JP6457481B2 - New vaccine against multiple subtypes of dengue virus - Google Patents

Description

  The present invention relates to improved dengue vaccines, improved methods for inducing an immune response, and for immunizing an individual prophylactically and / or therapeutically against dengue virus.

  Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes dengue fever (DF) and a serious life-threatening disease, dengue hemorrhagic fever / dengue fever shock syndrome (DHF / DSS). DENV is a small, enveloped, plus-strand RNA virus that belongs to the genus Flaviviridae of the Flaviviridae family. Four different subtypes or serotypes of dengue viruses (DV-1 to DV-4) are transmitted to humans by being bitten by Aedes aegypti and Aedes aegypti. It is estimated that 50 million to 100 million cases of DF and 250,000 to 500,000 cases of DHF occur every year. Dengue fever is an important international public health problem because two-fifths of the world's population lives in endemic areas of dengue and 50 to 100 million dengue infections occur annually. In addition, 2.5 billion people are at risk of infection in the world's subtropics and tropics where there is no effective intervention.

  More than 100 tropical countries have endemic dengue virus infections, and DHF has been recorded in more than 60 of these countries. Surveillance of DF / DHF is unsatisfactory in most countries and has focused primarily on DHF in the past; thus, the number of DF cases that occur each year can only be an estimate. However, in 1998, illness was widespread throughout Asia and the United States, and over 1.2 million cases of DF / DHF were reported to the World Health Organization (WHO). DHF global reports have increased on average by a factor of five over the past 20 years. At the beginning of the 21st century, it is estimated that 50 million to 100 million DF and hundreds of thousands of DHF occur each year depending on the activity of the epidemic. The mortality rate (CFR) varies from country to country, but in some countries it can be as high as 10-15% and in other countries it can be less than 1%.

  There are four subtypes of dengue virus: dengue-1 (DV-1), dengue-2 (DV-2), dengue-3 (DV-3), and dengue-4 (DV-4). Each of these subtypes forms different antigenic subgroups within the Flaviviridae family. They are enveloped RNA viruses that encode 10 proteins: 3 structural proteins and 7 nonstructural proteins. Structural proteins are capsid (C), envelope (E), and premembrane precursor (preM). The intracellular life cycle of DV begins with receptor-mediated endocytosis of the virus into the cell, followed by fusion of the viral envelope protein with the late endosomal membrane, thereby releasing the viral genome into the cytoplasm for replication .

  Infection with DV may be asymptomatic or may be characterized by fever, chills, frontal pain, muscle pain, joint pain, and rash. Subsequent infection with different serotypes may result in more severe disease signs with plasma leakage or bleeding (dengue hemorrhagic fever) and shock (dengue shock syndrome). Extensive research has been conducted over the years to understand the pathogenicity of DENV infection, but the development of specific anti-DV compounds has made little progress. There are currently no specific antiviral agents or vaccines for dengue infections approved in the United States.

  The envelope (E) glycosylated protein, the main structural protein present on the surface of mature dengue virus particles, is a type I integral membrane protein. The mature dengue E protein has been demonstrated to form homodimers in a reverse tandem fashion (head-to-tail orientation). Each monomer has three distinct domains: domain I (DI, major N-terminal domain), domain II (DII, dimerization domain), and domain III (PRM / E, immunoglobulin (Ig) -like C- It is folded into a terminal protein. The PRM / E domain of E protein consists of 100 amino acids at the C-terminus (residues 303-395). This domain has been suggested to be a receptor recognition and binding domain. Ig-like folds present in PRM / E proteins generally associate with structures that have an adhesion function. This domain extends perpendicular to the virus surface, with the tip protruding further from the surface of the virus particle than any other part of the E protein. In addition, studies have demonstrated that both recombinant PRM / E proteins and antibodies generated against PRM / E of flavivirus E protein can inhibit flavivirus entry into target cells. In addition, flaviviruses with mutations in the E protein PRM / E show attenuated toxicity or ability to escape immune neutralization.

  Development of a safe and effective vaccine against dengue virus infection remains a major public health goal. If it is believed that the presence of neutralizing antibodies is primarily associated with immunity against dengue virus, a prerequisite for comparing and optimizing vaccine candidates is to accurately neutralize vaccine-induced neutralizing antibody responses. Is the ability to measure. A combination of vaccines containing live attenuated viruses from all four serotypes has been shown to cause several complications (Guy B, Almond JW, Comp Immunol Microbiol Infect Dis. 2008 Mar. 31 (2-3): 239-52). In addition, there are few reports on adenovirus-based delivery of dengue antigens. Nevertheless, a well-recognized problem with the adenovirus system is that the majority of the population is known to have antibodies against one of the adenoviruses, and such existing antibodies are not compatible with these adeno-derived vaccines. There is a possibility of invalidating.

  Thus, there remains a need for the development of vaccines that provide broad immunity or universal immunity against all four, preferably all four serotypes of dengue virus, preferably economical and effective vaccines across all serotypes. In addition, there remains a need for effective methods of administering vaccines, such as DNA vaccines or DNA plasmid vaccines, to mammals to provide immunity against dengue virus, either prophylactically or therapeutically.

  One aspect of the invention provides a nucleic acid construct capable of expressing a polypeptide that elicits an immune response in a mammal against two or more subtypes of dengue virus. The nucleic acid construct includes an encoded nucleotide sequence and a promoter operably linked to the encoded nucleotide sequence. The encoded nucleotide sequence expresses a polypeptide comprising domain III (PRM / E domain or PRM / E) of an envelope protein from at least two different dengue virus subtypes. The promoter regulates the expression of the polypeptide in the mammal.

  Another aspect of the invention provides a DNA plasmid vaccine that can generate an immune response against multiple subtypes of dengue virus in a mammal. A DNA plasmid vaccine includes a DNA plasmid capable of expressing a common dengue antigen in a mammal and a pharmaceutically acceptable excipient. The DNA plasmid contains a promoter operably linked to a coding sequence that encodes a common dengue PRM / E antigen. The common dengue antigen comprises a common PRM / E domain of dengue virus-subtype 1, dengue virus-subtype 2, dengue virus-subtype 3, or dengue virus-subtype 4.

  Another aspect of the present invention is a process comprising delivering a DNA plasmid vaccine to mammalian tissue, wherein the DNA plasmid vaccine is adapted to induce viral responses in mammalian cells in order to elicit an immune response in the mammal. A DNA plasmid capable of expressing a plurality of common antigens derived from a plurality of subtypes, wherein the plurality of common antigens comprises antigen domains derived from at least two different subtypes of the virus; Electroporating tissue cells with a pulse of energy to allow entry of DNA plasmids into the cells, and a method of inducing an immune response against a plurality of viral subtypes in a mammal. provide.

