plants, or fungi) containing or produced from such cells. Methods of nucleic acid delivery can include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents (e.g., Transfectam™ and Lipofectin™) are commercially available. Delivery can be to a cell (eg, in vitro or ex vivo administration) or to a target tissue (eg, in vivo administration).

[0361] Various techniques and methods are known in the art for delivering nucleic acids to cells. For example, nucleic acids, such as ceDNA, for expression of peptides (e.g., antigens) can be formulated in lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core-shell nanoparticles. Typically, LNPs are composed of nucleic acid (e.g., ceDNA) molecules, one or more ionizable or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., phospholipids), a molecule that prevents aggregation (e.g., PEG or PEG-lipid conjugates), and optionally, a sterol (e.g., cholesterol).

[0362] Another method for delivering nucleic acids, such as ceDNA, into cells for expression of peptides (e.g., antigens) is by conjugating the nucleic acid to a ligand that is internalized by the cell. For example, the ligand can bind to a receptor on the cell surface and be internalized via endocytosis. The ligand can be covalently linked to a nucleotide in the nucleic acid. Exemplary conjugates for delivering nucleic acids into cells are described, for example, in WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326.

[0363] Nucleic acids, such as ceDNA vectors, for expression of peptides (e.g., antigens) can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS™ P protein transfection reagent (New England Biolabs), CHARIOT™ protein delivery reagent (Active Motif), PROTEOJUICE™ protein transfection reagent (EMD Millipore), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific), LIPOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECTIN™ (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), and CYP293FECTAMINE™ (Thermo Fisher Scientific). Scientific), LIPOFECTACE™, FUGENE™ (Roche, Basel, Switzerland), FUGENE™ HD (Roche), TRANSFECTAM™ (Transfectam, Promega, Madison, Wisconsin), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ (Promega), TRANSFECTIN™ (Bio-Rad, Hercules, California), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, California), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene TherapySystems, San Diego, CA), DHARMAFECT 1™ (Dharmacon, Lafayette, CO), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, MO), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids, such as ceDNA, can also be delivered to cells via microfluidic methods known to those of skill in the art.

[0364] The ceDNA vectors for expression of peptides (e.g., antigens) described herein can also be administered directly to an organism for transduction of cells in vivo. Administration can be by any of the routes typically used to introduce molecules so that they ultimately contact blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route can be used to administer a particular composition, certain routes can often provide a more immediate and effective response than another route.

[0365] ceDNA vectors for expression of peptides (e.g., antigens) according to the present disclosure can be incorporated into liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They function by fusing with cell membranes and rearranging their lipid structure to deliver the drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery are composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids. Exemplary liposomes and liposomal formulations, including, but not limited to, polyethylene glycol (PEG)-functionalized compounds, are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018; see, e.g., the section entitled "Pharmaceutical Formulations."

[0366] Various delivery methods known in the art, or modifications thereof, can be used to deliver ceDNA vectors in vitro or in vivo. For example, in some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) are delivered by creating transient permeation in cell membranes using mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy to facilitate DNA entry into targeted cells. For example, ceDNA vectors can be delivered by transiently disrupting cell membranes by squeezing cells through size-restricted channels or by other means known in the art. In some embodiments, ceDNA vectors alone are injected as naked DNA directly into any one or more tissues selected from the lung, liver, kidney, gallbladder, prostate, adrenal gland, heart, intestine, stomach, skin, thymus, cardiac muscle, or skeletal muscle. In some embodiments, ceDNA vectors are delivered by gene gun. Gold or tungsten spherical particles (1-3 μm in diameter) coated with capsid-free AAV vectors can be accelerated to high velocities by pressurized gas to penetrate into target tissue cells.

[0367] In some embodiments, the ceDNA vector is formulated using a lipid delivery system, such as the liposomes described herein. In some embodiments, such compositions are administered by any route desired by one of skill in the art. The compositions may be administered to a subject by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, and intraarticularly, or a combination thereof. For veterinary use, the compositions may be administered in an appropriately acceptable formulation according to standard veterinary practice. A veterinarian can easily determine the most suitable dosing regimen and route of administration for a particular animal. The compositions may be administered by a conventional syringe, a needleless injection device, a "microprojectile bombardment gene gun," or other physical methods, such as electroporation ("EP"), hydrodynamic methods, or ultrasound.

