EA045600B1 - CONSTRUCTS OF NUCLEIC ACIDS AND VACCINES FROM LARGE AND SMALL T-ANTIGENS OF MERKEL CELL POLIOMAVIRUS AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross-references related to the application

This application claims priority to US Provisional Application No. 62/619161, filed January 19, 2018, the contents of which are incorporated herein by reference in their entirety.

Technical field of the invention

The present invention relates to vaccines intended for inducing immune responses and treating individuals infected with Merkel cell polyomavirus (MCV) and/or for treating or preventing Merkel cell carcinoma (MCC). The present invention relates to the MCV large T antigen (LTAg) and small t antigen (STAg) consensus oncoproteins, and the nucleic acid molecules that encode them.

State of the art

Merkel cell polyomavirus (MCV) has received recent attention because it is associated with Merkel cell carcinoma (MCC), an aggressive human skin cancer. In the United States, approximately 1,500 new cases of MSS are diagnosed per year, and the mortality rate for subjects with MSS remains at 46%. MCC kills more patients than cutaneous T-cell lymphoma and chronic myelogenous leukemia. The majority (approximately 75%) of MCCs contain clonally integrated viral DNA and express viral T-antigen transcripts and protein.

Currently, there are no clinical trials of vaccines against MCC. Therefore, there is a need in the art for therapeutic vaccines against MCV and MCC. The present invention fills this missed need.

Disclosure of the Invention

In one embodiment, the invention relates to an immunogenic composition comprising a nucleic acid molecule encoding at least one modified Merkel cell polyomavirus T antigen, wherein the T antigen contains at least one mutation that disrupts at least one oncogenic function of the native T MCV antigen. In one embodiment, the at least one oncogenic signature is binding to at least one of CR1, DnaJ, pp2A phosphatase, Rb, ATPase activity, helicase activity, chaperone protein, hVam6p, Fbxw7, replication point binding, and transformation.

In one embodiment of the invention, the at least one mutation is a mutation of an amino acid of at least one of the following:

D44, W209, E216, L142, L91, k92, D93, Y94 or M95. In one embodiment, the at least one mutation is at least one of D44N, W209A, E216K, L142A, L91A, K92A, D93A, Y94A, or M95A mutations. In one embodiment, the modified MCV T antigen contains at least one of the D44N mutation, the W209A mutation, or the E216K mutation. In one embodiment, the modified MCV T antigen contains the D44N mutation, the W209A mutation, and the E216K mutation.

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

In one embodiment of the invention, the nucleic acid molecule encodes a peptide comprising: a) an amino acid sequence identical at least about 90% over the entire length of the amino acid sequence to at least one of SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, b) an immunogenic fragment that is at least approximately 90% identical 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.

In one embodiment of the invention, the nucleic acid molecule is a DNA molecule or an RNA molecule.

In one embodiment of the invention, the nucleic acid molecule contains a nucleotide sequence of at least one of: a) a nucleotide sequence that is at least about 90% identical over the entire length of the nucleotide sequence to 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 that is at least about 90% identical to at least 60% of the nucleotide sequence with 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.

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

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In one embodiment of the invention, the nucleic acid molecule encodes a peptide comprising an amino acid sequence of at least one of a) an amino acid sequence identical in at least about 90% along the entire length of the amino acid sequence to at least one of SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, b) an immunogenic fragment that is at least approximately 90% identical to at least 60% 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 with the amino acid sequence specified in SEQ ID No. 7.

In one embodiment of the invention, the nucleic acid molecule contains a nucleotide sequence of at least one of: a) a nucleotide sequence identical in at least about 90% along the entire length to 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 that is at least about 90% identical 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) nucleotide sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5, or d) an immunogenic fragment of the nucleotide sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5, operably linked to the nucleotide sequence encoding SEQ ID No. 7 .

In one embodiment of the invention, the nucleic acid molecule comprises an expression vector.

In one embodiment of the invention, the nucleic acid molecule is included in the viral particle.

In one embodiment of the invention, the immunogenic composition further comprises a pharmaceutically acceptable excipient.

In one embodiment of the invention, the immunogenic composition further comprises an adjuvant.

In one embodiment, the invention provides a nucleic acid molecule encoding a peptide comprising an amino acid sequence of at least one of: a) an amino acid sequence identical in at least about 90% along the entire length to 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 identical in at least about 90% to at least 60% amino acid sequence to 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.

In one embodiment of the invention, the nucleic acid molecule is a DNA molecule or an RNA molecule.

In one embodiment of the invention, the nucleic acid molecule contains a nucleotide sequence of at least one of: a) a nucleotide sequence that is at least about 90% identical throughout the entire 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 that is at least approximately 90% identical 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 SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5.

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

In one embodiment of the invention, the nucleic acid molecule encodes a peptide comprising an amino acid sequence of at least one of: a) an amino acid sequence identical in at least about 90% along the entire length to 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 identical in at least about 90% to at least 60% amino acid sequence to 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 with the amino acid sequence specified in SEQ ID No. 7.

In one embodiment of the invention, the nucleic acid molecule contains a nucleotide sequence of at least one of: a) a nucleotide sequence, at least

- 2 045600 at least approximately 90% identical over the entire 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 the nucleotide sequence that is at least approximately 90% identical in 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 the nucleotide sequence of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, operably linked to the nucleotide sequence encoding SEQ ID No. 7.

In one embodiment of the invention, the nucleic acid molecule comprises an expression vector.

In one embodiment of the invention, the nucleic acid molecule is included in the viral particle.

In one embodiment, the invention relates to an immunogenic composition containing a peptide, wherein the peptide comprises an amino acid sequence of at least one of: a) an amino acid sequence identical in at least about 90% along the entire length to the amino acid sequence of at least one from SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, b) an immunogenic fragment of an amino acid sequence that is at least approximately 90% identical 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 amino acid sequence fragment comprising at least 60% of the amino acid sequence of SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6.

In one embodiment, the invention provides a peptide comprising an amino acid sequence of at least one of: a) an amino acid sequence identical to at least about 90% along the entire 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 of an amino acid sequence that is at least approximately 90% identical 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 amino acid sequence fragment comprising at least 60% of the amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6.

In one embodiment, the invention relates to a method of inducing an immune response against MCV T antigen in a subject in need thereof, said method comprising administering an immunogenic composition comprising a nucleic acid molecule encoding a modified Merkel cell polyomavirus (MCV) T antigen, wherein The T antigen contains at least one mutation that disrupts at least one oncogenic feature of the subject's native MCV T antigen.

In one embodiment of the invention, the method of administration includes at least one of electroporation or injection.

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

In one embodiment of the invention, the method of administration includes at least one of the following: electroporation or injection.

In one embodiment of the invention, the pathology associated with MCV is at least one of the following: MCV infection or Merkel cell carcinoma.

Brief description of drawings

In fig. 1 including FIG. 1A and 1B show circuit diagrams of LTAg and STAg. In fig. Figure 1A shows the oncogenic signatures of LTAg and STAg. Figure 1B shows that the LTAg and STAg nucleic acid vaccine designs include several mutations to disrupt oncogenic traits. * D44N blocks binding to chaperone proteins; * W209A - blocks binding to hVam6p; * E216K - blocks binding to Rb and prevents transformation; * L142 A-blocks binding to PP2A; * 91-95LKDYM ^ АААА - blocks binding to Fbxw7 and prevents transformation.

In fig. 2, including FIG. 2A and 2B show schematic diagrams of consensus LTAg and STAg. Fig. 2A depicts a diagram of the LTAg consensus sequence constructed from all LTAg sequences available at NCBI. In fig. 2B shows a diagram of the STAg consensus sequence constructed from all available STAg sequences from NCBI. These antigenic sequences were synthesized and cloned into a mammalian expression plasmid, creating plasmid constructs

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DNA for expression of synthetic consensus antigens in vivo.

Fig. 3 depicts examples of experimental data demonstrating consensus MCC LTAg expression in vitro. Consensus MCC STAg expression was not detected due to the lack of effective anti-STAg antibodies.

Fig. 4, including FIG. 4A and 4B depict examples of experimental data demonstrating the induction of an immune response following vaccination with LTAg and STAg alone or in combination. Fig. 4A depicts the experimental design. Mice received plasmid DNA followed by intramuscular electroporation on day 0, day 14, and day 28. After a week, splenocytes were collected for analysis. Four groups of mice were vaccinated: group 1 - pVax-empty control vector; group 2 LTAg vaccine; Group 3 - STAg vaccine; group 4 - LTAg and STAg vaccines simultaneously. In fig. 4B depicts experimental data showing the induction of an immune response after vaccination with LTAg and STAg alone or in combination, and the lack of induction after vaccination with an empty control vector (pVax). To perform these experiments, peptides were matched to the corresponding sequences without inactivating mutations.

In fig. 5, including FIG. 5A and 5B provide examples of experimental data characterizing the immunodominant epitopes of LTAg and STAg. Fig. 5A depicts immunodominant epitopes for LTAg vaccination. Fig. 5B depicts immunodominant epitopes for STAg vaccination.

In fig. Figure 6 presents the results of an analysis of the degree of processing of large T-antigens MCC in samples of human Merkel cell carcinoma. Data were obtained from 42 large T antigen sequences downloaded from GenBank.

In fig. 7, including FIG. 7A-7F provide examples of experimental data demonstrating the levels of CD4+ and CD8+ T cell cytokine responses following vaccination and stimulation with LTAg peptides for 5 hours. In fig. 7A shows the levels of CD8+ T cell responses against IFNu. In fig. 7B shows the levels of CD8+ T cell response against TNFa. In fig. 7C shows the levels of CD8+T cell response against IL-2. In fig. 7D shows the levels of CD4+ T cell responses against IFNu. In fig. 7E shows the levels of CD4+ T cell response against TNFa. In fig. 7F shows the levels of CD4+ T cell responses against IL-2.

Fig. 8 depicts examples of experimental data demonstrating that LTAg vaccination induces robust polyfunctional CD8 T cells.

Fig. 9 depicts examples of experimental data demonstrating that LTAg vaccination induces robust polyfunctional CD4 T cells.

Fig. 10 depicts examples of experimental data demonstrating that LTAg vaccination induces CD8 T cells with cytotoxic potential coexpressing CD 107a, IFNu and T-bet.

Fig. 11 depicts examples of experimental data demonstrating that antigen vaccines based on large and small T antigens generate humoral responses, confirmed using mouse serum as a source of primary antibodies.

Fig. 12 depicts examples of experimental data demonstrating that the LTAg vaccine induces robust immune responses in genetically diverse outbred CD-1 mice.

Fig. 13 depicts examples of experimental data demonstrating that the STAg vaccine induces immune responses in genetically diverse outbred CD-1 mice.

In fig. 14, including FIG. 14A-14F provide examples of experimental data demonstrating the levels of CD4+H CD8+Ha T cell cytokine responses following vaccination in outbred mice with CD-1 and stimulation with LTAg peptides for 5 hours. In fig. 14A shows the levels of CD8+ T cell response against IFNu. In fig. 14B shows the levels of CD8+ T cell response against TNFa. In fig. 14C shows the levels of CD8+ T cell response against IL-2. In fig. 14D shows the levels of CD4+T cell response against IFNa. In fig. 14E shows the levels of CD4+T cell response against TNFa. In fig. 14F shows the levels of CD4+ T cell response against IL-2.

Fig. 15 containing FIG. 15A-15F provide examples of experimental data demonstrating the levels of CD4+H CD8+ T cell cytokine responses following vaccination in outbred mice with CD-1 and stimulation with STAg peptides for 5 hours. In fig. 15A shows the levels of CD8+ T cell response against IFNu. In fig. 15B shows the levels of CD8+ T cell response against TNFa. In fig. 15C shows the levels of CD8+ T cell response against IL-2. In fig. 15D shows the levels of CD4+ T cell response against IFNu. Fig. 15E depicts levels of CD4+ T cell response against TNFa. In fig. 15F shows the levels of CD4+ T cell response against IL-2.

Detailed description

Merkel cell polyomavirus (MCV) infection is associated with Merkel cell carcinoma (MCC) and currently has a 46% mortality rate.

