Large and small T antigens of Merkel cell polyomavirus, nucleic acid constructs, vaccines produced therefrom, and methods of use thereof

Abstract

Nucleic acid molecules and compositions comprising one or more nucleotide sequences encoding a consensus Merkel cell polyomavirus (MCV) T antigen. Immunomodulatory methods and methods of inducing an immune response to MCV are disclosed. Methods of treating MCV infection and methods of treating or preventing MCV-associated Merkel cell carcinoma are disclosed. Modified consensus MCV T antigens are disclosed.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 619,161, filed on January 19, 2018, which is incorporated herein by reference in its entirety. Technical field

[0003] The present invention relates to vaccines for inducing an immune response and treating individuals infected with MCV and / or treating or preventing Merkel cell carcinoma (MCC). The present invention relates to the common MCV large T antigen (LTAg) and small t antigen (STAg) oncoproteins and nucleic acid molecules encoding them. Background art

[0004] Merkel Cell Polyomavirus (MCV) has recently received attention due to its association with the aggressive human skin cancer Merkel Cell Carcinoma (MCC). In the United States, approximately 1,500 new MCC cases are diagnosed each year, and the mortality rate of MCC subjects remains at 46%. Compared with cutaneous T - cell lymphoma and chronic myeloid leukemia, MCC kills more patients. Most (about 75%) MCCs contain clonal integrated viral DNA and express viral T - antigen transcripts and proteins.

[0005] Currently, no vaccine for MCC is in clinical trials. Therefore, there is a need in the art for therapeutic vaccines against MCV and MCC. The present invention meets this unmet need. Summary of the invention

[0006] In one embodiment, the present invention relates to an immunogenic composition comprising a nucleic acid molecule encoding at least one modified Merkel Cell Polyomavirus (MCV) T antigen, wherein the T antigen comprises at least one mutation that disrupts at least one oncogenic characteristic of the native MCV T antigen. In one embodiment, the at least one oncogenic characteristic is at least one of CR1 binding, DnaJ binding, binding to phosphatase pp2A, Rb binding, ATPase activity, helicase activity, chaperone binding, hVam6p binding, Fbxw7 binding, origin binding, and transformation.

[0007] In one embodiment, the at least one mutation is an amino acid mutation, and the amino acid is located at least at one of D44, W209, E216, L142, L91, K92, D93, Y94 or M95. In one embodiment, the at least one mutation is at least one of D44N mutation, W209A, E216K mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation or M95A mutation. In one embodiment, the modified MCV T antigen comprises at least one of D44N mutation, W209A or E216K mutation. In one embodiment, the modified MCV T comprises D44N mutation, W209A and E216K mutations.

[0008] In one embodiment, at least one MCV T antigen is large T antigen (LTAg) or small t antigen (STAg). In one embodiment, at least one MCV T antigen is a combination of LTAg and STAg.

[0009] In one embodiment, the nucleic acid molecule encodes a peptide, and the peptide comprises the following amino acid sequences: a) an amino acid sequence having at least about 90% identity with the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity with at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0010] In one embodiment, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0011] In one embodiment, the nucleic acid molecule comprises at least one of the following nucleotide sequences: a) a nucleotide sequence having at least about 90% identity to the full length of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, b) an immunogenic fragment of a nucleotide sequence having at least about 90% identity to at least 60% of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, c) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or d) an immunogenic fragment of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0012] In one embodiment, the nucleotide sequence encoding the peptide is operably linked to at least one regulatory sequence. In one embodiment, the regulatory sequence is at least one of a start codon, an IgE leader sequence, or a stop codon.

[0013] In one embodiment, the nucleic acid molecule encodes a peptide that comprises at least one of the following amino acid sequences: a) an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity to at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, operably linked to the amino acid sequence shown in SEQ ID NO:7.

[0014] In one embodiment, the nucleic acid molecule comprises at least one of the following nucleotide sequences: a) a nucleotide sequence having at least about 90% identity to the full length of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, b) an immunogenic fragment of a nucleotide sequence having at least about 90% identity to at least 60% of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, c) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or d) an immunogenic fragment of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, operably linked to the amino acid sequence shown in SEQ ID NO:7.

[0015] In one embodiment, the nucleic acid molecule comprises an expression vector.

[0016] In one embodiment, the nucleic acid molecule is incorporated into virus particles.

[0017] In one embodiment, the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0018] In one embodiment, the immunogenic composition further comprises an adjuvant.

[0019] In one embodiment, the present invention relates to a nucleic acid molecule encoding a peptide comprising at least one of the following amino acid sequences: a) an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity to at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0020] In one embodiment, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0021] In one embodiment, the nucleic acid molecule comprises at least one of the following nucleotide sequences: a) a nucleotide sequence having at least about 90% identity to the full length of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, b) an immunogenic fragment of a nucleotide sequence having at least about 90% identity to at least 60% of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, c) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or d) an immunogenic fragment of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0022] In one embodiment, the nucleotide sequence encoding the peptide is operably linked to at least one regulatory sequence. In one embodiment, the regulatory sequence is at least one of a start codon, an IgE leader sequence, or a stop codon.

[0023] In one embodiment, the nucleic acid molecule encodes a peptide that comprises at least one of the following amino acid sequences: a) an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity to at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, operably linked to the amino acid sequence shown in SEQ ID NO:7.

[0024] In one embodiment, the nucleic acid molecule comprises at least one of the following nucleotide sequences: a) a nucleotide sequence having at least about 90% identity to the full length of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, b) an immunogenic fragment of a nucleotide sequence having at least about 90% identity to at least 60% of the nucleotide sequence of at least one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, c) the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or d) an immunogenic fragment of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, operably linked to the amino acid sequence shown in SEQ ID NO:7.

[0025] In one embodiment, the nucleic acid molecule comprises an expression vector.

[0026] In one embodiment, the nucleic acid molecule is incorporated into virus particles.

[0027] In one embodiment, the present invention relates to an immunogenic composition comprising a peptide, wherein the peptide comprises at least one of the following amino acid sequences: a) an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity to at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0028] In one embodiment, the present invention relates to a peptide, wherein the peptide comprises at least one of the following amino acid sequences: a) an amino acid sequence having at least about 90% identity to the full length of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90% identity to at least 60% of the amino acid sequence of at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragment comprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0029] In one embodiment, the present invention relates to a method of inducing an immune response against an MCV T antigen in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising a nucleic acid molecule encoding a modified Merkel cell polyomavirus (MCV) T antigen, wherein the T antigen comprises at least one mutation that disrupts at least one oncogenic feature of the native MCV T antigen.

[0030] In one embodiment, the method of administration comprises at least one of electroporation or injection.

[0031] In one embodiment, the present invention relates to a method of treating or preventing an MCV-related pathology in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising a nucleic acid molecule encoding a modified Merkel cell polyomavirus (MCV) T antigen, wherein the T antigen comprises at least one mutation that disrupts at least one oncogenic feature of the native MCV T antigen.

[0032] In one embodiment, the method of administration comprises at least one of electroporation or injection.

[0033] In one embodiment, the MCV-related pathology is at least one of MCV infection or Merkel cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1, which includes Figures 1A to 1B, provides schematic diagrams of LTAg and STAg. Figure 1A shows the oncogenic characteristics of LTAg and STAg. Figure 1B shows the design of LTAg and STAg of nucleic acid vaccines incorporated with several mutations to disrupt the oncogenic characteristics. *D44N - blocks the binding to chaperone proteins; *W209A - blocks the binding to hVam6p; *E216K - blocks the binding to Rb and prevents transformation; *L142A - blocks the binding to PP2A; *91 - 95LKDYM->AAAAA - blocks the binding to Fbxw7 and prevents transformation.

[0035] Figure 2, which includes Figures 2A to 2B, provides schematic diagrams of consensus LTAg and STAg. Figure 2A shows the LTAg consensus sequence diagram designed from all available NCBI LTAg sequences. Figure 2B shows the diagram of the consensus sequence of STAg designed from all available NCBI STAg sequences. These antigen sequences were synthesized and cloned into mammalian expression plasmids to generate plasmid DNA constructs for in vivo expression of synthetic consensus antigens.

[0036] Figure 3 Exemplary experimental data showing the expression of consensus MCC LTAg in vitro are presented. Due to the lack of effective antibodies targeting STAg, the expression of consensus MCC STAg was not detected.

[0037] Figure 4 includes Figures 4A to 4B, providing exemplary experimental data showing the induction of immune responses after vaccination with LTAg and STAg vaccines alone or in combination. Figure 4A shows the experimental design. On days 0, 14, and 28, mice received plasmid DNA followed by intramuscular electroporation. One week later, splenocytes were collected for analysis. Four groups of mice were vaccinated: Group 1 - pVax - empty vector control; Group 2 - LTAg vaccine; Group 3 - STAg vaccine; Group 4 - LTAg and STAg vaccines were co - administered at the same site. Figure 4B shows the experimental data demonstrating the induction of immune responses after vaccination with LTAg and STAg alone or in combination, while no immune response was induced after vaccination with the empty control vector (pVax). For these experiments, the peptides matched the corresponding sequences without inactivating mutations.

[0038] Figure 5 includes Figures 5A to 5B, providing exemplary experimental data characterizing the immunodominant epitopes of LTAg and STAg. Figure 5A shows the immunodominant epitopes of LTAg vaccination. Figure 5B shows the immunodominant epitopes of STAg vaccination.

[0039] Figure 6 The analysis results of the truncation degree of MCC large T in human Merkel cell carcinoma samples are presented. The data were compiled from 42 large T sequences in GenBank.

[0040] Figure 7 includes Figures 7A through 7F and provides exemplary experimental data showing the levels of cytokine production by CD4 + and CD8 + T cell responses 5 hours after LTAg peptide inoculation and stimulation. Figure 7A shows the level of CD8 + T cell response for IFNγ production. Figure 7B shows the level of CD8 + T cell response for TNFα production. Figure 7C shows the level of CD8 + T cell response for IL-2 production. Figure 7D shows the level of CD4 + T cell response for IFNγ production. Figure 7E shows the level of CD4 + T cell response for TNFα production. Figure 7F shows the level of CD4 + T cell response for IL-2 production.

[0041] Figure 8 shows exemplary experimental data indicating that LTAg inoculation induces robust multifunctional CD8 T cells.

[0042] Figure 9 shows exemplary experimental data indicating that LTAg inoculation induces robust multifunctional CD4 T cells.

[0043] Figure 10 shows exemplary experimental data indicating that LTAg inoculation induces CD8 T cells with cytotoxic potential co-expressing CD107a, IFNγ, and T-bet.

[0044] Figure 11 shows exemplary experimental data demonstrating the generation of a humoral response by the large T and small T antigen vaccines using mouse serum as the primary antibody.

[0045] Figure 12 shows exemplary experimental data indicating that the LTAg vaccine induces a robust immune response in genetically diverse CD-1 outbred mice.

[0046] Figure 13 shows exemplary experimental data indicating that the STAg vaccine induces an immune response in genetically diverse CD-1 outbred mice.

[0047] Figure 14 includes Figures 14A through 14F and provides exemplary experimental data showing the levels of cytokine production by CD4 + and CD8 + T cell responses 5 hours after LTAg peptide inoculation and stimulation in CD-1 outbred mice. Figure 14A shows the level of CD8 + T cell response for IFNγ production. Figure 14B shows the level of CD8+ The level of T cell response. Figure 14C shows CD8 production of IL-2 + The level of T cell response. Figure 14D shows CD4 production of IFNγ + The level of T cell response. Figure 14E shows CD4 production of TNFα + The level of T cell response. Figure 14F shows CD4 production of IL-2 + The level of T cell response.

[0048] Figure 15 includes Figures 15A to 15F, providing exemplary experimental data showing the levels of CD4 + and CD8 + T cell responses to cytokine production after inoculation of CD-1 outbred mice with STAg peptide and stimulation for 5 hours. Figure 15A shows CD8 production of IFNγ + The level of T cell response. Figure 15B shows CD8 production of TNFα + The level of T cell response. Figure 15C shows CD8 production of IL-2 + The level of T cell response. Figure 15D shows CD4 production of IFNγ + The level of T cell response. Figure 15E shows CD4 production of TNFα + The level of T cell response. Figure 15F shows CD4 production of IL-2 + The level of T cell response. Detailed Description

[0049] Merkel cell polyomavirus (MCV) infection is associated with Merkel cell carcinoma (MCC), and currently its mortality rate is 46%.

[0050] In one embodiment, the present invention includes a nucleic acid vaccine against MCV and MCC. In one embodiment, the vaccine comprises a plasmid encoding a consensus MCV T antigen. In one embodiment, the consensus MCV T antigen is large T antigen (LTAg). In one embodiment, the consensus MCV T antigen is small t antigen (STAg). In one embodiment, the consensus MCV T antigen further comprises a mutation that disrupts the oncogenic characteristics of the native T antigen. As a candidate vaccine, the enhanced DNA (DNA)-based platform has many advantages in terms of genetic optimization and delivery technology. Thus, each MCV T antigen can be genetically optimized, subcloned into a modified mammalian expression vector, and then delivered using in vivo electroporation (EP).

[0051] Vaccination in preclinical rodent studies is very effective because inoculation with a synthetic consensus MCV T antigen construct can generate a robust immune response.

[0052] In some embodiments, the strategy employs a synthetic consensus coding sequence of the MCV T antigen. Coding sequences for LTAg and STAg are provided. In some embodiments, the strategy employs a single synthetic consensus coding sequence of the MCV T antigen. In some embodiments, the strategy employs multiple synthetic consensus coding sequences of the MCV T antigen.

[0053] As a candidate vaccine, DNA vaccines exhibit multiple advantages, including rapid and low-cost large-scale production, stability at room temperature, and ease of transportation, all of which further enhance this platform from an economic and geographical perspective. Due to the synthetic nature of the plasmid, the antigen sequence can be rapidly and easily modified to respond to emerging strains and / or expanded to include other vaccine components.