[0368] In some cases, ceDNA vectors for expression of peptides (e.g., antigens) are delivered by hydrodynamic injection, which is a simple and highly efficient method for direct intracellular delivery of any water-soluble compound and particle into the visceral and skeletal muscles throughout the limb.

[0369] In some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) are delivered by ultrasound by creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles into cells of internal organs or tumors, and therefore the size and concentration of the plasmid DNA plays a major role in the efficiency of the system. In some embodiments, ceDNA vectors are delivered by magnetoinfection by using a magnetic field to concentrate nucleic acid-containing particles into target cells.

[0370] In some embodiments, chemical delivery systems can be used, for example, by using nanomer complexes, which involve compacting negatively charged nucleic acids with cationic liposomes / micelles or polycationic nanomer particles belonging to cationic polymers. Cationic lipids used for delivery methods include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol-derivatized compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[0371] Exosomes In some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are delivered by packaging them into exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the cell that produced the exosome, and display membrane proteins from the parent cell on their surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). In some embodiments, exosomes with diameters between 10 nm and 1 μm, between 20 nm and 500 nm, between 30 nm and 250 nm, and between 50 nm and 100 nm are contemplated for use. Exosomes can be isolated for delivery to target cells using their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing the capsid-free AAV vectors of the present disclosure.

[0372] Microparticles / Nanoparticles In some embodiments, the ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are delivered via lipid nanoparticles. Typically, lipid nanoparticles comprise an ionizable amino lipid (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and a coating lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), as disclosed, for example, by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0373] In some embodiments, the lipid nanoparticles have an average diameter of between about 10 and about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter that is less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of between about 10 and about 300 nm. In some embodiments, the lipid nanoparticles have a diameter that is less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of between about 25 and about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution with an average size (e.g., diameter) of between about 70 nm and about 200 nm, more typically with an average size of about 100 nm or less.

[0374] Various lipid nanoparticles known in the art can be used to deliver ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein. For example, various delivery methods using lipid nanoparticles are described in U.S. Patent Nos. 9,404,127, 9,006,417, and 9,518,272.

[0375] Conjugates According to some embodiments, ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are conjugated (e.g., covalently linked) to agents that increase cellular uptake. An "agent that increases cellular uptake" is a molecule that promotes the transport of nucleic acids across lipid membranes. For example, nucleic acids can be conjugated to lipophilic compounds (e.g., cholesterol, tocopherol, etc.), cell-penetrating peptides (CPPs) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine). Further examples of agents that increase cellular uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[0376] According to some embodiments, the ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are conjugated to a polymer (e.g., a polymer molecule) or a folic acid molecule (e.g., a folic acid molecule). In general, delivery of nucleic acids conjugated to polymers is known in the art, for example, as described in WO2000 / 34343 and WO2008 / 022309. According to some embodiments, the ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are conjugated to a poly(amide) polymer, for example, as described by U.S. Pat. No. 8,987,377. According to some embodiments, the nucleic acids described by the present disclosure are conjugated to a folic acid molecule, as described in U.S. Pat. No. 8,507,455.

[0377] According to some embodiments, the ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein are conjugated to sugars, for example, as described in U.S. Pat. No. 8,450,467.

[0378] nanocapsules Alternatively, nanocapsule formulations of ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein can be used. Nanocapsules can generally encapsulate substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0379] Liposomes ceDNA vectors for expression of peptides (e.g., antigens) according to the present disclosure can be incorporated into liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They function by fusing with cell membranes and rearranging their lipid structure to deliver the drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery are composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions can also contain other lipids.

[0380] The formation and use of liposomes are generally known to those skilled in the art. Liposomes have been developed with improved serum stability and circulatory half-life (see U.S. Patent No. 5,741,516). Furthermore, various methods for preparing liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868; and 5,795,587).

[0381] Exemplary Liposome and Lipid Nanoparticle (LNP) Compositions ceDNA vectors for expression of peptides (e.g., antigens) according to the present disclosure can be incorporated into liposomes for delivery to cells, e.g., cells requiring transgene expression. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They function by fusing with cell membranes and rearranging their lipid structure to deliver the drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery are composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0382] Lipid nanoparticles (LNPs) comprising ceDNA vectors are disclosed in International Application Nos. PCT / US2018 / 050042, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, which are incorporated herein in their entireties, and are contemplated for use in the methods and compositions of ceDNA vectors for expression of peptides (e.g., antigens) disclosed herein.