In one embodiment, the invention includes a nucleic acid vaccine against MCV and MCC. In one embodiment of the invention, the vaccine contains a plasmid encoding the MCV consensus T antigen. In one embodiment, the MCV consensus T antigen is a large T antigen (LTAg). In one embodiment of the invention, the MCV consensus T antigen is a small t antigen (STAg). In one embodiment, the MCV consensus T antigens further contain mutations that disrupt the oncogenic properties of the native T antigens. As a vaccine candidate, the advanced DNA-based platform provides many advantages for genetic optimization and delivery methods. As such, each MCV T antigen can be genetically optimized, subcloned into modified mammalian expression vectors, and then delivered using in vivo electroporation (IE).

Vaccination in preclinical studies in rodents was potent because vaccination with synthetic consensus MCV T-antigen constructs generated robust immune responses.

In some embodiments, the strategy includes a coding sequence for a synthetic consensus MCV T antigen. The coding sequence for LTAg and STAg is provided. In some embodiments, the strategy includes coding sequences for a single synthetic consensus MCV T antigen. In some embodiments, the strategy uses coding sequences for multiple synthetic consensus MCV T antigens.

As vaccine candidates, DNA vaccines offer many advantages, including rapid and inexpensive scale-up of production, stability at room temperature, and ease of transportation. All of the above advantages improve this platform from an economic and geographical point of view. Due to the synthetic nature of plasmids, antigenic sequences can be quickly and easily modified in response to emerging strains and/or expanded to include additional vaccine components.

Optimization of plasmid DNA vectors and the antigen genes they encode has led to increased immunogenicity in vivo. Cellular uptake and subsequent antigen expression are greatly enhanced when highly concentrated plasmid vaccine compositions are administered via in vivo electroporation, a technology that uses short, square-wave electrical pulses at the site of vaccination to deliver plasmids into transiently permeabilized cells. Theoretically, a cocktail of plasmid DNAs could be assembled to direct a highly specialized immune response against any number of variable antigens. In addition, immunity can be targeted by co-delivery of plasmid-based molecular adjuvants encoding species-specific cytokine genes, as well as consensus engineering of antigen amino acid sequences to bias vaccine-induced immunity to specific strains.

1. Definitions.

As used herein, all technical and scientific terms have the same meaning as commonly known to those skilled in the art unless otherwise noted. In the event of any conflict, this document, including definitions, shall control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may 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 are not intended to limit the invention.

The terms contains, includes, has, has, may, consists, and variations thereof, as used herein, are non-limiting transitional phrases, terms or words that do not exclude possible additional actions or structures. . Singular forms imply plurals unless the context clearly indicates otherwise. The present disclosure also contemplates other embodiments of the invention comprising, consisting of, and consisting substantially of the embodiments or elements provided herein, whether or not expressly set forth.

An adjuvant, as used herein, can mean any molecule added to nucleic acid vaccines to enhance the antigenicity of the vaccine.

Antibody may mean an antibody belonging to the classes IgG, IgM, IgA, IgD or IgE or fragments or derivatives thereof, including Fab, F(ab') ₂ , Fd and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity to the desired epitope or sequence derived therefrom.

As used herein, the terms antibody fragment or antibody fragment are used interchangeably and refer to the portion of an intact antibody containing an antigen binding site or variable region. This portion does not include the heavy chain constant domains (ie, CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of an intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single chain Fv molecules (scFv), single chain polypeptides containing

- 5 045600 only one light chain variable domain, single chain polypeptides containing three CDRs of a light chain variable domain, single chain polypeptides containing only one heavy chain variable region, and single chain polypeptides containing, but not limited to, three CDRs of a heavy chain variable region.

Antigen refers to proteins that have the ability to generate an immune response in the host. The antigen can be recognized and bound by the antibody. The antigen can come from the body or from the external environment.

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

The terms complementary or complementarity, as used herein, can mean that a nucleic acid can form pairs with other nucleotides, nucleotide analogues, or nucleic acid molecules according to the Watson-Crick rule (eg, A-T/U and C-G) or Hoogsteen.

Consensus or consensus sequence, as used herein, can mean a synthetic nucleotide sequence or a corresponding polypeptide sequence constructed based on multiple sequence alignment analysis (eg, multiple sequences of a particular viral antigen).

The term direct current, as used herein, describes the current that is sensed or experienced by the tissue or cells defining said tissue for the duration of an electrical impulse delivered to the same tissue. The electrical pulse is supplied from the electroporation devices described in this document. The current density remains constant in said tissue throughout the duration of exposure to the electrical pulse because the electroporation device proposed herein has a feedback element, preferably having instantaneous feedback. The feedback element allows the resistance of tissue (or cells) to be measured throughout the duration of the electrical pulse and to cause a change in the electrical output of the electroporation device (for example, increase the voltage) so that the current density in the same tissue remains constant throughout the duration. exposure to an electrical pulse (on the order of microseconds) and from pulse to pulse. In some embodiments of the invention, the feedback element comprises a controller.

The terms current feedback or feedback, as used herein, may be used interchangeably and may refer to the active response of the proposed electroporation devices, which involves sensing the current in the tissue between the electrodes and accordingly changing the energy supplied by the EC device to maintain the current at a constant level. This constant level is set by the user before pulsing or electrical stimulation begins. Feedback can be provided by an electroporator component, such as an electroporator controller, because its electrical circuitry is capable of continuously monitoring the tissue current between the electrodes and comparing this monitored current (or tissue current) to a set current and continuously adjusting the output energy to maintain the controlled current at given levels. The feedback loop can be instantaneous because it is analogous to closed-loop feedback.

The term decentralized current, as used herein, can mean the pattern of electrical currents delivered from the various needle electrode arrays of the electroporators described herein, in which the patterns minimize or preferentially eliminate the occurrence of thermal stress associated with electroporation in any area of the tissue being electroporated.

The terms electroporation, electropermeabilization, or electrokinetic enhancement (EP), as used herein, are used interchangeably and can refer to the application of a transmembrane electric field pulse to induce microscopic holes (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions and water to pass from one side of the cell membrane to the other.

An endogenous antibody, as used herein, may refer to an antibody produced by a subject to which an effective dose of an antigen is administered to induce a humoral immune response.

A feedback mechanism, as used herein, may refer to a process performed by software or hardware (or firmware) in which the impedance of the desired tissue is obtained and compared (before, during and/or after delivery

- 6 045600 pulse of energy) with the current value, preferably current, and regulate the pulse of energy delivered to achieve the set value. The feedback mechanism can be implemented as an analog closed loop.

A fragment can mean a percentage of the total length of a polypeptide sequence or a nucleotide sequence. Fragments may contain 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 percent of the total length of the original nucleotide sequence or amino acid sequence, or a variant thereof.

As used herein, the term genetic construct refers to DNA or RNA molecules that contain a nucleotide sequence encoding a protein, such as an antibody. A genetic construct can also refer to a DNA molecule that transcribes RNA. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including promoter and polyadenylation signals, capable of directing expression in cells of the subject to which the nucleic acid molecule is applied. As used herein, the term expressible form refers to a gene construct that contains necessary regulatory elements operably linked to a protein coding sequence in such a manner that the coding sequence will be expressed when found in a cell of a subject.

As used herein, the terms identical or identity, with respect to two or more nucleic acid or polypeptide sequences, can mean that the sequences have a certain percentage of residues that are the same in a specified region. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in a specified region, determining the number of positions at which the same residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more step breaks, and the specified comparison region includes only one sequence, the remainder of one sequence is included in the denominator but not the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

The term impedance, in the context of this document, can be used when considering in detail the feedback mechanism, where, in accordance with Ohm's law, impedance can be converted to the current value of the current, while allowing comparisons to be made with the specified current value

An immune response, as used herein, may mean the activation of the immune system of a host, for example a mammal, in response to administration of one or more consensus antigens by the proposed vaccines. The immune response can be in the form of a cellular or humoral response, or both.

The terms nucleic acid or oligonucleotide or polynucleotide, as used herein, can mean at least two nucleotides covalently linked to each other. The image of one strand also determines the sequence of the complementary strand. Thus, the nucleic acid also includes the complementary strand of the illustrated single strand. Many nucleic acid variants can be used for the same purpose as the given nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. The single strand also includes a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, the nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, either genomic, cDNA, RNA or hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids can be produced by chemical synthesis methods or by recombinant methods.

Functionally linked, as used herein, means that the expression of a gene is under the control of a promoter to which it is spatially linked. The promoter can be located at the 5' (upstream) or 3' (downstream) end of the controlled gene. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls and comes from. As is already known in the art, changing this distance can be accomplished without loss of promoter function.

The terms peptide, protein or polypeptide, as used herein, may refer to a linked sequence of amino acids and may be natural, synthetic or modified, or a combination of natural and synthetic.

A promoter, as used herein, can mean a synthetic or naturally occurring molecule that is capable of imparting, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or alter the spatial expression and/or temporal expression of a nucleic acid in a cell. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. The promoter can be obtained from sources including viral, bacterial, fungal, plant, insect and animal. A promoter may regulate expression of a gene component constitutively or differentially with respect to the cell, tissue or organ in which expression occurs, relative to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions or excitatory agents. Typical examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter.

Signal peptide and leader sequence are used interchangeably herein and refer to an amino acid sequence that can be linked to an amino group at the end of a protein set forth herein. Signal peptides/leader sequences typically direct protein localization. As used herein, signal peptides/leader sequences optimally facilitate secretion of a protein from the cell in which it is produced. When secreted from the cell, signal peptides/leader sequences are often cleaved from a remnant of the protein, commonly referred to as the mature protein. Signal peptides/leader sequences are associated with the N-terminus of the protein.

Stringent hybridization conditions, as used herein, may mean conditions under which a first nucleic acid molecule (eg, a probe) will hybridize to a second nucleic acid molecule (eg, a target), for example, in a complex mixture of nucleic acids. The stringent conditions depend on the sequence and will be different in different circumstances. Severe conditions can be selected to be approximately 5-10°C lower than the melting temperature (Tm) for a particular sequence at a particular pH ionic strength. Tm can be the temperature (at a certain ionic strength, pH and nuclein concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (since the target sequences are present in excess, at Tm, 50% of the probes are equilibrium bound) . Under severe conditions, the salt concentration may be less than about 1.0 M sodium ion, for example, a concentration of about 0.01-1.0 M sodium ion (or other salts) at a pH of 7.0-8.3, and a temperature of less than at least about 30°C for short probes (eg, about 10-50 nucleotides) and at least about 60°C for long probes (eg, greater than about 50 nucleotides). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. In selective or specific hybridization, the positive signal can be at least 2-10 times the background hybridization. Example stringent hybridization conditions include the following: 50% formamide, 5x standard saline and 1% SDS, incubated at 42°C or 5x standard saline, 1% SDS, incubated at 65°C, washed in 0.2x standard saline and 0.1% SDS at 65°C.

The terms subject and patient are used interchangeably herein and can refer to any vertebrate animal, including, without limitation, mammals (eg, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog , rat and mouse, nonhuman primate (eg, a monkey such as a cynomolgus or rhesus, a chimpanzee, etc., and a human).In some embodiments, the subject may or may not be a human.

The term substantially complementary, as used herein, may mean that the first sequence is 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 to the complement of the second sequence covering the 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, or that two sequences hybridize at harsh hybridization conditions.

The term substantially identical, as used herein, may mean that the first and second sequences are 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 covering an area 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 relative to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

The terms treat or treat, in the context of this document, may mean protecting the subject

- 8 045600 from a disease through means of preventing, suppressing, inhibiting or completely eliminating the disease. Prevention of the disease involves administering the vaccine described in the present invention to the subject before the onset of the disease. Disease suppression involves administering a vaccine described in the present invention to a subject after the induction of the disease but before its clinical manifestation. Disease suppression involves administering a vaccine described in the present invention to a subject after clinical manifestation of the disease.

As used herein, the term variant, with respect to a nucleic acid, may mean: (i) a portion or fragment of said nucleotide sequence; (ii) complement of the reference nucleotide sequence or part thereof; (iii) a nucleic acid that is substantially identical to the specified nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with said nucleic acid, its complement, or a sequence substantially identical to it.

