CN110055265A - For the novel vaccine of a variety of subtypes of dengue virus - Google Patents

Abstract

The present invention relates to the novel vaccines for being directed to a variety of subtypes of dengue virus.Specifically, one aspect of the present invention relates to express such as the nucleic acid construct of the polypeptide of shared Dengue prME and using their method, and the polypeptide causes the immune response for more than one subtypes of dengue virus of mammal.Additionally, there are that can generate the DNA plasmid vaccine for the immune response of a variety of subtypes of dengue virus and the method using them in mammals, the DNA plasmid vaccine includes DNA plasmid and pharmaceutically acceptable excipient.The amount that the DNA plasmid can effectively cause the immune response of the mammal in the cell of the mammal expresses shared Dengue antigen, and the immune response is cross reactivity for all 4 kinds of Dengue hypotypes.

Description

For the novel vaccine of a variety of subtypes of dengue virus

The application is entitled " for the novel vaccines of a variety of subtypes of dengue virus " for the applying date being on 03 14th, 2014 Chinese Patent Application No. 201480013820.2 divisional application.

Invention field

The immune response of dengue virus is directed to and for prophylactically the present invention relates to improved dengue vaccine, for inducing And/or therapeutically it is directed to the improved method of dengue virus immune body.

Background

Dengue virus (DENV) is a kind of emerging disease Dengue for causing dengue fever (DF) and serious life-threatening Mosquito matchmaker's cause of disease of Hemorrhagic fever/dengue shock syndrome (DHF/DSS).DENV is a kind of small, coating positive chain RNA virus, it Belong to the Flavivirus of flaviviridae (Flaviviridae) family.The four kinds of different hypotypes or serotype (DV- of dengue virus 1 to DV-4) propagation is bitten by mosquito kind Aedes aegypti (Aedes aegypti) and aedes albopictus (Aedes albopictus) To the mankind.Occur 5,000 ten thousand to 100,000,000 DF case and 250,000 to 500000 DHF case every year according to estimates.Dengue becomes Huge world public health problem, because 2/5ths of world population live in the epidemic regions of Dengue and occur every year Estimate 5,000 ten thousand to 100,000,000 Dengue cases of infection.In addition, there is no effectively interference, subtropical zone and heat in the world Region, 2,500,000,000 people are in the danger of infection.

More than 100 tropic countries have popular dengue virus infection, and are proved DHF and are being greater than 60 these states Exist in family.Lack the monitoring to DF/DHF in most countries and focuses primarily upon DHF in the past；Therefore can only estimate annual The number of the DF case of appearance.However in 1998, main epidemic disease occurs to spread Asia and the U.S., wherein to world health Tissue (WHO) reports greater than 1,200,000 DF/DHF cases.The Global Raport of DHF averagely increased by five times within past 20 years. At the beginning of 21 century, according to epidemic disease activity, the DF case and hundreds of thousands DHF case of annual appearance 5,000 ten thousand to 100,000,000 are estimated.It dies of illness Rate (CFR) is variant between country, but some countries can be up to 10-15% and in other country < 1%.

There are four kinds of subtypes of dengue virus: 1 type of Dengue (DV-1), 2 type of Dengue (DV-2), 3 type of Dengue (DV-3) and Dengue 4 Type (DV-4).Each of these hypotypes form subclass different on antigen in flaviviridae family.They are ten kinds of codings The RNA virus of the coating of albumen: three kinds of structural proteins and seven kinds of non-structural proteins.The structural proteins are capsid (C), coating (E) and pre- film precursor (preM).The life cycle of DV starts from the encytosis of receptor-mediated cell entry cell, then sick The envelope protein of poison is merged with advanced stage inclusion body film, this causes viral genome to be discharged into cytoplasm to replicate.

DV infection may be asymptomatic or characterized by fever, shiver with cold, metopodynia, myalgia, arthralgia and rash.Not with postoperative infection Same serotype can lead to including plasma leakage or bleeding (dengue hemorrhagic fever) and the more tight of (dengue shock syndrome) of suffering a shock The disease performance of weight.Although having carried out studying extensively for many years to understand the pathogenicity of DENV infection, in the anti-DVization of special efficacy It closes and what progress is not obtained in the development of object.At present the U.S. do not ratify for Dengue infection specific antivirotic or Vaccine.

Coating (E) glycosylation albumen be mature dengue virus particle surface present on major structural protein, be a kind of I Type integrated membrane protein.It is proved the E protein of mature dengue virus in a manner of antiparallel (head to tail direction) and forms homodimeric Body.Each monomer is folded into three kinds of different structural domains, i.e. structural domain I (DI, the N-terminal structural domain at center), domain II (DII, dimerization domain) and Domain III (PRM/E, the C-terminal structural domain of immunoglobulin (Ig) sample).The PRM/E of E protein Structural domain is made of 100 amino acid (residue 303-395) of C-terminal.This structural domain has shown it is Receptor recognition and combination knot Structure domain.Ig sample present in PRM/E albumen folds usually related with the structure with attached function.This structural domain vertically prolongs The surface for reaching virus, the farther tip of any other part for the ratio E protein stretched out with one from virion surface.This Outside, the antibody that research is proved the PRM/E of the E protein of the anti-flavivirus of recombination PRM/E albumen and generation can inhibit flavivirus Into target cell.In addition, the flavivirus of the mutation of the PRM/E with E protein shows the virulence of decrease or escapes immune neutralize Ability.

Developing the safe and efficient vaccine for dengue virus infection is still a main public health target.It is assumed that The primary association of immunity and dengue virus is considered as the presence of neutralizing antibody, then for comparing and optimizing vaccine candidate object Premise be accurately measure by vaccine induce neutralizing antibody reaction ability.Include decrease from all four serotypes The combination of vaccine of live virus have been displayed and lead to several complication (Guy B, Almond JW, Comp Immunol Microbiol Infect Dis.2008 March；31(2-3):239-52).Additionally, there are seldom about based on adenovirus Dengue antigen delivering report.Nevertheless, this of adenovirus system is it is a known problem that the most of mankind The known antibody with one of anti-adenovirus of group, and such pre-existing antibody can cause these based on adenovirus Vaccine failure.

Therefore, it provides it is still necessary to developing for the extensive immune of a variety of and preferably all four serotypes dengue virus Property or general immunisation (universal immunity) vaccine, and it is preferably economical and between all serotypes Effective vaccine.Vaccine such as DNA vaccination or DNA plasmid vaccine are applied in prevention to mammal in addition, there being needs Or the immune effective ways for being directed to dengue virus are provided in treatment.

Summary of the invention

One aspect of the present invention offer can express initiation mammal and exempt from for more than one subtypes of dengue virus The nucleic acid construct of the polypeptide of epidemic disease reaction.The nucleic acid construct by coding nucleotide sequence and with the coding nucleotide sequence The promoter composition being operably connected.The coding nucleotide sequence expresses the polypeptide, wherein the polypeptide includes coming from The Domain III (PRM/E structural domain or PRM/E) of the envelope protein of at least two different subtypes of dengue virus.The starting Son adjusts the expression of polypeptide described in mammal.

Another aspect of the present invention provides to generate in mammals to be exempted from for a variety of subtypes of dengue virus The DNA plasmid vaccine of epidemic disease reaction.The DNA plasmid vaccine in the mammal by that can express shared Dengue antigen DNA plasmid and pharmaceutically acceptable excipient composition.The DNA plasmid by with encode the shared Dengue PRM/E antigen The promoter composition that coded sequence is operably connected.The shared Dengue antigen is by subtypes of dengue virus 1, subtypes of dengue virus 2, the shared PRM/E structural domain of subtypes of dengue virus 3 or subtypes of dengue virus 4 forms.

Another aspect provides the method for causing immune response of the mammal for a variety of virus subtypes, institutes The method of stating includes the tissue delivery DNA plasmid vaccine to the mammal, and with energy pulse effectively to allow the DNA matter Grain enters the constant current of the cell for the cell electroporation of the tissue, and the DNA plasmid vaccine includes can be described A variety of shared antigens of the expression derived from the virus subtype are in the cell of mammal to cause the immune of the mammal The DNA plasmid of reaction, a variety of shared antigens include the antigenic domains from described at least two different virus subtypes.

Brief description

Fig. 1 show comparison from inoculation D1prME mouse serum with from be inoculated with DU mouse serum, be directed to The combination titre of all Dengue PRM/E structural domains from hypotype 1.

Fig. 2 shows comparison from inoculation D2prME mouse serum with from be inoculated with DU mouse serum, be directed to The combination titre of all Dengue PRM/E structural domains from hypotype 2.

Fig. 3 show comparison from inoculation D3prME mouse serum with from be inoculated with DU mouse serum, be directed to The combination titre of all Dengue PRM/E structural domains from hypotype 3.

Fig. 4 show comparison from inoculation D4prME mouse serum with from be inoculated with DU mouse serum, be directed to The combination titre of all Dengue PRM/E structural domains from hypotype 4.

Fig. 5 shows stained gel, and the stained gel displaying is exempted to the PRM/E structural domain from serotype 1,2,3 or 4 For the mouse of epidemic disease, for the binding antibody of prME protein types D1, D2, D3 and D4 generation.

Fig. 6, which is shown, to be shown for control guinea pig serum for each in Dengue PRM/E protein types (1 to 4) Neutralizing antibody figure.

Fig. 7, which is shown, to be shown for DU-DIII guinea pig serum for each in Dengue PRM/E protein types (1 to 4) The figure of the neutralizing antibody of kind.

Fig. 8 shows displaying, and for D1-D4prME guinea pig serum, (all four vaccine combinations are at a kind of mixture and together Application) for for the neutralizing antibody of each in Dengue PRM/E protein types (1 to 4) figure.

Fig. 9 shows displaying for D1-D4prME guinea pig serum (individually applying each vaccine) for Dengue The figure of the neutralizing antibody of each in PRM/E protein types (1 to 4).

Figure 10, which is shown, to be shown for D1prME guinea pig serum for each in Dengue PRM/E protein types (1 to 4) The figure of the neutralizing antibody of kind.