[0383] According to some embodiments, the present disclosure provides liposomal formulations containing one or more compounds with polyethylene glycol (PEG) functional groups (so-called PEGylated compounds), which can reduce the immunogenicity / antigenicity of the compounds, provide them with hydrophilic and hydrophobic properties, and reduce dosing frequency. Alternatively, the liposomal formulations simply contain polyethylene glycol (PEG) polymers as additional components. In such embodiments, the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.

[0384] According to some embodiments, the present disclosure provides liposomal formulations that deliver APIs with sustained or controlled release profiles over a period of hours to weeks. According to some related embodiments, the liposomal formulations can include aqueous chambers bounded by a lipid bilayer. In other related embodiments, the liposomal formulations encapsulate the API with components that undergo a physical transition at elevated temperatures, releasing the API over a period of hours to weeks.

[0385] According to some aspects, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. According to some aspects, the liposomal formulation comprises an Optisome.

[0386] According to some embodiments, the present disclosure provides liposomal formulations comprising one or more lipids selected from the following: N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol) conjugated lipid, HSPC (hydrogenated soy phosphatidylcholine), PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylcholine). dioleoylglycerol; EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylserine); POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxypolyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dioleoylphosphatidylcholine); DOPE (dioleoyl-sn-glycero-phosphoethanolamine). Cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or a combination thereof.

[0387] According to some embodiments, the present disclosure provides a liposome formulation comprising a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 56:38:5. According to some embodiments, the overall lipid content of the liposome formulation is 2-16 mg / mL. According to some embodiments, the present disclosure provides a liposome formulation comprising a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a PEGylated lipid. According to some embodiments, the present disclosure provides a liposome formulation comprising a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. According to some embodiments, the present disclosure provides a liposome formulation comprising a lipid comprising a phosphatidylcholine functional group, cholesterol, and a PEGylated lipid. According to some embodiments, the present disclosure provides a liposome formulation comprising a lipid comprising a phosphatidylcholine functional group and cholesterol. According to some embodiments, the PEGylated lipid is PEG-2000-DSPE. According to some aspects, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0388] According to some aspects, the present disclosure provides liposome formulations comprising one or more lipids comprising a phosphatidylcholine functional group and one or more lipids comprising an ethanolamine functional group. According to some aspects, the present disclosure provides liposome formulations comprising one or more of a lipid comprising a phosphatidylcholine functional group, a lipid comprising an ethanolamine functional group, and a sterol, e.g., cholesterol. According to some aspects, the liposome formulation comprises DOPC / DEPC; and DOPE.

[0389] According to some aspects, the present disclosure provides liposomal formulations further comprising one or more pharmaceutical excipients, for example, sucrose and / or glycine.

[0390] In some embodiments, the present disclosure provides liposome formulations that are either unilamellar or multilamellar in structure. In some embodiments, the present disclosure provides liposome formulations comprising multivesicular particles and / or foam-based particles. In some embodiments, the present disclosure provides liposome formulations that are larger in size relative to common nanoparticles, approximately 150-250 nm in size. In some embodiments, the liposome formulation is a lyophilized powder. In some embodiments, the present disclosure provides liposomal formulations prepared and loaded with the ceDNA vectors disclosed or described herein by adding a weak base to a mixture containing isolated ceDNA outside the liposomes. This addition increases the pH outside the liposomes to about 7.3, driving the API into the liposomes. In some embodiments, the present disclosure provides liposomal formulations having an acidic pH inside the liposomes. In such cases, the inside of the liposomes may be pH 4 to 6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides liposomal formulations prepared using intraliposomal drug stabilization techniques. In such cases, a polymeric or non-polymeric highly charged anion and an intraliposomal entrapment agent, such as polyphosphate or sucrose octasulfate, are utilized.

[0391] According to some embodiments, the present disclosure provides lipid nanoparticles containing ceDNA and ionizable lipids. For example, lipid nanoparticle formulations prepared and loaded with ceDNA obtained by the process disclosed in International Patent Application PCT / US2018 / 050042, filed September 7, 2018, are incorporated herein. This can be achieved by high-energy mixing of ethanolic lipids and aqueous ceDNA at low pH, which protonates the ionizable lipids and provides favorable energy for ceDNA / lipid association and particle nucleation. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to a desired level.