The binding titers for all dengue PRM / E domains from subtype 1 of sera from mice inoculated with D1prME against sera from mice inoculated with DU are shown. The binding titers for all dengue PRM / E domains from subtype 2 of sera from mice inoculated with D2prME against sera from mice inoculated with DU are shown. The binding titers for all dengue PRM / E domains from subtype 3 of sera from mice inoculated with D3prME against sera from mice inoculated with DU are shown. The binding titers for all dengue PRM / E domains from subtype 4 of sera from mice inoculated with D4prME against sera from mice inoculated with DU are shown. Staining gel showing bound antibodies made against prME proteins D1, D2, D3, and D4 types of mice immunized with PRM / E domains derived from serotypes 1, 2, 3, or 4 Show. FIG. 3 shows a graph showing neutralizing antibodies against one of each of dengue PRM / E proteins (types 1 to 4) for the control guinea pig serum. The graph which shows the neutralizing antibody with respect to each one of Dengue PRM / E protein (1 type-4 type) about the serum of DU-DIII guinea pig is shown. FIG. 7 shows graphs showing neutralizing antibodies against each one of dengue PRM / E proteins (types 1 to 4) for guinea pig sera with D1 to D4 prME (combining all four vaccines in one mixture and summing up) Administer). FIG. 2 shows graphs showing neutralizing antibodies against each one of dengue PRM / E proteins (types 1 to 4) for guinea pig sera of D1 to D4 prME (each vaccine is administered separately). The graph which shows the neutralizing antibody with respect to each one of dengue PRM / E protein (type 1-4 type) about the serum of D1 prME guinea pig is shown. The graph which shows the neutralizing antibody with respect to each one of dengue PRM / E protein (1 type-4 type) about the serum of D2prME guinea pig is shown. The graph which shows the neutralizing antibody with respect to each one of dengue PRM / E protein (1 type-4 type) about the serum of D3prME guinea pig is shown. The graph which shows the neutralizing antibody with respect to each one of dengue PRM / E protein (1 type-4 type) about the serum of D4prME guinea pig is shown. Figure 3 shows a graph showing neutralizing antibodies against Dengue 1 virus for sera from animals inoculated with all four D1-D4 prMEs. Figure 3 shows a graph showing neutralizing antibodies against dengue 2 virus for sera from animals inoculated with all four D1-D4 prMEs. FIG. 2 shows a graph showing neutralizing antibodies against dengue 3 virus for sera from animals inoculated with all four D1-D4 prMEs. Figure 3 shows a graph showing neutralizing antibodies against Dengue 4 virus for sera from animals inoculated with all four D1-D4 prMEs.

  The following simplified or shortened definitions are provided to aid in understanding the preferred embodiments of the present invention. The concise definitions provided herein are by no means exclusive and are consistent with definitions understood in the technical field or dictionary sense. Simplified definitions are provided herein to supplement or more clearly define definitions known in the art.

Definitions As used herein, nucleotide and amino acid sequence homologies are FASTA, BLAST, and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which are incorporated by reference in their entirety. Incorporated herein) and PAUP ^* 4.0b10 software (DL Swoffford, Sinauer Associates, Massachusetts). Briefly, the BLAST algorithm (abbreviation for Basic Local Alignment Search Tool) is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403). -410, the entire text of which is incorporated herein by reference). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the chance of a coincidence between two nucleotide sequences. . For example, if the minimum total probability in the comparison of the test nucleic acid with other nucleic acids is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, Is considered similar to another nucleic acid. “Percentage of similarity” can be calculated using PAUP ^* 4.0b10 software (DL Swoffford, Sinauer Associates, Massachusetts). The average similarity of consensus sequences is calculated by comparing all sequences in the phylogenetic tree.

  As used herein, the term “nucleic acid construct” refers to a DNA or RNA molecule that comprises a nucleotide sequence that encodes a protein. A coding sequence, or “encoding nucleic acid sequence”, contains initiation and termination signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered. May be included.

  As used herein, the term “expressible form” is operably linked to a coding sequence that encodes a protein such that the coding sequence is expressed when present in the cells of the individual. Refers to a nucleic acid construct containing essential regulatory elements.

  The term “constant current” is used herein to define the current that is received or felt by the same tissue or the cells that define the tissue during the duration of an electrical pulse delivered to the tissue. The electrical pulses are delivered from the electroporation device described herein. This current maintains a constant amperage in the tissue while the electrical pulse is maintained, because the electroporation device provided herein preferably has a feedback element with immediate feedback. The feedback element measures tissue (or cell) resistance during the duration of the pulse, and during the electrical pulse (in microseconds) within the same tissue, and between pulses The electric energy output can be changed (for example, the voltage can be increased) so as to keep constant. In some embodiments, the feedback element includes a controller.

  The terms “feedback” or “current feedback” are used interchangeably and refer to the active response of the provided electroporation device, which measures the current in the tissue between the electrodes and maintains the current at a constant level. Therefore, it includes changing the output of the energy delivered by the EP apparatus as appropriate. This constant level is preset by the user before the start of the pulse sequence or electrical processing. Preferably, the feedback is achieved by an electroporation component of the electroporation device, eg, a controller, because the electrical circuit therein continuously monitors the current in the tissue between the electrodes and the monitored current (or in the tissue Current) and a preset current, the energy output is continuously adjusted, and the monitored current is maintained at a preset level. In some embodiments, the feedback loop is immediate because it is an analog closed loop feedback.

  The terms “electroporation”, “electropermeabilization”, or “electro-kinetic enhancement” (“EP”) are used interchangeably herein and refer to micropaths (pores) in biological membranes. ), And the presence of their micropaths allows plasmids, oligonucleotides, siRNA, drugs, ions, and / or water to pass from one side of the cell membrane to the other. It becomes possible.

  The term “distributed current” is used herein to define a current pattern delivered from the various needle electrode arrays of the electroporation device described herein, the pattern being of the tissue to be electroporated. Minimize or preferably eliminate the occurrence of thermal stresses associated with electroporation in all areas.

  The term “feedback mechanism” as used herein refers to a process performed by either software or hardware (or firmware), which is the desired tissue impedance (before delivery of a pulse of energy, During and / or after) and comparing the current value, preferably the current, and adjusting the pulse of energy delivered to obtain a preset value. The term “impedance” is used herein when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thereby allowing comparison with a preset current. In a preferred embodiment, the “feedback mechanism” is implemented by an analog closed loop circuit.

  The term “immune response” is used herein to activate the host, eg, mammalian immune system, in response to the introduction of a dengue antigen, eg, a universal dengue antigen, via the provided DNA plasmid vaccine. Means. The immune response may be in the form of a cellular or humoral response, or both.

  The terms “common” or “consensus sequence” are used herein to analyze a sequence comparison of multiple strains of a particular dengue subtype (thus subtype-1, subtype-2, subtype-3, Means a synthetic nucleic acid sequence or a corresponding polypeptide sequence constructed on the basis of a subtype-4 common dengue sequence and the universal dengue described below). The common universal dengue can be used to broadly induce immunity against multiple subtypes or serotypes of dengue virus.

  The term “adjuvant” is used herein and is added to the DNA plasmid vaccine described herein to enhance the antigenicity of the dengue antigen encoded by the DNA plasmid and encoding nucleic acid sequence described below. Means the molecule.

  The terms “subtype” or “serotype” are used interchangeably herein and relate to a virus, eg, a dengue virus, such that one subtype is recognized by a different immune system than a different subtype. Means a genetic variant of a viral antigen. For example, dengue virus subtype 1 is immunologically distinguishable from dengue virus subtype 2.

  One aspect of the invention provides a nucleic acid construct capable of expressing a polypeptide that elicits a mammalian immune response against two or more subtypes of dengue virus. The nucleic acid construct includes an encoded nucleotide sequence and a promoter operably linked to the encoded nucleotide sequence. The encoded nucleotide sequence expresses a polypeptide comprising a PRM / E domain of at least two different subtypes of dengue virus. The promoter regulates the expression of the polypeptide in the mammal.

  In some embodiments, the nucleic acid construct may further include an IgE leader sequence operably linked to the N-terminus of the coding sequence and operably linked to the promoter. Preferably, the IgE leader has the sequence of SEQ ID NO: 11. The nucleic acid construct may also include a polyadenylation sequence attached to the C-terminus of the coding sequence. Preferably, the nucleic acid construct is codon optimized.

In some embodiments, the coding sequence encodes a polypeptide comprising a PRM / E domain from dengue virus-subtype 1, dengue virus-subtype 2, dengue virus subtype-3, and dengue virus subtype-4. Turn into. In a preferred embodiment, the encoded nucleotide sequence is
SEQ ID NO: Description 1 Den1-prME DNA sequence 2 Den1-prME protein sequence 3 Den2-prME DNA sequence 4 Den2-prME protein sequence 5 Den3-prME DNA sequence 6 Den3-prME protein sequence 7 Den4-prME DNA sequence 8 Den4-prME protein Selected from the group consisting of sequences.