Figure 11, which is shown, to be shown for D2prME guinea pig serum for each in Dengue PRM/E protein types (1 to 4) The figure of the neutralizing antibody of kind.

Figure 12, which is shown, to be shown for D3prME guinea pig serum for each in Dengue PRM/E protein types (1 to 4) The figure of the neutralizing antibody of kind.

Figure 13, which is shown, to be shown for D4prME guinea pig serum for each in Dengue PRM/E protein types (1 to 4) The figure of the neutralizing antibody of kind.

Figure 14, which is shown, to be shown for come the serum of the animal for all four D1-D4prME inoculations of using by oneself for Dengue 1 The figure of the neutralizing antibody of virus.

Figure 15, which is shown, to be shown for come the serum of the animal for all four D1-D4prME inoculations of using by oneself for Dengue 2 The figure of the neutralizing antibody of virus.

Figure 16, which is shown, to be shown for come the serum of the animal for all four D1-D4prME inoculations of using by oneself for Dengue 3 The figure of the neutralizing antibody of virus.

Figure 17, which is shown, to be shown for come the serum of the animal for all four D1-D4prME inoculations of using by oneself for Dengue 4 The figure of the neutralizing antibody of virus.

The detailed description of preferred embodiment

Following simple or brief definition is provided to help to understand the preferred embodiments of the invention.Letter described herein Definition slightly is definitely not detailed, definition that they and this field understand or dictionary meanings also never contradiction.Letter is provided herein Definition slightly is to supplement or more clearly define definition as known in the art.

Definition

As used herein, FASTA, BLAST and Gapped BLAST can be used in the sequence homology of nucleotide and amino acid (Altschul et al., Nuc.Acids Res., 1997,25,3389, be hereby incorporated by reference in its entirety.) and PAUP*4.0b10 software (D.L.Swofford, Sinauer Associates, Massachusetts) determines.In short, BLAST algorithm represents basic Local Alignment Search Tool (Basic Local Alignment Search Tool), it is suitable for Determining sequence similarity, (Altschul et al., J.Mol.Biol., 1990,215,403-410, passing through reference will be in its whole Appearance is incorporated herein).Software for executing BLAST analysis is public available by National Biotechnology Information Center.By The measurement of similitude that BLAST algorithm provides is minimum sum probability (P (N)) method, it provide two nucleotide sequences it Between the occurrent probability of matching instruction.For example, if minimum and general in detection nucleic acid is compared with another nucleic acid Rate is less than about 1, desirably less than about 0.1, preferably less than about 0.01 and most preferably less than about 0.001, that Think that the nucleic acid with another nucleic acid is similar.Can be used PAUP*4.0b10 software (D.L.Swofford, Sinauer Associates, Massachusetts) calculate " percentage of similitude ".Calculate consensus sequence and genealogical tree The average similarity that all sequences in (phylogenic tree) are compared.

As used herein, term " nucleic acid construct " refers to DNA or RNA points of the nucleotide sequence comprising coding protein Son.Coded sequence or " nucleic acid sequence encoding " may include the initial signal and termination signal being operably connected with controlling element, The controlling element includes the promoter that expression can be instructed in the cell for the individual that applied the nucleic acid molecules and poly- gland Nucleotide signal.

As used herein, term " effable form " refers to the nucleic acid construct comprising necessary controlling element, the tune Control element and the coded sequence of coding protein are operably connected to so that when the coded sequence is present in the cell of individual When middle, it will be expressed.

Identical group is being delivered to by the cell for organizing or defining the tissue using term " constant current " Lai Dingyi herein The electric current that the lasting period for the electric pulse knitted receives or undergoes.The electric pulse is passed from electroporation device as described herein It send.This electric current keeps constant amperage in the tissue in the validity period of electric pulse, because electricity provided herein is worn Aperture apparatus has feedback element, it is therefore preferred to have instantaneous feedback.The feedback element can measure group during entire pulse persistance The impedance of (or cell) is knitted, and electroporation device is caused to change its power output (such as increasing voltage), so in identical tissue Electric current in entire electric pulse (the delicate order of magnitude) and from pulse to pulse in keep constant.In some embodiments, instead Presenting element includes controller.

Term " feedback " or " current feedback " can be used interchangeably and indicate that the active of provided electroporation device is anti- It answers, the reaction includes the electric current in the tissue of measurement between the electrodes and therefore changes the energy output delivered by EP device To keep electric current in constant level.This constant level by user in pulse train or electric treatment before preset. Preferably, feedback is completed by the electroporation component such as controller of electroporation device, because circuit therein can be continuously Electric current in monitoring tissue between the electrodes, and by the electric current of the monitoring (or electric current in tissue) and predetermined current ratio Compared with, and energy output adjustment can be carried out continuously to keep the electric current of monitoring in preset level.In some embodiments, The feedback control loop is instantaneous, because it is simulation closed loop feedback.

As term " electroporation " used interchangeably herein, " electro-osmosis " or " electrodynamics enhancing " (" EP ") refer to use Cross-film electric field pulse induces the microcosmic approach (hole) in biomembrane；The presence of these microcosmic approach allows biomolecule such as matter Grain, oligonucleotides, siRNA, drug, ion and/or water are from the side of cell membrane by the other side.

Herein using term " scattered current " Lai Dingyi from each pin electrode array of electroporation device as described herein The current-mode of delivering, wherein the mode answers the relevant heat of electroporation on any region by the tissue of electroporation It is sharp to minimize, or preferably eliminated.

As used herein, term " feedback mechanism " refers to the process of through software or hardware (or firmware) execution, the mistake Journey receives the impedance (before delivering energy pulse, in the process and/or later) of expectation tissue and by itself and preset value (present value) (preferably electric current) is compared, and adjusts the energy pulse of delivering to reach preset value.It is anti-when discussing When infeed mechanism, term " impedance " by herein using and current value can be converted into according to Ohm's law, thus, it is possible to it is pre- If electric current compares.In a preferred embodiment, " feedback mechanism " is executed by simulation closed loop.

Swashing for the immune system of the immune system such as mammal of host is indicated using term " immune response " herein Living, the activation of the immune system is to introduce for example general Dengue antigen of Dengue antigen to by provided DNA plasmid vaccine Reaction.The form or both that immune response can be cell effect or humoral response has concurrently.

The comparison of multiple strains based on specific Dengue hypotype is indicated using term " shared " or " consensus sequence " herein Analysis and construct synthesis nucleic acid sequence or corresponding polypeptide sequence, generate hypotype 1, hypotype 2, hypotype 3, hypotype 4 and with The shared Dengue sequence of the lower general Dengue.It is described to share general Dengue to be used to induction sub- for a variety of dengue virus The wide in range immunity of type or serotype.

It indicates to be added to using term " adjuvant " herein in DNA plasmid vaccine as described herein to enhance by the DNA matter Antigenic any molecule of grain and the encoded Dengue antigen of nucleic acid sequence encoding described below.

Term " hypotype " or " serotype " can be used interchangeably herein and be related to a kind of virus such as dengue virus use, And the genetic variation of the viral antigen is indicated so that a hypotype is known with being different from a different subtype by immune system Not.For example, subtypes of dengue virus 1 can be different from subtypes of dengue virus 2 in immunology.

One aspect of the present invention offer can express initiation mammal and exempt from for more than one subtypes of dengue virus The nucleic acid construct of the polypeptide of epidemic disease reaction.The nucleic acid construct by coding nucleotide sequence and with the coding nucleotide sequence The promoter composition being operably connected.Coding nucleotide sequence expresses polypeptide, wherein the polypeptide includes coming from least two The PRM/E structural domain of different subtypes of dengue virus.The expression of polypeptide described in the promoter regulation mammal.

In some embodiments, nucleic acid construct can also include being operably connected with the N-terminal end of coded sequence And the IgE leader sequence being operably connected with promoter.Preferably, the IgE leader sequence is with SEQ ID NO:11's Sequence.The nucleic acid construct can also include the polyadenylation sequence being connected with the C-terminal end of the coded sequence.It is preferred that Ground, the nucleic acid construct are codon optimizations.

In some embodiments, coding nucleotide sequence coding includes coming from subtypes of dengue virus 1, subtypes of dengue virus 2, the polypeptide of the PRM/E structural domain of subtypes of dengue virus 3 and subtypes of dengue virus 4.In preferred embodiments, the coding Nucleotide sequence is selected from the group being made up of:

Another aspect of the present invention provides to generate in mammals to be exempted from for a variety of subtypes of dengue virus The DNA plasmid vaccine of epidemic disease reaction.The DNA plasmid vaccine in the mammal by that can express shared Dengue antigen DNA plasmid and pharmaceutically acceptable excipient composition.The DNA plasmid is by the code sequence with the coding shared Dengue antigen Arrange the promoter composition being operably connected.The shared Dengue antigen is by subtypes of dengue virus 1, subtypes of dengue virus 2, Dengue The shared PRM/E structural domain of virus subtype 3 or subtypes of dengue virus 4 composition.Preferably, the DNA plasmid includes that coding is shared The shared Dengue antigen of Dengue antigen, the shared Dengue antigen are selected from the group being made up 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 include be connected with the N-terminal end of the coded sequence and with starting The IgE leader sequence that son is operably connected.Preferably, the IgE leader sequence has sequence Met Arg Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val His Ser。

DNA plasmid can also include the polyadenylation sequence being connected with the C-terminal end of the coded sequence.Preferably, DNA plasmid is codon optimization.

In some embodiments, the pharmaceutically acceptable excipient is adjuvant.Preferably, adjuvant is selected from by IL- The group of 12 and IL-15 composition: in some embodiments, the pharmaceutically acceptable excipient is transfection.It is preferred that Ground, transfection are polyanion, polycation or lipid, and more preferably poly- L-Glu.Preferably, poly- L paddy ammonia Acid is the concentration less than 6mg/mL.Preferably, DNA plasmid vaccine has the total DNA plasmid concentration of 1mg/mL or bigger.