[0392] Typically, lipid nanoparticles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 60:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, or about 30:1 to about 60:1. In some embodiments, lipid particles (e.g., lipid nanoparticles) are prepared with a ceDNA (mass or weight) to total lipid ratio of about 60:1. In some embodiments, lipid particles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipid and ceDNA can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, or higher. Generally, the overall lipid content of lipid particle formulations can range from about 5 mg / mL to about 30 mg / mL.

[0393] Ionizable lipids are typically used to condense nucleic acid cargoes, such as ceDNA, at low pH and drive membrane association and fusogenicity. Generally, ionizable lipids are lipids that contain at least one amino group that is positively charged or protonated under acidic conditions, such as at pH 6.5 or below. Ionizable lipids are also referred to herein as cationic lipids.

[0394] Exemplary ionizable lipids are described in International PCT Patent Publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467...

Claims

1. 1. A method of inducing an immune response in a subject to a first peptide and a second peptide, comprising: administering to the subject a priming vaccine comprising deoxyribonucleic acid (DNA), the DNA encoding a first peptide; and (i) a ribonucleic acid (RNA), the RNA encoding the second peptide; or (ii) administering to the subject a boosting vaccine comprising a second peptide; thereby inducing in the subject an immune response to the first peptide and the second peptide.

2. 2. The method of claim 1, wherein the priming vaccine comprises DNA encoding the first peptide and the boosting vaccine comprises RNA encoding the second peptide.

3. 2. The method of claim 1, wherein the priming vaccine comprises DNA encoding the first peptide and the boosting vaccine comprises the second peptide.

4. 4. The method of any one of claims 1 to 3, wherein the DNA comprises a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone (dbDNA™) DNA, dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector or a non-viral vector.

5. The method of any one of claims 1 to 4, wherein the first and second peptides are derived from a bacterial, viral, fungal, or parasitic infectious agent.

6. The method of any one of claims 1 to 5, wherein the first and second peptides are derived from the same pathogenic organism.

7. The method of any one of claims 1 to 6, wherein the first and second peptides are the same in the priming vaccine and in the boosting vaccine.

8. The method of any one of claims 1 to 6, wherein at least one of the epitopes of the first and second peptides is different in the priming vaccine and the boosting vaccine.

9. 9. The method of any one of claims 1 to 8, wherein the DNA comprises a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between flanking inverted terminal repeats (ITRs), the at least one nucleic acid sequence encoding the peptide.

10. The method according to any one of claims 1 to 9, wherein the first and / or the second peptide is a tumor-associated antigen or is associated with an autoimmune condition.

11. The method of any one of claims 1 to 10, wherein the first and / or second peptide is selected from the group consisting of the peptides shown in Tables 1 to 8.

12. The method of any one of claims 1 to 11, wherein the DNA comprises a promoter sequence linked to the at least one nucleic acid sequence.

13. The method according to any one of claims 4 to 12, wherein the ceDNA vector comprises at least one polyA sequence.

14. The method of any one of claims 4 to 13, wherein the ceDNA vector comprises a 5'UTR and / or an intron sequence.

15. The method of any one of claims 4 to 14, wherein the ceDNA vector comprises a 3'UTR sequence.

16. The method of any one of claims 4 to 15, wherein the ceDNA vector comprises an enhancer sequence.

17. The method of any one of claims 9 to 16, wherein at least one of the flanking ITRs contains a functional terminal resolution site and a Rep binding site.

18. 18. The method of any one of claims 9 to 17, wherein one or both of the flanking ITRs are derived from a virus selected from the group consisting of parvovirus, dependovirus, and adeno-associated virus (AAV).

19. The method of any one of claims 9 to 18, wherein the adjacent ITRs are symmetric or asymmetric with respect to each other.

20. The method of any one of claims 9 to 19, wherein the adjacent ITRs are symmetric or substantially symmetric.

21. The method of any one of claims 9 to 19, wherein the adjacent ITRs are asymmetric.

22. The method of any one of claims 9 to 21, wherein one of the flanking ITRs is wild type or both of the flanking ITRs are wild type ITRs.

23. The method of any one of claims 9 to 22, wherein the flanking ITRs are from different viral serotypes.

24. The method of any one of claims 9 to 23, wherein the flanking ITRs are selected from the group consisting of pairs of viral serotypes shown in Table 8.