  Another aspect of the invention provides a DNA plasmid vaccine that can generate an immune response against multiple subtypes of dengue virus in a mammal. The DNA plasmid vaccine comprises a DNA plasmid capable of expressing a common dengue antigen in a mammal and a pharmaceutically acceptable excipient. The DNA plasmid includes a promoter operably linked to a coding sequence that encodes a common dengue antigen. The common dengue antigen comprises a common PRM / E domain of dengue virus-subtype 1, dengue virus-subtype 2, dengue virus-subtype 3, or dengue virus-subtype 4. Preferably, the DNA plasmid comprises a common dengue antigen encoding a common dengue antigen selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

  In some embodiments, the DNA plasmid further comprises an IgE leader sequence linked to the N-terminus of the coding sequence and operably linked to a promoter. Preferably, the IgE leader has the sequence Met Arg Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val His Ser.

  The DNA plasmid may further comprise a polyadenylation sequence linked to the C-terminus of the coding sequence. Preferably, the DNA plasmid is codon optimized.

  In some embodiments, the pharmaceutically acceptable excipient is an adjuvant. Preferably, the adjuvant is selected from the group consisting of IL-12 and IL-15. In some embodiments, the pharmaceutically acceptable excipient is a transfection facilitating agent. Preferably, the transfection facilitating agent is a polyanion, polycation, or lipid, more preferably poly-L-glutamic acid. Preferably, the concentration of poly-L-glutamic acid is less than 6 mg / mL. Preferably, the DNA plasmid vaccine has a total DNA plasmid concentration of 1 mg / mL or more.

  In some embodiments, the DNA plasmid encodes a polypeptide comprising each dengue virus-subtype 1, dengue virus-subtype 2, dengue virus-subtype 3, or dengue virus-subtype 4 of prM / E. Contains multiple unique DNA plasmids.

  The DNA plasmid vaccine comprises an encoded nucleotide sequence: SEQ ID NO: 1 which is a nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 3 which is a nucleotide sequence encoding SEQ ID NO: 4, and a nucleotide sequence which encodes SEQ ID NO: 6 DNA plasmids comprising SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8 can be included.

  In some embodiments, the DNA plasmid vaccine comprises at least two different DNA plasmids that express dengue virus prME. In some embodiments, the DNA plasmid vaccine may comprise four common dengue virus prM / E domains (subtypes 1-4).

  In some embodiments, the mammal against which the DNA plasmid vaccine produces an immune response is a primate. Preferably the mammal is a primate. The immune response may be either a humoral response or a cellular response, preferably both.

  Another aspect of the invention is the electroporation of tissue cells by delivering a DNA plasmid vaccine to mammalian tissue and a pulse of energy at a constant current effective to allow DNA plasmid entry into the cell. And a method of inducing an immune response against multiple subtypes of dengue virus in a mammal.

  In some embodiments, the method of eliciting an immune response includes a delivery step in which the DNA plasmid vaccine is injected into intradermal, subcutaneous, or muscle tissue.

  In some embodiments, a method of inducing an immune response includes pre-setting a current desired to be delivered to a tissue and electroporating tissue cells with a pulse of energy at a constant current equal to the preset current. And a step of performing.

  In some embodiments, the method of inducing an immune response includes measuring the impedance of an electroporated cell and measuring the impedance against the measured impedance to maintain a constant current in the electroporated cell. Adjusting the energy level of the energy pulse. The measurement and adjustment steps are preferably performed within the duration of the energy pulse.

  In some embodiments, the electroporation process includes delivering pulses of energy to the plurality of electrodes according to a pulse sequence pattern that delivers pulses of energy in a distributed pattern.

  In some embodiments of the invention, the DNA plasmid vaccine may further comprise an adjuvant. In some embodiments, the adjuvant is alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFα, including IL-15 that lacks a signal sequence and optionally comprises a signal peptide from IgE. , TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell attracting chemokine (CTACK), epithelial thymus-expressing chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, It is selected from the group consisting of CD80 and CD86. Other genes that may be useful adjuvants include MCP-1, MIP-1-alpha, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM -1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF IL-4, IL-18 mutant, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo- 1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, GKG2B, GKG, 2G Those encoding TAP1, TAP2, and functional fragments thereof. In some preferred embodiments, the adjuvant is selected from IL-12, IL-15, CTACK, TECK, or MEC.

  In some embodiments, the pharmaceutically acceptable excipient is a transfection facilitator, which is a LPS comprising a surfactant such as an immune stimulating complex (ISCOMS), Freund's incomplete adjuvant, monophosphoryl lipid A. Analogs, muramyl peptides, quinone analogs, vesicles like squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitation An agent may be included. Preferably, the transfection facilitating agent is a polyanion, a polycation containing poly-L-glutamic acid (LGS), or a lipid. Preferably, the transfection facilitating agent is poly-L-glutamic acid, more preferably poly-L-glutamic acid is present in the DNA plasmid vaccine at a concentration of less than 6 mg / mL. In some embodiments, the poly-L-glutamic acid concentration in the DNA plasmid vaccine is less than 4 mg / mL, less than 2 mg / mL, less than 1 mg / mL, less than 0.750 mg / mL, less than 0.500 mg / mL, Less than 0.250 mg / mL, less than 0.100 mg / mL, less than 0.050 mg / mL, or less than 0.010 mg / mL.

  In some embodiments, the DNA plasmid vaccine can be delivered to a mammal to elicit an immune response, preferably the mammal is a primate, including human and non-human primates, cows, pigs, chickens, A dog or a ferret. More preferably, the mammal is a human primate.

  One aspect of the invention relates to methods for inducing an immune response against multiple subtypes of viruses in a mammal. These methods include the steps of delivering a DNA plasmid vaccine to mammalian tissue, and electroporating the cells of the tissue with a pulse of energy at a constant current effective to allow entry of the DNA plasmid into the cells; including. The DNA plasmid vaccine comprises a DNA plasmid capable of expressing a plurality of common antigens derived from a plurality of viral subtypes in a mammalian cell in order to elicit an immune response in the mammal, Antigens include antigenic domains from at least two different subtypes of viruses.

  One aspect of the invention relates to methods for eliciting an immune response against multiple subtypes of dengue virus in a mammal. These methods involve delivering a DNA plasmid vaccine to mammalian tissue, wherein the DNA plasmid vaccine expresses a common dengue antigen in mammalian cells to elicit an immune response in the mammal. A concomitant dengue antigen comprising a consensus sequence encoding a prME protein from at least two dengue subtypes, preferably all four dengue subtypes. Examples of the dengue subtype include subtype-1, subtype-2, subtype-3, and subtype-4. The method of inducing an immune response includes the step of electroporating tissue cells with a pulse of energy at a constant current effective to allow DNA plasmid entry into the cell.

  In some embodiments, the methods of the invention include a delivery step, which involves injecting a DNA plasmid vaccine into intradermal, subcutaneous, or muscle tissue. Preferably, these methods use an in vivo electroporation device to preset a current that is desired to be delivered to the tissue, and to electrolyze tissue cells by a pulse of energy at a constant current equal to the preset current. Drilling. In some embodiments, the electroporation process is electroporated by measuring the impedance of the electroporated cell and adjusting the energy level of a pulse of energy relative to the measured impedance. Maintaining a constant current in the cells, wherein the measuring and adjusting step is performed within the duration of the energy pulse.

  In some embodiments, the electroporation process includes delivering pulses of energy to the plurality of electrodes according to a pulse sequence pattern that delivers pulses of energy in a distributed pattern.