[0054] Optimization of plasmid DNA vectors and their encoded antigen genes results in increased in vivo immunogenicity. When a high concentration of plasmid vaccine formulation is administered together with in vivo electroporation, cellular uptake and subsequent antigen expression will be greatly enhanced. This technique utilizes short square-wave electrical pulses at the vaccination site to drive the plasmid into transiently permeabilized cells. In theory, DNA plasmid mixtures can be assembled to direct a highly specific immune response to any number of variable antigens. Immunity can be further directed by co-delivery with plasmid molecular adjuvants encoding species-specific cytokine genes and "consensus engineering" of the antigen amino acid sequence to help bias vaccine-induced immunity towards specific strains.

[0055] Definitions.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0057] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)” and variations thereof are intended to be open transitional phrases, terms or words that do not preclude additional modalities or structural possibilities. Unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” include the plural forms. The present disclosure also contemplates other embodiments “including”, “consisting of” and “consisting essentially of”, whether or not explicitly recited, of the embodiments or elements shown herein.

[0058] As used herein, “adjuvant” can refer to any molecule added to a nucleic acid vaccine to enhance the antigenicity of the vaccine.

[0059] “Antibody” can refer to antibodies of the IgG, IgM, IgA, IgD or IgE classes, or fragments, or fragments or derivatives thereof, including Fab, F(ab')2, Fd and single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. Antibodies can be antibodies isolated from mammalian serum samples, polyclonal antibodies, affinity-purified antibodies or mixtures thereof that have sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0060] “Antibody fragment” or “fragment of an antibody” as used interchangeably herein refers to a portion of a full antibody that contains an antigen-binding site or variable region. This portion does not contain the constant heavy chain domains of the full antibody Fc region (i.e., CH2, CH3 or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing one light chain variable domain, single-chain polypeptides containing the three CDRs of the light chain variable binding domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0061] “Antigen” refers to a protein capable of eliciting an immune response in a host. Antigens can be recognized and bound by antibodies. Antigens can originate from within the body or the external environment.

[0062] As used herein, "coding sequence" or "coding nucleic acid" can refer to a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that encodes a protein. The coding sequence can further include start and stop signals operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can optionally further contain a start codon encoding an N-terminal methionine or a signal peptide (such as an IgE or IgG signal peptide).

[0063] As used herein, "complementary" or "complementary to" can mean a nucleic acid, and can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between the nucleotides or nucleotide analogs of a nucleic acid molecule.

[0064] As used herein, "consensus" or "consensus sequence" can mean a synthetic nucleotide sequence or the corresponding polypeptide sequence constructed based on the alignment analysis of multiple sequences (e.g., multiple sequences of a specific viral antigen).

[0065] As used herein, "constant current" is used to define the current received or experienced by a tissue or the cells defining the tissue during the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from the electroporation device described herein. Because the electroporation device provided herein has a feedback element, preferably with instantaneous feedback, the current remains constant in amperage in the tissue throughout the electrical pulse. The feedback element can measure the resistance of the tissue (or cells) throughout the pulse duration and cause the electroporation device to change its electrical energy output (e.g., increase the voltage), so that the current in the same tissue remains constant (at the microsecond level) throughout the electrical pulse and between pulses. In some embodiments, the feedback element includes a controller.

[0066] As used herein, "current feedback" or "feedback" can be used interchangeably and can mean the active response of the provided electroporation device, which includes measuring the current in the tissue between the electrodes and correspondingly changing the energy output delivered by the EP device to maintain a constant current. This constant level is preset by the user before the start of the pulse sequence or electrotherapy. This feedback can be achieved by the electroporation assembly (e.g., the controller) of the electroporation device because the circuit therein can continuously monitor the current in the tissue between the electrodes and compare the monitored current (or the current within the tissue) with the preset current, and continuously adjust the energy output to maintain the monitored current at the preset level. The feedback loop can be instantaneous because it is an analog closed-loop feedback.

[0067] As used herein, "dispersed current" may refer to the pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein, where the pattern minimizes or preferably eliminates electroporation occurrences associated with electroporation that occur on any region of the tissue.

[0068] As used interchangeably herein, "electrochemicalization", "electroporation", or "electroporation enhancement" ("EP") may refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence enables biomolecules (such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water) to cross from one side of the cell membrane to the other.

[0069] As used herein, "endogenous antibody" may refer to an antibody produced in a subject that has been administered an effective dose of an antigen to induce a humoral immune response.

[0070] As used herein, "feedback mechanism" may refer to a process performed by software or hardware (or firmware) that receives and compares the impedance of the expected tissue (before, during, and / or after energy pulse delivery) with the current value (preferably current) and adjusts the delivered energy pulse to reach a preset value. The feedback mechanism may be implemented by an analog closed-loop circuit.

[0071] "Fragment" may mean a percentage of a full-length polypeptide sequence or nucleotide sequence. A fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the full length of the parental nucleotide sequence or amino acid sequence or a variant thereof.

[0072] As used herein, "gene construct" refers to a DNA or RNA molecule that contains a nucleotide sequence encoding a protein (such as an antibody). A genetic construct may also refer to a DNA molecule that transcribes RNA. The coding sequence includes start and stop signals operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present in the cells of an individual.

[0073] As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" can mean that the sequences have a specified percentage of residues that are the same in a specified region. The sequence identity percentage can be determined by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residues occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and then multiplying the result by 100. If the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using computerized sequence algorithms such as BLAST or BLAST 2.0.

[0074] When discussing feedback mechanisms, the "impedance" used herein can be employed and can be converted to a current value according to Ohm's law so that it can be compared with a preset current.

[0075] As used herein, "immune response" can mean activation of the immune system (e.g., the immune system of a mammal) of a host in response to the introduction of one or more common antigens by a provided vaccine. The immune response can be in the form of a cellular response or a humoral response or both.

[0076] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently linked together. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the single strand. Many variants of a nucleic acid can be used for the same purpose as the established nucleic acid. Thus, a nucleic acid also encompasses nucleic acids that are substantially identical thereto and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0077] A nucleic acid can be single-stranded or double-stranded, or can contain portions of double-stranded and single-stranded sequences. A nucleic acid can be DNA (genomic and cDNA), RNA, or a hybrid, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. A nucleic acid can be obtained by chemical synthesis methods or recombinant methods.

[0078] As used herein, "operably linked" can mean that gene expression is controlled by a promoter that is spatially linked thereto. The promoter can be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be substantially the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0079] As used herein, "peptide", "protein" or "polypeptide" can mean a linked sequence of amino acids and can be natural, synthetic consensus or natural and synthetic modifications or combinations.

[0080] As used herein, "promoter" can mean a synthetic or natural source molecule capable of conferring, activating or enhancing nucleic acid expression in a cell. The promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. The promoter can also contain distal enhancer or repressor elements that can be located up to thousands of base pairs away from the transcription start site. Promoters can be from sources including viruses, bacteria, fungi, plants, insects and animals. The promoter can constitutively or differentially regulate the expression of gene components in response to the cell, tissue or organ in which expression occurs, or in response to the developmental stage at which expression occurs, or to external stimuli such as physiological stress, pathogens, metal ions or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0081] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of the proteins described herein. The signal peptide / leader sequence generally directs the localization of the protein. The signal peptide / leader sequence used herein preferably promotes the secretion of the protein from the cell in which it is produced. The signal peptide / leader sequence is generally cleaved from the remainder of the protein (commonly referred to as the mature protein) after secretion from the cell. The signal peptide / leader sequence is linked to the N-terminus of the protein.

[0082] As used herein, "stringent hybridization conditions" can mean conditions under which a first nucleic acid molecule (e.g., a probe) will hybridize to a second nucleic acid molecule (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary in different circumstances. Stringent conditions can be selected to be about 5-10 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm can be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence in equilibrium (due to the excess presence of the target sequence, at the Tm, 50% of the probes are in equilibrium) (at a defined ionic strength, pH, and nucleic acid concentration). Stringent conditions can be conditions in which the salt concentration is less than about 1.0 M sodium ion, such as at pH 7.0 to 8.3, for short probes (e.g., about 10-50 nucleotides) at at least about 30 °C, and for long probes (e.g., greater than about 50 nucleotides) at at least 60 °C, for a sodium ion concentration (or other salt) of about 0.01-1.0 M. Stringent conditions can also be achieved by adding destabilizers such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times that of background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42 °C, or 5x SSC, 1% SDS, incubated at 65 °C, and washed in 0.2x SSC and 0.1% SDS at 65 °C.

[0083] As used herein, "subject" and "patient" are used interchangeably to refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys, such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human.

[0084] As used herein, "substantially complementary" can mean that the complementary sequence of a first sequence has at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids with a second sequence, or the two sequences hybridize under stringent hybridization conditions.

[0085] As used herein, "substantially identical" can mean that the first sequence and the second sequence have at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in a region of 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides or amino acids, or for nucleic acids, if the first sequence is substantially complementary to the complementary sequence of the second sequence.

[0086] As used herein, "treatment" or "treating" means protecting a subject from a disease by preventing, inhibiting, suppressing or completely eliminating the disease. Preventing a disease includes administering a vaccine of the present invention to a subject before the onset of the disease. Inhibiting a disease includes administering a vaccine of the present invention to a subject after induction of the disease but before its clinical appearance. Suppressing a disease includes administering a vaccine of the present invention to a subject after the clinical appearance of the disease.

[0087] As used herein, a "variant" with respect to a nucleic acid can mean (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of the reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the reference nucleic acid or its complementary sequence; (iv) a nucleic acid that hybridizes to the reference nucleic acid, its complementary sequence or a sequence substantially identical thereto under stringent conditions.

[0088] "Variant" refers to a peptide or polypeptide whose amino acid sequence differs by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein having an amino acid sequence substantially the same as the reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor changes. As understood in the art, these minor changes can be partially identified by considering the hydrophilicity index of the amino acid. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices are known in the art to be interchangeable and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids and substitutions that will result in a protein retaining biological function can be demonstrated. Considering the hydrophilicity of amino acids in the peptide context, the maximum local average hydrophilicity of the peptide can be calculated, which is a useful method and has been reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in a peptide retaining biological activity, e.g., immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values are within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions compatible with biological function should be understood to depend on the relative similarity of the amino acids, particularly those amino acid side chains as indicated by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0089] A variant can be a nucleotide sequence that is substantially identical to the full length of the complete gene sequence or a fragment thereof. The nucleotide sequence can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical to the full length of the amino acid sequence or a fragment thereof. The amino acid sequence can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the full length of the amino acid sequence or a fragment thereof.

[0090] As used herein, "vector" can mean a nucleic acid molecule containing an origin of replication. The vector can be a plasmid, phage, bacterial artificial chromosome or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be an extrachromosomal vector that self-replicates or a vector integrated into the host genome.

[0091] In order to enumerate numerical ranges herein, every intermediate number having the same precision between them is clearly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated; for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are clearly contemplated.

[0092] Description

[0093] The present invention provides an optimized consensus sequence encoding the MCV T antigen. In one embodiment, the MCV T antigen encoded by the optimized consensus sequence is capable of eliciting an immune response in a mammal. In one embodiment, the MCV T antigen encoded by the optimized consensus sequence may comprise one or more epitopes, making it particularly effective as an immunogen capable of inducing an immune response thereto.

[0094] The optimized consensus sequence can be a consensus sequence derived from two or more MCV T antigens. The optimized consensus sequence can comprise a consensus sequence and / or modifications for improving expression. The modifications can include codon optimization, RNA optimization, addition of a kozak sequence to enhance translation initiation, and / or addition of an immunoglobulin leader sequence to enhance immunogenicity. The MCV T antigen encoded by the optimized consensus sequence can comprise a signal peptide, such as an immunoglobulin signal peptide, for example but not limited to immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the antigen encoded by the optimized consensus sequence can comprise a hemagglutinin (HA) tag. The antigen encoded by the optimized consensus sequence can be designed to elicit a stronger cellular and / or humoral immune response than the corresponding non-optimized antigen.

[0095] The present invention provides an MCV T antigen, which can be used to induce immunity to MCV in genetically diverse subjects suffering from MCV infection. In one embodiment, the present invention provides an immunogenic composition comprising one or more nucleic acid molecules capable of generating an immune response to the MCV T antigen in a mammal. The present invention also provides an isolated nucleic acid molecule capable of generating an immune response to the MCV T antigen in a mammal. In one embodiment, the nucleic acid molecule comprises an optimized nucleotide sequence encoding a consensus MCV T antigen.

[0096] In one embodiment, the MCV T antigen is modified to reduce or disrupt at least one oncogenic feature of the native MCV T antigen. In various embodiments, the MCV T antigen is modified to reduce or disrupt at least one of CR1 binding, DnaJ binding, binding to phosphatase pp2A, Rb binding, ATPase activity, helicase activity, chaperone binding, hVam6p binding, Fbxw7 binding, origin binding, and transformation. In one embodiment, the MCV T antigen comprises at least one mutation at D44, W209, E216, L142, L91, K92, D93, Y94, or M95 relative to the native T antigen sequence. In one embodiment, the MCV T antigen comprises at least one of the D44N mutation, W209A, E216K mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation, and M95A mutation. In one embodiment, the MCV LTAg comprises at least one of the D44N mutation, W209A, and E216K mutation. In one embodiment, the MCV LTAg comprises the D44N mutation, W209A, and E216K mutation. In one embodiment, the MCV STAg comprises at least one of the D44N mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation, and M95A mutation. In one embodiment, the MCV STAg comprises the D44N mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation, and M95A mutation.

[0097] The consensus amino acid sequence of the MCV T antigen includes SEQ ID NO:2, SEQ ID NO:4, and their variants, as well as fragments of SEQ ID NO:2, SEQ ID NO:4, and their variants. An exemplary amino acid sequence of the modified synthetic consensus MCV LTAg is provided as SEQ ID NO:2. An exemplary amino acid sequence of the modified synthetic consensus MCV STAg is provided as SEQ ID NO:2.

[0098] In one embodiment, the present invention provides a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a modified synthetic consensus MCV T antigen. In one embodiment, the nucleotide sequence encoding the modified synthetic consensus MCV LTAg is provided as SEQ ID NO:1, which encodes SEQ ID NO:2. In one embodiment, the nucleotide sequence encoding the modified synthetic consensus MCV STAg is provided as SEQ ID NO:3, which encodes SEQ ID NO:4.