25. 25. The method of any one of claims 9 to 24, wherein one or both of the flanking ITRs comprises a sequence selected from the group consisting of the sequences shown in Table 9.

26. 26. The method of any one of claims 9 to 25, wherein at least one of the flanking ITRs is modified from a wild-type AAV ITR sequence by a deletion, addition, or substitution that affects the overall three-dimensional structure of the ITR.

27. 27. The method of any one of claims 9 to 26, wherein one or both of the flanking ITRs are derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

28. 28. The method of any one of claims 9 to 27, wherein one or both of the flanking ITRs are synthetic.

29. The method of any one of claims 9 to 28, wherein one of the flanking ITRs is not a wild-type ITR or both of the ITRs are not wild-type ITRs.

30. 30. The method of any one of claims 9 to 29, wherein one or both of the flanking ITRs are modified by a deletion, insertion, and / or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D, and D'.

31. 31. The method of claim 30, wherein the deletion, insertion and / or substitution results in the deletion of all or part of the stem-loop structure formed by the A, A', B, B', C or C' regions.

32. 32. The method of any one of claims 9 to 31, wherein one or both of the flanking ITRs are modified by deletions, insertions and / or substitutions resulting in the deletion of all or part of the stem-loop structure formed by the B and B' regions.

33. 33. The method of any one of claims 9 to 32, wherein one or both of the flanking ITRs are modified by deletion, insertion and / or substitution resulting in the deletion of all or part of the stem-loop structure formed by the C and C' regions.

34. 34. The method of any one of claims 9 to 33, wherein one or both of the flanking ITRs are modified by a deletion, insertion and / or substitution resulting in the deletion of part of the stem-loop structure formed by the B and B' regions and / or part of the stem-loop structure formed by the C and C' regions.

35. 35. The method of any one of claims 9 to 34, wherein one or both of the flanking ITRs contain a single stem-loop structure in the region that would, in a wild-type ITR, contain a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

36. 36. The method of any one of claims 9 to 35, wherein one or both of the flanking ITRs comprises a single stem and two loops in a region that would, in a wild-type ITR, comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

37. 37. The method of any one of claims 9 to 36, wherein one or both of the flanking ITRs comprise a single stem and a single loop in the region that would, in a wild-type ITR, comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

38. The method of any one of claims 9 to 37, wherein both adjacent ITRs are modified in a manner that results in overall three-dimensional symmetry when said ITRs are inverted relative to each other.

39. The method of any one of claims 1 to 38, wherein the DNA is delivered in a lipid nanoparticle (LNP).

40. The method of any one of claims 1 to 39, wherein the RNA is delivered in an LNP.

41. 41. The method of any one of claims 1 to 40, wherein the RNA is messenger RNA (mRNA).

42. The method of any one of claims 1 to 41, wherein the RNA comprises at least one nucleotide analogue.

43. The method of any one of claims 1 to 42, wherein the immune response is an antibody response.

44. The method of any one of claims 1 to 43, wherein the immune response is a T cell response.

45. The immune response is memory (CD8 ＋ 45. The method of any one of claims 1 to 44, wherein the response is a T cell response.

46. 46. ​​The method of any one of claims 1-45, wherein the method comprises administering the boosting vaccine at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 1-2 weeks, at least about 2-3 weeks, at least about 3-4 weeks, at least about 4-5 weeks, at least about 5-6 weeks, at least about 6-7 weeks, at least about 7-8 weeks, at least about 8-9 weeks, at least about 9-10 weeks, at least about 10-11 weeks, at least about 11-12 weeks, at least about 12-13 weeks, at least 13-14 weeks, at least about 14-15 weeks, or at least about 15-16 weeks after administration of the priming vaccine.

47. 47. The method of any one of claims 1 to 46, wherein the method comprises administering the boosting vaccine about 8 weeks after administering the priming vaccine.