  The present invention also includes a plurality of DNA fragments encoding polypeptides that are capable of eliciting an immune response in a mammal that are substantially similar to those that are not fragments of at least one subtype of dengue virus. These DNA fragments are SEQ ID NO: 1 which is a nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 3 which is a nucleotide sequence encoding SEQ ID NO: 4, and SEQ ID NO: 5 which is a nucleotide sequence encoding SEQ ID NO: 6. , Selected from at least one of the various encoding nucleic acid sequences of the present invention, including SEQ ID NOs: 7 and 8, when applied as a particular encoding nucleic acid sequence provided herein, It may be any DNA fragment. In some embodiments, the DNA fragment is 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more. It may contain 270 or more, 300 or more, 320 or more, 340 or more, or 360 or more nucleotides. In some embodiments, the DNA fragment may comprise a coding sequence for an immunoglobulin E (IgE) leader sequence. In some embodiments, the DNA fragment is less than 60, less than 75, less than 90, less than 120, less than 150, less than 180, less than 210, less than 240, less than 270, less than 300. , Less than 320, less than 340, or less than 360 nucleotides.

  The present invention includes a polypeptide encoded by the encoded nucleotide sequence and may include a polypeptide having the amino acid sequence of SEQ ID NOs: 2, 4, 6, and 8. The invention also includes polypeptide fragments that are capable of eliciting an immune response in a mammal that are substantially similar to those that are not fragments of at least one subtype of dengue. These polypeptide fragments are selected from at least one of the various polypeptide sequences of the present invention, including SEQ ID NOs: 2, 4, 6, and 8, when applied to the specific polypeptide sequences provided herein. And any of the polypeptide fragments described below. In some embodiments, the polypeptide fragment is 15 or more, 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 100 or more, 110 or more, or 120 or more amino acids. May be included. In some embodiments, the polypeptide fragment may comprise less than 30, less than 45, less than 60, less than 75, less than 90, less than 100, less than 110, or less than 120 amino acids. .

  The determination of a functional fragment that elicits an immune response in a mammal that is substantially similar to a non-dengue fragment of at least one dengue can be readily determined by one skilled in the art. The fragment can also be analyzed to include at least one, and preferably more antigenic epitopes, as provided by a publicly available database, such as the National Center for Biotechnology Information (NCBI). In addition, immune response studies can be routinely assessed using mice and antibody titer and ELI spot analysis as shown in the examples below.

Vaccines In some embodiments, the present invention improves by providing a genetic construct encoding a protein and a protein having an epitope that makes the protein particularly effective as an immunogen against which an immune response can be elicited. Provided vaccines. Thus, a vaccine can be provided to induce a therapeutic or prophylactic immune response.

  According to some embodiments of the invention, the vaccine according to the invention is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response. When a nucleic acid molecule encoding a protein is taken up by an individual's cells, the nucleotide sequence is expressed in the cell, thereby delivering the protein to the individual. Aspects of the invention provide methods for delivering a coding sequence for a protein on a nucleic acid molecule, such as a plasmid.

  In accordance with some aspects of the present invention, compositions and methods for immunizing an individual prophylactically and / or therapeutically are provided.

  When taken up by a cell, the DNA plasmid can remain in the cell as separate genetic material. Alternatively, RNA may be administered to the cells. It is also contemplated to provide the gene construct as a linear minichromosome containing centromeres, telomeres, and origins of replication. A gene construct contains regulatory elements that are essential for gene expression of a nucleic acid molecule. Such elements include promoters, start codons, stop codons, and polyadenylation signals. In addition, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulating proteins. It is necessary that these elements be operably linked to the sequence encoding the desired protein and function within the individual to which the regulatory elements have been administered.

  Start and stop codons are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, these elements need to function within the mammal to which the nucleic acid construct is administered. These start and stop codons must be in frame with the coding sequence.

  The promoter and polyadenylation signal used must function in the cells of the individual.

  Examples of promoters useful in the practice of the present invention, particularly in the production of human genetic vaccines, include the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, and the bovine immunodeficiency virus (BIV). Human immunodeficiency virus (HIV) terminal repeat (LTR) promoter, Moloney virus, avian leukemia virus (ALV), cytomegalovirus (CMV) such as CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma Virus (RSV) and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein, including but not limited to, in other embodiments, the promoter is natural or Synthetic A tissue-specific promoters, such as muscle or skin-specific promoters may be. Examples of such promoters are described in US Patent Application Publication No. 20040175727, the entire text of which is incorporated herein by reference.

  Examples of polyadenylation signals useful in the practice of the invention, particularly in the generation of genetic vaccines for humans, include SV40 polyadenylation signals, LTR polyadenylation signals, bovine growth hormone (bGH) polyadenylation signals, human growth hormone (hGH ) Polyadenylation signal, and human β-globin polyadenylation signal. Specifically, the SV40 polyadenylation signal in the pCEP4 plasmid (Invitrogen, San Diego, CA), referred to as the SV40 polyadenylation signal, may be used.

  In addition to the regulatory elements required for DNA expression, other elements may be included in the DNA molecule. Such additional elements include enhancers. Such enhancers include, but are not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those derived from CMV, RSV, and EBV.

  The genetic construct may comprise a mammalian origin of replication to maintain the construct extrachromosomally and to generate multiple copies of the construct within the cell. Plasmids pVAX1, pCEP4, and pREP4 from Invitrogen (San Diego, Calif.) Contain the Epstein Barr virus origin of replication and the nuclear antigen EBNA-1 coding region that results in high copy episomal replication without integration.

  In order to maximize protein production, regulatory sequences that are very suitable for gene expression in the cells to which the construct is administered may be selected. In addition, a codon encoding the protein that is most efficiently transcribed in the host cell may be selected. One skilled in the art can create DNA constructs that function in cells.

  In some embodiments, a nucleic acid construct can be provided in which the coding sequence of a protein described herein is linked to an IgE signal peptide. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

  In some embodiments where proteins are used, for example, one of skill in the art can use known techniques to produce and isolate the proteins of the invention using known techniques. In some embodiments in which the protein is used, for example, those skilled in the art can use well-known techniques to place a DNA molecule encoding the protein of the invention into a commercially available expression vector for use in a well-known expression system. Can be inserted. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) May be used to produce proteins in E. coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) May be used, for example, for production in budding yeast strains of yeast. A commercially available MAXBAC ™ complete baculovirus expression system (Invitrogen, San Diego, Calif.) May be used, for example, for production in insect cells. Commercially available plasmid pcDNA or pcDNA3 (Invitrogen, San Diego, CA) may be used for production in mammalian cells such as, for example, Chinese hamster ovary (CHO) cells. One skilled in the art can use these commercially available expression vectors and systems, or others, to produce proteins using routine techniques and readily available starting materials (see, eg, Sambrook et al., Molecular (See Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)). Thus, the desired protein can be prepared in both nucleated and eukaryotic cell systems, resulting in a spectrum of processed forms of the protein.

  One of skill in the art may use other commercially available expression vectors and systems, or make vectors using well-known methods and readily available starting materials. Expression systems containing essential regulatory sequences such as promoters and polyadenylation signals, preferably enhancers, are readily available and are known in the art for a variety of hosts. For example, Sambrook et al. , Molecular Cloning a Laboratory Manual, Second Ed. See Cold Spring Harbor Press (1989). A genetic construct includes a protein coding sequence that is operably linked to a promoter that functions in a cell line or target tissue cell into which the construct is transfected. Examples of constitutive promoters include cytomegalovirus (CMV) or SV40 derived promoters. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoter. One of ordinary skill in the art can readily make genetic constructs useful for transfecting cells encoding DNA encoding the protein of the invention from readily available starting materials. An expression vector containing DNA encoding the protein is used to transform a compatible host that is subsequently cultured and maintained under conditions in which the foreign DNA is expressed.