In some embodiments, the DNA plasmid includes a variety of unique DNA plasmids, wherein a variety of unique DNA matter Each of grain all encodes sub- comprising prME subtypes of dengue virus 1, subtypes of dengue virus 2, subtypes of dengue virus 3 or dengue virus The polypeptide of type 4.

The DNA plasmid vaccine may include the DNA plasmid comprising following coding nucleotide sequence: SEQ ID NO:1；Coding The nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3；Encode SEQ ID NO:4, SEQ ID NO:5 nucleotide sequence；It compiles Code SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 nucleotide sequence.

In some embodiments, the DNA plasmid vaccine at least two comprising expression dengue virus prME is different DNA plasmid.In some embodiments, the DNA plasmid vaccine may include four kinds of shared dengue virus prME (hypotype 1-4).

In some embodiments, the mammal that wherein DNA plasmid vaccine generates immune response is primate.It is preferred that Ground, mammal are primates.Immune response can be humoral response or cell effect, and preferably the two has concurrently.

Another aspect provides the sides for causing immune response of the mammal for a variety of subtypes of dengue virus Method, the method includes the tissue delivery DNA plasmid vaccine to the mammal and with energy pulse effectively to allow institute DNA plasmid is stated into the constant current of the cell by the cell electroporation of the tissue.

In some embodiments, the method triggered an immune response includes by DNA plasmid vaccine injection to intradermal tissue, skin Delivery step in undertissue or musculature.

In some embodiments, the method triggered an immune response can also include presetting expectation to be delivered to tissue Electric current；And with energy pulse to be equal to the constant current of predetermined current for the cell electroporation of the tissue.

In some embodiments, the method triggered an immune response further includes the impedance in the cell measured by electroporation； The energy level of impedance adjustment energy pulse relative to measurement is to keep the constant current in electroporation of cells.It is described measurement and Set-up procedure is preferably occur in the validity period of the energy pulse.

In some embodiments, electroporation procedure includes according to the pulse train mould of decentralized model delivering energy pulse Formula send energy pulse delivery to multiple electrodes.

In some embodiments of the present invention, DNA plasmid vaccine can further include adjuvant.In some embodiments, Adjuvant is selected from the group being made up of: alpha-interferon, gamma interferon, platelet-derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell capture chemotactic factor (CF) (CTACK), epithelium thymus gland expression chemotactic because Son (TECK), mucous membrane associated epithelium chemotactic factor (CF) (MEC), IL-12, IL-15, MHC, CD80, CD86, including have and be deleted letter The IL-15 of number sequence simultaneously optionally includes the signal peptide from IgE.May be useful adjuvant other genes include coding with Under gene: MCP-1, MIP-l- α, MIP-1p, IL-8, RANTES, L-selectin, palatelet-selectin, E-Selectin, CD34, GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、 The mutant forms of LFA-3, M-CSF, G-CSF, IL-4, IL-18, CD40, CD40L, angiogenesis factor, fibroblast are raw The long 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, ifn response gene, 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 their function fragment.In some preferred embodiments, adjuvant be selected from IL-12, IL-15, CTACK, TECK or MEC.

In some embodiments, pharmaceutically acceptable excipient is transfection, it may include that following transfection promotees Into agent: surfactant, such as immunostimulating complex (ISCOMS)；Incomplete Freund's adjuvant；LPS analog, including single phosphorus Acyl lipid A；Muramyl peptide；Quinone analog；Vesicle, such as squalene and squalene (squalene and squalene)；It is transparent Matter acid；Lipid；Liposome；Calcium ion；Virus protein；Polyanion；Polycation；Or nano particle or other known turn Contaminate promotor.Preferably, transfection is polyanion, polycation including poly- L-Glu (LGS) or lipid.It is preferred that Ground, transfection are poly- L-Glus, and it is highly preferred that poly- L-Glu is present in DNA matter with the concentration less than 6mg/mL In grain vaccine.In some embodiments, the concentration of poly- L-Glu is less than 4mg/mL, is less than 2mg/ in DNA plasmid vaccine ML, it is less than 1mg/mL, is less than 0.750mg/mL, is less than 0.500mg/mL, is less than 0.250mg/mL, is less than 0.100mg/mL, is small In 0.050mg/mL or it is less than 0.010mg/mL.

In some embodiments, can by DNA plasmid vaccine delivery to mammal to trigger an immune response；It is preferred that Ground, mammal are primate, including the mankind and inhuman primate, ox, pig, chicken, dog or ferret.More preferably Ground, mammal are human primates.

One aspect of the present invention is related to causing the method for immune response of the mammal for a variety of virus subtypes.It is described Method includes the tissue delivery DNA plasmid vaccine to the mammal, and with energy pulse effectively to allow the DNA matter Grain enters the constant current of the cell for the cell electroporation of the tissue.DNA plasmid vaccine includes can be in the lactation A variety of shared antigens of hypotype of the expression derived from the virus are in the cell of animal to cause the immune anti-of the mammal The DNA plasmid answered, a variety of shared antigens include the antigenic domains from least two different virus hypotypes.

One aspect of the present invention is related to causing the method for immune response of the mammal for a variety of subtypes of dengue virus. The method includes the tissue delivery DNA plasmid vaccine to the mammal, the DNA plasmid vaccine includes can be described The DNA plasmid for sharing Dengue antigen to cause the immune response of the mammal is expressed in the cell of mammal, it is described total Having Dengue antigen includes the consensus sequence of coding prME albumen, and the prME albumen comes from least two Dengue hypotypes, Yi Jiyou Selection of land comes from all four Dengue hypotypes.Dengue hypotype includes hypotype 1, hypotype 2, hypotype 3 and hypotype 4.It triggers an immune response Method includes with energy pulse effectively to allow DNA plasmid to enter the constant current of cell for the cell electroporation of the tissue.

In some embodiments, the method for the present invention includes by DNA plasmid vaccine injection to intradermal tissue, subcutaneous tissue Or the delivery step of musculature.Preferably, these methods include presetting expectation using In vivo electroporation device to be passed It is sent to the electric current of tissue；And with energy pulse to be equal to the constant current of predetermined current for the cell electroporation of the tissue. In some embodiments, electroporation procedure further include: measure the impedance in the cell of electroporation；Impedance tune relative to measurement The energy level of whole energy pulse is to keep the constant current in electroporation of cells；Wherein the measurement and set-up procedure occur In the validity period of energy pulse.

In some embodiments, electroporation procedure includes according to the pulse train mould of decentralized model delivering energy pulse Formula send energy pulse delivery to multiple electrodes.

It is described immune the invention also includes the DNA fragmentation for the polypeptide that coding can trigger an immune response in mammals Reaction is substantially similar to the un-segmented immune response of at least one subtypes of dengue virus.When the DNA fragmentation is applied to this When the specific coding nucleic acid sequence that text provides, it is selected from various coding nucleotide sequences of the invention (including SEQ ID NO:1；Encode the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3；Encode SEQ ID NO:4, SEQ ID NO:5 nucleosides Acid sequence；Encode SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 nucleotide sequence) at least one segment, and Any one of and can be DNA fragmentation as described below.In some embodiments, DNA fragmentation may include 30 or more, 45 A or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more It is more, 210 or more, 240 or more, 270 or more, 300 or more, 320 or more, 340 or more or Person 360 or more amino acid.In some embodiments, DNA fragmentation may include immunoglobulin E (IgE) leader sequence Coded sequence.In some embodiments, DNA fragmentation may include less than 60, less than 75, less than 90, less than 120, be less than 150,180 are less than, 210 is less than, is less than 240, is less than 270, is less than 300, is less than 320, less than 340 or less than 360 nucleotide.

The present invention includes by the encoded polypeptide of coding nucleotide sequence and may include with the and of SEQ ID NO:2,4,6 The polypeptide of 8 amino acid sequence.It is described to exempt from the invention also includes the polypeptide fragment that can be triggered an immune response in mammals Epidemic disease reacts the un-segmented immune response for being substantially similar at least one Dengue hypotype.When the polypeptide fragment is applied to herein When the particular polypeptide sequence of offer, they are selected from various polypeptide sequences of the invention (including SEQ ID NO:2,4,6 and 8) Any one of at least one, and can be polypeptide fragment as described below.In some embodiments, polypeptide fragment may include 15 or more, 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 100 or more More, 110 or more or 120 or more amino acid.In some embodiments, polypeptide fragment may include less than 30, Less than 45, less than 60, less than 75, less than 90, less than 100, be less than 110 or less than 120 amino acid.

Cause substantially similar immune of un-segmented immune response at least one Dengue hypotype in mammals The determination of the function fragment of reaction can be readily determined by those of ordinary skill in the art.Such as by public available database Such as National Biotechnology Information Center (NCBI) provided by, can analyze the segment with comprising at least one, preferably more Multiple epitopes.In addition, mouse and antibody titer and enzyme-linked immunospot assay (ELISpots) can be used routinely to comment Estimate immune response research, such as shown in embodiments below.

Vaccine

In some embodiments, the present invention is by providing the heredity of the protein of protein and coding with following epitope Construct and provide improved vaccine: the epitope make protein particularly effectively as induction immune response it is targeted Immunogene.Therefore, it is possible to provide vaccine is with inducing therapeutic or preventative immune response.

Vaccine delivery according to the present invention is adjusted individual immunity system to individual by some embodiments according to the present invention The activity of system and thus enhancing immune response.When cellular uptake of the nucleic acid molecules of code for said proteins by the individual, The nucleotides sequence, which is listed in cell, to be expressed and thus the protein is delivered to individual.Each aspect of the present invention provides The method coded sequence of protein being delivered on nucleic acid molecules such as plasmid.It is according to the present invention in some terms, provide Preventative and/or individual therapeutic immunization composition and method.

When by cellular uptake, DNA plasmid can stay in cell as isolated inhereditary material.Selectively, RNA can It is administered to cell.It further include providing genetic constructs as linear minichromosome, including centromere, telomere and replicate Point.Genetic constructs include controlling element necessary to the gene expression of nucleic acid molecules.The element includes: promoter, starting Codon, terminator codon and polyadenylation signal.In addition, the gene table of coding target protein or the sequence of immune modulator Up to usually requiring enhancer.These elements must be operably connected with the sequence of encoding desired proteins, and regulating element must It must be operationally in the individual for applying them.