48. and / or wherein the interval between said administration of said priming vaccine and said administration of said boosting vaccine is at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, at least about 66 days, at least about 67 days, at least 7 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, at least about 66 days, at least about 67 days, at least about 68 days, at least about 69 days, at least about 70 days, at least about 71 days, at least about 72 days, at least about 73 days, at least about 74 days, at least about 75 days, at least about 76 days, At least about 77 days, at least about 78 days, at least about 79 days, at least about 80 days, at least about 81 days, at least about 82 days, at least about 83 days, at least about 84 days, at least about 85 days, at least about 86 days, at least about 87 days, at least about 88 days, at least about 89 days, at least about 90 days, at least about 91 days, at least about 92 days, at least about 93 days, at least about 94 days, at least about 95 days, at least about 96 days, at least about 97 days, at least about 98 days, at least about 99 days, at least about 100 days, at least about 101 days,48. The method of any one of claims 1 to 47, wherein the incubation time is at least about 102 days, at least about 103 days, at least about 104 days, at least about 105 days, at least about 106 days, at least about 107 days, at least about 108 days, at least about 109 days, at least about 110 days, at least about 111 days, or at least about 112 days.

49. 49. The method of any one of claims 1 to 48, wherein the interval between the administration of the priming vaccine and the administration of the boosting vaccine is about 64 days.

50. The method of any one of claims 1 to 49, wherein the method comprises administering to the subject two or more doses of the boosting vaccine.

51. 51. The method of any one of claims 1-50, wherein the method comprises administering each dose of the boosting vaccine at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 1-2 weeks, at least about 2-3 weeks, at least about 3-4 weeks, at least about 4-5 weeks, at least about 5-6 weeks, at least about 6-7 weeks, at least about 7-8 weeks, at least about 8-9 weeks, at least about 9-10 weeks, at least about 10-11 weeks, at least about 11-12 weeks, at least about 12-13 weeks, at least 13-14 weeks, at least about 14-15 weeks, or at least about 15-16 weeks after administration of the previous vaccine.

52. The interval between administration of each dose of boosting vaccine and administration of the previous vaccine is at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, at least about 43 days, at least about 44 days, at least about 45 days, at least about 46 days, at least about 47 days, at least about 48 days, at least about 49 days, at least about 50 days, at least about 51 days, at least about 52 days, at least about 53 days, at least about 54 days, at least about 55 days, at least about 56 days, at least about 57 days, at least about 58 days, at least about 59 days, at least about 60 days, at least about 61 days, at least about 62 days, at least about 63 days, at least about 64 days, at least about 65 days, at least about 66 days, at least about 67 days, at least about 68 days, at least about 69 days, at least about 70 days, at least about 71 days, at least about 72 days, at least about 73 days, at least about 74 days, at least about 75 days, at least about 76 days, at least about 77 days, at least about 78 days, at least about 79 days, at least about 80 days, at least about 81 days, at least about 82 days, at least about 83 days, at least about 84 days, at least about 85 days, at least about 86 days, at least about 87 days, at least about 88 days, at least about 89 days, at least about 90 days, at least about 91 days, at least about 92 days, at least about 93 days, at least about 94 days, at least about 95 days, at least about 96 days, at least about 97 days, at least about 98 days, at least about 99 days, at least about 100 days, at least about 101 days, at least about 102 days,52. The method of any one of claims 1 to 51, wherein the incubation time is at least about 103 days, at least about 104 days, at least about 105 days, at least about 106 days, at least about 107 days, at least about 108 days, at least about 109 days, at least about 110 days, at least about 111 days, or at least about 112 days.

53. 53. The method of any one of claims 1 to 52, wherein the subject has a bacterial, viral, parasitic, or fungal infection.

54. The method of any one of claims 1 to 53, wherein the subject has cancer.

55. The method of any one of claims 1 to 54, wherein the subject has an autoimmune disease or disorder.

56. 56. The method of any one of claims 1 to 55, wherein one or more of the priming vaccine or the boosting vaccine comprises a pharma- ceutically acceptable carrier.

57. 57. The method of claim 56, wherein at least one of the priming vaccine and boosting vaccine compositions further comprises an adjuvant.

58. 58. The method of any one of claims 1 to 57, wherein at least one of the priming vaccine and the boosting vaccine is administered by a route selected from the group consisting of intramuscular, intraperitoneal, buccal, inhalation, intranasal, intrathecal, intravenous, subcutaneous, intradermal, and intratumoral, or is administered into the interstitial space of a tissue.

59. 1. A vaccine regimen comprising a priming vaccine comprising deoxyribonucleic acid (DNA), the DNA encoding a first peptide, followed by a boosting vaccine comprising (i) a ribonucleic acid (RNA) encoding a second peptide, or (ii) the second peptide.