  The produced protein is recovered from the culture by lysing the cells or, if necessary, from a culture medium known in the art. One skilled in the art can isolate proteins produced using such expression systems using well-known techniques. A method for purifying a protein derived from a natural material using an antibody that specifically binds to a specific protein as described above may be similarly applied to purify a protein produced by recombinant DNA methodology.

  In addition to protein production by recombinant techniques, an automated peptide synthesizer can also be used to make isolated essentially pure protein. Such techniques are well known to those of skill in the art and are useful when no derivative is provided in the production of the DNA encoded protein.

  Nucleic acid molecules can be injected into DNA with or without in vivo electroporation, liposome-mediated, nanoparticle-enhanced, recombinant vectors such as recombinant adenoviruses, recombinant adenovirus-related viruses and recombinant vaccinia viruses ( It may be delivered using any of several well-known techniques, including DNA vaccination). Preferably, nucleic acid molecules such as the DNA plasmids described herein are delivered via DNA injection along with in vivo electroporation.

  The routes of administration are intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, and topically, transdermally, by inhalation or suppository, or vagina, rectum, urethra. The administration route includes, but is not limited to, washing of mucosal tissues such as oral cavity and sublingual tissue. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The gene construct can be a conventional syringe, needle-free injection device, “microprojectile bombardment gun”, or other physical methods such as electroporation (EP), “fluid mechanics”, or ultrasound. Can be administered by means not limited to these.

  Examples of preferred electroporation devices and electroporation methods for facilitating delivery of the DNA vaccines of the present invention include US Pat. No. 7,245,963 (Draghia-Akli et al.), US Patent Application Publication No. 2005/0052630. (Smith et al.), The entire contents of which are incorporated herein by reference. Also, a co-pending and co-owned US patent application No. 11/874072 filed on October 17, 2007 (US provisional application filed on October 17, 2006 under section 119 (e) of the US Patent Act) No. 60 / 852,149, and No. 60 / 978,982 filed Oct. 10, 2007, all of which are incorporated herein by reference) Also preferred are electroporation devices and electroporation methods to facilitate the delivery of the DNA vaccines that have been made.

  US Pat. No. 7,245,963 (Draghia-Akli et al.) Describes a modular electrode system and its use to facilitate the introduction of biomolecules into cells of selected tissues in the body or plant. Yes. The module electrode system includes a plurality of needle electrodes, a hypodermic needle, an electrical connector that provides a conductive connection from the programmable constant current pulse controller to the plurality of needle electrodes, and a power source. An operator can grab a plurality of needle electrodes mounted on a support structure and securely insert them into selected tissue in the body or plant. The biomolecule is then delivered to the selected tissue via a hypodermic needle. A programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electric pulse promotes the introduction of biomolecules between the plurality of electrodes into the cell. The entire text of US Pat. No. 7,245,963 is incorporated herein by reference.

  US Patent Application Publication No. 2005/0052630 (Smith et al.) Describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells into selected tissues in the body or plants. doing. The electroporation apparatus includes an electrodynamic device (“EKD device”) whose operation is determined by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between arranged electrodes based on user control and input of pulse parameters, allowing the storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disk having a series of needle electrodes, a central injection channel for the injection needle, and a removable guide disk. The entire text of US Patent Application Publication No. 2005/0052630 is incorporated herein by reference.

  The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005/0052630 are adapted to penetrate deeply into other tissues or organs as well as tissues such as muscle. ing. Due to the configuration of the electrode array, the injection needle (for delivering the selected biomolecule) is also fully inserted into the target organ and the injection is administered perpendicular to the target tissue in the area previously drawn by the electrode. Is done. The electrodes described in US Pat. No. 7,245,963 and US Patent Application Publication No. 2005/005263 are preferably 21 gauge, 20 mm long.

  The following is an example of the method of the present invention and is discussed in more detail in the above referenced patent: an electroporation device provides a constant current similar to a current input preset by a user to a desired tissue in a mammal. It can be configured to deliver a pulse of energy to be generated. The electroporation device comprises an electroporation component and an electrode assembly or handle assembly. The electroporation component includes one of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a storage component, a power supply, and a power switch A plurality of various elements may be included or incorporated. The electroporation component can function as one element of the electroporation device, and the other element is another element (or component) in communication with the electroporation component. In some embodiments, the electroporation component can function as two or more elements of the electroporation device, which can communicate with further elements of the electroporation device remote from the electroporation component. The present invention is not limited by the elements of an electroporation device that exist as part of one electromechanical or mechanical device, as the elements can function as one device or as separate elements that communicate with each other. The electroporation component can deliver a pulse of energy that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, the electrode assembly receiving a pulse of energy from an electroporation component and delivering it through the electrodes to the desired tissue. At least one of the plurality of electrodes is neutral during the delivery of the pulse of energy, measures the impedance in the desired tissue, and transmits the impedance to the electroporation component. The feedback mechanism can receive the measured impedance and can adjust the pulse of energy delivered by the electroporation component to maintain a constant current.

  In some embodiments, the plurality of electrodes can deliver pulses of energy in a distributed pattern. In some embodiments, the plurality of electrodes can deliver pulses of energy in a distributed pattern through the control of the electrodes under a programmed sequence, the programmed sequence being input to the electroporation component by a user . In some embodiments, the programmed sequence includes a plurality of pulses delivered in sequence, each pulse of the plurality of pulses delivered by at least two active electrodes by one neutral electrode measuring impedance, The next pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes by one neutral electrode measuring impedance.

  In some embodiments, the feedback mechanism is implemented by either hardware or software. Preferably, the feedback mechanism is implemented by an analog closed loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or simultaneous (ie, substantially simultaneous as determined by techniques available to determine response time). It is. In some embodiments, the neutral electrode measures the impedance in the desired tissue and communicates the impedance to a feedback mechanism that responds to the impedance and adjusts the pulse of energy to provide a preset current. Maintain a constant current at a value similar to. In some embodiments, the feedback mechanism maintains a constant current continuously and immediately during the delivery of a pulse of energy.

  Pharmaceutically acceptable excipients may include functional molecules such as vehicles, adjuvants, carriers, or diluents, which are known and generally readily available. Preferably, the pharmaceutically acceptable excipient is an adjuvant or transfection facilitating agent. In some embodiments, the nucleic acid molecule, or DNA plasmid, is delivered to the cell in conjunction with administration of a polynucleotide function enhancer or gene vaccine enhancer (ie, transfection enhancer). Polynucleotide function enhancers are described in US Pat. Nos. 5,593,972, 5,962,428, and International Application No. PCT / US94 / 00899 (filed Jan. 26, 1994). Each of which is incorporated herein by reference. Genetic vaccine promoters are described in US Provisional Patent Application No. 021,579 (filed April 1, 1994), which is incorporated herein by reference. The transfection facilitating agent may be administered in combination with the nucleic acid molecule as a mixture with the nucleic acid molecule, or may be administered separately, simultaneously with, before or after administration of the nucleic acid molecule. Examples of transfection facilitating agents include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and squalene and squalene. And hyaluronic acid may be administered in combination with the gene construct. In some embodiments, the DNA plasmid vaccine also includes liposomes, including other liposomes known in the art, such as lipids, lecithin liposomes or DNA-liposome mixtures (see, eg, WO 9324640), Transfection enhancers such as calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection enhancers may be included. Preferably, the transfection facilitating agent is a polyanion, a polycation containing poly-L-glutamic acid (LGS), or a lipid.

  In some preferred embodiments, the DNA plasmid is delivered with an adjuvant that is a gene for a protein that further enhances the immune response to such target protein. Examples of such genes are alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4. IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL, including IL-15, optionally including a signal peptide from IgE It encodes other cytokines such as -15 and lymphokines. Other genes that may be useful include MCP-1, MIP-1α, MIP-1p, 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, IL-4 , Mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, R6, 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 Gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2B, NKG2B, NK2C And those encoding functional fragments thereof.