As used herein, the term variant, with respect to a peptide or polypeptide, means a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative amino acid substitution, but retains at least one biological activity. A variant may also mean a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative amino acid substitution, that is, the replacement of an amino acid with another amino acid with similar properties (eg, hydrophilicity, degree and distribution of charged regions), as known in the art, usually involves minor changes. These minor changes can be identified, in part, by taking into account the hydropathic amino acid index, which is known in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be replaced with each other while maintaining protein function. In one aspect, amino acids having hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that preserve the biological function of proteins. Considering the hydrophilicity of amino acids in the context of a peptide allows one to calculate the highest local average hydrophilicity of that peptide, which is a useful evaluation criterion that, as mentioned, correlates well with antigenicity and immunogenicity. US Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values may result in the peptides retaining the biological activity, such as immunogenicity, implied by the art. Amino acids having hydrophilicity values within ± 2 of each other can be used for replacement. The hydrophobicity index and hydrophilicity value of amino acids are influenced by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are thought to depend on the relative similarity of the amino acids and, in particular, on the side chains of those amino acids, as reflected in hydrophobicity, hydrophilicity, charge, size, and other properties.

A variant may be a nucleic acid sequence that is substantially identical throughout the entire length of the complete sequence of a gene or fragment thereof. The nucleic acid sequence may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical throughout gene sequence or its fragment. A variant may be an amino acid sequence that is substantially identical over the entire length of an amino acid sequence or a fragment thereof. The amino acid sequence may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical along the entire length of the amino acid sequence or fragment thereof.

The term vector, as used herein, can mean a nucleic acid molecule containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

To list numeric ranges, this document treats each number in between with the same degree of precision. For example, for the number range 6-9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range 6.0-7.0 the numbers 6.0, 6.1, 6.2, 6.3, 6 are considered ,4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

2. Description.

The invention describes an optimized consensus sequence encoding the MCV Tantigen. 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 contain epitope(s) that make it particularly effective as a

- 9 045600 immunogen against which an immune response can be induced.

The optimized consensus sequence may be a consensus sequence derived from two or more MCV T antigens. The optimized consensus sequence may contain the consensus sequence and/or modification(s) to improve expression. Modification may include codon optimization, RNA optimization, addition of a Kozak sequence to increase translation initiation, and/or addition of an immunoglobulin leader sequence to increase immunogenicity. The MCV T antigen encoded by the optimized consensus sequence may comprise a signal peptide, such as, for example, but not limited to, an immunoglobulin signal peptide, namely, immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the antigen encoded by the optimized consensus sequence may contain a hemagglutinin (HA) tag. An antigen encoded by an optimized consensus sequence can be designed to elicit stronger cellular and/or humoral immune responses compared to the corresponding non-optimized antigen.

Provided herein are MCV T antigens that can be used to induce immunity against MCV in genetically diverse MCV-infected subjects. In one embodiment, the present invention provides an immunogenic composition comprising one or more nucleic acid molecules capable of generating an immune response against MCV T antigen in a mammal. The present invention also provides isolated nucleic acid molecules capable of generating an immune response against MCV T antigen in a mammal. In one embodiment of the invention, the nucleic acid molecule contains an optimized nucleotide sequence encoding the MCV consensus T antigen.

In one embodiment, the MCV T antigens are modified to reduce or destroy at least one oncogenic feature of the native MCV T antigen. In various embodiments, the MCV T antigens are modified to reduce or disrupt at least one of CR1 binding, DnaJ binding, pp2A phosphatase binding, Rb binding, ATPase activity, helicase activity, chaperone protein binding, hVam6p binding, Fbxw7 binding, original binding and transformation. In one embodiment, the MCV T antigen contains at least one mutation in D44, W209, E216, L142, L91, K92, D93, Y94, or M95 relative to the native T antigen sequence. In one embodiment, the MCV T antigen contains at least one of the following mutations: D44N mutation, W209A mutation, E216K mutation, L142A mutation, L91A mutation, K92A mutation, D93A mutation, Y94A mutation, and M95A mutation. In one embodiment, the MCV LTAg contains at least one of the D44N mutation, the W209A mutation, and the E216K mutation. In one embodiment, the MCV LTAg contains the D44N mutation, the W209A mutation, and the E216K mutation. In one embodiment, the MCV STAg contains 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 contains the D44N mutation, the L142A mutation, the L91A mutation, the K92A mutation, the D93A mutation, the Y94A mutation, and the M95A mutation.

The consensus amino acid sequences of the MCV T antigens include SEQ ID No. 2, SEQ ID No. 4, variants thereof, and fragments of SEQ ID No. 2, SEQ ID No. 4, and variants thereof. An example amino acid sequence of a modified synthetic consensus LTAg MCV is provided as SEQ ID NO:2. An example amino acid sequence of a modified synthetic consensus MCV STAg is provided as SEQ ID NO:2.

In one embodiment, the invention provides compositions comprising a nucleic acid molecule comprising a nucleotide sequence that encodes a modified synthetic MCV consensus T antigen. In one embodiment, the nucleotide sequence that encodes the modified synthetic MCV consensus LTAg is provided as SEQ ID No. 1 encoding SEQ ID No. 2. In one embodiment, the nucleotide sequence that encodes the modified synthetic consensus MCV STAg is provided as SEQ ID No. 3 encoding SEQ ID No. 4.

In various embodiments, the invention provides compositions containing a combination of a modified LTAg and a modified STAg, or one or more nucleic acid molecules encoding them. The compositions may contain multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids.

The compositions may contain a single nucleic acid molecule, such as a plasmid, which contains the coding sequence for multiple MCV consensus T antigens. In one embodiment of the invention, the compositions may contain a single nucleic acid molecule comprising nucleotide sequences encoding MCV LTAg and MCV STAg. In one embodiment of the invention, each coding sequence of each consensus MCV Tantigen is located on a separate plasmid.

Accordingly, compositions that contain one or more nucleotide sequences

- 10,045,600 articles encoding multiple MCV consensus T antigens can be contained in a single plasmid. In one embodiment of the invention, the composition contains a single plasmid that encodes MCV LTAg and MCV STAg under the influence of a single promoter. In such an embodiment, the MCV LTAg coding sequence and the MCV STAg coding sequence may be linked by a fusion peptide sequence, such as a furin cleavage sequence. An example amino acid sequence of one construct containing 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 of the invention, a single nucleotide sequence encoding a modified synthetic consensus MCV LTAg and MCV STAg linked by a furin cleavage sequence is provided as SEQ ID No. 5 encoding SEQ ID No. 6.

In one embodiment of the invention, the optimized consensus-encoded MCV T antigen is operably linked to one or more regulatory elements. In one embodiment, the regulatory element is a leader sequence. In one embodiment of the invention, the leader sequence is an IgE leader sequence. In one embodiment of the invention, the IgE leader sequence has the amino acid sequence set forth in SEQ ID No. 7. Therefore, in one embodiment, the invention provides an amino acid sequence set forth in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6 operably related to the amino acid sequence set forth in SEQ ID No. 7. In one embodiment, the invention provides a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6 operably linked to the amino acid sequence set forth in SEQ ID No. 7.

In one embodiment of the invention, the regulatory element is a start codon. Therefore, in one embodiment, the invention provides a nucleotide sequence set forth in SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, or a fragment or homologue thereof operably linked to a nucleotide sequence containing a 5' start codon end. In one embodiment, the invention provides an amino acid sequence set forth in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, or a fragment or homolog thereof operably linked to the amino acid encoded by the start codon (e.g., methionine) at N -end.

In one embodiment, the regulatory element is at least one stop codon. Therefore, in one embodiment, the invention provides a nucleotide sequence set forth in SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, or a fragment or homologue thereof operably linked to a nucleotide sequence comprising at least one stop -codon at the 3' end. In one embodiment, to improve the efficiency of translation termination, the nucleotide sequence is operably linked to two stop codons.

In one embodiment, the nucleic acid molecule may encode a peptide having the amino acid sequence set forth in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6. In one embodiment of the invention, the nucleic acid molecule contains the nucleotide sequence set forth in SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5. In some embodiments, the sequence may be a nucleotide sequence identical in 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% over the entire length of the nucleotide sequence presented in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5. In other embodiments, the sequence may be a nucleotide sequence encoding an amino acid sequence identical in 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% over the entire length of the amino acid sequence represented in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6.

In some embodiments, the nucleic acid molecule contains an RNA sequence that is a transcript of a DNA sequence identical to 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% over the entire length of the nucleotide sequence specified in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5. In some embodiments, the nucleic acid molecule contains an RNA sequence encoding an amino acid sequence that is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 identical to , 94, 95, 96, 97, 98, 99, or 100% over the entire length of the amino acid sequence represented in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6.

In some embodiments, the nucleic acid molecule may comprise a nucleotide sequence encoding a full-length MCV consensus T antigen. The nucleic acid molecules may contain a sequence encoding SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6. The nucleic acid molecules may contain the nucleotide sequence of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5. The nucleic acid molecule may optionally contain coding sequences that encode a signal peptide, such as, for example

- 11 045600 measures, IgE or IgG signal peptide.

The MCV consensus 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 identical to 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% over the entire length of the amino acid sequence presented in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6.

Immunogenic fragments of SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6 may be provided. The immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 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 fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain a leader sequence.

Immunogenic protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6 may be provided. Such immunogenic fragments may constitute 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 proteins that are 95% homologous to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences provided herein. Some embodiments provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences provided herein. In some embodiments, the immunogenic fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain a leader sequence.

In one embodiment, the immunogenic nucleic acid molecule fragment encodes at least one immunodominant or subimmunodominant epitope of a full-length optimized MCV consensus T antigen.

Some embodiments of the invention provide immunogenic fragments of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5 comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 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 fragments may be at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5. In some embodiments, the immunogenic fragments include sequences encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence.

In one embodiment, the nucleic acid molecule contains a sequence that is at least 90% homologous to SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5.

In one embodiment of the invention, the nucleic acid molecule contains an RNA sequence encoding the consensus sequence of the MCV T antigen described herein. For example, the nucleic acids may comprise an RNA sequence encoding one or more of SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, a variant thereof, a fragment thereof, or any combination thereof.

In some embodiments, the nucleic acid molecule includes a sequence encoding an MCV T antigen without an 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.

The nucleic acid molecule may further include a polyadenylation sequence appended at the C-terminus of the coding sequence. In one embodiment

- 12 045600 invention, the nucleic acid molecule is codon-optimized.

Vaccines and immunogenic compositions.

Immunogenic compositions are provided, such as vaccines containing an optimized consensus sequence, an optimized consensus-encoded antigen, a fragment thereof, a variant thereof, or a combination thereof. The immunogenic composition can significantly induce an immune response in a subject to which the immunogenic composition against MCV T antigen is administered. The vaccine may contain multiple nucleic acid molecules or combinations thereof. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

The immunogenic composition may be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination vaccine. The vaccine may include an optimized consensus nucleotide sequence encoding an antigen. The nucleotide sequence may be DNA, RNA, a DNA variant thereof, a fragment thereof, or a combination thereof. The nucleotide sequence may also include additional sequences encoding linker, leader or tag sequences linked to the antigen by peptide bond. The peptide vaccine may include an antigen, a variant thereof, a fragment thereof, or a combination thereof. The combination of DNA and peptide vaccine may include the optimized consensus nucleotide sequence described above and the encoded antigen.

The vaccine may be a DNA vaccine. DNA vaccines are disclosed in US 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 5,676,954, references to which are incorporated herein in their entirety. The DNA vaccine may additionally contain elements or reagents that prevent its integration into the chromosome.

The vaccine may be RNA of one or more MCV T antigens. An RNA vaccine can be introduced into a cell.

The vaccine may be an attenuated live vaccine, a vaccine using recombinant antigen delivery vectors, a subunit vaccine, and a glycoprotein vaccine, for example, the vaccine described in US Pat. 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; 5.3893.68; 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, but is not limited to.

The vaccine of the present invention may meet the properties recommended for effective vaccines, such as, for example, safety, meaning that the vaccine does not cause disease or death by itself; protection from diseases; induction of protective T-cell response; and ensuring ease of use, few side effects, biological stability and low cost per dose.

Provided herein is an immunogenic composition capable of generating an immune response against MCV in a mammal. The immunogenic composition may contain any of the plasmids discussed above. The immunogenic composition may contain multiple plasmids or combinations thereof. The immunogenic composition may be provided to induce a therapeutic or prophylactic immune response.