It is generally acknowledged that initiation codon and terminator codon are a part of the nucleotide sequence of encoding desired proteins.So And these elements must be functional in the mammal of administration of nucleic acid construct.Initiation codon and terminator codon It must be in frame with coded sequence.

Used promoter and polyadenylation signal must be functional in individual cells.

The example for implementing the especially promoter used in the production of people's genetic vaccine of the invention includes but is not limited to: coming From the promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) example of simian virus 40 (SV40) As the long end of bovine immunodeficiency virus (BIV) repeats (LTR) promoter, Moloney (Moloney) virus, Avian Leukosis Virus (ALV), cytomegalovirus (CMV) such as CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoter, and come from human gene such as human actin, human myoglobulin, human hemoglobin, people's muscle creatin With the promoter of human metal thioalbumen (metalothionein)；In other embodiments, promoter can be it is natural or The tissue-specific promoter of synthesis such as muscle or skin-specific promoter.It is special that the example of such promoter is described in the U.S. In sharp Shen Qing Publication the US20040175727th, to be incorporated by herein.

Implement the especially polyadenylation signal used in the production of people's genetic vaccine of the invention example include but It is not limited to: SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, people Growth hormone (hGH) polyadenylation signal and human beta-globin polyadenylation signal.Specifically, pCEP4 matter can be used SV40 polyadenylation signal in grain (Invitrogen, San Diego, CA), referred to as SV40 polyadenylation signal.

Other than controlling element needed for DNA expression, other elements be may also be included in that in DNA molecular.It is such its His element includes enhancer.Enhancer can be selected from including but not limited to below group: human actin, human myoglobulin, people Hemoglobin, people's muscle creatin and virus enhancer, such as the enhancer from CMV, RSV and EBV.

Genetic constructs can be provided for being maintained at construct outside chromosome together with mammalian origin of replication, and And multiple copies of construct are generated in cell.Plasmid pVAX1, pCEP4 from Invitrogen (San Diego, CA) It include the replication orgin of Epstein-Barr virus and the nuclear antigen EBNA-1 coding for generating the high copy episomal replication that do not integrate with pREP4 Area.

In order to maximize the generation of protein, it may be selected to be well suited for gene expression in the cell of application construct Regulating and controlling sequence.In addition, the codon for the code for said proteins by full blast transcribed in the host cell may be selected. Those of ordinary skill in the art can produce the functional DNA construct in cell.

In some embodiments, it is possible to provide wherein the coded sequence of protein described herein is connected with IgE signal peptide Nucleic acid construct.In some embodiments, protein as described herein is connected with IgE signal peptide.

In some embodiments using protein, for example, those of ordinary skill in the art can be used well-known technique raw It produces and separates protein of the invention using well-known technique.In some embodiments using protein, for example, this field is general Well-known technique can be used to be inserted into the DNA molecular for encoding protein of the invention for logical technical staff to be made in known expression system In commercially available expression vector.For example, commercially available plasmid pSE420 (Invitrogen, San Diego, CA) can be used for producing albumen in Escherichia coli (E.coli).For example, commercially available plasmid pYES2 (Invitrogen, San Diego, CA) can be used for saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterium in yeast Production in strain.For example, commercially available MAXBAC^TMComplete baculovirus expression system (Invitrogen, San Diego, CA it) can be used for the production in insect cell.For example, commercially available plasmid pcDNA or pcDNA3 (Invitrogen, San Diego, CA) it can be used for the production in mammalian cell, such as Chinese Hamster Ovary (CHO) cell.The common skill in this field These commercially available expression vectors and system or other carriers and system can be used to pass through routine techniques and readily available for art personnel Starting material produces protein (see, for example, Sambrook et al., Molecular Cloning a Laboratory Manual (Molecular Cloning:A Laboratory guide), the second edition.Cold Spring Harbor Press(1989)).It therefore, can be in prokaryotic system With preparation expectation albumen in eukaryotic system, a series of protedogenous form processings are produced.

The expression vector and system that those of ordinary skill in the art can be used other commercially available, or use known side Method and the starting material being easy to get produce carrier.Include necessary regulating and controlling sequence such as promoter and polyadenylation signal And it preferably includes the expression system of enhancer and can be readily available and be known in the art for various hosts. See, for example, Sambrook et al., (Molecular Cloning: A Laboratory refers to Molecular Cloning a Laboratory Manual South), the second edition.Cold Spring Harbor Press(1989).Gene construct includes being operably connected with promoter Protein coding sequence, the promoter transfection construct cell line or target tissue cell in be functional.Group The example of constitutive promoter includes the promoter from cytomegalovirus (CMV) or SV40.The example of inducible promoter includes The promoter of mammary gland of mouse leukemia virus or metallothionein.Those of ordinary skill in the art can be from the starting material being easy to get It is readily produced in material for the genetic constructs with the DNA transfection cell for encoding protein of the invention.It will include coding egg The expression vector of the DNA of white matter is used to convert compatible host, then by the host in the item that exogenous DNA expression wherein occurs It cultivates and keeps under part.

By dissolving cell or from culture medium appropriate and well known by persons skilled in the art by the protein of generation It is recycled from culture.Well-known technique can be used to separate the egg produced using such expression system in those of ordinary skill in the art White matter.Using the antibody specifically bound with above-described specific protein, method for purifying proteins can be same from natural source It is applied to the protein purification generated by recombinant DNA method to sample.

Except through recombinant technique produce protein except, can also be produced using automated peptide synthesizer separation, Substantially pure albumen.Such technology is well known for those of ordinary skill in the art, and if has the derivative of substitution If object provides not in the production of the protein of DNA encoding, such technology is useful.

Any one of several well-known techniques can be used to deliver nucleic acid molecules, the technology includes: with and without internal electricity The DNA of perforation injects (also referred to as DNA inoculation), liposome-mediated, and nano particle promotes, recombinant vector such as recombined adhenovirus, Virus related to rocombinant adenovirus and recombinant bovine bovine vaccine.Preferably, nucleic acid molecules as described herein such as DNA plasmid is to pass through DNA Injection is delivered together with In vivo electroporation.

Administration method includes but is not limited to: it is intramuscular, intranasal, peritonaeum is interior, intradermal, subcutaneous, intravenous, intra-arterial, it is intraocular and It is oral and locally, percutaneously, by sucking or suppository or to mucosal tissue for example by lavation to vagina, rectum, urethra, Cheek or sublingual tissue.Preferred administration method include it is intramuscular, peritonaeum is interior, intradermal and be subcutaneously injected.Genetic constructs can lead to Following means application is crossed, including but not limited to: conventional syringe, Needleless injection device, " micropellet bombardment particle gun (microprojectile bombardment gone gun) " or other physical methods such as electroporation (" EP "), " fluid is dynamic Mechanics method " or ultrasound.

Preferred electroporation device and the example of electroporation method for promoting the delivering of DNA vaccination of the invention include In the U.S. Patent Application Publication that the U.S. Patent No. 7,245,963 of Draghia-Akli et al., Smith et al. are submitted Their full content is incorporated herein by the content of example described in No. 2005/0052630, two documents by quoting.Also It is preferred that the electroporation device and electroporation method of the delivering for promoting DNA vaccination provided in following documents: in October, 2007 The co-pending and shared U.S. Patent Application Serial 11/874072 possessed of No. 17 submissions, it is according to United States Code No. 35 119 sections of item (e) requires to enjoy the U.S. Provisional Application Serial No. 60/852,149 and 2007 year 10 submitted on October 17th, 2006 Entire contents are incorporated to this by the equity for the U.S. Provisional Application Serial No. 60/978,982 that the moon is submitted, all these documents Text.

U.S. Patent No. 7,245,963 of Draghia-Akli et al. describe standard electrode systems and they are used to promote Into the purposes imported biomolecule in the cell for selecting tissue in body or plant.Standard electrode systems include multiple needle-shaped electricity Pole；Hypodermic needle；The electrical connection of conduction connection from sequencing constant current pulses controller to the multiple needle electrode is provided Device；And power supply.Operator can catch the multiple needle electrodes filled on the support structure and they are securely inserted into body In body or endophytic selected tissue.Then biomolecule is delivered in selected tissue by hypodermic needle.The constant electricity of sequencing Stream impulse controller is activated, and the electric pulse of constant current is applied to multiple needle electrodes.The constant current electrical of application Pulse promotes biomolecule to imported into the cell between multiple electrodes.The full content that U.S. Patent No. 7,245,963 is logical It crosses and is incorporated herein by reference.

The U.S. Patent Application Publication that Smith et al. is submitted the 2005/0052630th describes and can be used to effectively facilitate Biomolecule imported into body or plant the electroporation device in the cell for selecting tissue.The electroporation device includes by soft Part or the electric device (" EKD device ") of the specified operation of firmware.The input of control and pulse parameter of the EKD device based on user A series of constant current pulses mode of programmables is generated between electrode in an array, and allows current waveform data Storage and acquisition.Electroporation device further includes the alternative electrode disk with needle electrode array, the center for injection needle Injection canal and removable guidance disk.The full content of U.S. Patent Publication 2005/0052630 is incorporated by reference into this Text.

Electrode array described in U.S. Patent No. No. 7,245,963 and U.S. Patent Application Publication the 2005/0052630th Column and method are suitable for deeply penetrating the tissue of such as muscle and its hetero-organization or organ.Because of the configuration of electrod-array, Injection needle (biomolecule for delivering selection) is also fully inserted into target organ, and inject by vertically be applied to it is pre- by electrode The target tissue in region first delimited.In U.S. Patent No. 7,245,963 and U.S. Patent Application Publication 2005/005263 The electrode of description is preferably 20mm long and 21 thickness (gauge).