  The DNA plasmid vaccine according to the present invention has about 1 nanogram to 10 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 micrograms to about 10 milligrams, or more preferably about 100 micrograms to about 1 milligrams. In the amount of DNA. In some preferred embodiments, a DNA plasmid vaccine according to the present invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine comprises about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine comprises about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine comprises about 1 to about 350 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine comprises about 25 to about 250 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccine comprises about 100 micrograms to about 1 milligram of DNA.

  The DNA plasmid vaccine according to the present invention is formulated according to the administration method used. If the DNA plasmid vaccines are injectable compositions, they are sterile and / or free of pyrogens and / or free of microparticles. Isotonic preparations are preferably used. In general, tonicity agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation. In some embodiments, stabilizers are added to the formulation that allow the formulation to be stable for long periods at room temperature or ambient temperature, such as LGS or other polycations, or polyanions.

  In some embodiments, methods of inducing an immune response against a common dengue antigen in a mammal include methods of inducing a mucosal immune response. Such methods include CTACK protein, TECK protein, MEC protein, and functional fragments thereof, or their expressible coding sequences combined with a DNA plasmid containing a common dengue antigen as described above. Of these, administration of one or more species to mammals can be mentioned. One or more of CTACK protein, TECK protein, MEC protein, and functional fragments thereof may be administered before, simultaneously with, or after administration of the DNA plasmid dengue vaccine provided herein. . In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC, and functional fragments thereof is administered to the mammal.

  The invention is further described in the following examples. While these examples illustrate preferred embodiments of the present invention, it should be understood that they are provided for illustration only. From the above discussion and these examples, those skilled in the art will elucidate the essential features of the present invention and make various changes and modifications thereto without departing from the spirit and scope of the present invention. Can be adapted to different applications and conditions. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

  Preferably, the DNA formulation for use with the muscle or skin EP device described herein has a high DNA concentration, preferably a small volume, preferably a small injection volume, ideal for delivery to the skin. Specifically, it has a concentration containing DNA in an amount of 1 microgram to several tens of milligrams, preferably in an amount of milligrams, in 25 to 200 microliters (μL). In some embodiments, the DNA formulation has a high DNA concentration, such as 1 mg / mL or more (mg (DNA) / volume of formulation). More preferably, the DNA formulation has a DNA concentration that provides a gram amount of DNA in a 200 μL formula, more preferably a gram amount of DNA in a 100 μL formula.

  DNA plasmids for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferably on January 1, 2009, US Patent Application Publication No. 20090004716. As described in US patent application Ser. No. 12 / 126,611. In some examples, the DNA plasmid used in these studies may be formulated at a concentration of 10 mg / mL or higher. Manufacturing techniques also include those described in U.S. Patent Application Publication No. 20090004716 and those described in U.S. Pat. No. 7,238,522, issued July 3, 2007, It includes or incorporates various devices and protocols generally known to those skilled in the art. The high concentration of plasmid used in conjunction with the EP device and delivery technology described herein enables administration of the plasmid in the ID / SD space at a reasonably low volume to enhance expression and immune effects. Useful. Publications, US Patent Application Publication No. 20090004716, and US Patent No. 7,238,522 are incorporated herein in their entirety.

Example Example 1
prM prevents premature fusion of the E protein during virus maturation by forming a prM-E heterodimeric complex of non-infectious immature virus particles. Immature particles migrate through the low pH environment of the Golgi region and a reversible conformational change occurs in the E protein prior to prM treatment. Cleavage of prM to M by cellular serine protease in the trans-Golgi network results in an irreversible conformational change in E that maintains the integrity of the neutralizing epitope.

  D1prME, D2prME, D3prME, and D4prME contain consensus sequences for both pRM (pre-membrane precursor) protein and E (envelope) protein for each of serotypes D1, D2, D3, or D4 It was a construct encoding an antigen.

  DU is the consensus sequence of DIII domain of E (envelope) protein of serotype D1, the consensus sequence of DIII domain of E (envelope) protein of serotype D2, and the consensus sequence of DIII domain of E (envelope) protein of serotype D3 , And an antigen encoding a consensus sequence of the DIII domain of the serotype D4 E (envelope) protein, each separated by a linker. A construct combining all four DIII domains (universal or universal DIII) separated by proteolytic cleavage sequences was constructed and called DU (SEQ ID NO: 9). The common DIII domain DU having SEQ ID NO: 10 is a linker sequence (DV1-DIII, DV-2-DIII, DV-3-DIII, and DV-4-DIII) that links to each of the four subtypes of DIII. Consists of. This linker has the sequence RGRKRRS, which makes it possible to cleave the DU with a separate DIII sequence of a specific subtype. However, the linker sequence can be any other linker sequence available to one of ordinary skill in the art with similar characteristics, and replacement with a linker such as RGRKRRS currently in use is routine for those skilled in the art. Is within the scope of knowledge.

  Mice were immunized with 100 μg D1prME, D2prME, D3prME, D4prME, or DU. Mice were immunized 3 times using 3P Cellectra (ID), each immunization was performed 3 weeks apart and bled every 3 weeks at the same injection site. Each group included 5 animals (n = 5).

  Serial dilutions (1:50, 1: 150, 1: 450, 1: 1350, and 1: 4050) were made from sera from each mouse. This serum was contacted with DIII (domain III of envelope protein E) from each of the four serotypes D1, D2, D3, or D4. The binding of antibodies in serum to DIII protein was determined by measuring absorbance at 450 nm. BSA was used as a control.

The outline of the experimental design is as follows.

  The results are shown in FIGS. As shown in FIG. 1, all mice immunized with D1prME produced antibodies that bound to DIII derived from D1, and all mice immunized with DU similarly received antibodies bound to DIII derived from D1. Produced. As shown in FIG. 2, all mice immunized with D2prME produced antibodies that bound DIII derived from D2, and all mice immunized with DU similarly received antibodies bound to DIII derived from D2. Produced. As shown in FIG. 3, mice 3, 4, and 5 immunized with D3prME produced antibodies that bound to D3-derived DIII, but were not as binding as shown in FIGS. Mice 1 and 2 immunized with D3prME did not produce antibodies that bound well with D3-derived DIII. All mice immunized with DU produced antibodies that bound DIII from D3. As shown in FIG. 4, mice 4 and 5 immunized with D1prME produced antibodies that bound DIII derived from D4, whereas mice 1, 2, and 3 did not produce this antibody. All mice immunized with DU produced antibodies that bound DIII from D4.

  Sera from mice immunized with D1prME, D2prME, D3prME, or D4prME were analyzed using Western blot analysis for protein E and protein prM. As shown in FIG. 5, proteins E and prM were separated and were present in sera from all mice.

Example 2: Dengue Li-Cor Assay Guinea pigs were immunized with 100 μg of D1prME, D2prME, D3prME, D4prME, or DU. In some of these groups, each guinea pig was immunized with all four D1prME, D2prME, D3prME, or D4prME at either a single or separate immunization site, as shown below. At the single dose site, all four were mixed together in a single dose. For separate immunization sites, each was administered separately. In the case of Example 2, D1prME, D2prME, D3prME, and D4prME are antigens that contain both pRM protein (pre-membrane precursor) protein and E (envelope) protein derived from serotypes D1, D2, D3, or D4, respectively. Was a construct that encodes DU is the consensus sequence of DIII domain of E (envelope) protein of serotype D1, the consensus sequence of DIII domain of E (envelope) protein of serotype D2, and the consensus sequence of DIII domain of E (envelope) protein of serotype D3 , And an antigen encoding a consensus sequence of the DIII domain of the serotype D4 E (envelope) protein, each separated by a linker as shown in Example 1.