Immunogenic compositions can be used to deliver nucleic acid molecules encoding one or more MCV consensus T antigens. Preferably, the immunogenic compositions are compositions containing plasmids.

The immunogenic composition may further contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as carriers, adjuvants, vehicles or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LIS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known agents that facilitate transfection.

The transfection facilitating agent is a polyanion, polycation including poly-Lglutamate (LGS) or lipid. The transfection facilitator is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the immunogenic composition at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LIS analog including monophosphoryl lipid A, muramyl peptides, quinone analogues and vesicles such as squalene and squalene, and hyaluronic acid may also used in combination with a genetic construct. In some embodiments, the immunogenic compositions may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes, or other liposomes known in the art as a mixture of DNA liposomes (see, for example,

- 13 045600

W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known agents that facilitate transfection. Preferably, the transfection facilitating agent 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 0.050 mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be one or more adjuvants. The adjuvant may be other genes expressed on the same or an alternative plasmid or delivered as proteins in combination with the above plasmid in an immunogenic composition. One or more adjuvants may be proteins and/or nucleic acid molecules that encode proteins selected from the group consisting of CCL20, α-interferon (IFN-±), β-interferon (IFNβ), γ-interferon, platelet-derived growth factor ( PDGF), TNFa, TNF, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus epithelial expressing chemokine (TECK), mucosal epithelial chemokine (MEC), IL -12, IL-15, including IL-15 with a signal or coding sequence encoding a deleted signal sequence, and optionally including another signal peptide, such as from IgE, or a coding sequence that encodes a differential 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, MIP1α, MIP-1a, IL-8 , 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, mutant forms of IL18, 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, INK, interferon response 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 adjuvant may be one or more proteins and/or protein-coding nucleic acid molecules selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MES or RANTES. Examples of IL-12 constructs and sequences are disclosed in Patent Application No. PCT/US1997/019502 and corresponding US Application Serial No. 08/956865 and US Provisional Application Serial No. 61/569600 filed December 12, 2011, referenced to each which are included in this document. Examples of IL-15 constructs and sequences are disclosed in Patent Application No. PCT/US04/18962 and corresponding US Application Serial No. 10/560650, and Patent Application No. PCT/US07/00886 and corresponding US Application Serial No. 12/160766 and patent application PCT/US10/048827, references to each of which are included herein. Examples of IL-28 constructs and sequences are disclosed in Patent Application No. PCT/US09/039648 and corresponding US Application Serial No. 12/936192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in Patent Application No. PCT/US1999/004332 and corresponding US Patent Application Serial No. 09/622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in Patent Application No. PCT/US 11/024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in Patent Application No. PCT/US1999/004332 and related US Application Serial No. 09/622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in Patent Application No. PCT/US 11/024098, which is incorporated herein by reference. Examples of chemokines, constructs and sequences CTACK, TECK and MEC are disclosed in patent application No. PCT/US 2005/042231 and corresponding US application Serial No. 11/719 646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulators are disclosed in US Application No. 10/560653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in US Application Serial No. 09/622452, which is incorporated herein by reference.

The immunogenic composition may further include a genetic vaccine delivery promoting agent described in US Application Serial No. 021579, filed April 1, 1994, which is incorporated herein by reference in its entirety.

The immunogenic composition may contain consensus antigens and plasmids in amounts from about 1 ng to 100 mg; from about 1 μg to about 10 mg; or preferably from about 0.1 μg to about 10 mg; or more preferably from about 1 mg to about 2 mg. In some preferred embodiments, the pharmaceutical compositions of the present invention contain from about 5 ng to

- 14 045600 approximately 1000 μg of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 10 ng to about 800 μg of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 μg of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 1 to about 350 μg of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 25 to about 250 μg, from about 100 to about 200 μg, from about 1 ng to 100 mg; from about 1 μg to about 10 mg; from about 0.1 μg to about 10 mg; from about 1 mg to about 2 mg, from about 5 ng to about 1000 micrograms, from about 10 ng 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, about 100 to about 200 micrograms of consensus antigen or plasmid thereof.

In some embodiments, the pharmaceutical compositions of the present invention contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95 or 100 ng of a nucleic acid molecule described in the invention. In some embodiments, the pharmaceutical compositions may contain 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, 21 5 , 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 μg of a nucleic acid molecule described in the invention. In some embodiments, the pharmaceutical composition may include 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 molecules described in the invention.

In other embodiments, the pharmaceutical composition may contain up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 8θ, 85, 90, 95, or 100 ng molecules nucleic acid described in the invention. In some embodiments, the pharmaceutical composition may contain up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, PO, 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 μg inclusive of nucleic acid molecules described in the invention. In some embodiments, the pharmaceutical composition may contain up to 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 inclusive of nucleic acid molecules described in the invention.

The immunogenic composition can be formulated according to the method of administration. The pharmaceutical composition for the injectable immunogenic composition may be sterile, pyrogen-free and particulate-free. An isotonic composition or solution can be used. Isotonic supplements may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The immunogenic composition may contain a vasoconstrictor agent. Isotonic solutions may include phosphate buffered saline. The immunogenic composition may further contain stabilizers, including gelatin and albumin. Stabilization by components such as LGS or polycations or polyanions in the immunogenic composition can cause it to become stable at room or ambient temperature for extended periods of time.

The immunogenic composition may be stable at room temperature (25°C) for more than 1 week, in some embodiments for more than 2 weeks, in some embodiments for more than 3 weeks, in some embodiments for more than 4 weeks, in in some embodiments for more than 5 weeks and in some embodiments for 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

- 15 045600 tsev. In some embodiments, the vaccine is stable for more than 1 year, more than 2 years, more than 3 years, or more than 5 years. In one embodiment of the invention, the immunogenic composition is stable when refrigerated (2-8°C). Accordingly, in one embodiment of the invention, the immunogenic composition does not require a cold chain. An immunogenic composition is stable if it retains its biological activity for a sufficient period of time without loss of properties necessary for its intended use (eg, to generate an immune response in a subject). For example, for immunogenic compositions that must be stored, shipped, etc., their stability may be advantageous over months or years.

Immune response.

The immunogenic composition may induce an immune response in the subject to whom the composition is administered. The induced immune response may be specific for MCV T antigen. The induced immune response may be reactive against MCV T antigen bound to an optimized consensus-encoded antigen. In various embodiments, related antigens include antigens with amino acid sequences having 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, related antigens include antigens encoded by nucleotide sequences having 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 with the optimized consensus nucleotide sequences disclosed herein. The immunogenic composition may induce a humoral immune response in a subject to whom the immunogenic composition is administered. The induced humoral immune response may be specific against MCV T antigen. The induced humoral immune response may be reactive against MCV T antigen bound to an optimized consensus-encoded antigen. The humoral immune response may be induced in a subject administered the immunogenic composition by about 1.5 to about 16 times, about 2 to about 12 times, or about 3 to about 10 times. The humoral immune response may be induced in a subject administered the immunogenic composition by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold .0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5 .5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 12.5 times 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5-fold or at least about 16.0-fold compared to a subject not administered the immunogenic composition or a subject administered a non-optimized MCV T antigen.

The humoral immune response induced by the immunogenic composition may include an increased level of IgG antibodies in a subject administered the immunogenic composition compared to a subject not administered the immunogenic composition. These IgG antibodies may be specific for the MCV T antigen genetically related to the optimized consensus antigen. These IgG antibodies may be reactive against an MCV T antigen genetically related to the optimized consensus antigen. The level of IgG antibodies of a subject administered the immunogenic composition may be increased from about 1.5 times to about 16 times, from about 2 times to about 12 times, or from about 3 times to about 10 times the with a subject not receiving the immunogenic composition. The IgG antibody level of a subject to whom an immunogenic composition is administered may be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold. 0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5, 5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8, 0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, less

- 16 045600 at least approximately 10.0 times, at least approximately 10.5 times, at least approximately 11.0 times, at least approximately 11.5 times, at least approximately 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14 times .5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times compared to a subject not administered the immunogenic composition or a subject administered unoptimized Tantigen MCV.

The immunogenic composition may induce a cellular immune response in a subject to whom the immunogenic composition is administered. The induced cellular immune response may be specific for MCV T antigen associated with an optimized consensus-encoded antigen. The induced cellular immune response may be reactive against MCV T antigen bound to an optimized consensus-encoded antigen. The induced cellular immune response may include the elicitation of a CD8+ T cell response. The evoked CD8+ T cell response may be reactive against the MCV T antigen genetically linked to the optimized consensus antigen. The resulting CD8+ T cell response may be multifunctional. The induced cellular immune response may include eliciting a CD8+T cell response in which the CD8+T cells produce interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

The induced cellular immune response may include an increased CD8+T cell response in a subject who was administered the immunogenic composition compared to a subject who was not administered the immunogenic composition. The CD8+ T cell response of a subject administered the immunogenic composition may be increased by about 2-fold to about 30-fold, by about 3-fold to about 25-fold, or by about 4-fold to about 20-fold compared with a subject to whom the immunogenic composition is not administered. The CD8+ T cell response of a subject administered the immunogenic composition may 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 times, at least about 5.0 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15 times, at least about 16.0 times, at least about 17.0 times, at least about 18.0 times, at least about 19.0 times, at least about 20 .0 times, at least about 21.0 times, at least about 22.0 times, at least about 23.0 times, at least about 24.0 times, at least about 25 .0 times, at least about 26.0 times, at least about 27.0 times, at least about 28.0 times, at least about 29.0 times, or at least about 30.0 times compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T antigen.

The induced cellular immune response may include an increased frequency of CD107a/INFγ/T-bet triple positive CD8 T cells that are reactive against MCV Tantigen. The frequency of CD107a/IFNγ/T-bet triple positive CD8 T cells from a subject administered the immunogenic composition may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20-fold compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T antigen.

The induced cellular immune response may include an increased frequency of CD107a/IFNγ double-positive CD8 T cells that are reactive to MCV T antigen. The frequency of double positive CD107a/IFNγ CD8 T cells from a subject administered an immunogenic composition may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold times, 9 times, 10 times, 11 times, 12 times, 13 times or 14 times compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T antigen.

The cellular immune response induced by the immunogenic composition may include the induction of a CD4+ T cell response. The elicited CD4+ T cell response may be reactive against the MCV Tantigen genetically linked to the optimized consensus antigen. The resulting CD4+ T cell response can be multifunctional. The induced cellular immune response may involve eliciting a CD4+T cell response in which the CD4+T cells produce IFN-γ, TNFα, IL-2, or a combination of IFN-γ and TNF-α.

The induced cellular immune response may include an increase in the frequency of CD4+ T cells producing IFN-γ. The frequency of CD4+IFN-γ+T cells of a subject administered the immunogenic composition may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T antigen.

The induced cellular immune response may include an increase in the frequency of TNF-α-producing CD4+ T cells. The frequency of CD4+TNF-α+T cells of a subject administered an immunogenic composition may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times , 21-fold or 22-fold compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T-antigen.

The induced cellular immune response may include an increase in the frequency of CD4 + T cells producing both IFN-γ and TNF-α. The frequency of CD4+IFN-γ+TNF-α+subject administered the immunogenic composition can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold. 0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times , 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, 14.0 times, 14.5 times, 15.0 times, 15.5 times, 16.0 times, 16, 5 times, 17.0 times, 17.5 times, 18.0 times, 18.5 times, 19.0 times, 19.5 times, 20.0 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times or 35 times compared to a subject who was not administered the immunogenic composition or a subject who was administered a non-optimized MCV T antigen.

The immunogenic composition may further induce an immune response when administered to various tissues, such as muscle or skin. The immunogenic composition may further elicit an immune response when administered by electroporation, or injection, or subcutaneously or intramuscularly.

Vector.

The nucleotide construct described above can be placed into one or more vectors. One or more vectors may contain a replication origin. The one or more vectors may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The one or more vectors may be either a self-propagating extrachromosomal vector or a vector that integrates into the host genome.

Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, recombinant naked DNA vector of any form, and the like. The vector includes a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. It has been found that the vector can be a naked nucleic acid or a nucleic acid complexed with a protein or lipid. The vector optionally contains viral or bacterial nucleic acids and/or proteins and/or membranes (eg, cell membrane, viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (eg, RNA replicons, bacteriophages), to which DNA fragments can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (eg, plasmids, viruses and the like, see US Pat. No. 5,217,879), and include both expression and non-expression plasmids. When a recombinant microorganism or cell culture is described as an expression vector acceptor, both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s) are meant. If the vector is maintained by a host cell, the vector can either be stably replicated by cells during mitosis as an autonomous structure, or incorporated into the host genome.

One or more vectors may be an expression construct, which is typically a plasmid used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein encoded by the gene is produced by ribosomal cellular transcription and translation complexes. The plasmid is often designed to contain regulatory sequences that act as enhancer and promoter regions leading to efficient transcription of the gene carried by the expression vector. According to the present invention, the vectors express large amounts of stable messenger RNA and, therefore, proteins.

Vectors may include expression signals such as a strong promoter, a strong termination codon, regulation of the distance between the promoter and the cloned gene, and insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

(1) Expression vector.

One or more vectors may be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing the expression of a specific nucleotide sequence in a corresponding cell of a subject. One or more vectors containing the recombinant nucleic acid construct may be chimeric, meaning that at least one of their components is heterologous with respect to at least one of their other components.

(2) Plasmid.

One or more vectors may be plasmids. The plasmid can be used to transfect cells with a recombinant nucleic acid construct. The plasmid can be used to introduce a recombinant nucleic acid construct into a subject. The plasmid may also contain a regulatory sequence that may be well suited for gene expression in the cell into which the plasmid is introduced.

The plasmid may also contain a mammalian origin of replication to maintain the plasmid's extrachromosomal existence and to produce multiple copies of the plasmid within the cell. The plasmid may be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, Calif.), which may include the Epstein-Barr virus origin of replication and the EBNA-1 nuclear antigen coding region, and can produce high-copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication-defective adenovirus type 5 (Ad5) plasmid.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce protein in Escherichia coli (E. coli). The plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce protein in strains of the yeast Saccharomyces cerevisiae. The plasmid may also be a complete baculovirus expression system MAHBAS™ (Invitrogen, San Diego, Calif.), which can be used to produce the protein in insect cells. The plasmids may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce the protein in mammalian cells, such as Chinese hamster ovary (CHO) cells.

(3) RNA.

In one embodiment of the invention, the nucleic acid is an RNA molecule. In one embodiment of the invention, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by a DNA sequence that is at least 90% homologous 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 includes an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence with 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. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more MCV T antigens. RNA can be a plus strand. Accordingly, in some embodiments, the RNA molecule can be translated by cells without the need to introduce any intermediate replication steps, such as reverse transcription. The RNA molecule used in the invention may be 5'-capped (eg, 7-methylguanosine). Capping can enhance RNA translation in vivo. The 5' nucleotide of the RNA molecule used in the invention may have a 5'-triphosphate group. In capped RNA it can be linked to 7methylguanosine through a 5'-5' bridge. An RNA molecule may have a 3' poly-A tail. It may also include a polyA polymerase recognition sequence (eg, AAUAAA) at approximately its 3' end. The RNA molecule used in the invention may be single-stranded. The RNA molecule used in the invention may contain synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment of the invention, the RNA molecule is contained in a vector.

In one embodiment of the invention, the RNA has 5' and 3' UTRs. In one embodiment of the invention, the length of the 5'-UTR is in the range of zero to 3000 nucleotides. The length of the 5' and 3' UTR sequences added to the coding region can be changed in various ways, including, but not limited to, designing PCR primers to anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be the endogenous natural 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by including UTR sequences in the forward and reverse primers or through any other template modifications. The use of UTR sequences that are not endogenous to the gene of interest can be useful to modify the stability and/or translation efficiency of RNA. For example, it is known that AU-rich elements at 3' UTR sequences can reduce RNA stability. Therefore, 3'UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of the UTRs that are well known in the art.

In one embodiment of the invention, the 5'-UTR may contain a Kozak sequence of an endogenous gene. Alternatively, in the case where a 5'UTR that is not endogenous to the gene of interest is added by PNR as described above, the Kozak consensus sequence can be modified by adding the 5'UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but there appears to be no need to ensure efficient translation for all RNAs. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to interfere with RNA exonuclease degradation.

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

In one embodiment of the invention, the RNA is nucleoside-modified RNA. Nucleoside-modified RNA has particular advantages over unmodified RNA, including, for example, increased stability, low or no inherent immunogenicity, and improved translation.

(4) Ring and linear vectors.

One or more vectors may be a circular plasmid that can transform a target cell by integration into the cellular genome or exist extrachromosomal (eg, an autonomously replicating plasmid with an origin of replication). The vector may 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.

Also provided herein is a linear nucleic acid or linear expression cassette (LEC) that is capable of being efficiently delivered to a subject by electroporation and expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid construct. LEC can be any linear DNA lacking any phosphate backbone. LEC may not contain any antibiotic resistance and/or phosphate backbone genes. The LEC may not contain other nucleotide sequences that are not relevant to the desired gene expression.

LEC can be obtained from any plasmid capable of linearization. The plasmid may be capable of expressing a heavy chain polypeptide and/or a light chain polypeptide encoded by the recombinant nucleic acid construct. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may 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 the recombinant nucleic acid construct.

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

(5) Viral vectors.

In one embodiment, the invention provides viral vectors capable of delivering the nucleic acid of the invention into a cell. The expression vector may be provided to the cell 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) and in Ausubel et al. (1997) and other manuals on virology and molecular biology. Viruses used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease convenience sites, and one or more selectable markers (see, for example, WO 01/96584; WO 01/29058; and US Pat. No. 6326193). Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Patent Nos. 5,350,674 and 5,585,362.

(6) Method for preparing vectors.

Provided herein is a method for producing one or more vectors into which a recombinant nucleic acid construct is placed. After the final subcloning step, the vector can be used to inoculate a cell culture in a large-scale fermentation tank using methods known in the art.

- 20 045600

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

One or more vectors may be formulated or manufactured using a combination of known devices and technologies, but they are preferably made using plasmid manufacturing technology, which is described in licensed, pending US provisional application Serial No. 60/939792, filed May 23, 2007 In some examples, the plasmid DNA described herein can be prepared in concentrations greater than or equal to 10 mg/ml. The manufacturing techniques also include various devices and protocols that are generally known to those skilled in the art, in addition to those described in US Serial No. 60/939,792, including those described in US Pat. No. 7,238,522 issued 3 July 2007 The above application and patent, Serial No. 60/939792 and US Patent No. 7238522, respectively, are incorporated herein in their entirety.

Multiple vectors.

The immunogenic composition may contain multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, an immunogenic composition may contain a plurality of two, three, four, five, six, seven, eight, nine or ten or more different nucleic acid molecules. Such compositions may contain a plurality of two, three, four, five, six or more different plasmids.

Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the MCV T antigen coding sequence. Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the coding sequence of several antigens. In one embodiment, the antigens are MCV T antigen and one or more additional cancer antigens. Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the coding sequence of one or more MCV Tantigens and one or more cancer antigens.

Cancer antigens.

The immunogenic composition may contain one or more cancer antigens, such as WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, MAGE A3, p53 (non-mutant), NY-ESO-1, PSMA, GD2 , CEA, MelanA/MARTl, 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 Al, 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 membrane proteins, AKAP-4, SSX2, XAGE 1, B7H3 , Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2 and FOS-related antigen 1 for the treatment or prevention of tumor-related pathology. The immunogenic composition may additionally combine one or more cancer antigens WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, MAGE A3, p53 (non-mutant), 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 membrane proteins, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2 and FOS-related antigen with optimized consensus-encoded MCV T-antigen for the treatment or prevention of tumor pathology. Other combinations of cancer antigens may also be used to treat or prevent tumor pathology.

Ways.

Provided herein are methods of treating, protecting, and/or preventing disease associated with MCV in a subject in need thereof by administering one or more immunogenic compositions described herein to the subject. Administration of an immunogenic composition to a subject may induce or elicit an immune response in the subject. The induced immune response can be used to treat, prevent and/or protect against disease, for example, MCV infections or MCC associated with MCV infection.

Described herein is a method of delivering an immunogenic composition to provide genetic constructs and consensus antigen proteins containing epitopes making them particularly effective against MCV or MCC against which an immune response can be induced. A method of delivering an immunogenic composition or vaccine may be provided to induce a therapeutic and prophylactic immune response. The vaccination process can induce an immune response in the mammal against MCV or MCC. The immunogenic composition may be delivered to an individual to modulate the activity of the mammal's immune system and enhance the immune response. Delivery of the immunogenic composition may be the transfection of a consensus antigen into a nucleic acid molecule that is expressed in the cell and delivered to the cell surface, at which the immune system recognizes and induces a cellular, humoral, or cellular and humoral response. Delivery of an immunogenic composition can be used to induce or detect an immune response in mammals against MCV or MCC by administering the immunogenic composition to the mammals in the manner described above.

When the immunogenic composition and plasmid are delivered to mammalian cells, the transfected cells will express and secrete consensus antigens for each of the plasmids injected in the immunogenic composition. These proteins will be identified by the immune system as foreign, and antibodies will be produced against them. These antibodies will be maintained by the immune system and allow it to respond effectively to subsequent MCV infections.

The immunogenic composition may be administered to a mammal to elicit an immune response. A mammal can be human, primate, lesser primate, cow, cattle, sheep, goat, antelope, bison, buffalo, buffalo, ox, deer, hedgehog, elephant, llama, alpaca, mice, rats and chicken.

The induced immune response may include an induced humoral immune response and/or an induced cellular immune response. The humoral immune response can be induced by about 1.5 to about 16 times, from about 2 to about 12 times, or from about 3 to about 10 times. The induced cellular immune response may include a CDS+T cell response that is induced by about 2-30-fold, about 3-25-fold, or about 4-20-fold.

The dose of the immunogenic composition may be from 1 μg to 10 mg of active component/kg body weight/time and may be from 20 μg to 10 mg of component/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 doses of the immunogenic composition for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The immunogenic composition can be formulated in accordance with standard techniques well known to those skilled in the art of pharmaceuticals. Such compositions can be administered in dosages and by methods well known to those skilled in the art of medicine, taking into account factors such as the age, sex, weight and condition of the particular subject, and the route of administration.

The immunogenic composition can be administered prophylactically or therapeutically. When administered prophylactically, immunogenic compositions can be administered in an amount sufficient to induce an immune response. When used therapeutically, the immunogenic compositions are administered to a subject in need thereof in an amount sufficient to achieve a therapeutic effect. The amount sufficient to achieve a therapeutic effect is defined as the therapeutically effective dose. Amounts effective for a given use will depend, for example, on the specific composition of the regimen for administering the immunogenic composition, the route of administration, the stage and severity of the disease, the general health of the subject, and the judgment of the prescriber.

The immunogenic composition can be administered by methods well known in the art, as described by Donnelly et al. (Ann. Rev. Immunol. 15: 617-648 (1997)); Feigner et al. (US Patent No. 5,580,859, issued December 3, 1996); Feigner (US Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), the contents of which are incorporated herein by reference in their entirety. The DNA of the immunogenic composition can form complexes with particles or beads that can be administered to an individual, for example, using a vaccine pistol. One skilled in the art will be aware that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

The immunogenic composition can be delivered in various ways. Typical routes of delivery include parenteral administration, such as intradermal, intramuscular or subcutaneous administration. Other routes include oral, intranasal and intravaginal administration. In particular with respect to DNA immunogenic composition, the immunogenic composition can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., US Patent Nos. 5,580,859 and 5,703,055, the contents of which are incorporated herein in their entirety). The immunogenic composition may also be administered into the muscle, or may be administered by intradermal or subcutaneous injection, or transdermally, for example, by iontophoresis. Epidermal administration of the immunogenic composition can also be used. Epidermal administration may involve mechanical or chemical stimulation of the outer layer of the epidermis to stimulate an immune response to the stimulus (Carson et al., US Pat. No. 5,679,647, the contents of which are incorporated by reference in their entirety).