An example of method of the invention below, and in patent references discussed above in more detail by It discusses: the expectation tissue that the energy pulse of the constant current of generation is delivered to mammal by electroporation device can be configured, The predetermined current that the constant current is similar to user inputs.Electroporation device includes electroporation component and electrode assembly or hand Handle component.One or more of the various elements that electroporation component may include and mix electroporation device, comprising: controller, Current waveform generator, impedance testing device, kymograph, input element, status reporting element, communication port, memory component, Power supply and power switch.Electroporation component can as an elements act of electroporation device, and other elements be with The separated element (or component) that electroporation component is kept in touch.In some embodiments, electroporation component can be used as more In the elements act of an electroporation device, it can also keep in touch with the other elements of electroporation device, it is described other Element is to separate with electroporation component.The present invention is not filled by electroporation existing for the part as electromechanical assembly or mechanical device The element limitation set, because the element can be acted as a device or as the separated element kept in touch each other With.Electroporation component can deliver the energy pulse that constant current is generated in desired tissue, and including feedback mechanism.Electrode Component includes the electrod-array in spatial arrangement with multiple electrodes, wherein the electrode assembly, which receives, comes from electroporation component Energy pulse and this energy pulse is delivered to desired tissue by electrode.At least one of multiple electrodes are in delivering energy It is neutral during measuring pulse, and measures the impedance in desired tissue, and the impedance is transmitted to electroporation component. Feedback mechanism can receive the impedance of measurement and can adjust by the energy pulse of electroporation parts delivery to keep constant electric current.

In some embodiments, the multiple electrode can deliver energy pulse with decentralized model.In some embodiment party In case, the multiple electrode can by under agenda coordination electrode energy pulse delivered with decentralized model, and it is described Agenda is input in electroporation component by user.In some embodiments, agenda includes delivering in order Multiple pulses, each pulse in plurality of pulse is a neutrality by least two active electrodes and measurement impedance Electrode delivering, and in plurality of pulse be by one different at least two active electrodes and measurement with afterpulse One neutral electrode of impedance delivers.

In some embodiments, feedback mechanism is executed by hardware or software.Preferably, feedback mechanism is to pass through Simulate what closed loop executed.Preferably, every 50 μ s of this feedback, 20 μ s, 10 μ s or 1 μ s occur, it is preferred that anti-in real time Feedback or instantaneous feedback (i.e. as by for determining feedback substantially instantaneous determined by the available technology in reaction time).? In some embodiments, neutral electrode measurement it is expected the impedance in tissue and the impedance is transferred to feedback mechanism, the feedback Mechanism responds the impedance, and adjusts energy pulse so that constant current is maintained at the value similar with predetermined current.In some implementations In scheme, feedback mechanism continuously and instantaneously keeps constant electric current during delivering energy pulse.

Pharmaceutically acceptable excipient may include for example carrier, adjuvant, carrier or diluent it is this kind of it is public it is known simultaneously And readily available functional molecular.Preferably, pharmaceutically acceptable excipient is adjuvant or transfection.In some realities It applies in scheme, nucleic acid molecules or DNA plasmid is delivered to cell, and apply polynucleotide function enhancers or genetic vaccine rush Into agent (or transfection).Polynucleotide function enhancers are described in U.S. Patent No. 5,593,972, U.S. Patent No. 5, In 962, No. 428 and the international patent application series number PCT/US94/00899 that submits on January 26th, 1994, these documents are respectively It is incorporated herein by reference.Genetic vaccine promotor is described in U.S. Serial the 021st, 579 submitted on April 1st, 1994, It is incorporated herein by reference.Transfection can be applied with nucleic acid molecules together as with the mixture of nucleic acid molecules, or Person nucleic acid molecules apply while, before or after separate administration.The example of transfection includes surfactant, such as Immunostimulating complex (ISCOMS)；Incomplete Freund's adjuvant；LPS analog, including monophosphoryl lipid A；Muramyl peptide；Quinones Like object and vesicle such as squalene and squalene；And hyaluronic acid also can be used and genetic constructs are administered in combination.? In some embodiments, DNA plasmid vaccine can also include transfection, such as lipid；Liposome, including lecithin lipid Body or other known liposomes of this field are DNA liposomal mixtures (see, for example, W09324640)；Calcium ion；Viral egg It is white；Polyanion；Polycation or nano particle；Or other known transfection.Preferably, transfection is poly- Anion, polycation, including poly- L-Glu (LGS) or lipid.

In some preferred embodiments, DNA plasmid and adjuvant are delivered together, the adjuvant is to further enhance needle To the gene of the protein of the immune response of such target protein.The example of this genoid is that those encode other cell factors and leaching The gene of Ba Yinzi, the cell factor and lymphokine for example alpha-interferon, gamma interferon, platelet-derived growth because Sub (PDGF), TNF α, TNF β, 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-15, including being deleted the IL-15 of signal sequence and optionally including from IgE Signal peptide.Other genes to come in handy include encoding gene below: MCP-1, MIP-l α, MIP-1p, IL-8, RANTES, L-selectin, palatelet-selectin, E-Selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, The mutation bodily form of pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18 Formula, CD40, CD40L, angiogenesis factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth The 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, ifn response gene, 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 their function fragment.

The amount of DNA that DNA plasmid vaccine according to the present invention includes is from about 1 nanogram to 10 milligrams；About 1 microgram to about 10 millis Gram；Or preferably about 0.1 microgram is to about 10 milligrams；Or more preferably about 100 micrograms are to about 1 milligram.Some preferred In embodiment, DNA plasmid vaccine according to the present invention includes the DNA of about 5 nanograms to about 1000 micrograms.In some preferred realities It applies in scheme, DNA plasmid vaccine contains about 10 nanograms to the DNA of about 800 micrograms.In some preferred embodiments, DNA matter Grain vaccine contains the DNA of about 0.1 microgram to about 500 micrograms.In some preferred embodiments, DNA plasmid vaccine contains about 1 DNA of the microgram to about 350 micrograms.In some preferred embodiments, it is micro- to about 250 to contain about 25 micrograms for DNA plasmid vaccine Gram DNA.In some preferred embodiments, DNA plasmid vaccine contains about 100 micrograms to about 1 milligram of DNA.

DNA plasmid vaccine according to the present invention is prepared according to method of application ready for use.It is injectable in DNA plasmid vaccine Composition in the case where, they be sterilizing and/or it is pyrogen-free and/or agranular.Isotonic preparation is preferably used.One As for, the additive of isotonicity may include sodium chloride, dextrose, mannitol, sorbierite and lactose.In some cases, excellent Select isotonic solution, such as phosphate buffered saline (PBS).Stabilizer includes gelatin and albumin.In some embodiments, by blood vessel Contracting agent is added in preparation.In some embodiments, so that at preparation continue in room temperature or ambient temperature stable one section Between stabilizer such as LGS or other polycations or polyanion be added in preparation.

In some embodiments, the method for causing the immune response that mammal is directed to shared Dengue antigen includes induction The method of mucosal immunoreaction.Such method includes applying one or more CTACK albumen, TECK egg to the mammal White, MEC albumen and their function fragment or their effable coded sequence and including above-described shared Dengue The DNA plasmid of antigen.One or more CTACK albumen, TECK albumen, MEC albumen and their function fragment can be herein It is applied prior to, concurrently with, or after the application of the DNA plasmid dengue vaccine of offer.In some embodiments, it is applied to mammal With isolated nucleic acid molecules, the one or more protein selected from the group being made up of of the nucleic acid molecule encoding: CTACK, TECK, MEC and their function fragment.

The present invention is further illustrated in the examples below.It should be understood that these embodiments point out it is of the invention excellent When selecting embodiment, only it is illustrated with mode and is presented.By described above and these embodiments, those skilled in the art's energy Enough determine essential feature of the invention, and without departing from the spirit and scope of the present invention, can to the present invention into The various variations of row and improvement are to make it fit in various utilizations and condition.Therefore, by foregoing description, various improvement of the invention with And improvement those of illustrated and described herein will be apparent those skilled in the art.Such improvement, which is also intended to, falls into appended power Within the scope of sharp claim.

Preferably, have the high DNA of small size dense for the DNA preparation that muscle as described herein or skin EP device use Degree preferably includes microgram to the DNA concentration of tens of micrograms amount and preferred milligram quantities, and the small size is for being delivered to skin Skin is optimal, preferably small volume injected, it is generally desirable to 25-200 microlitres (μ L).In some embodiments, the DNA preparation With high DNA concentration, such as 1mg/mL or bigger (mg DNA/ volumes of formulation).It is highly preferred that the DNA concentration that DNA preparation has Provide the DNA of the gram quantity in the formula of 200 μ L, and more preferably in the formula of 100 μ L gram quantity DNA.

The formulated in combination of known devices and technology or the DNA plasmid that manufacture is used for EP device of the invention can be used, but The plasmid manufacturing technology of optimization described in following documents is preferably employed in manufacture them: U.S. Patent Application No. 12/ No. 126611, it announces No. 20090004716 as U.S. Patent Application Publication on January 1st, 2009 and discloses.Some In example, the DNA plasmid used in these researchs can be prepared with the concentration more than or equal to 10mg/mL.In addition in the U.S. The U.S. Patent No. 7,238,522 submitted described in patent application publication the 20090004716th and on July 3rd, 2007 Except device and scheme described in number, manufacturing technology further includes or combines generally known each of those of ordinary skill in the art Kind device and scheme.Allow plasmid at a fairly low for the high concentration plasmid that skin EP device described herein and delivery technique use Volume be administered in the space ID/SC and help Enhanced expressing and immunization.Disclosure U.S. Patent Application Publication No. 20090004716 are integrally incorporated to it with U.S. Patent No. 7,238,522 herein.

Embodiment

Embodiment 1

PrM prevents E protein during virus maturation due to forming the non-infectious i.e. prM-E of immature virion heterologous two The too early fusion of poly- compound.Immature particle is changed by the low ph conditions of golgiosome chamber, and in this stage, Before the processing of prM, reversible conformation change occurs in E protein.Pass through in reverse side golgiosome reticular structure in prM Cell serine protease is cut into after M, produces the irreversible conformation change of E, to maintain the complete of Neutralization and crystallization Whole property.