  Serial dilutions (1:50, 1: 150, 1: 450, 1: 1350, and 1: 4050) were made from sera from each guinea pig. The binding of antibodies in serum to dengue virus was determined by measuring absorbance at 450 nm.

Two-fold serial dilutions of plasma were made and placed in 96 well plates at 50 μl per well. 50 pfu (50 μl per well) of dengue virus was added to each well. Plates were incubated for 1 hour at 37 ° C. to allow virus neutralization with serum-derived antibodies. The entire mixture (100 μl) was added to VERO cells (seeding 1.5 × 10 ⁴ / well). Plates were incubated for 4 days at 37 ° C. Cells were fixed for 30 minutes using 3.7% formaldehyde. Cells were washed and permeabilized using 0.1% Triton X-100 / PBS. Cell line ELISA was performed using mouse 4G2 mAb and using biotinylated anti-mouse IgG and IRDye 800CW streptavidin + 5 mM DRAQ5 cocktail solution (cocktail solution). Plates were washed, dried and scanned using a Li-Cor Aerous system. The ratio of 800 nm / 700 nm was calculated.

  The results are shown in FIGS. As shown in FIG. 6, sera from two control guinea pigs (no immunization) did not bind to DIII from Dl, D2, D3, or D4 serotypes. As shown in FIG. 7, of the guinea pigs immunized with DU, only guinea pig 4 produced antibodies that bound to DIII proteins derived from Dl, D2, D3, and D4 serotypes. As shown in FIG. 8, five guinea pigs were immunized with a mixture of 100 μg D1prME, 100 μg D2prME, 100 μg D3prME, and 100 μg D4prME for a total of all 400 μg at one immune site per guinea pig. All guinea pigs produced antibodies that bound DIII protein from D1, D2, and D3. However, only two guinea pigs produced antibodies that bound DIII protein from D4, and this binding was weak compared to other serotypes. As shown in FIG. 9, 5 guinea pigs were immunized at 4 separate sites with 100 μg D1prME, 100 μg D2prME, 100 μg D3prME, and 100 μg D4prME for a total of 400 μg per guinea pig. All guinea pigs produced antibodies that bound DIII protein from D1, D2, and D3. However, only 3 or 4 guinea pigs produced antibodies that bound DIII protein from D4, and this binding was weak compared to other serotypes. As shown in FIG. 10, 5 guinea pigs were each immunized with 100 μg D1prME. All guinea pigs produced antibodies that bound to DIII protein from D1. However, only a few guinea pigs produced antibodies that bound to DIII protein from D2, D3, or D4, and this binding was weak compared to D1. As shown in FIG. 11, 5 guinea pigs were each immunized with 100 μg D2prME. All guinea pigs produced antibodies that bound to D2-derived DIII protein. However, only one guinea pig produced an antibody that bound to DIII protein from D1, and this binding was weak compared to D1. None of the guinea pigs produced antibodies bound to DIII protein from D3 or D4. As shown in FIG. 12, 5 guinea pigs were each immunized with 100 μg D3prME. All guinea pigs produced antibodies that bound to DIII proteins from D1 and D3. However, only one guinea pig produced an antibody that bound to the DIII protein from D2. None of the guinea pigs produced antibodies bound to D4-derived DIII protein. As shown in FIG. 13, 5 guinea pigs were each immunized with 100 μg D4prME. Four guinea pigs produced antibodies that bound D4 derived DIII protein. Guinea pig 1 produced an antibody that bound to DIII protein from D1 and D2. Only a few guinea pigs produced antibodies that (minimally) bound to DIII protein from D2 and D3.

  Comparison of FIG. 7 with FIGS. 8 and 9 shows that immunization using a combination of D1prME, D2prME, D3prME, and D4prME is performed using DU even if immunization is performed separately or in a mixture. It showed good immunogenicity against DIII.

Example 3: Dengue PRNT ₅₀ and FRNT assays Guinea pigs were immunized with 100 μg DU, D1prME, D2prME, D3prME, or D4prME. In some of these groups, each guinea pig was immunized with all four D1prME, D2prME, D3prME, or D4prME at either a single or separate immunization site, as detailed below. . In Examples 2 and 3, D1prME, D2prME, D3prME, and D4prME are antigens that contain both pRM (pre-membrane precursor) and E (envelope) proteins derived from serotypes D1, D2, D3, or D4, respectively. Was a construct that encodes DU is the consensus sequence of DIII domain of E (envelope) protein of serotype D1, the consensus sequence of DIII domain of E (envelope) protein of serotype D2, and the consensus sequence of DIII domain of E (envelope) protein of serotype D3 , And an antigen encoding a consensus sequence of the DIII domain of the serotype D4 E (envelope) protein, each separated by a linker as shown in Example 2.

For the PRNT50 assay, VERO cells were plated on day 1 in a 6-well format (7.5 × 10 ^{5 to} 1.0 × 10 ⁶ cells / well). On the second day, 50 pfu dengue virus / well was incubated with serum from guinea pigs for 1 hour at 37 ° C. The dengue virus used was one of Hawaii, NGC, H87, or H241. This mixture was added to a single membrane of cells in the plate and incubated at 37 ° C. for 1 hour. Cells were overlaid with 2% 199 medium containing 1% methylcellulose. Cells were incubated at 37 ° C. for 5 days after infection. On day 7, cells were fixed and stained with a crystal violet / 20% methanol mixture (2 hour incubation). The plate was washed with dH ₂ O and dried. The number of plaques was counted. Neutralizing titer was defined as the reciprocal of the highest serum dilution that reduced the plaque count by more than 50% compared to a control without serum.

  For the Focus Diagnostics FRNT assay, 24-well plates were used with 4-fold diluted serum. Plates were incubated for 4 days. The occurrence of plaque was determined and measured using an immunofocus.

The results are shown in the following Table 2 and FIGS. As shown in FIG. 14, guinea pigs immunized with all of the D1prME, D2prME, D3prME, or D4prME constructs, and sera from guinea pigs immunized with only D1prME produced antibodies that bind to proteins from Hawaiian dengue virus. It was not seen in immunization with DU. As shown in FIG. 15, guinea pigs immunized with all of the D1prME, D2prME, D3prME, or D4prME constructs, and sera from guinea pigs immunized with only D2prME produced antibodies that bound to proteins derived from NGC dengue virus. It was not seen in immunization with DU. As shown in FIG. 16, guinea pigs immunized with all of the D1prME, D2prME, D3prME, or D4prME constructs, and guinea pig-derived sera immunized with only D3prME produced antibodies that bound to proteins derived from H87 dengue virus. The titer observed after immunization with DU was minimal. As shown in FIG. 17, guinea pigs immunized with all of D1prME, D2prME, D3prME, or D4prME, and guinea pigs immunized with only D4prME produced antibodies that bound to proteins derived from H241 dengue virus. It was not seen in immunization with DU.