[0099] In multiple embodiments, the present invention provides compositions comprising a combination of a modified LTAg and a modified STAg or one or more nucleic acid molecules encoding them. The compositions can comprise multiple copies of a single nucleic acid molecule, such as a single plasmid, or two copies of two or more different nucleic acid molecules, such as two or more different plasmids.

[0100] The composition can comprise a single nucleic acid molecule, such as a plasmid, which contains multiple coding sequences of the consensus MCV T antigen. In one embodiment, the composition can comprise a single nucleic acid molecule which contains nucleotide sequences encoding the MCV LTAg and the MCV STAg. In one embodiment, each coding sequence of each consensus MCV T antigen is on a separate plasmid.

[0101] Thus, a composition comprising one or more nucleotide sequences encoding multiple consensus MCV T antigens can be on a single plasmid. In one embodiment, the composition comprises a single plasmid encoding the MCV LTAg and the MCV STAg under a single promoter. In such an embodiment, the sequence encoding the MCV LTAg and the sequence encoding the MCV STAg can be linked by a fusion peptide sequence (e.g., a furin cleavage sequence). An exemplary amino acid sequence of a single construct comprising a modified synthetic consensus MCV LTAg and MCV STAg linked by a furin cleavage site is provided as SEQ ID NO:6. In one embodiment, the single nucleotide sequence encoding the modified synthetic consensus MCV LTAg and MCV STAg linked by a furin cleavage sequence is provided as SEQ ID NO:5, which encodes SEQ ID NO:6.

[0102] In one embodiment, the MCV T antigen encoded by an optimized consensus sequence is operably linked to one or more regulatory elements. In one embodiment, the regulatory element is a leader sequence. In one embodiment, the leader sequence is an IgE leader sequence. In one embodiment, the IgE leader sequence has the amino acid sequence shown in SEQ ID NO:7. Thus, in one embodiment, the present invention relates to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, which is operably linked to the amino acid sequence shown in SEQ ID NO:7. In one embodiment, the present invention relates to a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, which is operably linked to the amino acid sequence shown in SEQ ID NO:7.

[0103] In one embodiment, the regulatory element is a start codon. Thus, in one embodiment, the present invention relates to a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or a fragment or homolog thereof, which is operably linked to a nucleotide sequence containing a start codon at the 5'-end. In one embodiment, the present invention relates to an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a fragment or homolog thereof, which is operably linked to an amino acid encoded by a start codon (e.g., methionine) at the N-terminus.

[0104] In one embodiment, the regulatory element is at least one stop codon. Thus, in one embodiment, the present invention relates to a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or a fragment or homolog thereof, which is operably linked to a nucleotide sequence containing at least one stop codon at the 3'-end. In one embodiment, the nucleotide sequence is operably linked to two stop codons to increase the efficiency of translation termination.

[0105] In one embodiment, the nucleic acid molecule can encode a peptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In one embodiment, the nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the sequence can have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. In other embodiments, the sequence can be a nucleotide sequence encoding the amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0106] In some embodiments, the nucleic acid molecule comprises an RNA sequence that is a transcript of a DNA sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length of the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the nucleic acid molecule comprises an RNA sequence encoding an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0107] In some embodiments, the nucleic acid molecule may comprise a nucleotide sequence encoding a full-length consensus MCV T antigen. The nucleic acid molecule may comprise a sequence encoding SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. The nucleic acid molecule may comprise the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. The nucleic acid molecule may optionally comprise a coding sequence encoding a signal peptide (such as, for example, an IgE or IgG signal peptide).

[0108] The consensus MCV T antigen may be a peptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In some embodiments, the antigen may have an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0109] An immunogenic fragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 can be provided. The immunogenic fragment can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In some embodiments, the immunogenic fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the immunogenic fragment does not contain a leader sequence.

[0110] An immunogenic fragment of a protein having an amino acid sequence homologous to the immunogenic fragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 can be provided. Such immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein having 95% homology to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic fragments having 96% homology to the immunogenic fragments of the protein sequences common herein. Some embodiments relate to immunogenic fragments having 97% homology to the immunogenic fragments of the protein sequences common herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the protein sequences common herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the protein sequences common herein. In some embodiments, the immunogenic fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the immunogenic fragment does not contain a leader sequence.

[0111] In one embodiment, the immunogenic fragment of the nucleic acid molecule encodes at least one immunodominant or sub-immunodominant epitope of the full-length optimized common MCV T antigen.

[0112] Some embodiments relate to immunogenic fragments of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, which comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. The immunogenic fragment may have at least 96%, at least 97%, at least 98%, or at least 99% homology with a fragment of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In some embodiments, the immunogenic fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain the coding sequence encoding the leader sequence.

[0113] In one embodiment, the nucleic acid molecule comprises a sequence having at least 90% homology with SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0114] In one embodiment, the nucleic acid molecule comprises an RNA sequence encoding the consensus MCV T antigen sequence described herein. For example, the nucleic acid may comprise an RNA sequence encoding one or more of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, variants thereof, fragments thereof, or any combination thereof.

[0115] In some embodiments, the nucleic acid molecule includes a sequence encoding the MCV T antigen, minus the IgE leader sequence at the N-terminus of the coding sequence. In some embodiments, the DNA nucleic acid molecule further comprises an IgE leader sequence attached to the N-terminus of the coding sequence and operably linked to a promoter.

[0116] The nucleic acid molecule may further include a polyadenylation sequence attached to the C-terminus of the coding sequence. In one embodiment, the nucleic acid molecule is codon-optimized.

[0117] Vaccines and immunogenic compositions

[0118] There are provided immunogenic compositions (such as vaccines) comprising an optimized consensus sequence, an optimized consensus-encoded antigen, fragments thereof, variants thereof, or combinations thereof. The immunogenic composition can significantly induce an immune response against the MCV T antigen in a subject to whom the immunogenic composition is administered. The vaccine may comprise a plurality of nucleic acid molecules or combinations thereof. The vaccine can be provided to induce a therapeutic or prophylactic immune response.

[0119] An immunogenic composition can be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination vaccine. The vaccine can comprise an optimized consensus nucleotide sequence encoding an antigen. The nucleotide sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleotide sequence can further include other sequences encoding a linker, a leader sequence, or a tag sequence that is linked to the antigen by a peptide bond. The peptide vaccine can include the antigen, a variant thereof, a fragment thereof, or a combination thereof. The combination of the DNA and peptide vaccines can include the above-mentioned optimized consensus nucleotide sequence and the encoded antigen.

[0120] The vaccine can be a DNA vaccine. DNA vaccines are disclosed in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are hereby incorporated by reference in their entirety. The DNA vaccine can further comprise an element or reagent that inhibits its integration into the chromosome.

[0121] The vaccine can be RNA of one or more MCV T antigens. The RNA vaccine can be introduced into cells.

[0122] The vaccine can be a live attenuated vaccine, a vaccine using a recombinant vector to deliver an antigen, a subunit vaccine, and a glycoprotein vaccine, such as but not limited to U.S. Patent Nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0123] The vaccine of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause disease or death; preventing diseases; inducing a protective T cell response; and providing characteristics of being easy to administer, having few side effects, biological stability, and low cost per dose.

[0124] The present disclosure provides immunogenic compositions capable of generating an immune response against MCV in mammals. The immunogenic compositions can comprise each plasmid as described above. The immunogenic compositions can comprise multiple plasmids, or combinations thereof. The immunogenic compositions can be provided to induce a therapeutic or prophylactic immune response.

[0125] The immunogenic compositions can be used to deliver nucleic acid molecules encoding one or more consensus MCV T antigens. The immunogenic compositions are preferably compositions comprising plasmids.

[0126] The immunogenic compositions can further comprise pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients can be functional molecules such as solvents, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipients can be transfection facilitators, which can include surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, vesicles (such as squalene and squalene), hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitators.

[0127] The transfection facilitator is a polyanion, polycation, including poly-L-glutamate (LGS) or lipid. The transfection facilitator is poly-L-glutamic acid, and more preferably, poly-L-glutamic acid is present in the immunogenic composition at a concentration less than 6 mg / ml. The transfection facilitator can also include surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, and vesicles (such as squalene and squalene), hyaluronic acid, and can be co-administered with the gene construct. In some embodiments, the immunogenic composition can further comprise a transfection facilitator such as a lipid, liposome (including lecithin liposomes or other liposomes known in the art, such as DNA-liposome mixtures (see, e.g., W09324640)), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitators. Preferably, the transfection facilitator is a polyanion, polycation, including poly-L-glutamate (LGS) or lipid. The concentration of the transfection agent in the immunogenic composition is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0128] A pharmaceutically acceptable excipient can be one or more adjuvants. The adjuvant can be other genes expressed from the same or alternative plasmids, or other genes combined with the above-mentioned plasmids in the immunogenic composition for protein delivery. The one or more adjuvants can be proteins and / or nucleic acid molecules encoding proteins selected from the group consisting of: CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine in epithelium (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15 (including IL-15 with a signal sequence or a coding sequence encoding a deleted signal sequence, and optionally including a different signal peptide (such as from IgE) or a coding sequence encoding a different signal peptide (such as from IgE)), IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-lα, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof, or combinations thereof.

[0129] In some embodiments, the adjuvant can be one or more proteins and / or nucleic acid molecules encoding proteins selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, or RANTES. Examples of IL-12 constructs and sequences are disclosed in PCT application number PCT / US1997 / 019502 and corresponding US application serial number 08 / 956,865, and US provisional application serial number 61 / 569600 filed on December 12, 2011, each of which is incorporated herein by reference. Examples of IL-15 constructs and sequences are disclosed in PCT application number PCT / US04 / 18962 and corresponding US application serial number 10 / 560,650, and PCT application number PCT / US07 / 00886 and corresponding US application serial number 12 / 160,766, and PCT application number PCT / US10 / 048827, each of which is incorporated herein by reference. Examples of IL-28 constructs and sequences are disclosed in PCT application number PCT / US09 / 039648 and corresponding US application serial number 12 / 936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT application number PCT / US1999 / 004332 and corresponding US application serial number 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT application number PCT / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT application number PCT / US1999 / 004332 and corresponding US application serial number 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT application number PCT / US11 / 024098, which is incorporated herein by reference. Examples of chemokine CTACK, TECK, and MEC constructs and sequences are disclosed in PCT application number PCT / US2005 / 042231 and corresponding US application serial number 11 / 719,646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulators are disclosed in US application serial number 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in US application serial number 09 / 622452, which is incorporated herein by reference.

[0130] The immunogenic composition can further comprise a gene vaccine promoter, as described in US serial number 021,579 filed on April 1, 1994, which is incorporated herein by reference in its entirety.

[0131] The immunogenic composition may comprise an amount of the common antigen and plasmid of from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; or preferably from about 0.1 microgram to about 10 milligrams; or more preferably from about 1 milligram to about 2 milligrams. In some preferred embodiments, the pharmaceutical composition according to the invention comprises from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises from about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises from about 25 to about 250 micrograms, from about 100 to about 200 micrograms, from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; from about 0.1 microgram to about 10 milligrams; from about 1 milligram to about 2 milligrams, from about 5 nanograms to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 micrograms of the common antigen or its plasmid.

[0132] In some embodiments, the pharmaceutical composition according to the present invention comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms of the nucleic acid molecule of the present invention. In some embodiments, the pharmaceutical composition may comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of the nucleic acid molecule of the present invention. In some embodiments, the pharmaceutical composition may comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg or more of the nucleic acid molecule of the present invention.

[0133] In other embodiments, the pharmaceutical composition may comprise up to (including) 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms of the nucleic acid molecule of the present invention. In some embodiments, the pharmaceutical composition may comprise up to (including) 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of the nucleic acid molecule of the present invention. In some embodiments, the pharmaceutical composition may comprise up to (including) 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg of the nucleic acid molecule of the present invention.

[0134] An immunogenic composition can be formulated according to the mode of administration used. An injectable immunogenic composition pharmaceutical composition can be sterile, pyrogen-free, and particulate-free. Isotonic preparations or solutions can be used. Isotonic additives can include sodium chloride, glucose, mannitol, sorbitol, and lactose. The immunogenic composition can contain a vasoconstrictor. Isotonic solutions can include phosphate buffer. The immunogenic composition can further contain stabilizers, including gelatin and albumin. Stabilization can render the formulation stable for an extended period at room temperature or ambient temperature, such as LGS or polycations or polyanions for immunogenic composition formulations.

[0135] The immunogenic composition can be stable for more than 1 week at room temperature (25 °C), in some embodiments more than 2 weeks, in some embodiments more than 3 weeks, in some embodiments more than 4 weeks, in some embodiments more than 5 weeks, and in some embodiments more than 6 weeks. In some embodiments, the vaccine is stable for more than one month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months. In some embodiments, the vaccine is stable for more than 1 year, more than 2 years, more than several years, or more than 5 years. In one embodiment, the immunogenic composition is stable upon refrigeration (2 - 8 °C). Thus, in one embodiment, the immunogenic composition does not require a frozen cold chain. An immunogenic composition is stable if it retains its biological activity for a sufficient length of time to permit its intended use (e.g., to generate an immune response in a subject). For example, for an immunogenic composition that is to be stored, transported, etc., it may be desirable for the immunogenic composition to remain stable for months to years.

[0136] Immune response

[0137] An immunogenic composition can induce an immune response in a subject to which the composition is administered. The induced immune response can be specific for the MCV T antigen. The induced immune response can react with the MCV T antigen associated with an optimized consensus-encoded antigen. In various embodiments, the related antigen includes an antigen having an amino acid sequence that has 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 100% homology to the amino acid sequence of the optimized consensus-encoded antigen. In various embodiments, the related antigen includes an antigen encoded by a nucleotide sequence that has 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 100% homology to the optimized consensus nucleotide sequence disclosed herein.

[0138] An immunogenic composition can induce a humoral immune response in a subject to which the immunogenic composition is administered. The induced humoral immune response can be specific for the MCV T antigen. The induced humoral immune response can react with the MCV T antigen associated with an optimized consensus-encoded antigen. A humoral immune response of about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold can be induced in a subject to which the immunogenic composition is administered. As compared to a subject not administered the immunogenic composition or a subject administered an unoptimized MCV T antigen, a humoral immune response of at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold can be induced in a subject to which the immunogenic composition is administered.