D1prME, D2prME, D3prME and D4prME are that coding includes pRM (pre- film precursor) albumen and E (coating) albumen The construct of the antigen of the consensus sequence of serotype D1, D2, D3 or D4 is directed in the two respectively.

DU is the construct of antigen of the coding comprising following consensus sequence: for E (coating) albumen of serotype D1 The consensus sequence of DIII structural domain, for serotype D2 E (coating) albumen DIII structural domain consensus sequence, be directed to serum The DIII structure of the consensus sequence of the DIII structural domain of E (coating) albumen of type D3 and E (coating) albumen for serotype D4 The consensus sequence in domain, each free connector of consensus sequence separate.It is (general to construct all four DIII structural domains of combination Or general DIII) construct and be referred to as DU (SEQ ID NO:9), four kinds of DIII structural domains are cut by proteolysis Sequence is cut to separate.Shared DIII structural domain DU with sequence SEQ ID NO:10 is made of catenation sequence, the catenation sequence Connect each in four kinds of hypotypes (DV-1-DIII, DV-2-DIII, DV-3-DIII and DV-4-DIII) of DIII.The company Junctor has sequence RGRKRRS, it is known cleavage site and allows for DU to be cut into the independent of specific subtype DIII sequence.However, the catenation sequence can be available any other connection sequence in this field with similar features Column, and the ordinary skill that the RGRKRRS currently utilized belongs to this field is substituted for this connector.

Mouse is immunized with D1prME, D2prME, D3prME, D4prME or DU of 100 μ g.Mouse receives and uses 3P 3 immune, each immunization intervals 3 weeks that Cellectra (ID) is carried out, and take a blood sample at same injection site every 3 weeks.It is each Group includes 5 animals (n=5).

Serial dilution (1:50,1:150,1:450,1:1350 and 1:4050) is carried out to the serum from each mouse.Make Each DIII (Domain III of envelope protein E) of the serum exposure in four kinds of serotypes D1, D2, D3 or D4.Pass through Measure absorbance at 450 nm to measure the combination of Serum Antibody and DIII albumen.BSA is used as control.

It is the general introduction of experimental design below.

1. experimental design of table

Result is shown in Fig. 1-5.The DIII from D1 is combined as shown in Figure 1, being generated with all mouse that D1prME is immunized Antibody, and with DU be immunized all mouse also generates combination the DIII from D1 antibody.As shown in Fig. 2, using D2prME Immune all mouse generate the antibody for combining the DIII from D2, and all mouse being immunized with DU also generate to combine and come from The antibody of the DIII of D2.As shown in figure 3, the antibody for combining the DIII from D3 is generated with the mouse 3,4 and 5 that D3prME is immunized, But not with shown in Fig. 1 and 2 combine it is equally good.Good combination is not generated from D3 with the mouse 1 and 2 that D3prME is immunized DIII antibody.The antibody for combining the DIII from D3 is generated with all mouse that DU is immunized.As shown in figure 4, using D1prME Immune mouse 4 and 5 generates the antibody for combining the DIII from D4, but the antibody from mouse 1,2 and 3 does not generate combination. The antibody for combining the DIII from D4 is generated with all mouse that DU is immunized.

With western blot analysis to come the serum for D1prME, D2prME, D3prME or D4prME immune mouse of using by oneself Analyze albumen E and albumen prM.As shown in figure 5, albumen E and prM are individual, and it is present in the serum from all mouse In.

Embodiment 2: Dengue Li-Cor measurement

With D1prME, D2prME, D3prME, D4prME or DU immune guinea pig of 100 μ g.As described in detail below, one In a little groups, exempted from single immunization sites or independent immunization sites with all four D1prME, D2prME, D3prME or D4prME The each cavy of epidemic disease.In individually application site, mixed in a manner of single administration by all four.For position is individually immunized For point, each is individually applied.As in example 2, D1prME, D2prME, D3prME and D4prME are that coding includes Respectively from the construct of the antigen of both pRM (the pre- film precursor) albumen of serotype D1, D2, D3 or D4 and E (coating) albumen. DU is the construct of antigen of the coding comprising following consensus sequence: for the DIII structural domain of E (coating) albumen of serotype D1 Consensus sequence, for serotype D2 E (coating) albumen DIII structural domain consensus sequence, for serotype D3 E (packet Film) albumen DIII structural domain consensus sequence and for serotype D4 E (coating) albumen DIII structural domain shared sequence Column, each free connector of consensus sequence separate, and are such as described in detail in embodiment 1.

Serial dilution (1:50,1:150,1:450,1:1350 and 1:4050) is carried out to the serum from each cavy.It is logical It crosses and measures absorbance at 450 nm to measure the combination of Serum Antibody and dengue fever virus.

2 times of serial dilutions of serum are carried out, and are placed it in 96 orifice plates, every 50 μ L of hole.By 50pfu (every 50 μ of hole L dengue fever virus) is added in each hole.Plate is incubated for 1 hour at 37 DEG C, to allow virus and the antibody from serum It neutralizes.VERO cell (seed 1.5x10 is added in entire mixture (100 μ L)⁴/ hole).Plate is incubated for 4 days at 37 DEG C.It uses The cells are fixed 30 minutes for 3.7% formaldehyde.Use 0.1%TritonX-100/PBS washing and permeabilization cell.Use mouse 4G2mAb, biontnylated anti-mouse IgG and IRDye800CW streptavidin+5mM DRAQ5 mixture solution are come Execute the ELISA based on cell.It is scanned by plate washing, drying, and using Li-Cor Aerius system.Calculate 800nm/ 700nm ratio.

Fig. 6-13 shows result.As shown in fig. 6, from two control cavy (not being immunized) serum do not combine from D1, The DIII of D2, D3 or D4 serotype.As shown in fig. 7, with DU immune guinea pig, and only, cavy 4 generates combination from D1, D2, D3 With the antibody of the DIII albumen of D4 serotype.As shown in figure 8, with the D1prME of 100 μ g, the D2prME of 100 μ g, 100 μ g The mixture of the D4prME of D3prME and 100 μ g is immunized five cavys, every cavy 400 μ g in total, all an immune position Point.All cavys generate the antibody for combining the DIII albumen from D1, D2 and D3.However, only two cavys generation combinations come from The antibody of the DIII albumen of D4, and combine and weaken for other serotypes.As shown in figure 9, with 100 μ g's D1prME, the D2prME of 100 μ g, the D3prME of 100 μ g and 100 μ g D4prME five globefish are immunized at four independent sites Mouse, every cavy 400 μ g in total.All cavys generate the antibody for combining the DIII albumen from D1, D2 and D3.However, only three Or four cavys generate the antibody for combining the DIII albumen from D4, and combine and weaken for other serotypes.Such as Shown in Figure 10, five cavys are immunized with the D1prME of every 100 μ g.All cavys, which generate, combines the anti-of the DIII albumen from D1 Body.However, only several cavys generate the antibody for combining the DIII albumen from D2, D3 or D4, and combine for D1 Weaken.As shown in figure 11, five cavys are immunized with the D2prME of every 100 μ g.All cavys, which generate, combines the DIII from D2 The antibody of albumen.However, only a cavy generates the antibody for combining the DIII albumen from D1, and combine relative to other D1 For weaken.No cavy generates the antibody for combining the DIII albumen from D3 or D4.As shown in figure 12, with every 100 μ g's Five cavys are immunized in D3prME.All cavys generate the antibody for combining the DIII albumen from D1 and D3.However, an only cavy Generate the antibody for combining the DIII albumen from D2.No cavy generates the antibody for combining the DIII albumen from D4.Such as Figure 13 institute Show, five cavys are immunized with the D4prME of every 100 μ g.Four cavys generate the antibody for combining the DIII albumen from D4.Globefish Mouse 1 generates the antibody for combining the DIII albumen from D1 and D2.Only several cavys generate combine (minimally) from D2 and The antibody of the DIII albumen of D3.

Fig. 8 and 9 is indicated compared with Fig. 7: (no matter individually being gone back with the combination of D1prME, D2prME, D3prME and D4prME Mixing) carry out it is immune than carrying out immune generation to the better immunogenicity of DIII with DU.

Embodiment 3: Dengue PRNT₅₀It is measured with FRNT

With DU, D1prME, D2prME, D3prME or D4prME immune guinea pig of 100 μ g.As described in detail below, one In a little groups, exempted from single immunization sites or independent immunization sites with all four D1prME, D2prME, D3prME or D4prME The each cavy of epidemic disease.Such as in embodiment 2 and 3, D1prME, D2prME, D3prME and D4prME are codings comprising respectively from blood The construct of the antigen of pRM (the pre- film precursor) albumen and both E (coating) albumen of clear type D1, D2, D3 or D4.DU is coding packet The construct of antigen containing following consensus sequence: for the DIII structural domain of E (coating) albumen of serotype D1 consensus sequence, Consensus sequence for the DIII structural domain of E (coating) albumen of serotype D2, E (coating) albumen for serotype D3 The consensus sequence of DIII structural domain and for serotype D4 E (coating) albumen DIII structural domain consensus sequence, it is described total There is sequence respectively to be separated by connector, is such as described in detail in example 2.

For Dengue PRNT50 measurement, VERO cell is inoculated with (7.5x10 with 6 hole formats on day 1⁵To 1.0x10⁶ A cells/well).On day 2, at 37 DEG C, dengue fever virus/hole of 50pfu is incubated for up to 1 hour with the serum from cavy. Used dengue fever virus is one of bacterial strain Hawaii, NGC, H87 or H241.Feed the mixture into the cell list in plate Layer, and be incubated for 1 hour at 37 DEG C.1% methylcellulose in 2% culture medium 199 covers cell.It will at 37 DEG C Cell incubation was up to 5 days after infection.At the 7th day, simultaneously staining cell (being incubated for 2 hours) was fixed with crystal violet/20% carbinol mixture. Use dH₂O washs plate and is subject to drying.To plaque counting.Dilution factor is defined as: compared with the control of serum-free, plaque Count the inverse of the highest serum dilution of reduction > 50%.