The immunogenic composition may also be formulated for administration through the nasal passages. Compositions suitable for nasal administration wherein the carrier is a solid

- 22 045600 society may include a coarse powder having a particle size, for example, in the range of from about 10 to about 500 microns, administered in the same way as snuff is consumed, i.e. by rapidly inhaling from a container of powder held close to the nose through the nasal passage. The composition may be a nasal spray, nasal drops, or aerosol administration by nebulizer. The composition may include aqueous or oily solutions of the immunogenic composition.

The immunogenic composition may be a liquid preparation such as a suspension, syrup or elixir. The immunogenic composition may also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (eg, injection), such as a sterile suspension or emulsion.

The immunogenic composition may be included in liposomes, microspheres or other polymer matrices (Feigner et al., US Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein in their entirety completeness). Liposomes may be composed of phospholipids or other lipids and may be non-toxic, physiologically acceptable and metabolizable carriers that are relatively easy to manufacture and use.

A method of treating cancer using a vaccine.

The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against a cancer or tumor (eg, MCC) of the mammal or subject in need thereof. The immune response generated can prevent cancer or tumor growth.

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

In some embodiments, the administered vaccine may mediate the clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses, generating antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting the cells myeloid-derived suppressors (MDSC) and suppressing tumor growth; (2) increasing cytotoxic T lymphocytes such as CD8+ (CTL) to attack and kill tumor cells; (3) increasing T helper cell responses; (4) and enhancing inflammatory responses through IFN-γ and TNF-α, or, preferably, all of the above.

In some embodiments, the immune response may generate a humoral immune response and/or antigen-specific cytotoxic T lymphocytes (CTLs) that do not cause damage or inflammation of various tissues or systems (e.g., brain or neurological system, etc.) in the subject, who is receiving the vaccine.

In some embodiments, the administered vaccine may increase tumor-free survival, reduce tumor burden, increase tumor-bearing survival, or a combination thereof in a subject. The administered vaccine can increase tumor-free survival 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 and 60% or more percent. The administered vaccine can reduce tumor burden in a subject following immunization 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 percent. The administered vaccine can prevent and block increases in a subject's levels of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. In some embodiments, the administered vaccine can prevent and block the increase of MCP-1 in cancer or tumor tissue in a subject, thereby reducing the vascularization of the cancer or tumor tissue in the subject.

The administered vaccine can increase the survival rate of a subject with a tumor 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 percent. In some embodiments, the vaccine can be administered peripherally (as described in more detail below) to establish an antigen-specific immune response directed at cancer or tumor cells or tissues to clear or eliminate cancer or tumor expressing one or more MCV T antigens, not harming and not causing illness or death in the subject to whom the vaccine is administered.

The vaccine administered may enhance the cellular immune response in a 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 5000-fold 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times, or about 300 times to approximately 6000 times. In some embodiments, the vaccine administered may enhance the cellular immune response in a subject by approximately 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold,

- 23 045600

400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times , 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times , 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

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

The vaccine dose may range from 1 μg to 10 mg of active ingredient/kg body weight/time and may range 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 doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Combination Therapy with Checkpoint Inhibitors The present invention also provides a method of enhancing the immune response in a mammal using a vaccine, such as described above, in combination with one or more checkpoint inhibitors. In one embodiment of the invention, the vaccine described above may contain MCV T antigen and an antibody to a control protein. As used herein, the term checkpoint inhibitor includes inhibitors or molecules that block immune checkpoints commonly known in the field of cancer immunotherapy. Most often, checkpoint inhibitors are antibodies that block immune checkpoint proteins. Immune checkpoint proteins include PD1, PDL1, PDL2, CTLA4, LAG3, TIM3, B7-H3, BTLA, VISTA, CD40, CEACAM1, CD80, CD86, OX40, CD27, GITR, DNAM-1, TIGIT, TMIGD2 and DC-SIGN , but are not limited to them. Some examples of known checkpoint inhibitors include, but are not limited to, ipilimumab, pembrolizumab, nivolumab, pidilizumab, avelumab, and others.

The combination may be in one composition or may be separate and administered sequentially (either MCV T antigen first and then a checkpoint inhibitor, or a checkpoint inhibitor first and then MCV T antigen). In some embodiments, Tantigen MCV can be administered to a subject after approximately 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 o'clock, 9 o'clock, 10 o'clock, 11 o'clock, 12 o'clock, 13 o'clock, 14 o'clock, 15 o'clock, 16 o'clock, 17 o'clock, 18 o'clock, 19 o'clock, 20 o'clock, 21 o'clock, 22 o'clock, 23 o'clock, 24 o'clock , 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 administration of the checkpoint inhibitor to the subject. In other embodiments, the checkpoint inhibitor can be administered to the subject at approximately 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 o'clock, 9 o'clock, 10 o'clock, 11 o'clock, 12 o'clock, 13 o'clock, 14 o'clock, 15 o'clock, 16 o'clock, 17 o'clock, 18 o'clock, 19 o'clock, 20 o'clock, 21 o'clock, 22 o'clock, 23 o'clock, 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 Tantigen MCV to the subject.

The combination of MCV T antigen and a checkpoint inhibitor induces the immune system more effectively than a vaccine containing only MCV T antigen. This more effective immune response provides increased effectiveness in treating and/or preventing a particular type of cancer.

In some embodiments, the immune response may be increased by approximately

- 24 045600 exactly 0.5-15 times, approximately 0.5-10 times or approximately 0.5-8 times. Alternatively, the immune response of the subject receiving the vaccine may 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-fold, 0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4, 5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7 times ,0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9 times .5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12 times ,0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14, 5 times or at least about 15.0 times.

In other alternative embodiments, the immune response of the subject receiving the vaccine may be increased by about 50% to about 1500%, from about 50% to about 1000%, or from about 50% to about 800%. In other embodiments, the immune response of a subject receiving the vaccine may be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, by 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 by about 550%, by at least about 600%, by at least about 650%, by at least about 700%, by at least about 750%, by at least about 800%, by 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 %, by at least about 1200%, by at least about 1250%, by at least about 1300%, by at least about 1350%, by at least about 1450%, or by at least about 1500 %.

The vaccine dose may range from 1 μg to 10 mg of active ingredient/kg body weight/time and may range 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 doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Merkel cell carcinoma.

The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against Merkel cell carcinoma (MCC) in the mammal or subject in need thereof. The induced immune response may prevent the growth of MCC. The induced immune response may reduce the growth of MCC. The induced immune response can prevent and/or reduce metastasis of cancerous or MCC tumor cells. Accordingly, the vaccine can be used in a manner that treats and/or prevents MCC in a mammal or subject to which the vaccine is administered.

In some embodiments, the administered vaccine may mediate clearance or prevent the growth of MCC by inducing (1) humoral immunity through Bcellular responses to generate antibodies targeting MCV T antigen expressed by MCC cells; (2) increasing cytotoxic T lymphocytes such as CD8 + (CTL) to attack and kill MCC cells; (3) increased T helper cell responses; and (4) enhancing inflammatory responses through IFN-γ and TNF-α, or through all of the above.

In some embodiments, the administered vaccine may increase MCC-free survival in a subject, reduce MCC weight, increase MCC-free survival, or a combination thereof. The administered vaccine may increase the survival rate of a subject without MCC by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45% or more. The administered vaccine can reduce a subject's MCC weight after immunization 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 more. The administered vaccine can prevent and block increases in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells in a subject. In some embodiments, the administered vaccine can prevent and block the increase of MCP-1 in the subject's MCC tissue, thereby reducing the vascularization of the subject's MCC tissue. The administered vaccine can increase the survival rate of a subject with MCC 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 more.

- 25 045600

Combined treatment.

The immunogenic composition can be administered in combination with other proteins and/or genes encoding CCL20, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell chemoattaractant (CTAC), thymic epithelial expressed chemokine (TESC), mucosal associated epithelial chemokine (MEC), IL-12, IL-15, including IL-15 with the signal sequence removed and optionally including another signal peptide such as IgE, MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP -1a, MIP-1e, IL-8, RANTES, 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, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7 , nerve growth factor, vascular endothelial growth factor, Fas, FIO receptor, Fit, 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 response 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 containing a coding sequence that encodes 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.

The immunogenic composition can be administered by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and intraarticular, or combinations thereof. For veterinary use, the composition can be administered in a form acceptable in accordance with normal veterinary practices. The veterinarian can easily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The immunogenic composition can be administered using traditional syringes, needleless injection devices, microparticle bombardment guns, or other physical methods such as electroporation (EP), fluid dynamics, or ultrasound.

The immunogenic composition plasmid can be delivered to a mammal by several known technologies, including DNA injection (also called DNA vaccination) with and without in vivo electroporation, liposomal delivery, nanoparticle delivery, recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus , and recombinant variola virus. The consensus antigen can be delivered by DNA injection and coupled with in vivo electroporation.

Electroporation.

Administration of an immunogenic composition by electroporation can be accomplished using electroporation devices configured to deliver a pulse of energy into a desired tissue of a mammal effective to form reversible pores in cell membranes, where preferably the pulse of energy is a direct current corresponding to an input current specified by the user. The electroporation device may include an electroporation component and an electrode assembly or a manual control assembly. An electroporation component may include and be composed of one or more different electroporation device elements, including: a controller, a current signal generator, an impedance tester, a signal recorder, an input element, a status message element, a communication port, a memory component, a power supply, and switch. Electroporation can be performed using an in vivo electroporation device, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA) to facilitate transfection of cells with the plasmid .

The electroporator component may function as one element of the electroporation device, and the other elements are separate elements (or components) associated with the electroporator component. The electroporator component may function as more than one electroporation device element, which may be coupled to other electroporation device elements separate from the electroporator component. Elements of electroporation devices existing as parts of a single electromechanical or mechanical device cannot be limited, since the elements can function as a single device or as separate elements coupled to each other. The electroporator component may be capable of delivering a pulse of energy producing

- 26 045600 direct current, in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives a pulse of energy from the electroporator component and delivers it to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during application of the energy pulse and senses impedance in the desired tissue and transmits the impedance to the electroporator component. The feedback mechanism can receive the measured impedance and. accordingly, can adjust the pulse of energy supplied by the electroporator component to maintain a given constant current strength.

Multiple electrodes can deliver a pulse of energy in a decentralized manner. The plurality of electrodes may deliver a pulse of energy in a decentralized manner by driving the electrodes in a programmed sequence, and the programmed sequence is input by the user into the electroporator component. The programmed sequence may comprise a plurality of pulses delivered sequentially, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode that senses impedance, and in which a subsequent pulse of the plurality of pulses is delivered by another of the at least two active electrodes with one neutral electrode that measures impedance.

The feedback mechanism can be implemented in either hardware or software. The feedback mechanism can be implemented using an analog closed circuit. Feedback readings occur every 50 µs, 20 µs, 10 µs or 1 µs, but preferably the feedback should be real-time or instantaneous (that is, essentially instantaneous as determined by available response timing methods). The neutral electrode can sense the impedance in the desired tissue and communicate the impedance to the feedback mechanism, wherein the feedback mechanism responds to the impedance value and adjusts the energy pulse to maintain a constant current at a value corresponding to the target current. The feedback mechanism can maintain a constant current continuously and instantaneously during the delivery of a pulse of energy.

Examples of electroporation devices and electroporation methods that may facilitate administration of the immunogenic compositions of the present invention include those described in US Pat. No. 7,245,963 to Draghia-Akli et al., US Patent Publication 2005/0052630 to Smith et al., referenced in are fully included in this description. Other electroporation devices and electroporation methods that can be used to facilitate the administration of immunogenic compositions include those disclosed in co-pending US Patent Application Serial No. 11/874,072, filed October 17, 2007, which claims benefit under 35 USC 119(e) for US provisional applications Ser. 60/852149, filed October 17, 2006, and 60/978982, filed October 10, 2007, incorporated herein in their entirety.

US Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use to facilitate the introduction of a biomolecule into the cells of a selected body or plant tissue. Modular electrode systems can contain multiple needle electrodes; hypodermic needle; an electrical connector that provides conductive communication from the programmable DC pulse controller to the plurality of needle electrodes; and power supply. The operator can grasp a plurality of needle electrodes mounted on a support structure and insert them firmly into the selected body or plant tissue. The biomolecules are then injected through a hypodermic needle into the selected tissue. The programmable DC pulse controller is activated and a DC electrical pulse is applied to a plurality of needle electrodes. An applied DC electrical pulse facilitates the introduction of the biomolecule into the cell between the electrode array. References to the entire contents of US Patent No. 7,245,963 are incorporated in this specification in their entirety.