[0139] Compared to subjects who have not received the immunogenic composition, the humoral immune response induced by the immunogenic composition can include an increase in the level of IgG antibodies associated with the subjects who have received the immunogenic composition. These IgG antibodies can be specific for the MCV T antigen genetically related to the optimized consensus antigen. These IgG antibodies can react with the MCV T antigen genetically related to the optimized consensus antigen. Compared to subjects who have not received the immunogenic composition, the level of IgG antibodies associated with the subjects who have received the immunogenic composition can increase by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. Compared to subjects who have not received the immunogenic composition or subjects who have received an unoptimized MCV T antigen, the level of IgG antibodies associated with the subjects who have received the immunogenic composition can increase by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold or at least about 16.0-fold.

[0140] The immunogenic composition can induce a cellular immune response in the subjects who have received the immunogenic composition. The induced cellular immune response can be specific for the MCV T antigen related to the optimized consensus coding antigen. The induced cellular immune response can react with the MCV T antigen related to the optimized consensus coding antigen. The induced cellular immune response can include eliciting a CD8 + T cell response. The elicited CD8 + T cell response can react with the MCV T antigen genetically related to the optimized consensus antigen. The elicited CD8 + T cell response can be multifunctional. The induced cellular immune response can include eliciting a CD8 + T cell response, wherein the CD8 + cells produce interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 2 (IL-2) or a combination of IFN-γ and TNF-α.

[0141] Compared to subjects who have not received the immunogenic composition, the induced cellular immune response can include CD8 +The T cell response is increased. The CD8 T cell response associated with subjects administered an immunogenic composition can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold compared to subjects not administered the immunogenic composition. The CD8 T cell response associated with subjects administered an immunogenic composition can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold compared to subjects not administered the immunogenic composition or subjects administered an unoptimized MCV T antigen. + The CD8 T cell response can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold. The CD8 T cell response can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold compared to subjects not administered the immunogenic composition or subjects administered an unoptimized MCV T antigen. + The CD8 T cell response can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold compared to subjects not administered the immunogenic composition or subjects administered an unoptimized MCV T antigen.

[0142] The induced cellular immune response can include an increase in the frequency of CD107a / IFNγ / T-bet triple-positive CD8 T cells reactive to the MCV T antigen. The frequency of CD107a / IFNγ / T-bet triple-positive CD8 T cells associated with subjects administered an immunogenic composition can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold compared to subjects not administered the immunogenic composition or subjects administered an unoptimized MCV T antigen.

[0143] The induced cellular immune response can include an increase in the frequency of CD107a / IFNγ double-positive CD8 T cells reactive to the MCV T antigen. The frequency of CD107a / IFNγ double-positive CD8 T cells associated with subjects administered an immunogenic composition can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold compared to subjects not administered the immunogenic composition or subjects administered an unoptimized MCV T antigen.

[0144] The cellular immune responses induced by the immunogenic composition can include eliciting CD4 + T cell responses. The elicited CD4 + cell responses can react with the MCV T antigen genetically related to the optimized consensus antigen. The elicited CD4 + T cell responses can be multifunctional. The induced cellular immune responses can include eliciting CD4 + T cell responses, wherein the CD4 + T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0145] The induced cellular immune responses can include an increased frequency of CD4 + T cells that produce IFN-γ. Compared with subjects who have not received the immunogenic composition or subjects who have received an unoptimized MCV T antigen, the frequency of CD4 + IFN-γ + T cells associated with subjects who have received the immunogenic composition can increase by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0146] The induced cellular immune responses can include an increased frequency of CD4 + T cells that produce TNF-α. Compared with subjects who have not received the immunogenic composition or subjects who have received an unoptimized MCV T antigen, the frequency of CD4 + TNF-α + T cells associated with subjects who have received the immunogenic composition can increase by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22-fold.

[0147] The induced cellular immune responses can include an increased frequency of CD4 + T cells that produce IFN-γ and TNF-α. Compared with subjects who have not received the immunogenic composition or subjects who have received an unoptimized MCV T antigen, the frequency of CD4 + IFN-γ + TNF-α +The frequency can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold or 35-fold.

[0148] When administered to different tissues (such as muscle or skin), the immunogenic composition can further induce an immune response. When administered by electroporation or injection, or subcutaneously or intramuscularly, the immunogenic composition can further induce an immune response.

[0149] vector

[0150] The above nucleotide constructs can be placed in one or more vectors. One or more vectors can contain an origin of replication. One or more vectors can be plasmids, phages, bacterial artificial chromosomes or yeast artificial chromosomes. One or more vectors can be extrachromosomal self-replicating vectors or vectors integrated into the host genome.

[0151] Vectors include but are not limited to plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vectors, etc. A "vector" contains nucleic acids that can infect, transfect, transiently or permanently transduce cells. It will be appreciated that a vector can be naked nucleic acid or nucleic acid complexed with protein or lipid. Optionally, the vector contains viral or bacterial nucleic acids and / or proteins, and / or membranes (e.g., cell membranes, viral lipid envelopes, etc.). Vectors include but are not limited to replicons (e.g., RNA replicons, phages) into which DNA fragments can be ligated and replicated. Thus, vectors include but are not limited to RNA, autonomously self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Patent No. 5,217,879), and include both expressing and non-expressing plasmids. When a recombinant microorganism or cell culture is described as a host "expression vector", it includes both extrachromosomal circular and linear DNA and DNA incorporated into the host chromosome. In cases where the host cell maintains the vector, the vector can be stably replicated by the cell as an autonomous structure during mitosis or incorporated into the genome of the host.

[0152] One or more vectors can be expression constructs, which are typically plasmids used to introduce a specific gene into a target cell. Once the expression vector enters the interior of the cell, the protein encoded by the gene is produced by the ribosomal complex of the cell's transcription and translation machinery. Plasmids are typically engineered to contain regulatory sequences that act as enhancer and promoter regions and result in efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and thus express protein.

[0153] Vectors can have expression signals (such as strong promoters, strong stop codons), regulate the distance between the promoter and the cloned gene, and the insertion of transcription termination sequences and PTIS (simple translation initiation sequences).

[0154] Expression vector

[0155] One or more vectors can be circular plasmids or linear nucleic acids. Circular plasmids and linear nucleic acids are capable of directing the expression of a specific nucleotide sequence in a suitable subject cell. One or more vectors containing a recombinant nucleic acid construct can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.

[0156] Plasmid

[0157] One or more vectors can be plasmids. Plasmids can be used to transfect cells with a recombinant nucleic acid construct. Plasmids can be used to introduce a recombinant nucleic acid construct into a subject. Plasmids can also contain regulatory sequences that can be well-suited for expressing a gene in the cells to which the plasmid is administered.

[0158] Plasmids can also contain an origin of replication for mammals to maintain the plasmid episomally and produce multiple copies of the plasmid in the cell. Plasmids can be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain an origin of replication of Epstein Barr virus and the nuclear antigen EBNA-1 coding region, which can produce high-copy episomal replication without integration. The backbone of the plasmid can be pAV0242. Plasmids can be replication-deficient type 5 adenovirus (Ad5) plasmids.

[0159] The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in Escherichia coli (E. coli). The plasmid can be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in Saccharomyces cerevisiae yeast strains. The plasmid can also be the MAXBACTM Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which can be used to produce proteins in insect cells. The plasmid can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in mammalian cells (such as Chinese hamster ovary (CHO) cells).

[0160] RNA

[0161] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from the DNA sequences described herein. For example, in some embodiments, the RNA molecule is encoded by a DNA sequence having at least 90% homology to one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a variant or fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence encoding a polypeptide sequence having at least 90% homology to one of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or a variant or fragment thereof. Thus, in one embodiment, the present invention provides an RNA molecule encoding one or more MCV T antigens. The RNA can be positive-stranded. Thus, in some embodiments, the RNA molecule can be translated by cells without any intermediate replication step, such as reverse transcription. The RNA molecules useful in the present invention can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of the RNA molecules useful in the present invention can have a 5' triphosphate group. In a capped RNA, it can be linked to 7-methylguanosine by a 5' to 5' bridge. The RNA molecule can have a 3' poly-A tail. It can also contain a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecules useful in the present invention can be single-stranded. The RNA molecules useful in the present invention can comprise synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained within a carrier.

[0162] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the length of the 5' UTR is between 0 and 3000 nucleotides. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including but not limited to, designing PCR primers for annealing to different regions of the UTR. Using this method, one of ordinary skill in the art can modify the lengths of the 5' and 3' UTRs required for optimal translation efficiency after transfection of the transcribed RNA.

[0163] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Optionally, a UTR sequence can be added by incorporating the UTR sequence into the forward and reverse primers or by any other modification of the template to add a UTR sequence that is not endogenous to the gene of interest. Using a UTR sequence that is not endogenous to the gene of interest can be used to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence reduce the stability of the RNA. Thus, based on the properties of UTRs well known in the art, a 3' UTR can be selected or designed to increase the stability of the transcribed RNA.

[0164] In one embodiment, the 5' UTR can contain the Kozak sequence of an endogenous gene. Optionally, when adding a 5' UTR that is not endogenous to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. The Kozak sequence can enhance the translation efficiency of certain RNA transcripts, but it appears that not all RNAs are efficiently translated. The requirements of many RNAs for the Kozak sequence are known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used for the 3' or 5' UTR to prevent exonucleolytic degradation of the RNA.

[0165] In one embodiment, the RNA has a cap at both the 5' end and the 3' poly(A) tail, which determines ribosome binding, initiation of translation, and the stability of the RNA in cells.

[0166] In one embodiment, the RNA is a nucleoside-modified RNA. Nucleoside-modified RNAs have particular advantages compared to unmodified RNAs, such as higher stability, low or no innate immunogenicity, and enhanced translation.

[0167] Circular and linear vectors

[0168] One or more vectors can be circular plasmids, which can be integrated into the cellular genome to transform target cells or exist episomally (e.g., autonomously replicating plasmids with an origin of replication). The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid construct.

[0169] The present invention also provides linear nucleic acids or linear expression cassettes ("LECs") that can be effectively delivered to a subject by electroporation and express a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid construct. The LEC can be any linear DNA without any phosphate backbone. The LEC may not contain any antibiotic resistance genes and / or phosphate backbone. The LEC may not contain other nucleotide sequences unrelated to the expected gene expression.

[0170] The LEC can be derived from any plasmid that can be linearized. The plasmid may be capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid construct. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid construct.

[0171] The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0172] Viral vectors

[0173] In one embodiment, provided herein are viral vectors capable of delivering the nucleic acids of the present invention to cells. The expression vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), as well as in Ausubel et al. (1997), and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.)

[0174] Method for preparing vectors

[0175] Provided herein is a method for preparing one or more vectors into which a recombinant nucleic acid construct has been inserted. After the final subcloning step, the vectors can be used to inoculate cell cultures in a large-scale fermenter using methods known in the art.

[0176] In other embodiments, after the final subcloning step, the vectors can be used with one or more electroporation (EP) devices. The EP devices are described in more detail below.

[0177] One or more vectors can be formulated or produced using a combination of known devices and techniques, but preferably, they are produced using the plasmid production techniques described in the licensed, co-pending U.S. Provisional Application U.S. Serial No. 60 / 939,792, filed on May 23, 2007. In some instances, the DNA plasmids described herein can be formulated at a concentration greater than or equal to 10 mg / mL. In addition to those described in U.S. Serial No. 60 / 939,792 (including those described in the licensed patent, U.S. Patent No. 7,238,522, which was published on July 3, 2007), the production techniques also include or incorporate various devices and protocols commonly known to those of ordinary skill in the art. The above-cited applications and patents, U.S. Serial No. 60 / 939,792 and U.S. Patent No. 7,238,522, respectively, are incorporated herein by reference in their entirety.

[0178] Multiple vectors

[0179] An immunogenic composition can comprise multiple copies of a single nucleic acid molecule, such as a single plasmid, or two copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, the immunogenic composition can comprise two, three, four, five, six, seven, eight, nine, ten or more different nucleic acid molecules. Such compositions can comprise multiple two, three, four, five, six or more different plasmids.

[0180] An immunogenic composition can comprise nucleic acid molecules, such as plasmids, that together comprise the coding sequence of an MCV T antigen. An immunogenic composition can comprise nucleic acid molecules, such as plasmids, that together comprise the coding sequences of multiple antigens. In one embodiment, the antigen is an MCV T antigen and one or more other cancer antigens. An immunogenic composition can comprise nucleic acid molecules, such as plasmids, that together comprise the coding sequences of one or more MCV T antigens and one or more cancer antigens.

[0181] Cancer antigen

[0182] The immunogenic composition may comprise one or more cancer antigens, such as WT1, MUC1, LMP2, HPV E6E7, EGFRvIII, HER-2 / neu, individual genotype, MAGE A3, p53 (non-mutated), NY-ESO-1, PSMA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, proteinase 3 (PR1), Bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, TRP-2, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3 ganglioside, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, sperm fibrous sheath protein, AKAP-4, SSX2, XAGE1, B7H3, podoplanin, Tie 2, Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2 and FOS-related antigen 1 for treating or preventing tumor-related pathologies.The immunogenic composition may further comprise one or more cancer antigens WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, individual genotype, MAGE A3, p53 (non-mutated), NY-ESO-1, PSMA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, proteinase 3 (PR1), Bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, TRP-2, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3 ganglioside, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, sperm fibrous sheath protein, AKAP-4, SSX2, XAGE 1, B7H3, podoplanin, Tie 2, Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2 and FOS-related antigen, as well as an optimized consensus-encoded MCV T antigen for treating or preventing tumor-related pathologies. Other combinations of cancer antigens may also be used for treating or preventing tumor-related pathologies.

[0183] Method

[0184] Provided herein are methods for treating, preventing, and / or precluding MCV-related diseases in a subject in need thereof by administering to the subject one or more of the immunogenic compositions described herein. Administering the immunogenic composition to the subject may induce or elicit an immune response in the subject. The induced immune response may be used to treat, prevent, and / or preclude a disease, e.g., an MCV infection or MCC associated with an MCV infection.