For Focus Diagnostics FRNT measurement, 4 times of dilutions of 24 orifice plates and serum are used.Plate is incubated for 4 days.The development of plaque is measured and counted using immune focusing.

Result is shown in following table 2 and in Figure 14-17.As shown in figure 14, come all D1prME, D2prME that use by oneself, The cavy and come from from only generating to combine with the serum of the D1prME cavy being immunized that D3prME or D4prME construct is immunized The antibody of the protein of Hawaii dengue fever virus.It is carried out immune, not generated with DU.As shown in figure 15, all to use by oneself The immune cavy of D1prME, D2prME, D3prME or D4prME construct and from only with the blood of the D2prME cavy being immunized It is clear to generate the antibody for combining the protein from NGC dengue fever virus.It is carried out immune, not generated with DU.As shown in figure 16, Come the cavy and come from only immune with D3prME that all D1prME, D2prME, D3prME or D4prME constructs are immunized of using by oneself Cavy serum generate combine the protein from H87 dengue fever virus antibody.In the immune observation later carried out with DU To titre be the smallest.As shown in figure 17, immune come all D1prME, D2prME, D3prME or D4prME constructs of using by oneself Cavy and combine protein from H241 dengue fever virus from only being generated with the serum of the D4prME cavy being immunized Antibody.It is carried out immune, not generated with DU.

The 1 phase clinical research virucidin that table 2. measures to measure by LiCor, FRNT and PRNT reacts.

Sequence table

<110>UNIV PENNSYLVANIA

David Wei Na

Yan Jian

Ni Lanzhan thayer desai

<120>it is directed to the novel vaccine of a variety of subtypes of dengue virus

<130> UPVG0055 WO2

<150> 61801972

<151> 2013-03-15

<160> 10

<170>PatentIn 3.5 editions

<210> 1

<211> 2100

<212> DNA

<213>artificial sequence

<220>

<223>Dengue 1-prME DNA sequence dna

<400> 1

atggactgga cctggattct gtttctggtc gccgccgcaa ctagagtgca ttcaaatagg 60

aggaagagga gcgtcacaat gctgctgatg ctgatgccca ccgccctggc tttccacctg 120

accacacggg gcggggagcc tcatatgatc gtgtccaagc aggaaagagg gaaatccctg 180

ctgtttaaga cttctgccgg agtgaacatg tgcaccctga ttgctatgga cctgggcgag 240

ctgtgcgaag ataccatgac atacaagtgt ccaaggatca cagaggccga acccgacgat 300

gtggactgct ggtgtaatgc tactgatacc tgggtgacct atgggacatg ttcacagacc 360

ggagagcacc ggagagacaa gaggagcgtg gcactggccc ctcacgtcgg actgggactg 420

gagacacgca ctgaaacctg gatgagctcc gagggggcct ggaaacagat tcagagagtg 480

gaaacctggg ctctgaggca ccctggattc acagtgatcg cactgtttct ggctcatgca 540

attggaactt ctatcaccca gaagggcatc attttcattc tgctgatgct ggtgacccca 600

agtatggcaa tgcgatgcgt gggaatcgga aaccgagact ttgtcgaggg cctgtccggg 660

gctacatggg tggatgtggt cctggaacac ggctcttgtg tcactaccat ggcaaaggac 720

aaaccaaccc tggatatcga gctgctgaag acagaagtga ctaaccccgc agtcctgcga 780

aaactgtgca ttgaggccaa gatcagtaat acaactaccg attcacgctg tcccactcag 840

ggcgaagcta ccctggtgga ggaacaggac gcaaacttcg tgtgcaggcg cacctttgtc 900

gatcgcggat ggggcaatgg gtgtggactg ttcggcaagg ggtccctgat cacatgcgcc 960

aagtttaaat gtgtgactaa gctggagggc aaaattgtcc agtacgaaaa cctgaaatat 1020

tcagtcatcg tgaccgtcca cacaggcgac cagcatcaag tggggaatga gtctaccgaa 1080

cacgggacaa ctgcaacaat tactcctcag gccccaacaa gcgagatcca gctgactgac 1140

tacggagccc tgaccctgga ttgctcccct cggaccggac tggatttcaa cgagatggtg 1200

ctgctgacaa tgaaggaaaa aagttggctg gtgcataagc agtggtttct ggacctgcca 1260

ctgccctgga catctggcgc ctcaacaagc caggagactt ggaatagaca ggatctgctg 1320

gtgactttca agaccgccca cgctaagaaa caggaggtgg tcgtgctggg cagccaggaa 1380

ggagctatgc atacagcact gactggcgcc accgagattc agaccagcgg gaccacaact 1440

atcttcgccg gacacctgaa gtgccggctg aagatggaca aactgacact gaaaggaatg 1500

agctacgtga tgtgtactgg ctcctttaag ctggagaaag aagtggctga gacccagcat 1560

ggcacagtgc tggtccaggt gaaatatgaa gggaccgacg ccccctgtaa gatccctttc 1620

agcacccagg atgagaaagg agtgacacag aacggcaggc tgattacagc aaatcctatc 1680

gtgactgata aggaaaaacc agtcaacatt gaggccgaac ccccttttgg cgagagttac 1740

atcgtcgtgg gagctggcga aaaggcactg aaactgtcat ggttcaagaa agggtctagt 1800

attggaaaga tgtttgaggc aaccgccaga ggcgcccgac gaatggctat tctgggcgac 1860

actgcttggg atttcgggtc tatcggaggc gtctttacca gtgtgggcaa gctggtccac 1920

cagatcttcg gcacagccta tggggtgctg ttttcagggg tcagctggac tatgaaaatc 1980

gggattggaa tcctgctgac ttggctggga ctgaattcca gatctaccag tctgagcatg 2040

acttgtattg ccgtcggact ggtgacactg tatctgggcg tgatggtgca ggcctgataa 2100

<210> 2

<211> 698

<212> PRT

<213>artificial sequence

<220>

<223>Dengue 1-prME protein sequence

<400> 2

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Asn Arg Arg Lys Arg Ser Val Thr Met Leu Leu Met Leu Met