Claims (33)

At least two encoding nucleotide sequences that express a polypeptide, wherein the encoding nucleotide sequence encodes a nucleotide sequence that encodes SEQ ID NO: 2, a nucleotide sequence that encodes SEQ ID NO: 4, and SEQ ID NO: 6 An encoded nucleotide sequence selected from the group consisting of a nucleotide sequence that encodes and a nucleotide sequence that encodes SEQ ID NO: 8 ;
Expressing a polypeptide that elicits an immune response against two or more subtypes of dengue virus in a mammal, comprising a promoter that regulates expression of the polypeptide in the mammal and is operably linked to the encoded nucleotide sequence A nucleic acid construct that can. 2. The nucleic acid construct of claim 1, further comprising a nucleotide sequence encoding an IgE leader sequence operably linked to the 5' -end of the coding sequence and operably linked to the promoter. The nucleic acid construct of any one of claims 1-2, further comprising a nucleotide sequence encoding a polyadenylation sequence linked to the 3' -end of the coding sequence.   The nucleic acid construct according to any one of claims 1 to 3, wherein codons of the nucleic acid construct are optimized. The nucleic acid construct according to any one of claims 1 to 4, wherein the at least one encoded nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 . At least two encoding nucleotide sequence, SEQ ID NO: 1, SEQ ID NO 3, SEQ ID NO 5, and is selected from the group consisting of SEQ ID NO: 7, a nucleic acid according to any one of claims 1 to 5 construct. An at least one DNA plasmid capable of expressing in a mammalian cell an amount of a common PRM and E protein from at least two dengue virus subtypes effective to elicit an immune response in the mammal; The at least two common PRM and E proteins are dengue virus-subtype 1 common PRM and E protein , dengue virus-subtype 2 common PRM and E protein , dengue virus-subtype 3 common PRM and E protein , or A DNA plasmid selected from the group consisting of dengue virus-subtype 4 common PRM and E proteins , or combinations thereof;
A DNA plasmid vaccine capable of generating an immune response against multiple dengue virus subtypes in a mammal comprising a pharmaceutically acceptable excipient,
The DNA plasmid, see contains a promoter operably linked to a coding sequence encoding the common PRM and E protein,
A group consisting of a nucleotide sequence encoding SEQ ID NO: 2, a nucleotide sequence encoding SEQ ID NO: 4, a nucleotide sequence encoding SEQ ID NO: 6, and a nucleotide sequence encoding SEQ ID NO: 8; A DNA plasmid vaccine comprising one or more sequences selected from   8. The DNA plasmid vaccine of claim 7, wherein the DNA plasmid further comprises an IgE leader sequence that binds to the N-terminus of the coding sequence and is operably linked to a promoter.   The DNA plasmid vaccine of any one of claims 7 to 8, wherein the DNA plasmid further comprises a polyadenylation sequence linked to the C-terminus of the coding sequence.   The DNA plasmid vaccine according to any one of claims 7 to 9, wherein codons of the DNA plasmid are optimized.   The DNA plasmid vaccine according to any one of claims 7 to 10, wherein the pharmaceutically acceptable excipient is an adjuvant.   12. A DNA plasmid vaccine according to claim 11 wherein the adjuvant is selected from the group consisting of IL-12 and IL-15.   The DNA plasmid vaccine according to any one of claims 7 to 10, wherein the pharmaceutically acceptable excipient is a transfection promoting agent.   The DNA plasmid vaccine according to claim 13, wherein the transfection facilitating agent is a polyanion, a polycation, or a lipid.   14. The DNA plasmid vaccine of claim 13, wherein the transfection facilitating agent is poly-L-glutamic acid at a concentration of less than 6 mg / mL.   The DNA plasmid vaccine according to any one of claims 7 to 15, wherein the DNA plasmid vaccine has a total DNA plasmid concentration of 1 mg / mL or more. Wherein said DNA plasmid vaccine a plurality of distinct DNA plasmids, wherein each of the plurality of distinct DNA plasmids, Dengue virus - subtype 1, dengue virus - from subtype 4 - subtype 2, dengue virus - subtype 3, and dengue virus The DNA plasmid vaccine according to any one of claims 7 to 16, which encodes a polypeptide comprising at least one common PRM and E protein selected from the group consisting of: At least one encoding nucleotide sequence, SEQ ID NO: 1, SEQ ID NO 3, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO 5, and SEQ ID NO: 7, any one of claims 7 to 17 A DNA plasmid vaccine according to 1. Comprising at least two different DNA plasmids expressing dengue virus PRM / E domains, said plasmid comprising:
A DNA plasmid comprising sequences encoding common dengue virus-subtype 1 PRM and E proteins ;
A DNA plasmid comprising sequences encoding the common dengue virus-subtype 2 PRM and E proteins ;
A DNA plasmid comprising a sequence encoding a common dengue virus-subtype 3 PRM and E protein , and a DNA plasmid comprising a sequence encoding a common dengue virus-subtype 4 PRM and E protein ;
The DNA plasmid vaccine according to any one of claims 7 to 18. A DNA plasmid comprising sequences encoding the common dengue virus-subtype 1 PRM and E proteins ;
A DNA plasmid comprising sequences encoding the common dengue virus-subtype 2 PRM and E proteins ;
8. A DNA plasmid comprising a sequence encoding the common dengue virus-subtype 3 PRM and E protein , and a DNA plasmid comprising a sequence encoding the common dengue virus-subtype 4 P-RM and E protein. The DNA plasmid vaccine of any one of -19. 21. The DNA plasmid vaccine of any one of claims 7 to 20, wherein the common dengue virus-subtype 1 PRM and E proteins comprise the amino acid sequence of SEQ ID NO: 2 . The DNA plasmid vaccine of any one of claims 7 to 21, wherein the common dengue virus-subtype 2 PRM and E proteins comprise the amino acid sequence of SEQ ID NO: 4 . 23. The DNA plasmid vaccine of any one of claims 7 to 22, wherein the common dengue virus-subtype 3 PRM and E proteins comprise the amino acid sequence of SEQ ID NO: 6 . 24. The DNA plasmid vaccine of any one of claims 7 to 23, wherein the common dengue virus-subtype 4 PRM and E proteins comprise the amino acid sequence of SEQ ID NO: 8 .   The DNA plasmid vaccine according to any one of claims 7 to 24, wherein the mammal is a non-human primate.   The DNA plasmid vaccine according to any one of claims 7 to 25, wherein the immune reaction is a humoral reaction.   The DNA plasmid vaccine according to any one of claims 7 to 25, wherein the immune reaction is a cellular reaction.   26. The DNA plasmid vaccine according to any one of claims 7 to 25, wherein the immune response is a combination of a humoral response and a cellular response. Use of a DNA plasmid vaccine in the preparation of a medicament for use in a therapeutic or prophylactic method for inducing an immune response against multiple subtypes of dengue virus in a mammal, said method comprising:
Delivering a DNA plasmid vaccine to mammalian tissue, wherein the DNA plasmid vaccine expresses a plurality of common antigens derived from viral subtypes in mammalian cells and elicits an immune response in the mammal A plurality of common antigens comprising antigenic domains derived from at least two different subtypes of the virus;
Look containing a to electroporated tissue cells by pulse energy valid constant current to permit entry into a cell of the DNA plasmid,
The DNA plasmid vaccine comprises an encoding nucleotide sequence encoding at least two common dengue PRM and E proteins, wherein the encoding nucleotide sequence encodes a nucleotide sequence that encodes SEQ ID NO: 2, SEQ ID NO: 4 Use, comprising at least two sequences selected from the group consisting of a nucleotide sequence encoding, a nucleotide sequence encoding SEQ ID NO: 6, and a nucleotide sequence encoding SEQ ID NO: 8 . 30. Use according to claim 29 , wherein the DNA plasmid vaccine is for administration to the transdermal, subcutaneous or muscle tissue. Presetting a desired current to be delivered to the tissue by the method ;
Electroporating the cells of the tissue with a pulse of energy at a constant current equal to the preset current;
30. The use of claim 29 , further comprising: The electroporation process includes
Measuring impedance in the electroporated cell;
Adjusting the energy level of a pulse of energy relative to the measured impedance to maintain a constant current in the electroporated cell;
The measuring and adjusting steps are performed within the duration of the pulse of energy,
30. Use according to claim 29 . 30. The use of claim 29 , wherein the electroporation process comprises delivering pulses of energy to a plurality of electrodes according to a pulse sequence pattern that delivers pulses of energy in a distributed pattern.