[0185] The present invention provides methods for delivering immunogenic compositions for providing gene constructs and proteins of common antigens, which comprise epitopes that render them particularly effective in inducing an immune response against MCV or MCC. Methods for delivering immunogenic compositions or vaccinating can be provided to induce therapeutic and prophylactic immune responses. The vaccination process may generate an immune response against MCV or MCC in a mammal. The immunogenic composition can be delivered to an individual to modulate the activity of the mammalian immune system and enhance the immune response. Delivery of the immunogenic composition can be transfection of the common antigen as a nucleic acid molecule, which is expressed in cells and delivered to the cell surface, where the immune system recognizes it and induces a cellular, humoral, or cellular and humoral response. By administering the immunogenic composition as described above to a mammal, delivery of the immunogenic composition can be used to induce or elicit an immune response against MCV or MCC in the mammal.

[0186] After delivering the immunogenic composition and plasmid into mammalian cells, the transfected cells will express and secrete the common antigen against each plasmid injected from the immunogenic composition. These proteins will be recognized as foreign by the immune system, and antibodies will be generated against them. These antibodies will be maintained by the immune system and can produce an effective response against subsequent MCV infections.

[0187] An immunogenic composition can be administered to a mammal to elicit an immune response in the mammal. The mammal can be a human, primate, non-human primate, cow, bovine, sheep, goat, antelope, bison, buffalo, wild ox, bovine animal, deer, hedgehog, elephant, llama, alpaca, mouse, rat, and chicken.

[0188] The induced immune response can include an induced humoral immune response and / or an induced cellular immune response. A humoral immune response can be induced at about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold. The induced cellular immune response can include CD8 + T cell responses, which are induced at about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold.

[0189] The dose of the immunogenic composition can be 1 μg to 10 mg of active ingredient / kg body weight / time, and can be 20 μg to 10 mg of ingredient / kg body weight / time. The immunogenic composition can be administered every 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, or 31 days. The number of administrations of the immunogenic composition for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0190] Immunogenic compositions can be formulated according to standard techniques well known to those skilled in the art of pharmacy. Taking into account factors such as age, sex, body weight, and the medical condition of a particular subject, as well as the route of administration, such compositions can be administered in doses and by techniques well known to those skilled in the medical arts.

[0191] Immunogenic compositions can be administered prophylactically or therapeutically. In prophylactic administration, the immunogenic composition can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the immunogenic composition is administered to a subject in need thereof in an amount sufficient to effect a therapeutic result. The amount sufficient to accomplish this is defined as a "therapeutically effective dose". The amount effective for this use will depend, for example, on the particular composition of the immunogenic composition regimen being administered, the mode of administration, the stage and severity of the disease, the overall health of the subject, and the judgment of the prescribing physician.

[0192] Immunogenic compositions can be administered by methods well known in the art, such as Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Patent No. 5,580,859, published Dec. 3, 1996); Felgner (U.S. Patent 5,703,055, published Dec. 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, published Oct. 21, 1997), the entire contents of which are incorporated herein by reference in their entirety. The DNA of the immunogenic composition can be complexed to particles or beads, which can be administered to an individual, for example, using a vaccine gun. Those skilled in the art will know that the choice of a pharmaceutically acceptable carrier, including physiologically acceptable compounds, depends, for example, on the route of administration of the expression vector.

[0193] Immunogenic compositions can be delivered by a variety of routes. Typical routes of delivery include parenteral administration, for example, intradermal, intramuscular, or subcutaneous delivery. Other routes include oral, intranasal, and intravaginal routes. Particularly for the DNA of immunogenic compositions, the immunogenic composition can be delivered into the interstitial space of an individual's tissue (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055, the entire contents of which are incorporated herein by reference in their entirety). The immunogenic composition can also be administered to muscle, or can be administered by intradermal or subcutaneous injection or transdermally (such as by iontophoresis). Epidermal administration of the immunogenic composition can also be employed. Epidermal administration can involve mechanical or chemical stimulation of the outermost layer of the epidermis to stimulate an immune response to the stimulant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference in their entirety.

[0194] The immunogenic composition can also be formulated for administration via the nasal passage. A formulation suitable for nasal administration in which the carrier is a solid can include a particle size for administration in the manner of, for example, snuff, e.g., in the range of about 10 to about 500 microns, which is administered in the manner of snuff, i.e., by rapid inhalation through the nasal passage from a powder container held close to the nose. The formulation can be a nasal spray, nasal drops, or administered by nebulizer aerosol. The formulation can include an aqueous or oily solution of the immunogenic composition.

[0195] The immunogenic composition can be a liquid formulation, such as a suspension, syrup, or elixir. The immunogenic composition can also be a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injection administration), such as a sterile suspension or emulsion.

[0196] The immunogenic composition can be incorporated into liposomes, microspheres, or other polymeric matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0197] Method for treating cancer with vaccine

[0198] The vaccine can be used to generate or elicit an immune response in a mammal that is reactive or specific to a cancer or tumor (e.g., MCC) in the mammal or subject in need thereof. The elicited immune response can prevent the growth of the cancer or tumor.

[0199] The elicited immune response can prevent and / or reduce the metastasis of cancer cells or tumor cells. Thus, the vaccine can be used in a method for treating and / or preventing cancer or tumors in the mammal or subject to which the vaccine is administered.

[0200] In some embodiments, the administered vaccine can induce (1) a humoral immune response by B cells to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying myeloid-derived suppressor cells (MDSC) and inhibiting tumor growth; (2) increase cytotoxic T lymphocytes, such as CD8+ (CTL), to attack and kill tumor cells; (3) increase T helper cell responses; and (4) increase the inflammatory response through IFN-γ and TFN-α or preferably all of the foregoing.

[0201] In some embodiments, the immune response can generate a humoral immune response and / or an antigen-specific cytotoxic T lymphocyte (CTL) response that does not result in damage or inflammation to various tissues or systems (e.g., the brain or nervous system, etc.) of the vaccinated subject.

[0202] In some embodiments, the administered vaccine can increase the tumor-free survival rate, reduce the tumor mass, increase the tumor survival rate, or a combination thereof in a subject. The administered vaccine can increase the tumor-free survival rate of the subject by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% or more. The administered vaccine can reduce the tumor mass of the immunized subject by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70% or more. The administered vaccine can prevent and inhibit the increase of monocyte chemoattractant protein 1 (MCP-1) in a subject, and MCP-1 is a cytokine secreted by myeloid-derived suppressor cells. In some embodiments, the administered vaccine can prevent and inhibit the increase of MCP-1 in the cancerous tissue or tumor tissue of a subject, thereby reducing the angiogenesis of the cancerous tissue or tumor tissue of the subject.

[0203] The administered vaccine can increase the tumor survival rate of the subject by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70% or more. In some embodiments, the vaccine can be administered peripherally (described in more detail below) to establish an antigen-specific immune response targeting cancer cells or tumor cells or tissues to eliminate or eradicate a cancer or tumor expressing one or more MCV T antigens without harming the vaccinated subject or causing their disease or death.

[0204] The administered vaccine can increase the cellular immune response of the subject by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold. In some embodiments, the administered vaccine can increase the cellular immune response of the subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold or 6000-fold.

[0205] The administered vaccine can increase the interferon gamma (IFN-γ) level in the subject by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold. In some embodiments, the administered vaccine can increase the IFN-γ level in the subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold or 6000-fold.

[0206] The vaccine dose can be from 1 μg to 10 mg of active ingredient / kg body weight / time, and can be from 20 μg to 10 mg of ingredient / kg body weight / time. The vaccine can be administered every 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 or 31 days. The number of vaccine administrations for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0207] Checkpoint inhibitor combination therapy

[0208] The present invention also relates to a method of using the vaccine as described above in combination with one or more checkpoint inhibitors to enhance the immune response in a mammal. In one embodiment, the vaccine as described above may comprise an MCV T antigen and an antibody against a checkpoint protein. As used herein, "checkpoint inhibitor" includes inhibitors or molecules that block immune checkpoints, as commonly understood in the field of cancer immunotherapy. More generally, a checkpoint inhibitor is an antibody that blocks an immune checkpoint protein. Immune checkpoint proteins include, but are not limited to, PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, B7-H3, BTLA, VISTA, CD40, CEACAM1, CD80, CD86, OX40, CD27, GITR, DNAM-1, TIGIT, TMIGD2, and DC-SIGN. Some examples of known checkpoint inhibitors include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, pidilizumab, avelumab, and the like.

[0209] The combination can be a single formulation or separate and administered sequentially (first the MCV T antigen and then the checkpoint inhibitor, or first the checkpoint inhibitor and then the MCV T antigen). In some embodiments, the MCV T antigen can be administered to the subject about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks before administering the checkpoint inhibitor to the subject. In other embodiments, the checkpoint inhibitor can be administered to the subject about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks before administering the MCV T antigen to the subject.

[0210] The combination of the MCV T antigen and a checkpoint inhibitor induces the immune system more effectively than a vaccine containing only the MCV T antigen. This more effective immune response provides increased efficacy in the treatment and / or prevention of specific cancers.

[0211] In some embodiments, the immune response can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold. Optionally, the immune response of a subject administered the vaccine can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0212] In other alternative embodiments, the immune response of a subject administered the vaccine can be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response of a subject administered the vaccine can be increased by at least about 50%, 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 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0213] The vaccine dose can be from 1 μg to 10 mg of active ingredient / kg body weight / time and can be from 20 μg to 10 mg of ingredient / kg body weight / time. The vaccine can be administered every 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, or 31 days. The number of vaccine administrations for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0214] Merkel cell carcinoma

[0215] The vaccine can be used to generate or elicit in a mammal an immune response reactive or specific to Merkel cell carcinoma (MCC) of the mammal or a subject in need thereof. The elicited immune response can prevent MCC growth. The elicited immune response can reduce the growth of MCC. The elicited immune response can prevent and / or reduce the metastasis of cancer cells or tumor cells from MCC. Accordingly, the vaccine can be used in a method for treating and / or preventing MCC in a mammal or subject to which the vaccine is administered.

[0216] In some embodiments, the administered vaccine can mediate clearance or prevent growth of MCC by inducing (1) a humoral immune response by B cells to produce antibodies targeting the MCV T antigen expressed by MCC cells; (2) increasing cytotoxic T lymphocytes, such as CD8+ (CTL), to attack and kill MCC cells; (3) increasing T helper cell responses; (4) increasing an inflammatory response by IFN-γ and TFN-α or all of the above.

[0217] In some embodiments, the administered vaccine can increase the survival rate without MCC, reduce the MCC mass, increase the MCC survival rate, or a combination thereof in a subject. The administered vaccine can increase the survival rate without MCC by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% or higher levels. The administered vaccine can reduce the MCC mass in a subject after vaccination by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% or higher. The administered vaccine can prevent and block the increase of monocyte chemoattractant protein 1 (MCP-1) in a subject, and MCP-1 is a cytokine secreted by myeloid-derived suppressor cells. In some embodiments, the administered vaccine can prevent and block the increase of MCP-1 within the MCC tissue of a subject, thereby reducing angiogenesis in the MCC tissue of the subject. The administered vaccine can increase the MCC survival rate in a subject by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% or higher.

[0218] Combined therapy

[0219] The immunogenic composition can be co-administered with the following: other proteins and / or genes encoding CCL20, interferon-alpha, interferon-gamma, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15 (including IL-15 lacking the signal sequence and optionally including a different signal peptide such as the IgE signal peptide), MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8, L-selectin, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-18, CD40, CD40L, angiogenic growth factors, fibroblast growth factors, IL-7, nerve growth factors, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof, or combinations thereof.In some embodiments, the immunogenic composition is administered in combination with one or more of the following nucleic acid molecules and / or proteins: nucleic acid molecules selected from the group consisting of nucleic acid molecules comprising a coding sequence encoding one or more of CCL20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, and RANTES or functional fragments thereof, and proteins selected from the group consisting of CCL02, IL-12 protein, IL-15 protein, IL-28 protein, CTACK protein, TECK protein, MEC protein, or RANTES protein or functional fragments thereof.

[0220] The immunogenic composition can be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccally, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly or combinations thereof. For veterinary use, the composition can be administered as a suitably acceptable formulation in accordance with normal veterinary practice. A veterinarian can readily determine the dosing regimen and route of administration most suitable for a particular animal. The immunogenic composition can be administered by conventional syringes, needleless injection devices, "particle bombardment guns", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or sonication.

[0221] The plasmid of the immunogenic composition can be delivered to a mammal by several well-known techniques, including DNA injection (also known as DNA vaccination) with or without in vivo electroporation, liposome-mediated, nanoparticle-facilitated recombinant vectors such as recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccines. Conserved antigens can be delivered together by DNA injection and in vivo electroporation.

[0222] Electroporation

[0223] Administration of an immunogenic composition via electroporation can be accomplished using an electroporation device that can be configured to deliver an energy pulse to a desired tissue of a mammal that effectively causes the formation of reversible pores in the cell membrane, and preferably, the energy pulse is a constant current similar to a preset current input by the user. The electroporation device can include an electroporation assembly and an electrode member or a handle member. The electroporation assembly can include and incorporate one or more of the various elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a storage element, a power supply, and a power switch. In vivo electroporation devices, such as the CELLECTRA EP System (Inovio Pharmaceuticals, Plymouth Meeting, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA), can be used to accomplish electroporation to facilitate plasmid transfection of cells.

[0224] The electroporation assembly can be used as one element of the electroporation device, while the other elements are separate elements (or assemblies) in communication with the electroporation assembly. The electroporation assembly can be used as more than one element of the electroporation device, which can be in communication with other elements of the electroporation device that are separate from the electroporation assembly. The elements of the electroporation device that are present as part of an electromechanical or mechanical device can be unrestricted as these elements can be used as one device or as independent elements in communication with each other. The electroporation assembly may be capable of delivering an energy pulse that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode member can include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode member receives the energy pulse from the electroporation assembly and delivers it to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during the delivery of the energy pulse and measures the impedance in the desired tissue and transfers the impedance to the electroporation assembly. The feedback mechanism can receive the measured impedance and can adjust the pulse of energy delivered by the electroporation assembly to maintain a constant current.

[0225] The plurality of electrodes can deliver the energy pulse in a dispersed mode. The plurality of electrodes can deliver the energy pulse in a dispersed mode under the control of the electrodes in a programmed sequence, and the user inputs the programmed sequence into the electroporation assembly. The programmed sequence can include a plurality of pulses delivered sequentially, wherein each of the plurality of pulses is delivered by at least two active electrodes together with a neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by another of the at least two active electrodes together with one of the neutral electrodes having impedance measurement.