US Patent Publication 2005/0052630 by Smith et al. describes an electroporation device that can be used to effectively facilitate the introduction of a biomolecule into the cells of a selected body or plant tissue. The electroporation device contains an electrokinetic device (EKD device), the operation of which is controlled by external software or firmware. The EKD device generates a series of programmable patterned DC pulses between the electrode array based on user control and input pulse parameters, and allows current waveform data to be stored and retrieved. The electroporation device also includes a replaceable electrode disk having a row of needle electrodes, a central channel for an injection needle, and a removable guide disk. References to the entire contents of US Pat. No. 2005/0052630 are incorporated herein in their entirety.

The electrode array and methods described in US Pat. No. 7,245,963 and 2005/0052630 can be adapted to penetrate deeply not only into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle (to deliver the desired biomolecule) is also fully inserted into the target organ, and the injection is administered perpendicular to the target tissue in the area that is previously delineated by the electrodes. Electrodes that are described in the patent

US No. 7,245,963 and US Patent 2005/005263 are preferably 20 mm long and 21 gauge.

In addition, certain embodiments that include electroporation devices and their use include electroporation devices that are described in the following patents: US Patent 5,273,525, issued December 28, 1993, US Patents 6,110,161, issued August 29, 2000, 6 261,281, issued on July 17, 2001 and 6,958,060, issued on October 25, 2005, and US Patent 6,939,862, issued on September 6, 2005. In addition, patents covered by this document cover the subject matter provided in US Patent 6,697,669, issued on 24 February 2004, which covers the injection of DNA using any of a variety of devices, and US Patent 7,328,064, issued February 5, 2008, for a method of DNA injection. References to the above patents are incorporated herein in their entirety.

Generation of antigens in vitro and ex vivo.

In one embodiment of the invention, an optimized MCV consensus T antigen is generated in vitro or ex vivo. For example, in one embodiment of the invention, a nucleic acid encoding an optimized MCV consensus T antigen can be introduced and expressed in a cell in vitro or ex vivo.

Methods for introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means.

Physical methods of introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing 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). The preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Patent Nos. 5,350,674 and 5,585,362.

Chemical means of introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).

When a non-viral delivery system is used, a typical delivery vehicle is a liposome. It is contemplated that lipid formulations may be used to introduce nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-bound nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed in the lipid bilayer of the liposome, attached to the liposome through a linking molecule that is associated with both the liposome and the oligonucleotide entrapped in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, mix with a lipid, combine with a lipid, be suspended in a lipid, be contained or complexed with a micelle, or otherwise be associated with a lipid. Compositions associated with a lipid, lipid/DNA complex, or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present as a bilayer structure, as micelles, or as a compressed structure. They may also simply be incorporated into the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that either occur naturally or are synthetic. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Examples

The present invention is further illustrated in the following example. It should be understood that while these examples indicate preferred embodiments of the invention, they are provided by way of illustration only. One skilled in the art can determine the essential characteristics of this invention from the above discussions and given examples and, without departing from the spirit and scope of the invention, can make various changes and modifications to the invention to adapt it to various applications and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications also fall within the scope of the appended claims.

Example 1 Nucleic acid vaccine targeting Merkel cell polyomavirus.

A nucleic acid vaccine targeting Merkel cell polyomavirus (MCV) T antigens was developed (Figs. 1 and 2). Optimized synthetic MCV T antigen consensus sequences representing large T antigen (LTAg) and small t antigen (STAg) were separately cloned into mammalian expression plasmid DNA (Figure 3) and delivered to mice via intramuscular electroporation (Figure 4A). . Following immunization, the DNA vaccine constructs generated robust antibody and T cell responses against MCV T antigen peptides (FIGS. 4B-15).

Fig. 4B, 7, 12 and 14 demonstrate that the LTAg vaccine is highly immunogenic in outbred C57B1/6 and CD-1 mice. Fig. 8–10 demonstrate that LTAg vaccination results in robust generation of multifunctional CD4 and CD8 T cells and cytotoxic CD8 T cells.

Fig. 4B and 15 demonstrate that the STAg vaccine is immunogenic in C57B1/6 and CD-1 mice. In fig. 15 shows that both CD4 and CD8 responses were detected for IFNa/TNFa in CD-1 mice.

Fig. 11 demonstrates that both vaccines induce an antibody response in C57B1/6 mice.

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Example 2. Sequences.

SEQ ID No. 1: Nucleotide sequence encoding a modified synthetic consensus LTAg MCV

ATGGACCTGGTGCTGAACAGGAAGGAGAGAGAGGCCCTGTGCAAGCTGCTG GAGATCGCCCCCAACTGTTACGGCAATATCCCTCTGATGAAGGCCGCCTTCAAGCGG AGCTGCCTGAAGCACCACCCCAACAAGGGCGGCAACCCTGTGATCATGATGGAGCT GAATACCCTGTGGTCCAAGTTTCAGCAGAATATCCACAAGCTGCGGTCCGATTTCTC TATGTTTGACGAGGTGGA TGAGGCCCCTATCTACGGCACCACCAAGTTCAAGGAGT GGTGGCGCTCCGGCGGCTTCTCTTTTGGCAAGGCCTACGAGTACGGCCCTAACCCAC ACGGCACCAATAGCAGGTCCAGAAAGCCAAGCTCCAACGCCAGCAGGGGAGCACC ATCCGGATCTAGCCCACCTCACAGCCAGTCCTCTAGCTCCGGCTACGGCTCTTTTAG CGCCTCCCAGGCCTCTGACAGCCAGTCCAGAGGCC CCGATATCCCACCCGAGCACC ACGAGGAGCCTACCTCTAGCTCCGGCTCTAGCTCCCGGGAGGAGACAACCAACAGC GGCAGGGAGTCTAGCACCCCAAACGGCACCTCCGTGCCAAGGAATTCCTCTAGGAC CGACGGAACCGCCGAGGACCTGTTCTGCGATAAGTCCCTGAGCTCCCCTGAGCCTCC ATCTAGCTCCGAGGAGCCAGAGGAGCCCCCTTCTAGCAGGTCCTCTCCCAGACA GCC ACCAAGCTCCTCTGCCGAGGAGGCAAGCTCCTCTCAGTTCACCGACGAGGAGTACA GGAGCTCCTCTTTTACCACCCCTAAGACCCCTCCACCCTTCTCCCGGAAGCGCAAGT TTGGAGGCTCTAGGAGCTCCGCCTCTAGCGCCTCCTCTGCCAGCTTCACCTCCACCC CTCCAAAGCCCAAGAAGAACAGAGACACCCGTGCCTACCGACTTTCCTATCGAC CTGAGCGATTACC TGTCCCACGCCGTGTACTCTAATAAGACCGTGAGCTGTTTCGCC ATCTACACCACCAGCGACAAGGCCATCGAGCTGTACGATAAGATCGAGAAGTTCAA GGTGGACTTCAAGTCCAGGCACGCATGCGAGCTGGGATGTATCCTGCTGTTCATCAC CCTGTCCAAGCACCGCGTGTCTGCCATCAAGAACTTCTGCAGCACCTTTTGTACCAT CTCCTTTCTGATCTGCAAGGGCGTGA ATAAGATGCCTGAGATGTACAACAACCTGTG CAAGCCCCCTTACAAGCTGCTGCAGGAGAACAAGCCACTGCTGAATTACGAGTTCC AGGAAGAGGAGAAGGAGGCCAGCTGCAACTGGAATCTGGTGGCCGAGTTCGCCTGT GAGTACGAGCTGGACGATCACTTTATCATCCTGGCCCACTACCTGGACTTCGCCAAG CCATTTCCCTGCCAGAAGTGTGAGAACAGGTCTAGACTGAAGCCACACAA GGCCCA CGAGGCCCACCACTCCAATGCCAAGCTGTTTTACGAGTCTAAGAGCCAGAAGACCA TCTGCCAGCAGGCAGCAGACACCGTGCTGGCAAAGAGGAGACTGGAGATGCTGGAG ATGACCAGGACCGAGATGCTGTGCAAGAAGTTCAAGAAGCACCTGGAGCGGCTGCG CGACCTGGATACCATCGATCTGCTGTACTACATGGGCGGCGTGGCCTGGTACTGCTG TCTGTTCGA GGAGTTTGAGAAGAAGCTGCAGAAGATCATCCAGCTGCTGACCGAGA ACATCCCAAAGTACAGAAATATCTGGTTCAAGGGCCCCATCAACTCTGGCAAGACC AGCTTCGCCGCCGCCCTGATCGACCTGCTGGAGGGCAAGGCCCTGAACATCAATTGC CCTAGCGATAAGCTGCCATTCGAGCTGGGCTGTGCCCTGGACAAGTTCATGGTGGTG TTTGAGGATGTGAAGGGCCAGAACTCC CTGAATAAGGACCTGCAGCCCGGCCAGGG CATCAACAATCTGGATAACCTGCGGGACCACCTGGATGGAGCAGTGGCCGTGAGCC TGGAGAAGAAGCACGTGAACAAGAAGCACCAGATCTTCCCACCCTGCATCGTGACC GCCAATGACTACTTTATCCCAAAGACCCTGATCGCCCGCTTCTCTTACACCCTGCACT TTAGCCCCAAGGCCAACCTGAGGGACAGCCTGGATCAGAATATGGAGATCAG AAAG AGGCGCATCCTGCAGTCCGGAACCACCCTGCTGCTGTGCCTGATCTGGTGTCTGCCT GACACCACCTTCAAGCCATGCCTGCAGGAGGAGATCAAACTGGAAGCAGATCCT GCAGTCTGAGATCAGCTACGGCAAGTTTTGTCAGATGATCGAGAACGTGGAGGCCG GCCAGGACCCCCTGCTGAATATCCTGATCGAGGAGGAGGGCCCAGAGGAGACAGAG GAGACA CAGGACTCCGGCACCTCTCTCAG

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SEQ ID No. 2: Amino acid sequence of modified synthetic consensus LTAg MCV

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMME LNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPH GTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSS SGSSSREETTNSGRESSTPNGTSVPRNSSRTDG TAEDLFCDKSLSSPEPPSSSEEPEEPPSSR SSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTP PKPKKNRETPVPTDFPIDLSDYLSHAVYSNKTVSCFAIYTTSDKAIELYDKIEKFKVDFKS RHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYK LLQE NKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSR LKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKK HLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSG KTSFAAALIDLEGKAL NINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQG INNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPK ANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGK FCQMIENVEAGQDPLLNILIEEEGPEETEETQDSGTF S.Q.