60. 60. The vaccine regimen of claim 59, wherein the priming vaccine comprises an immunologically effective amount of DNA encoding the first peptide, and the boosting vaccine comprises an immunologically effective amount of RNA encoding the second peptide.

61. 60. The vaccine regimen of claim 59, wherein the priming vaccine comprises an immunologically effective amount of DNA encoding the first peptide and the boosting vaccine comprises an immunologically effective amount of the second peptide.

62. 62. The vaccine regimen of any one of claims 59 to 61, wherein the DNA comprises a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), Doggybone (dbDNA™) DNA, dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector or a non-viral vector.

63. 63. The vaccine regimen of any one of claims 59 to 62, wherein the first peptide and the second peptide are derived from a bacterial, viral, fungal, or parasitic infectious agent.

64. 64. The vaccine regimen of any one of claims 59 to 63, wherein the first peptide and the second peptide are derived from the same pathogenic organism.

65. 65. The vaccine regimen of any one of claims 59 to 64, wherein the first peptide and the second peptide are the same in the priming vaccine and in the boosting vaccine.

66. 65. The vaccine regimen of any one of claims 59 to 64, wherein at least one epitope of the first peptide and the second peptide is different in the priming vaccine and the boosting vaccine.

67. 67. The vaccine regimen of any one of claims 59 to 66, wherein the DNA comprises a capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleic acid sequence between inverted repeat translocations (ITRs) flanking the vector, the at least one nucleic acid sequence encoding the first peptide.

68. 68. A vaccine regimen according to any one of claims 59 to 62 or 65 to 67, wherein the first and / or the second peptide is a tumor associated antigen or is associated with an autoimmune condition.

69. 69. The vaccine regimen of any one of claims 59 to 68, wherein the first and / or second peptide is selected from the group consisting of the peptides shown in Tables 1 to 8.

70. 70. The vaccine regimen of any one of claims 59 to 69, wherein the DNA comprises a promoter sequence linked to the at least one nucleic acid sequence.

71. A vaccine regimen according to any one of claims 62 to 70, wherein the ceDNA vector comprises at least one polyA sequence.

72. 72. The vaccine regimen of any one of claims 62 to 71, wherein the ceDNA vector comprises a 5'UTR and / or an intron sequence.

73. 73. The vaccine regimen of any one of claims 62 to 72, wherein the ceDNA vector comprises a 3'UTR sequence.

74. 74. The vaccine regimen of any one of claims 62 to 73, wherein the ceDNA vector comprises an enhancer sequence.

75. 75. The vaccine regimen of any one of claims 67 to 74, wherein at least one of the flanking ITRs contains a functional terminal resolution site and a Rep binding site.

76. 76. The vaccine regimen of any one of claims 67 to 75, wherein one or both of the flanking ITRs are derived from a virus selected from a parvovirus, a dependovirus, and an adeno-associated virus (AAV).

77. 77. The vaccine regimen of any one of claims 67 to 76, wherein the adjacent ITRs are symmetric or asymmetric with respect to each other.

78. 78. The vaccine regimen of any one of claims 67 to 77, wherein the adjacent ITRs are symmetric or substantially symmetric.

79. 78. The vaccine regimen of any one of claims 67 to 77, wherein the flanking ITRs are asymmetric.

80. 80. The vaccine regimen of any one of claims 67 to 79, wherein one of the flanking ITRs is wild type or both of the ITRs are wild type ITRs.

81. 81. The vaccine regimen of any one of claims 67 to 80, wherein the flanking ITRs are derived from different viral serotypes.

82. 82. The vaccine regimen of any one of claims 67-81, wherein the flanking ITRs are selected from the group consisting of the viral serotypes shown in Table 8.

83. 83. The vaccine regimen of any one of claims 67-82, wherein one or both of the flanking ITRs comprises a sequence selected from the group consisting of the sequences shown in Table 9.

84. 84. The vaccine regimen of any one of claims 67-83, wherein at least one of the flanking ITRs is modified from a wild-type AAV ITR sequence by a deletion, addition, or substitution that affects the overall three-dimensional structure of the ITR.

85. 85. The vaccine regimen of any one of claims 67-84, wherein one or both of the flanking ITRs are derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

86. 86. The vaccine regimen of any one of claims 67 to 85, wherein one or both of the flanking ITRs are synthetic.

87. 87. The vaccine regimen of any one of claims 67 to 86, wherein one of the flanking ITRs is not a wild-type ITR or both of the flanking ITRs are not wild-type ITRs.