[0226] The feedback mechanism can be implemented by hardware or software. The feedback mechanism can be performed by an analog closed-loop circuit. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but preferably, real-time feedback or instantaneous feedback (i.e., substantially instantaneous as determined by the available techniques for determining the response time). The neutral electrode can measure the impedance in the expected tissue and transmit the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain a constant current at a value similar to a preset current. The feedback mechanism can continuously and instantaneously maintain a constant current during the delivery of the energy pulse.

[0227] Examples of electroporation devices and electroporation methods that can facilitate the delivery of the immunogenic compositions of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the delivery of immunogenic compositions include those provided in co-pending and co-owned U.S. Patent Application Serial No. 11 / 874,072 filed on October 17, 2007, which claims the benefit of U.S. Provisional Application Serial No. 60 / 852,149 filed on October 17, 2006, and U.S. Provisional Application Serial No. 60 / 978,982 filed on October 10, 2007, all of which are incorporated herein by reference in their entirety.

[0228] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use in facilitating the introduction of biomolecules into the cells of selected tissues in vivo or in plants. The modular electrode system can include a plurality of needle electrodes; a subcutaneous injection needle; an electrical connector that provides an electrical connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue in the body or in a plant. Then, the biomolecules are delivered to the selected tissue through the subcutaneous injection needle. The programmable constant current pulse controller is activated, and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse helps to introduce the biomolecules into the cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0229] U.S. Patent Publication 2005 / 0052630 to Smith et al. describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues in a human or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes in an array according to user control and input of pulse parameters, and allows storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disk having a needle electrode array, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Publication 2005 / 0052630 is incorporated herein by reference.

[0230] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005 / 0052630 can be applied to penetrate not only deep into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle (for delivering selected biomolecules) is also fully inserted into the target organ, and injection is performed perpendicular to the target problem within a pre-defined area at the target site. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005 / 005263 are preferably 20 mm long and of gauge 21.

[0231] In addition, in certain embodiments incorporating the electroporation device and its uses, the electroporation devices described in the following patents are considered: U.S. Patent 5,273,525, issued December 28, 1993; U.S. Patent 6,110,161, issued August 29, 2000; 6,261,281, issued July 17, 2001; and 6,958,060, issued October 25, 2005, as well as U.S. Patent 6,939,862, issued September 6, 2005. Further, patents covering the subject matter provided in U.S. Patent 6,697,669, issued February 24, 2004, which relates to delivering DNA using any of a variety of devices, and U.S. Patent 7,328,064, issued February 5, 2008, are considered herein. The above patents are incorporated herein by reference in their entirety.

[0232] In vitro and ex vivo antigen production

[0233] In one embodiment, an optimized consensus MCV T antigen is produced in vitro or ex vivo. For example, in one embodiment, a nucleic acid encoding the optimized consensus MCV T antigen can be introduced and expressed in in vitro or ex vivo cells.

[0234] Methods for introducing genes into cells and expressing them in cells are known in the art. In the case of expression vectors, the vector can be easily introduced into a host cell by any method in the art, such as mammalian, bacterial, yeast, or insect cells. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0235] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0236] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0237] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0238] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). On the other hand, nucleic acids can associate with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, linked to liposomes by a linking molecule that associates with both the liposome and the oligonucleotide, encapsulated in liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, bound to lipids, included as a suspension of lipids in lipids, or complexed with micelles, or bound to lipids. Compositions associated with lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they can exist in bilayer structures, micelles, or "collapsed" structures. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0239] Examples

[0240] The following examples further illustrate the invention. It should be understood that these examples, while showing preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, those skilled in the art can determine the basic features of the invention and, without departing from the spirit and scope of the invention, make various changes and modifications to adapt the invention to various uses and conditions. Thus, as previously mentioned, various modifications of the invention will be apparent to those skilled in the art other than those shown and described herein. Such modifications are also intended to fall within the scope of the appended claims.

[0241] Example 1: Nucleic Acid Vaccine Against Merkel Cell Polyomavirus

[0242] A nucleic acid vaccine against the Merkel cell polyomavirus (MCV) T antigen has been developed (Figures 1 and 2). Optimized synthetic consensus MCV T antigen sequences representing the large T antigen (LTAg) and small T antigen (STAg) were separately cloned into mammalian expression plasmid DNA ( Figure 3 ), and delivered to mice by intramuscular electroporation (Figure 4A). After immunization, the DNA vaccine constructs generated robust antibody and T cell responses against MCV T antigen peptides (Figures 4B to 15).

[0243] Figures 4B, 7, Figure 12 and 14 show that the LTAg vaccine is highly immunogenic in C57Bl / 6 and CD-1 outbred mice. Figures 8 to 10It is shown that LTAg vaccination can generate robust multifunctional CD4 and CD8 T cells as well as cytotoxic CD8 T cells.

[0244] Figures 4B and 15 show that the STAg vaccine is immunogenic in C57Bl / 6 and CD-1 mice. Figure 15 shows that for CD-1 mice, CD4 and CD8 responses to IFNγ / TNFα were detected.

[0245] Figure 11 It is shown that both vaccines generate a humoral response in C57Bl / 6 mice.

[0246] Example 2: Sequences

[0247] SEQ ID NO:1: Nucleotide sequences encoding a modified synthetic consensus MCV LTAg

[0248]

[0249] SEQ ID NO:2: Modified synthetic consensus MCV LTAg amino acid sequence

[0250] MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPHGTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSSSGSSSREETTNSGRESSTPNGTSVPRNSSRTDGTAEDLFCDKSLSSPEPPSSSEEPEEPPSSRSSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTPPKPKKNRETPVPTDFPIDLSDYLSHAVYSNKTVSCFAIYTTSDKAIELYDKIEKFKVDFKSRHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYKLLQENKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSRLKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKKHLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSGKTSFAAALIDLLEGKALNINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQGINNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPKANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGKFCQMIENVEAGQDPLLNILIEEEGPEETEETQDSGTFSQ

[0251] SEQ ID NO:3: Nucleotide sequence encoding modified synthetic consensus MCV STAg

[0252] ATGGACCTGGTGCTGAACCGAAAGGAGAGGGAGGCCCTGTGCAAGCTGCTGGAGATCGCCCCTAACTGTTACGGCAATATCCCACTGATGAAGGCCGCCTTCAAGAGGTCTTGCCTGAAGCACCACCCAAACAAGGGCGGCAATCCCGTGATCATGATGGAGCTGAACACCCTGTGGAGCAAGTTTCAGCAGAATATCCACAAGCTGCGGAGCGACTTCTCCATGTTTGATGAGGTGAGCACCAAGTTCCCCTGGGAGGAGTACGGAACAGCAGCAGCAGCAGCACAGTCCGGCTATAACGCCAGGTTTTGCAGAGGCCCTGGCTGTATGCTGAAGCAGCTGCGGGACTCCAAGTGCGCCTGTATCTCTTGCAAGCTGAGCCGCCAGCACTGTTCTCTGAAGACCCTGAAGCAGAAGAATTGCGCCACATGGGGCGAGTGCTTCTGTTATCAGTGTTTTATCCTGTGGTTCGGCTTTCCCCCTACATGGGAGTCCTTCGATTGGTGGCAGAAAACCCTGGAAGAAACCGACTACTGTCTGCTGCATCTGCATCTGTTC

[0253] SEQ ID NO:4: Amino acid sequence of modified synthetic consensus MCV STAg

[0254] MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVSTKFPWEEYGTAAAAAQSGYNARFCRGPGCMLKQLRDSKCACISCKLSRQHCSLKTLKQKNCATWGECFCYQCFILWFGFPPTWESFDWWQKTLEETDYCLLHLHLF

[0255] SEQ ID NO:5: Nucleotide sequence encoding modified synthetic consensus LTAg and STAg linked to a furin cleavage site

[0256]

[0257] SEQ ID NO:6: Amino acid sequences of modified synthetic consensus LTAg and STAg linked to a furin cleavage site.

[0258]

[0259] SEQ ID NO:7: Amino acid sequence of the IgE leader sequence

[0260] MDWTWILFLVAAATRVHS

[0261] It should be understood that the foregoing and the appended examples are merely exemplary and should not be regarded as limiting the scope of the present invention, which is defined only by the appended claims and their equivalents.