SEQ ID No. 3: nucleotide sequence encoding the modified synthetic consensus STAg MCV ATGGACCTGGTGCTGAACCGAAAGGAGAGGGAGGCCCTGTGCAAGCTGCTG

GAGATCGCCCCTAACTGTTACGGCAATATCCCACTGATGAAGGCCGCCTTCAAGAG GTCTTGCCTGAAGCACCACCCAAACAAGGGCGGCAATCCCGTGATCATGATGGAGC TGAACACCCTGTGGAGCAAGTTTCAGCAGAATATCCACAAGCTGCGGAGCGACTTCTCCATGTTTGATGAGGTGAGCACCAAGTTCCCCTGGGAGGAGTACGGAACAGCAGC AGCAGCAGCACAGTCCGG CTATAACGCCAGGTTTTGCAGAGGCCCTGGCTGTATGCI GAAGCAGCTGCGGGACTCCAAGTGCGCCTGTATCTCTTGCAAGCTGAGCCGCCAGC ACTGTTCTCTGAAGACCCTGAAGCAGAAGAATTGCGCCACATGGGGCGAGTGCTTCI GTTATCAGTGTTTTATCCTGTGGTTCGGCTTTCCCCCTACATGGGAGTCCTTCGATTG GTGGCAGAAAACCCTGGAAGAAACCGACTACTGTCTG CTGCATCTGCATCTGTTC

SEQ ID No. 4: Amino acid sequence of modified synthetic consensus STAg MCV

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMME LNTLWSKFQQNIHKLRSDFSMFDEVSTKFPWEEYGTAAAAAQSGYNARFCRGPGCMLK QLRDSKCACISCKLSRQHCSLKTLKQKNCATWGECFCYQCFILWFGFPPTWESFDWWQ KTLEETDYCLLHLHLF

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SEQ ID No. 5: nucleotide sequence encoding modified synthetic consensus LTAg and STAg associated with a furin cleavage site

ATGGACCTGGTGCTGAACAGGAAGGAGAGAGAGGCCCTGTGCAAGCTGCTG

GAGATCGCCCCCAACTGTTACGGCAATATCCCTCTGATGAAGGCCGCCTTCAAGCGG AGCTGCCTGAAGCACCACCCCAACAAGGGCGGCAACCCTGTGATCATGATGGAGCT GAATACCCTGTGGTCCAAGTTTCAGCAGAATATCCACAAGCTGCGGTCCGATTTCTC TATGTTTGACGAGGTGGATGAGGCCCCTATCTACGGCACCACCAAGTTCAAGGAGT GGTGGCGCTC CGGCGGCTTCTCTTTTGGCAAGGCCTACGAGTACGGCCCTAACCCAC ACGGCACCAATAGCAGGTCCAGAAAGCCAAGCTCCAACGCCAGCAGGGGAGCACC ATCCGGATCTAGCCCACCTCACAGCCAGTCCTCTAGCTCCGGCTACGGCTCTTTTAG CGCCTCCCAGGCCTCTGACAGCCAGTCCAGAGGCCCCGATATCCCACCCGAGCACC ACGAGGAGCCTACCTCTAGCTCCGGCTCTA GCTCCCGGGAGGAGACAACCAACAGC GGCAGGGAGTCTAGCACCCCAAACGGCACCTCCGTGCCAAGGAATTCCTCTAGGAC CGACGGAACCGCCGAGGACCTGTTCTGCGATAAGTCCCTGAGCTCCCCTGAGCCTCC ATCTAGCTCCGAGGAGCCAGAGGAGCCCCCTTCTAGCAGGTCCTCTCCCAGACAGCC ACCAAGCTCCTCTGCCGAGGAGGCAAGCTCCTCCAGTTCACCGACGAG GAGTACA GGAGCTCCTCTTTTACCACCCCTAAGACCCCTCCACCCTTCCCGGAAGCGCAAGT TTGGAGGCTCTAGGAGCTCCGCCTCTAGCGCCTCCTCTGCCAGCTTCACCTCCACCC CTCCAAAGCCCAAGAAGAACAGAGAGACACCCGTGCCTACCGACTTTCCTATCGAC CTGAGCGATTACCTGTCCCACGCCGTGTACTCTAATAAGACCGTGAGCTGTTTCGCC ATCTA CACCACCAGCGACAAGGCCATCGAGCTGTACGATAAGATCGAGAAGTTCAA GGTGGACTTCAAGTCCAGGCACGCATGCGAGCTGGGATGTATCCTGCTGTTCATCAC CCTGTCCAAGCACCGCGTGTCTGCCATCAAGAACTTCTGCAGCACCTTTTGTACCAT CTCCTTTCTGATCTGCAAGGGCGTGAATAAGATGCCTGAGATGTACAACAACCTGTG CAAGCCCCCTTACAAGCTGCTG CAGGAGAACAAGCCACTGCTGAATTACGAGTTCC AGGAAGAGGAGAAGGAGGCCAGCTGCAACTGGAATCTGGTGGCCGAGTTCGCCTGT GAGTACGAGCTGGACGATCACTTTATCATCCTGGCCCACTACCTGGACTTCGCCAAG CCATTTCCCTGCCAGAAGTGTGAGAACAGGTCTAGACTGAAGCCACACAAGGCCCA CGAGGCCCACCACTCCAATGCCAAGCTGTTTTACGAGTCTAAGAG CCAGAAGACCA TCTGCCAGCAGGCAGCAGACACCGTGCTGGCAAAGAGGAGACTGGGATGCTGGAG ATGACCAGGACCGAGATGCTGTGCAAGAAGTTCAAGAAGCACCTGGAGCGGCTGCG CGACCTGGATACCATCGATCTGCTGTACTACATGGGCGGCGTGGCCTGGTACTGCTG TCTGTTCGAGGAGTTTGAGAAGAAGCTGCAGAAGATCATCCAGCTGCTGACCGAGA ACAT CCCAAAGTACAGAAATATCTGGTTCAAGGGCCCCATCAACTCTGGCAAGACC AGCTTCGCCGCCGCCCTGATCGACCTGCTGGAGGGCAAGGCCCTGAACATCAATTGC CCTAGCGATAAGCTGCCATTCGAGCTGGGCTGTGCCCTGGACAAGTTCATGGTGGTG TTTGAGGATGTGAAGGGCCAGAACTCCCTGAATAAGGACCTGCAGCCCGGCCAGGG CATCAACAATGGATAACCTGC GACCACCTGGATGGAGCAGTGGCCGTGAGCC TGGAGAAGAAGCACGTGAACAAGAAGCACCAGATCTTCCCACCCTGCATCGTGACC GCCAATGACTACTTTATCCCAAAGACCCTGATCGCCCGCTTCTCTTACACCCTGCACT TTAGCCCCAAGGCCAACCTGAGGGACAGCCTGGATCAGAATATGGAGATCAGAAAG

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AGGCGCATCCTGCAGTCCGGAACCACCCTGCTGCTGTGCCTGATCTGGTGTCTGCCT GACACCACCTTCAAGCCATGCCTGCAGGAGGAGATCAAGAACTGGAAGCAGATCCT GCAGTCTGAGATCAGCTACGGCAAGTTTTGTCAGATGATCGAGAACGTGGAGGCCG GCCAGGACCCCCTGCTGAATATCCTGATCGAGGAGGAGGGCCCAGAGGAGACAGAG GAGACACA GGACTCCGGCACCTTCTCTCAGAGAGGCCGCAAAAGGAGGTCTGATCT GGTGCTGAATCGGAAAGAGAGAAGCCCTGTGCAAACTGCTGGAAATCGCCCCAA ACTGTTACGGCAACATCCCCCTGATGAAGGCCGCCTTCAAGAGGTCTTGCCTGAAGC ACCACCCAAACAAGGGCGGCAATCCCGTGATCATGATGGAGCTGAACACCCTGTGG AGCAAGTTTCAGCAGAATATCCACAAGCT GCGGAGCGACTTCTCCATGTTTGATGAG GTGAGCACCAAGTTCCCTTGGGAGGAGTACGGAACAGCAGCAGCAGCAGCACAGTC CGGCTATAACGCCAGGTTTTGCAGAGGCCCAGGCTGTATGCTGAAGCAGCTGCGGG ACTCCAAGTGCGCCTGTATCTCTTGCAAGCTGAGCCGCCAGCACTGTTCTCTGAAGA CCCTGAAGCAGAAGAATTGCGCCACATGGGGCGAGTGCTTCTGTTATCAG TGTTTTA TCCTGTGGTTCGGCTTTCCCCCTACATGGGAGTCCTTCGATTGGTGGCAGAAAACCC TGGAGGAAACTGATTACTGTCTGCTGCACCTGCACCTGTTC

SEQ ID No. 6: Amino acid sequence of modified synthetic consensus LTAg and STAg associated with furin cleavage site

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMME LNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPH GTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSS SGSSSREETTNSGRESSTPNGTSVPRNSSRTDG TAEDLFCDKSLSSPEPPSSSEEPEEPPSSR SSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTP PKPKKNRETPVPTDFPIDLSDYLSHAVYSNKTVSCFAIYTTSDKAIELYDKIEKFKVDFKS RHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYK LLQE NKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSR LKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKK HLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSG KTSFAAALIDLEGKAL NINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQG INNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPK ANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGK FCQMIENVEAGQDPLLNILIEEEGPEETEETQDSGTF SQRGRKRRSDLVLNRKEREALCK LLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFS MFDEVSTKFPWEEYGTAAAAAQSGYNARFCRGPGCMLKQLRDSKCACISCKLSRQHCS LKTLKQKNCATWGECFCYQCFILWFGFPPTWESFDWWQKTLEETDYCLLHLHLF SE Q ID No. 7: amino acid sequence of IgE leader sequence MDWTWILFLVAAATRVHS

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be construed as limiting the scope of the invention as defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to changes relating to chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of application of the invention, may be made without departing from the spirit and scope of the invention.

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Claims (19)

CLAIM 1. An immunogenic composition containing a nucleic acid molecule encoding at least one modified T-antigen of the Merkel cell polyomavirus (MCV), wherein the T-antigen contains at least one mutation that disrupts at least one oncogenic feature of the native MCV T-antigen wherein at least one oncogenic trait is selected from the group consisting of CR1 binding, DnaJ binding, pp2A phosphatase binding, Rb binding, ATPase activity, helicase activity, chaperone protein binding, hVam6p binding, Fbxw7 binding, origin of replication binding, and transformation , and where: (I) the nucleic acid molecule encodes a peptide containing an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of an amino acid sequence that is at least 99.7% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 2, an amino acid sequence that is at least 98% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence identical to at least 90% over the entire length of the amino acid sequence of SEQ ID NO: 6; b) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; And c) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; and/or (II) the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence that is at least 90% identical over the entire length to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; b) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; And c) an immunogenic fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. 2. The immunogenic composition according to claim 1, characterized in that at least one mutation is a mutation in 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, characterized in that at least one mutation is selected from the group consisting of the D44N mutation, the W209A mutation, the E216K mutation, the L142A mutation, the L91A mutation, the K92A mutation, the D93A mutation, the Y94A mutation and the M95A mutation . 4. The immunogenic composition according to claim 1, characterized in that the MCV T-antigen is selected from the group consisting of a large T-antigen (LTAg), a small t-antigen (STAg) and a combination thereof. 5. An immunogenic composition according to claim 1, characterized in that the nucleic acid molecule encodes a peptide containing an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of an amino acid sequence identical to at least 99.7% over the entire length of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, an amino acid sequence identical to at least 98 % over the entire length of the amino acid sequence SEQ ID NO: 4, or an amino acid sequence identical to at least 90% over the entire length of the amino acid sequence SEQ ID NO: 6; b) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; And c) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. 6. An immunogenic composition according to claim 1, characterized in that the nucleic acid molecule is selected from the group consisting of a DNA molecule and an RNA molecule. 7. An immunogenic composition according to claim 1, characterized in that the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence that is at least 90% identical over the entire length to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, b) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, and c) an immunogenic fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. - 34 045600 8. An immunogenic composition according to claim 1, characterized in that 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. 9. An immunogenic composition according to claim 8, characterized in that the nucleic acid molecule encodes a peptide containing an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of an amino acid sequence that is at least 99.7% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 2, an amino acid sequence that is at least 98% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence identical to at least 90% over the entire length of the amino acid sequence SEQ ID NO: 6, b) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and c) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, operably related to the amino acid sequence specified in SEQ ID NO: 7. 10. An immunogenic composition according to claim 9, characterized in that the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence that is at least 90% identical over the entire length to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 b) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, and c) an immunogenic fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, operably linked to the nucleotide sequence encoding SEQ ID NO: 7. 11. An immunogenic composition according to claim 1, characterized in that the nucleic acid molecule contains an expression vector. 12. The immunogenic composition according to claim 1, characterized in that the immunogenic composition further comprises a viral particle, wherein the nucleic acid molecule is included in the viral particle. 13. An immunogenic composition according to claim 1, characterized in that it additionally contains a pharmaceutically acceptable excipient. 14. Immunogenic composition according to claim 1, characterized in that it additionally contains an adjuvant. 15. Use of an immunogenic composition according to claim 1 in a method of inducing an immune response against MCV T-antigen in a subject in need thereof, which comprises administering the immunogenic composition of the invention. 16. Use according to claim 15, characterized in that the administration includes at least one of the following methods: electroporation and injection. 17. Use of an immunogenic composition according to claim 1 in a method of treating or preventing MCV-associated pathology in a subject in need thereof, which includes administering the immunogenic composition to the subject. 18. Use according to claim 17, characterized in that the administration includes at least one of the following methods: electroporation and injection. 19. The use of claim 18, wherein the MCV-associated pathology is at least one of MCV infection and Merkel cell carcinoma. -