88. 88. The vaccine regimen of any one of claims 67 to 87, wherein one or both of the flanking ITRs are modified by a deletion, insertion, and / or substitution in at least one of the ITR regions selected from A, A', B, B', C, C', D, and D'.

89. 89. The vaccine regimen of claim 88, wherein the deletion, insertion and / or substitution results in the deletion of all or part of the stem-loop structure formed by the A, A', B, B', C, or C' regions.

90. 90. A vaccine regimen according to any one of claims 67 to 89, wherein one or both of the flanking ITRs are modified by deletion, insertion and / or substitution resulting in the deletion of all or part of the stem-loop structure formed by the B and B' regions.

91. A vaccine regimen according to any one of claims 67 to 90, wherein one or both of the flanking ITRs are modified by deletion, insertion and / or substitution resulting in the deletion of all or part of the stem-loop structure formed by the C and C' regions.

92. 92. The vaccine regimen of any one of claims 67 to 91, wherein one or both of the flanking ITRs are modified by a deletion, insertion and / or substitution resulting in the deletion of part of the stem-loop structure formed by the B and B' regions and / or part of the stem-loop structure formed by the C and C' regions.

93. 93. The vaccine regimen of any one of claims 67 to 92, wherein one or both of the flanking ITRs contain a single stem-loop structure in the region that would, in a wild-type ITR, contain a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

94. 94. The vaccine regimen of any one of claims 67 to 93, wherein one or both of the flanking ITRs comprises a single stem and two loops in the region that would, in a wild-type ITR, comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

95. 95. The vaccine regimen of any one of claims 67 to 94, wherein one or both of the flanking ITRs comprise a single stem and a single loop in the region that would, in a wild-type ITR, comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions.

96. 96. The vaccine regimen of any one of claims 67 to 95, wherein both adjacent ITRs are modified in a manner that results in overall three-dimensional symmetry when the ITRs are inverted relative to each other.

97. 97. A method of treating a subject with a bacterial, viral, parasitic or fungal infection comprising carrying out a method according to any one of claims 1 to 58 or administering to said subject a vaccine regimen according to any one of claims 59 to 96.

98. 97. A method of treating a subject with cancer comprising carrying out a method according to any one of claims 1 to 58 or administering to said subject a vaccine regimen according to any one of claims 59 to 96.

99. 97. A method of treating a subject with an autoimmune disease or disorder comprising carrying out a method according to any one of claims 1 to 58 or administering to said subject a vaccine regimen according to any one of claims 59 to 96.

100. 97. A method of preventing a bacterial, viral, parasitic or fungal infection in a subject comprising carrying out a method according to any one of claims 1 to 58 or administering to the subject a vaccine regimen according to any one of claims 59 to 96.

101. 97. A method of preventing cancer in a subject comprising carrying out a method according to any one of claims 1 to 58 or administering to said subject a vaccine regimen according to any one of claims 59 to 96.

102. 97. A method for preventing an autoimmune disease in a subject comprising carrying out a method according to any one of claims 1 to 58 or administering to said subject a vaccine regimen according to any one of claims 59 to 96.

103. 103. The method of any one of claims 97 to 102, wherein the method comprises administering to the subject two or more doses of the boosting vaccine.

104. 104. The method of any one of claims 97-103, wherein the method comprises administering the boosting vaccine about 8 weeks after administering the priming vaccine.

105. 105. The method of any one of claims 97 to 104, further comprising administering to the subject one or more additional therapeutic agents.

106. 97. The vaccine regimen of any one of claims 59 to 96, wherein the priming vaccine and the boosting vaccine are each formulated in a pharmaceutical composition.

107. 107. The vaccine regimen of claim 106, wherein one or both of the priming vaccine and the boosting vaccine further comprise one or more additional therapeutic agents.

108. 108. The vaccine regimen of any one of claims 59-96 or 106-107, wherein one or both of the priming vaccine and the boosting vaccine further comprise a lipid.

109. 109. The vaccine regimen of claim 108, wherein the lipid is a lipid nanoparticle (LNP).

110. 110. The vaccine regimen of any one of claims 106 to 109, wherein one or both of the priming vaccine and the boosting vaccine are lyophilized.

111. A kit comprising the vaccine regimen of any one of claims 59-96 or 106-110 and instructions for use.

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