[0262] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Changes and modifications that can be made without departing from the spirit and scope of the present invention include, but are not limited to, those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the present invention. Sequence Listing <110> The Wistar Institute of Anatomy and Biology David B. Weiner Elizabeth DiPelle <120> Large and small T antigens of Merkel cell polyomavirus, nucleic acid constructs, and vaccines produced therefrom and methods of use thereof <130> 206194-0021-00-WO.608420 <150> 62619161 <151> 2018-01-19 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 2451 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding a modified synthetic consensus MCV LTAg <400> 1 atggacctgg tgctgaacag gaaggagaga gaggccctgt gcaagctgct ggagatcgcc 60 cccaactgtt acggcaatat ccctctgatg aaggccgcct tcaagcggag ctgcctgaag 120 caccacccca acaagggcgg caaccctgtg atcatgatgg agctgaatac cctgtggtcc 180 aagtttcagc agaatatcca caagctgcgg tccgatttct ctatgtttga cgaggtggat 240 gaggccccta tctacggcac caccaagttc aaggagtggt ggcgctccgg cggcttctct 300 tttggcaagg cctacgagta cggccctaac ccacacggca ccaatagcag gtccagaaag 360 ccaagctcca acgccagcag gggagcacca tccggatcta gcccacctca cagccagtcc 420 tctagctccg gctacggctc ttttagcgcc tcccaggcct ctgacagcca gtccagaggc 480 cccgatatcc cacccgagca ccacgaggag cctacctcta gctccggctc tagctcccgg 540 gaggagacaa ccaacagcgg cagggagtct agcaccccaa acggcacctc cgtgccaagg 600 aattcctcta ggaccgacgg aaccgccgag gacctgttct gcgataagtc cctgagctcc 660 cctgagcctc catctagctc cgaggagcca gaggagcccc cttctagcag gtcctctccc 720 agacagccac caagctcctc tgccgaggag gcaagctcct ctcagttcac cgacgaggag 780 tacaggagct cctcttttac cacccctaag acccctccac ccttctcccg gaagcgcaag 840 tttggaggct ctaggagctc cgcctctagc gcctcctctg ccagcttcac ctccacccct 900 ccaaagccca agaagaacag agagacaccc gtgcctaccg actttcctat cgacctgagc 960 gattacctgt cccacgccgt gtactctaat aagaccgtga gctgtttcgc catctacacc 1020 accagcgaca aggccatcga gctgtacgat aagatcgaga agttcaaggt ggacttcaag 1080 tccaggcacg catgcgagct gggatgtatc ctgctgttca tcaccctgtc caagcaccgc 1140 gtgtctgcca tcaagaactt ctgcagcacc ttttgtacca tctcctttct gatctgcaag 1200 ggcgtgaata agatgcctga gatgtacaac aacctgtgca agccccctta caagctgctg 1260 caggagaaca agccactgct gaattacgag ttccaggaga aggagaagga ggccagctgc 1320 aactggaatc tggtggccga gttcgcctgt gagtacgagc tggacgatca ctttatcatc 1380 ctggcccact acctggactt cgccaagcca tttccctgcc agaagtgtga gaacaggtct 1440 agactgaagc cacacaaggc ccacgaggcc caccactcca atgccaagct gttttacgag 1500 tctaagagcc agaagaccat ctgccagcag gcagcagaca ccgtgctggc aaagaggaga 1560 ctggagatgc tggagatgac caggaccgag atgctgtgca agaagttcaa gaagcacctg 1620 ctggagatgc tggagatgac caggaccgag atgctgtgca agaagttcaa gaagcacctg 1620 gagcggctgc gcgacctgga taccatcgat ctgctgtact acatgggcgg cgtggcctgg 1680 gagcggctgc gcgacctgga taccatcgat ctgctgtact acatgggcgg cgtggcctgg 1680 tactgctgtc tgttcgagga gtttgagaag aagctgcaga agatcatcca gctgctgacc 1740 tactgctgtc tgttcgagga gtttgagaag aagctgcaga agatcatcca gctgctgacc 1740 gagaacatcc caaagtacag aaatatctgg ttcaagggcc ccatcaactc tggcaagacc 1800 gagaacatcc caaagtacag aaatatctgg ttcaagggcc ccatcaactc tggcaagacc 1800 agcttcgccg ccgccctgat cgacctgctg gagggcaagg ccctgaacat caattgccct 1860 agcttcgccg ccgccctgat cgacctgctg gagggcaagg ccctgaacat caattgccct 1860 agcgataagc tgccattcga gctgggctgt gccctggaca agttcatggt ggtgtttgag 1920 agcgataagc tgccattcga gctgggctgt gccctggaca agttcatggt ggtgtttgag 1920 gatgtgaagg gccagaactc cctgaataag gacctgcagc ccggccaggg catcaacaat 1980 gatgtgaagg gccagaactc cctgaataag gacctgcagc ccggccaggg catcaacaat 1980 ctggataacc tgcgggacca cctggatgga gcagtggccg tgagcctgga gaagaagcac 2040 ctggataacc tgcgggacca cctggatgga gcagtggccg tgagcctgga gaagaagcac 2040 gtgaacaaga agcaccagat cttcccaccc tgcatcgtga ccgccaatga ctactttatc 2100 gtgaacaaga agcaccagat cttcccaccc tgcatcgtga ccgccaatga ctactttatc 2100 ccaaagaccc tgatcgcccg cttctcttac accctgcact ttagccccaa ggccaacctg 2160 ccaaagaccc tgatcgcccg cttctcttac accctgcact ttagccccaa ggccaacctg 2160 agggacagcc tggatcagaa tatggagatc agaaagaggc gcatcctgca gtccggaacc 2220 agggacagcc tggatcagaa tatggagatc agaaagaggc gcatcctgca gtccggaacc 2220 accctgctgc tgtgcctgat ctggtgtctg cctgacacca ccttcaagcc atgcctgcag 2280 accctgctgc tgtgcctgat ctggtgtctg cctgacacca ccttcaagcc atgcctgcag 2280 gaggagatca agaactggaa gcagatcctg cagtctgaga tcagctacgg caagttttgt 2340 cagatgatcg agaacgtgga ggccggccag gaccccctgc tgaatatcct gatcgaggag 2400 gagggcccag aggagacaga ggagacacag gactccggca ccttctctca g 2451 <210> 2 <211> 817 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of modified synthetic consensus MCV LTAg <400> 2 Met Asp Leu Val Leu Asn Arg Lys Glu Arg Glu Ala Leu Cys Lys Leu 1 5 10 15 Leu Glu Ile Ala Pro Asn Cys Tyr Gly Asn Ile Pro Leu Met Lys Ala 20 25 30 Ala Phe Lys Arg Ser Cys Leu Lys His His Pro Asn Lys Gly Gly Asn 35 40 45 Pro Val Ile Met Met Glu Leu Asn Thr Leu Trp Ser Lys Phe Gln Gln 50 55 60 Asn Ile His Lys Leu Arg Ser Asp Phe Ser Met Phe Asp Glu Val Asp 65 70 75 80 Glu Ala Pro Ile Tyr Gly Thr Thr Lys Phe Lys Glu Trp Trp Arg Ser 85 90 95 Gly Gly Phe Ser Phe Gly Lys Ala Tyr Glu Tyr Gly Pro Asn Pro His 100 105 110 Gly Thr Asn Ser Arg Ser Arg Lys Pro Ser Ser Asn Ala Ser Arg Gly 115 120 125 Ala Pro Ser Gly Ser Ser Pro Pro His Ser Gln Ser Ser Ser Ser Gly 130 135 140 Tyr Gly Ser Phe Ser Ala Ser Gln Ala Ser Asp Ser Gln Ser Arg Gly 145 150 155 160 Pro Asp Ile Pro Pro Glu His His Glu Glu Pro Thr Ser Ser Ser Gly 165 170 175 Ser Ser Ser Arg Glu Glu Thr Thr Asn Ser Gly Arg Glu Ser Ser Thr 180 185 190 Pro Asn Gly Thr Ser Val Pro Arg Asn Ser Ser Arg Thr Asp Gly Thr 195 200 205 Ala Glu Asp Leu Phe Cys Asp Lys Ser Leu Ser Ser Pro Glu Pro Pro 210 215 220 Ser Ser Ser Glu Glu Pro Glu Glu Pro Pro Ser Ser Arg Ser Ser Pro 225 230 235 240 Arg Gln Pro Pro Ser Ser Ser Ala Glu Glu Ala Ser Ser Ser Gln Phe 245 250 255 Thr Asp Glu Glu Tyr Arg Ser Ser Ser Phe Thr Thr Pro Lys Thr Pro 260 265 270 Pro Pro Phe Ser Arg Lys Arg Lys Phe Gly Gly Ser Arg Ser Ser Ala 275 280 285 Ser Ser Ala Ser Ser Ala Ser Phe Thr Ser Thr Pro Pro Lys Pro Lys 290 295 300 Lys Asn Arg Glu Thr Pro Val Pro Thr Asp Phe Pro Ile Asp Leu Ser 305 310 315 320 Asp Tyr Leu Ser His Ala Val Tyr Ser Asn Lys Thr Val Ser Cys Phe 325 330 335 Ala Ile Tyr Thr Thr Ser Asp Lys Ala Ile Glu Leu Tyr Asp Lys Ile 340 345 350 Glu Lys Phe Lys Val Asp Phe Lys Ser Arg His Ala Cys Glu Leu Gly 355 360 365 Cys Ile Leu Leu Phe Ile Thr Leu Ser Lys His Arg Val Ser Ala Ile 370 375 380 Lys Asn Phe Cys Ser Thr Phe Cys Thr Ile Ser Phe Leu Ile Cys Lys 385 390 395 400 Gly Val Asn Lys Met Pro Glu Met Tyr Asn Asn Leu Cys Lys Pro Pro 405 410 415 Tyr Lys Leu Leu Gln Glu Asn Lys Pro Leu Leu Asn Tyr Glu Phe Gln 420 425 430 Glu Lys Glu Lys Glu Ala Ser Cys Asn Trp Asn Leu Val Ala Glu Phe 435 440 445 Ala Cys Glu Tyr Glu Leu Asp Asp His Phe Ile Ile Leu Ala His Tyr 450 455 460 Leu Asp Phe Ala Lys Pro Phe Pro Cys Gln Lys Cys Glu Asn Arg Ser 465 470 475 480 Arg Leu Lys Pro His Lys Ala His Glu Ala His His Ser Asn Ala Lys 485 490 495 Leu Phe Tyr Glu Ser Lys Ser Gln Lys Thr Ile Cys Gln Gln Ala Ala 500 505 510 Asp Thr Val Leu Ala Lys Arg Arg Leu Glu Met Leu Glu Met Thr Arg 515 520 525 Thr Glu Met Leu Cys Lys Lys Phe Lys Lys His Leu Glu Arg Leu Arg 530 535 540 Asp Leu Asp Thr Ile Asp Leu Leu Tyr Tyr Met Gly Gly Val Ala Trp 545 550 555 560 Tyr Cys Cys Leu Phe Glu Glu Phe Glu Lys Lys Leu Gln Lys Ile Ile 565 570 575 Gln Leu Leu Thr Glu Asn Ile Pro Lys Tyr Arg Asn Ile Trp Phe Lys 580 585 590 Gly Pro Ile Asn Ser Gly Lys Thr Ser Phe Ala Ala Ala Leu Ile Asp 595 600 605 Leu Leu Glu Gly Lys Ala Leu Asn Ile Asn Cys Pro Ser Asp Lys Leu 610 615 620 Pro Phe Glu Leu Gly Cys Ala Leu Asp Lys Phe Met Val Val Phe Glu 625 630 635 640 Asp Val Lys Gly Gln Asn Ser Leu Asn Lys Asp Leu Gln Pro Gly Gln 645 650 655 Gly Ile Asn Asn Leu Asp Asn Leu Arg Asp His Leu Asp Gly Ala Val 660 665 670 Ala Val Ser Leu Glu Lys Lys His Val Asn Lys Lys His Gln Ile Phe 675 680 685 Pro Pro Cys Ile Val Thr Ala Asn Asp Tyr Phe Ile Pro Lys Thr Leu 690 695 700 Ile Ala Arg Phe Ser Tyr Thr Leu His Phe Ser Pro Lys Ala Asn Leu 705 710 715 720 Arg Asp Ser Leu Asp Gln Asn Met Glu Ile Arg Lys Arg Arg Ile Leu 725 730 735 Gln Ser Gly Thr Thr Leu Leu Leu Cys Leu Ile Trp Cys Leu Pro Asp 740 745 750 Thr Thr Phe Lys Pro Cys Leu Gln Glu Glu Ile Lys Asn Trp Lys Gln 755 760 765 Ile Leu Gln Ser Glu Ile Ser Tyr Gly Lys Phe Cys Gln Met Ile Glu 770 775 780 Asn Val Glu Ala Gly Gln Asp Pro Leu Leu Asn Ile Leu Ile Glu Glu 785 790 795 800 Glu Gly Pro Glu Glu Thr Glu Glu Thr Gln Asp Ser Gly Thr Phe Ser 805 810 815 Gln <210> 3 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding a modified synthetic consensus MCV STAg <400> 3 atggacctgg tgctgaaccg aaaggagagg gaggccctgt gcaagctgct ggagatcgcc 60 cctaactgtt acggcaatat cccactgatg aaggccgcct tcaagaggtc ttgcctgaag 120 caccacccaa acaagggcgg caatcccgtg atcatgatgg agctgaacac cctgtggagc 180 aagtttcagc agaatatcca caagctgcgg agcgacttct ccatgtttga tgaggtgagc 240 accaagttcc cctgggagga gtacggaaca gcagcagcag cagcacagtc cggctataac 300 gccaggtttt gcagaggccc tggctgtatg ctgaagcagc tgcgggactc caagtgcgcc 360 tgtatctctt gcaagctgag ccgccagcac tgttctctga agaccctgaa gcagaagaat 420 tgcgccacat ggggcgagtg cttctgttat cagtgtttta tcctgtggtt cggctttccc 480 cctacatggg agtccttcga ttggtggcag aaaaccctgg aagaaaccga ctactgtctg 540 ctgcatctgc atctgttc 558 <210> 4 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of modified synthetic consensus MCV STAg <400> 4 Met Asp Leu Val Leu Asn Arg Lys Glu Arg Glu Ala Leu Cys Lys Leu 1 5 10 15 Leu Glu Ile Ala Pro Asn Cys Tyr Gly Asn Ile Pro Leu Met Lys Ala 20 25 30 Ala Phe Lys Arg Ser Cys Leu Lys His His Pro Asn Lys Gly Gly Asn 35 40 45 Pro Val Ile Met Met Glu Leu Asn Thr Leu Trp Ser Lys Phe Gln Gln 50 55 60 Asn Ile His Lys Leu Arg Ser Asp Phe Ser Met Phe Asp Glu Val Ser 65 70 75 80 Thr Lys Phe Pro Trp Glu Glu Tyr Gly Thr Ala Ala Ala Ala Ala Gln 85 90 95 Ser Gly Tyr Asn Ala Arg Phe Cys Arg Gly Pro Gly Cys Met Leu Lys 100 105 110 Gln Leu Arg Asp Ser Lys Cys Ala Cys Ile Ser Cys Lys Leu Ser Arg 115 120 125 Gln His Cys Ser Leu Lys Thr Leu Lys Gln Lys Asn Cys Ala Thr Trp 130 135 140 Gly Glu Cys Phe Cys Tyr Gln Cys Phe Ile Leu Trp Phe Gly Phe Pro 145 150 155 160 Pro Thr Trp Glu Ser Phe Asp Trp Trp Gln Lys Thr Leu Glu Glu Thr 165 170 175 Asp Tyr Cys Leu Leu His Leu His Leu Phe 180 185 <210> 5 <211> 3027 <212> DNA <213> Artificial Sequence <220> <223> Encodes a modified synthetic consensus LTAg and STAg nucleotide sequence linked to a furin cleavage site <400> 5 atggacctgg tgctgaacag gaaggagaga gaggccctgt gcaagctgct ggagatcgcc 60 cccaactgtt acggcaatat ccctctgatg aaggccgcct tcaagcggag ctgcctgaag 120 caccacccca acaagggcgg caaccctgtg atcatgatgg agctgaatac cctgtggtcc 180 aagtttcagc agaatatcca caagctgcgg tccgatttct ctatgtttga cgaggtggat 240 gaggccccta tctacggcac caccaagttc aaggagtggt ggcgctccgg cggcttctct 300 tttggcaagg cctacgagta cggccctaac ccacacggca ccaatagcag gtccagaaag 360 ccaagctcca acgccagcag gggagcacca tccggatcta gcccacctca cagccagtcc 420 tctagctccg gctacggctc ttttagcgcc tcccaggcct ctgacagcca gtccagaggc 480 cccgatatcc cacccgagca ccacgaggag cctacctcta gctccggctc tagctcccgg 540 gaggagacaa ccaacagcgg cagggagtct agcaccccaa acggcacctc cgtgccaagg 600 aattcctcta ggaccgacgg aaccgccgag gacctgttct gcgataagtc cctgagctcc 660 cctgagcctc catctagctc cgaggagcca gaggagcccc cttctagcag gtcctctccc 720 agacagccac caagctcctc tgccgaggag gcaagctcct ctcagttcac cgacgaggag 780 tacaggagct cctcttttac cacccctaag acccctccac ccttctcccg gaagcgcaag 840 tttggaggct ctaggagctc cgcctctagc gcctcctctg ccagcttcac ctccacccct 900 ccaaagccca agaagaacag agagacaccc gtgcctaccg actttcctat cgacctgagc 960 gattacctgt cccacgccgt gtactctaat aagaccgtga gctgtttcgc catctacacc 1020 accagcgaca aggccatcga gctgtacgat aagatcgaga agttcaaggt ggacttcaag 1080 tccaggcacg catgcgagct gggatgtatc ctgctgttca tcaccctgtc caagcaccgc 1140 gtgtctgcca tcaagaactt ctgcagcacc ttttgtacca tctcctttct gatctgcaag 1200 ggcgtgaata agatgcctga gatgtacaac aacctgtgca agccccctta caagctgctg 1260 caggagaaca agccactgct gaattacgag ttccaggaga aggagaagga ggccagctgc 1320 aactggaatc tggtggccga gttcgcctgt gagtacgagc tggacgatca ctttatcatc 1380 ctggcccact acctggactt cgccaagcca tttccctgcc agaagtgtga gaacaggtct 1440 agactgaagc cacacaaggc ccacgaggcc caccactcca atgccaagct gttttacgag 1500 tctaagagcc agaagaccat ctgccagcag gcagcagaca ccgtgctggc aaagaggaga 1560 ctggagatgc tggagatgac caggaccgag atgctgtgca agaagttcaa gaagcacctg 1620 gagcggctgc gcgacctgga taccatcgat ctgctgtact acatgggcgg cgtggcctgg 1680 tactgctgtc tgttcgagga gtttgagaag aagctgcaga agatcatcca gctgctgacc 1740 gagaacatcc caaagtacag aaatatctgg ttcaagggcc ccatcaactc tggcaagacc 1800 agcttcgccg ccgccctgat cgacctgctg gagggcaagg ccctgaacat caattgccct 1860 agcgataagc tgccattcga gctgggctgt gccctggaca agttcatggt ggtgtttgag 1920 gatgtgaagg gccagaactc cctgaataag gacctgcagc ccggccaggg catcaacaat 1980 ctggataacc tgcgggacca cctggatgga gcagtggccg tgagcctgga gaagaagcac 2040 gtgaacaaga agcaccagat cttcccaccc tgcatcgtga ccgccaatga ctactttatc 2100 ccaaagaccc tgatcgcccg cttctcttac accctgcact ttagccccaa ggccaacctg 2160 agggacagcc tggatcagaa tatggagatc agaaagaggc gcatcctgca gtccggaacc 2220 accctgctgc tgtgcctgat ctggtgtctg cctgacacca ccttcaagcc atgcctgcag 2280 gaggagatca agaactggaa gcagatcctg cagtctgaga tcagctacgg caagttttgt 2340 cagatgatcg agaacgtgga ggccggccag gaccccctgc tgaatatcct gatcgaggag 2400 gagggcccag aggagacaga ggagacacag gactccggca ccttctctca gagaggccgc 2460 aaaaggaggt ctgatctggt gctgaatcgg aaagagagag aagccctgtg caaactgctg 2520 gaaatcgccc caaactgtta cggcaacatc cccctgatga aggccgcctt caagaggtct 2580 tgcctgaagc accacccaaa caagggcggc aatcccgtga tcatgatgga gctgaacacc 2640 ctgtggagca agtttcagca gaatatccac aagctgcgga gcgacttctc catgtttgat 2700 gaggtgagca ccaagttccc ttgggaggag tacggaacag cagcagcagc agcacagtcc 2760 ggctataacg ccaggttttg cagaggccca ggctgtatgc tgaagcagct gcgggactcc 2820 aagtgcgcct gtatctcttg caagctgagc cgccagcact gttctctgaa gaccctgaag 2880 cagaagaatt gcgccacatg gggcgagtgc ttctgttatc agtgttttat cctgtggttc 2940 ggctttcccc ctacatggga gtccttcgat tggtggcaga aaaccctgga ggaaactgat 3000 tactgtctgc tgcacctgca cctgttc 3027 <210> 6 <211> 1009 <212> PRT <213> Artificial Sequence <220> <223> Modified synthetic consensus LTAg and STAg amino acid sequences linked to furin cleavage site <400> 6 Met Asp Leu Val Leu Asn Arg Lys Glu Arg Glu Ala Leu Cys Lys Leu 1 5 10 15 Leu Glu Ile Ala Pro Asn Cys Tyr Gly Asn Ile Pro Leu Met Lys Ala 20 25 30 Ala Phe Lys Arg Ser Cys Leu Lys His His Pro Asn Lys Gly Gly Asn 35 40 45 Pro Val Ile Met Met Glu Leu Asn Thr Leu Trp Ser Lys Phe Gln Gln 50 55 60 Asn Ile His Lys Leu Arg Ser Asp Phe Ser Met Phe Asp Glu Val Asp 65 70 75 80 Glu Ala Pro Ile Tyr Gly Thr Thr Lys Phe Lys Glu Trp Trp Arg Ser 85 90 95 Gly Gly Phe Ser Phe Gly Lys Ala Tyr Glu Tyr Gly Pro Asn Pro His 100 105 110 Gly Thr Asn Ser Arg Ser Arg Lys Pro Ser Ser Asn Ala Ser Arg Gly 115 120 125 Ala Pro Ser Gly Ser Ser Pro Pro His Ser Gln Ser Ser Ser Ser Gly 130 135 140 Tyr Gly Ser Phe Ser Ala Ser Gln Ala Ser Asp Ser Gln Ser Arg Gly 145 150 155 160 Pro Asp Ile Pro Pro Glu His His Glu Glu Pro Thr Ser Ser Ser Gly 165 170 175 Ser Ser Ser Arg Glu Glu Thr Thr Asn Ser Gly Arg Glu Ser Ser Thr 180 185 190 Pro Asn Gly Thr Ser Val Pro Arg Asn Ser Ser Arg Thr Asp Gly Thr 195 200 205 Ala Glu Asp Leu Phe Cys Asp Lys Ser Leu Ser Ser Pro Glu Pro Pro 210 215 220 Ser Ser Ser Glu Glu Pro Glu Glu Pro Pro Ser Ser Arg Ser Ser Pro 225 230 235 240 Arg Gln Pro Pro Ser Ser Ser Ala Glu Glu Ala Ser Ser Ser Gln Phe 245 250 255 Thr Asp Glu Glu Tyr Arg Ser Ser Ser Phe Thr Thr Pro Lys Thr Pro 260 265 270 Pro Pro Phe Ser Arg Lys Arg Lys Phe Gly Gly Ser Arg Ser Ser Ala 275 280 285 Ser Ser Ala Ser Ser Ala Ser Phe Thr Ser Thr Pro Pro Lys Pro Lys 290 295 300 Lys Asn Arg Glu Thr Pro Val Pro Thr Asp Phe Pro Ile Asp Leu Ser 305 310 315 320 Asp Tyr Leu Ser His Ala Val Tyr Ser Asn Lys Thr Val Ser Cys Phe 325 330 335 Ala Ile Tyr Thr Thr Ser Asp Lys Ala Ile Glu Leu Tyr Asp Lys Ile 340 345 350 Glu Lys Phe Lys Val Asp Phe Lys Ser Arg His Ala Cys Glu Leu Gly 355 360 365 Cys Ile Leu Leu Phe Ile Thr Leu Ser Lys His Arg Val Ser Ala Ile 370 375 380 Lys Asn Phe Cys Ser Thr Phe Cys Thr Ile Ser Phe Leu Ile Cys Lys 385 390 395 400 Gly Val Asn Lys Met Pro Glu Met Tyr Asn Asn Leu Cys Lys Pro Pro 405 410 415 Tyr Lys Leu Leu Gln Glu Asn Lys Pro Leu Leu Asn Tyr Glu Phe Gln 420 425 430 Glu Lys Glu Lys Glu Ala Ser Cys Asn Trp Asn Leu Val Ala Glu Phe 435 440 445 Ala Cys Glu Tyr Glu Leu Asp Asp His Phe Ile Ile Leu Ala His Tyr 450 455 460 Leu Asp Phe Ala Lys Pro Phe Pro Cys Gln Lys Cys Glu Asn Arg Ser 465 470 475 480 Arg Leu Lys Pro His Lys Ala His Glu Ala His His Ser Asn Ala Lys 485 490 495 Leu Phe Tyr Glu Ser Lys Ser Gln Lys Thr Ile Cys Gln Gln Ala Ala 500 505 510 Asp Thr Val Leu Ala Lys Arg Arg Leu Glu Met Leu Glu Met Thr Arg 515 520 525 Thr Glu Met Leu Cys Lys Lys Phe Lys Lys His Leu Glu Arg Leu Arg 530 535 540 Asp Leu Asp Thr Ile Asp Leu Leu Tyr Tyr Met Gly Gly Val Ala Trp 545 550 555 560 Tyr Cys Cys Leu Phe Glu Glu Phe Glu Lys Lys Leu Gln Lys Ile Ile 565 570 575 Gln Leu Leu Thr Glu Asn Ile Pro Lys Tyr Arg Asn Ile Trp Phe Lys 580 585 590 Gly Pro Ile Asn Ser Gly Lys Thr Ser Phe Ala Ala Ala Leu Ile Asp 595 600 605 Leu Leu Glu Gly Lys Ala Leu Asn Ile Asn Cys Pro Ser Asp Lys Leu 610 615 620 Pro Phe Glu Leu Gly Cys Ala Leu Asp Lys Phe Met Val Val Phe Glu 625 630 635 640 Asp Val Lys Gly Gln Asn Ser Leu Asn Lys Asp Leu Gln Pro Gly Gln 645 650 655 Gly Ile Asn Asn Leu Asp Asn Leu Arg Asp His Leu Asp Gly Ala Val 660 665 670 Ala Val Ser Leu Glu Lys Lys His Val Asn Lys Lys His Gln Ile Phe 675 680 685 Pro Pro Cys Ile Val Thr Ala Asn Asp Tyr Phe Ile Pro Lys Thr Leu 690 695 700 Ile Ala Arg Phe Ser Tyr Thr Leu His Phe Ser Pro Lys Ala Asn Leu 705 710 715 720 Arg Asp Ser Leu Asp Gln Asn Met Glu Ile Arg Lys Arg Arg Ile Leu 725 730 735 Gln Ser Gly Thr Thr Leu Leu Leu Cys Leu Ile Trp Cys Leu Pro Asp 740 745 750 Thr Thr Phe Lys Pro Cys Leu Gln Glu Glu Ile Lys Asn Trp Lys Gln 755 760 765 Ile Leu Gln Ser Glu Ile Ser Tyr Gly Lys Phe Cys Gln Met Ile Glu 770 775 780 Asn Val Glu Ala Gly Gln Asp Pro Leu Leu Asn Ile Leu Ile Glu Glu 785 790 795 800 Glu Gly Pro Glu Glu Thr Glu Glu Thr Gln Asp Ser Gly Thr Phe Ser 805 810 815 Gln Arg Gly Arg Lys Arg Arg Ser Asp Leu Val Leu Asn Arg Lys Glu 820 825 830 Arg Glu Ala Leu Cys Lys Leu Leu Glu Ile Ala Pro Asn Cys Tyr Gly 835 840 845 Asn Ile Pro Leu Met Lys Ala Ala Phe Lys Arg Ser Cys Leu Lys His 850 855 860 His Pro Asn Lys Gly Gly Asn Pro Val Ile Met Met Glu Leu Asn Thr 865 870 875 880 Leu Trp Ser Lys Phe Gln Gln Asn Ile His Lys Leu Arg Ser Asp Phe 885 890 895 Ser Met Phe Asp Glu Val Ser Thr Lys Phe Pro Trp Glu Glu Tyr Gly 900 905 910 Thr Ala Ala Ala Ala Ala Gln Ser Gly Tyr Asn Ala Arg Phe Cys Arg 915 920 925 Gly Pro Gly Cys Met Leu Lys Gln Leu Arg Asp Ser Lys Cys Ala Cys 930 935 940 Ile Ser Cys Lys Leu Ser Arg Gln His Cys Ser Leu Lys Thr Leu Lys 945 950 955 960 Gln Lys Asn Cys Ala Thr Trp Gly Glu Cys Phe Cys Tyr Gln Cys Phe 965 970 975 Ile Leu Trp Phe Gly Phe Pro Pro Thr Trp Glu Ser Phe Asp Trp Trp 980 985 990 Gln Lys Thr Leu Glu Glu Thr Asp Tyr Cys Leu Leu His Leu His Leu 995 1000 1005 Phe <210> 7 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of IgE leader sequence <400> 7 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser

Claims

1. An immunogenic composition comprising a nucleic acid molecule encoding at least one modified Merkel cell polyomavirus (MCV) T antigen, wherein, the T antigen comprises at least one mutation that disrupts at least one oncogenic feature of the native MCV T antigen; wherein the at least one oncogenic feature is selected from the group consisting of binding to phosphatase pp2A, Rb binding, chaperone binding, hVam6p binding, Fbxw7 binding; and wherein (I) the nucleic acid molecule encodes a peptide consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, and / or (II) the nucleic acid molecule consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:

5.

2. The immunogenic composition according to claim 1, wherein, the at least one mutation is a mutation at an amino acid selected from the group consisting of D44, W209, E216, L142, L91, K92, D93, Y94, and M95.

3. The immunogenic composition according to claim 1, wherein, the at least one mutation is selected from the group consisting of D44N mutation, W209A, E216K mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation, and M95A mutation.

4. The immunogenic composition according to claim 1, wherein, the MCV T antigen is selected from the group consisting of large T antigen (LTAg), small t antigen (STAg), and combinations thereof.

5. The immunogenic composition according to claim 1, wherein, the nucleic acid molecule is selected from the group consisting of DNA molecules and RNA molecules.

6. The immunogenic composition according to claim 1, wherein, the nucleic acid molecule consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:

5.

7. The immunogenic composition according to claim 1, wherein, the nucleotide sequence encoding the peptide is operably linked to at least one regulatory sequence selected from the group consisting of a start codon, an IgE leader sequence, and a stop codon.

8. The immunogenic composition according to claim 7, wherein, the nucleic acid molecule encodes a peptide consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, and is operably linked to the amino acid sequence shown in SEQ ID NO:

7.

9. The immunogenic composition according to claim 8, wherein, the nucleic acid molecule consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, and is operably linked to the nucleotide sequence encoding SEQ ID NO:

7.

10. The immunogenic composition according to claim 1, wherein, the nucleic acid molecule is cloned into an expression vector.

11. The immunogenic composition according to claim 1, wherein, the nucleic acid molecule is contained in a virus particle.

12. The immunogenic composition according to claim 1, which further comprises a pharmaceutically acceptable excipient.

13. The immunogenic composition according to claim 1, which further comprises an adjuvant.

14. A nucleic acid molecule encoding a peptide, the peptide consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

6.

15. The nucleic acid molecule according to claim 14, wherein, the nucleic acid molecule is selected from the group consisting of a DNA molecule and an RNA molecule.

16. The nucleic acid molecule according to claim 14, wherein, the nucleic acid molecule consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:

5.

17. The nucleic acid molecule according to claim 14, wherein, the encoded peptide is operably linked to at least one regulatory sequence selected from the group consisting of a start codon, an IgE leader sequence, and a stop codon.

18. The nucleic acid molecule according to claim 17, wherein, the nucleic acid molecule encodes a peptide consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, and is operably linked to the amino acid sequence shown in SEQ ID NO:

7.

19. The nucleic acid molecule according to claim 18, wherein, the nucleic acid molecule consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, and is operably linked to the nucleotide sequence encoding SEQ ID NO:

7.

20. The nucleic acid molecule according to claim 14, wherein, the nucleic acid molecule is cloned into an expression vector.

21. The nucleic acid molecule according to claim 14, wherein, the nucleic acid molecule is contained in a virus particle.

22. An immunogenic composition comprising a peptide, wherein, the peptide consists of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

6.

23. A peptide consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

6.

24. Use of the immunogenic composition according to any one of claims 1 to 13, the nucleic acid molecule according to any one of claims 14 to 21, or the immunogenic composition according to claim 22 in the preparation of a medicament for inducing an immune response against an MCVT antigen in a subject in need thereof, comprising administering the immunogenic composition to the subject.

25. The use according to claim 24, wherein, the administration comprises at least one of electroporation and injection.

26. Use of an immunogenic composition according to any one of claims 1 to 13, a nucleic acid molecule according to any one of claims 14 to 21, or an immunogenic composition according to claim 22 in the preparation of a medicament for treating or preventing an MCV-related pathology in a subject in need thereof, comprising administering the immunogenic composition to the subject.

27. The use according to claim 26, wherein, the administration comprises at least one of electroporation and injection.

28. The use according to claim 26, wherein, the MCV-related pathology is at least one of MCV infection and Merkel cell carcinoma.

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