20 25 30

Pro Thr Ala Leu Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His

35 40 45

Met Ile Val Ser Lys Gln Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr

50 55 60

Ser Ala Gly Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu

65 70 75 80

Leu Cys Glu Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala

85 90 95

Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val

100 105 110

Thr Tyr Gly Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg

115 120 125

Ser Val Ala Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Thr

130 135 140

Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys Gln Ile Gln Arg Val

145 150 155 160

Glu Thr Trp Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe

165 170 175

Leu Ala His Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe

180 185 190

Ile Leu Leu Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly

195 200 205

Ile Gly Asn Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val

210 215 220

Asp Val Val Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asp

225 230 235 240

Lys Pro Thr Leu Asp Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro

245 250 255

Ala Val Leu Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr

260 265 270

Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu

275 280 285

Gln Asp Ala Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp

290 295 300

Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala

305 310 315 320

Lys Phe Lys Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu

325 330 335

Asn Leu Lys Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His

340 345 350

Gln Val Gly Asn Glu Ser Thr Glu His Gly Thr Thr Ala Thr Ile Thr

355 360 365

Pro Gln Ala Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr Gly Ala Leu

370 375 380

Thr Leu Asp Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val

385 390 395 400

Leu Leu Thr Met Lys Glu Lys Ser Trp Leu Val His Lys Gln Trp Phe

405 410 415

Leu Asp Leu Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu

420 425 430

Thr Trp Asn Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala

435 440 445

Lys Lys Gln Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His

450 455 460

Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr

465 470 475 480

Ile Phe Ala Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr

485 490 495

Leu Lys Gly Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu

500 505 510

Lys Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys

515 520 525

Tyr Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp

530 535 540

Glu Lys Gly Val Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile

545 550 555 560

Val Thr Asp Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Pro Phe

565 570 575

Gly Glu Ser Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu

580 585 590

Ser Trp Phe Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr

595 600 605

Ala Arg Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp

610 615 620

Phe Gly Ser Ile Gly Gly Val Phe Thr Ser Val Gly Lys Leu Val His

625 630 635 640

Gln Ile Phe Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp

645 650 655

Thr Met Lys Ile Gly Ile Gly Ile Leu Leu Thr Trp Leu Gly Leu Asn

660 665 670

Ser Arg Ser Thr Ser Leu Ser Met Thr Cys Ile Ala Val Gly Leu Val

675 680 685

Thr Leu Tyr Leu Gly Val Met Val Gln Ala

690 695

<210> 3

<211> 2100

<212> DNA

<213>artificial sequence

<220>

<223>Dengue 2-prME DNA sequence dna

<400> 3

atggactgga catggattct gttcctggtc gccgctgcta cacgggtgca ttcaaataga 60

cggagacgga gtgccgggat gattatcatg ctgattccaa ccgtgatggc tttccacctg 120

accacaagga acggcgagcc ccatatgatc gtgggacgcc aggaaaaggg caaatccctg 180

ctgtttaaaa ctgaggacgg agtgaatatg tgcaccctga tggcaattga cctgggcgag 240

ctgtgcgaag atactatcac ctacaagtgt ccactgctga ggcagaacga gcccgaagac 300

atcgattgct ggtgtaatag tacatcaact tgggtgactt atggcacctg tactaccaca 360

ggggagcacc ggagagaaaa gagatctgtc gctctggtgc cccatgtcgg catggggctg 420

gagaccagga cagaaacttg gatgagctcc gagggcgcat ggaagcacgt gcagcgcatt 480

gaaacatgga ttctgcgaca tcctgggttc actattatgg ccgctatcct ggcctacacc 540

attggaacta cccacttcca gcgcgctctg atttttatcc tgctgacagc tgtggcacca 600

tccatgacta tgcggtgcat tggcatctct aacagagact tcgtggaggg ggtcagcggc 660

gggtcctggg tggatatcgt cctggaacat ggcagctgtg tgacaactat ggcaaagaac 720

aagcctaccc tggattttga gctgatcaag accgaagcca agcagccagc tacactgcgc 780

aaatattgca tcgaggccaa gctgaccaac accacaactg agagtcgatg tcccacacag 840

ggggaacctt cactgaatga ggaacaggac aaacgatttg tgtgcaagca cagcatggtc 900

gatcggggat ggggcaacgg gtgtggactg ttcggaaaag gaggcattgt gacatgcgcc 960

atgtttactt gtaagaaaaa catggagggc aagatcgtgc agcccgagaa tctggaatac 1020

accattgtca tcacacctca ctccggagag gaacatgccg tgggcaatga cactgggaag 1080

cacggaaaag agattaaggt cacccctcag tctagtatca ccgaggctga actgacaggc 1140

tatgggaccg tgacaatgga atgctctcct cggacaggcc tggatttcaa cgagatggtg 1200

ctgctgcaga tggaaaataa ggcatggctg gtccatagac agtggtttct ggacctgcca 1260

ctgccatggc tgccaggagc agatacccag ggatctaact ggattcagaa agagacactg 1320

gtgactttca agaatcccca cgccaagaaa caggacgtgg tcgtgctggg cagtcaggag 1380

ggagcaatgc ataccgccct gacaggcgct actgaaatcc agatgtcaag cgggaacctg 1440

ctgttcacag gacacctgaa atgcaggctg cgcatggata aactgcagct gaaggggatg 1500

agctactcca tgtgtaccgg aaagtttaaa gtcgtgaagg agatcgccga aactcagcac 1560

ggcaccattg tgatccgggt ccagtatgag ggagacggca gcccttgtaa aattccattc 1620

gagatcatgg atctggaaaa gagacatgtg ctggggaggc tgattactgt gaaccctatc 1680

gtcaccgaga aggacagccc agtgaatatc gaggctgaac ccccttttgg agattcctac 1740

atcattatcg gagtggagcc tggccagctg aaactgaact ggttcaagaa agggtcctct 1800

attggacaga tgtttgaaac cacaatgcga ggcgcaaagc ggatggccat cctgggcgac 1860

acagcctggg atttcgggtc actgggcggc gtgttcacca gcattggcaa agctctgcac 1920

caggtcttcg gcgcaatcta tggggcagcc ttttctgggg tgagttggac catgaagatt 1980

ctgatcggag tcattatcac atggatcggc atgaattcta gaagtacttc actgtccgtg 2040

agcctggtcc tggtcggcgt ggtgacactg tatctgggcg tgatggtgca ggcctgataa 2100

<210> 4

<211> 698

<212> PRT

<213>artificial sequence

<220>

<223>Dengue 2-prME protein sequence

<400> 4

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Asn Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile

20 25 30

Pro Thr Val Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His

35 40 45

Met Ile Val Gly Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr

50 55 60

Glu Asp Gly Val Asn Met Cys Thr Leu Met Ala Ile Asp Leu Gly Glu

65 70 75 80

Leu Cys Glu Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn

85 90 95

Glu Pro Glu Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val

100 105 110

Thr Tyr Gly Thr Cys Thr Thr Thr Gly Glu His Arg Arg Glu Lys Arg

115 120 125

Ser Val Ala Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr

130 135 140

Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile

145 150 155 160

Glu Thr Trp Ile Leu Arg His Pro Gly Phe Thr Ile Met Ala Ala Ile

165 170 175

Leu Ala Tyr Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe

180 185 190

Ile Leu Leu Thr Ala Val Ala Pro Ser Met Thr Met Arg Cys Ile Gly

195 200 205

Ile Ser Asn Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val

210 215 220

Asp Ile Val Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn

225 230 235 240

Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro

245 250 255

Ala Thr Leu Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr

260 265 270

Thr Glu Ser Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu

275 280 285

Gln Asp Lys Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp

290 295 300

Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala

305 310 315 320

Met Phe Thr Cys Lys Lys Asn Met Glu Gly Lys Ile Val Gln Pro Glu

325 330 335

Asn Leu Glu Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His

340 345 350

Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Val Thr

355 360 365

Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val

370 375 380

Thr Met Glu Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val

385 390 395 400

Leu Leu Gln Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe

405 410 415

Leu Asp Leu Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser

420 425 430

Asn Trp Ile Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala

435 440 445

Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His

450 455 460

Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu

465 470 475 480

Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln

485 490 495

Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val

500 505 510

Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln

515 520 525

Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp

530 535 540

Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile

545 550 555 560

Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe

565 570 575

Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu

580 585 590

Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr

595 600 605

Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp

610 615 620

Phe Gly Ser Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His

625 630 635 640

Gln Val Phe Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp

645 650 655

Thr Met Lys Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn

660 665 670

Ser Arg Ser Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly Val Val

675 680 685

Thr Leu Tyr Leu Gly Val Met Val Gln Ala

690 695

<210> 5

<211> 2094

<212> DNA

<213>artificial sequence

<220>

<223>Dengue 3-prME DNA sequence dna

<400> 5

atggactgga cctggattct gttcctggtc gccgccgcaa cccgcgtgca ttcaaacaaa 60

agaaagaaaa cttcactgtg cctgatgatg atgctgccag ccactctggc tttccacctg 120

accagccgag acggagaacc acggatgatc gtgggcaaga acgagagggg gaaaagtctg 180

ctgtttaaga ccgcttcagg cattaatatg tgcacactga tcgcaatgga tctgggggag 240

atgtgcgacg ataccgtcac atacaagtgt ccccatatta ccgaggtgga acctgaggac 300

atcgattgct ggtgtaacct gactagtacc tgggtgactt atgggacctg taatcaggcc 360

ggagagcacc ggagagacaa gagatcagtc gccctggctc ctcatgtggg catggggctg 420

gatacaagaa ctcagacctg gatgagcgca gagggagcat ggcgacaggt cgaaaaagtg 480

gagacttggg ccctgcgaca ccctggattc accattctgg ccctgtttct ggctcattac 540

atcggcacat cactgactca gaaggtggtc atcttcattc tgctgatgct ggtgacacca 600

agcatgacta tgagatgcgt cggagtgggc aacagggact ttgtcgaagg gctgtccgga 660

gccacctggg tggatgtggt cctggagcac ggcggatgtg tgaccacaat ggctaagaac 720

aagccaaccc tggacattga actgcagaag accgaggcaa cacagctggc cacactgagg 780

aaactgtgca tcgaagggaa gattactaac atcactaccg attcccgctg tccaacccag 840

ggagaggctg tgctgcccga ggaacaggac cagaactacg tctgcaagca tacatatgtg 900

gatagagggt ggggaaatgg ctgtgggctg ttcggaaaag gctctctggt gacctgcgcc 960

aagtttcagt gtctggaacc catcgaggga aaagtggtcc agtacgagaa cctgaagtat 1020

acagtcatca ttactgtgca caccggcgac cagcatcagg tcggaaatga aacccagggc 1080

gtgacagccg agattactcc ccaggcctcc accgtggaag ctatcctgcc tgagtatggc 1140

acactggggc tggaatgctc tccccgaact ggcctggact tcaacgagat gatcctgctg 1200

acaatgaaga acaaggcttg gatggtgcac cgccagtggt tctttgatct gccactgccc 1260

tggacttccg gcgcaacaac tgaaacacct acttggaacc ggaaagagct gctggtgacc 1320

tttaagaatg cacacgccaa gaaacaggaa gtggtcgtgc tgggatctca ggagggcgct 1380

atgcatacag cactgactgg cgccaccgaa attcagaact caggaggcac cagcatcttc 1440

gctgggcacc tgaaatgcag actgaagatg gacaaactgg agctgaaggg aatgtcttac 1500

gccatgtgta ccaatacatt tgtcctgaag aaagaagtga gtgagaccca gcacgggaca 1560

atcctgatta aggtggaata taaaggagag gacgcccctt gtaaaatccc attcagtacc 1620

gaggatgggc agggaaaggc acataacggg aggctgatta cagccaatcc tgtcgtgact 1680

aagaaagagg aaccagtgaa catcgaagca gagccccctt ttggcgaaag caatatcgtg 1740

attggcatcg gggataaggc cctgaaaatt aactggtaca agaaagggag ctccatcgga 1800

aaaatgttcg aggctacagc acgcggcgct aggcgaatgg caattctggg cgacactgcc 1860

tgggattttg ggagcgtcgg gggagtgctg aattccctgg gaaagatggt gcaccagatc 1920

ttcggcagcg cttataccgc actgttttct ggcgtcagtt ggattatgaa aattggaatc 1980

ggcgtgctgc tgacctggat cgggctgaac tccaagaata catctatgtc cttttcatgt 2040

attgctattg gaattattac tctgtatctg ggagccgtgg tgcaggcctg ataa 2094

<210> 6

<211> 696

<212> PRT

<213>artificial sequence

<220>

<223>Dengue 3-prME protein sequence

<400> 6

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Asn Lys Arg Lys Lys Thr Ser Leu Cys Leu Met Met Met Leu

20 25 30

Pro Ala Thr Leu Ala Phe His Leu Thr Ser Arg Asp Gly Glu Pro Arg

35 40 45

Met Ile Val Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr

50 55 60

Ala Ser Gly Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu

65 70 75 80

Met Cys Asp Asp Thr Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val

85 90 95

Glu Pro Glu Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val

100 105 110

Thr Tyr Gly Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg

115 120 125

Ser Val Ala Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr

130 135 140

Gln Thr Trp Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val

145 150 155 160

Glu Thr Trp Ala Leu Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe

165 170 175

Leu Ala His Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe

180 185 190

Ile Leu Leu Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly

195 200 205

Val Gly Asn Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val

210 215 220

Asp Val Val Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn

225 230 235 240

Lys Pro Thr Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu

245 250 255

Ala Thr Leu Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr

260 265 270

Thr Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala Val Leu Pro Glu Glu

275 280 285

Gln Asp Gln Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp

290 295 300

Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala

305 310 315 320

Lys Phe Gln Cys Leu Glu Pro Ile Glu Gly Lys Val Val Gln Tyr Glu

325 330 335

Asn Leu Lys Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His

340 345 350

Gln Val Gly Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln

355 360 365

Ala Ser Thr Val Glu Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu

370 375 380

Glu Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Leu Leu

385 390 395 400

Thr Met Lys Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp

405 410 415

Leu Pro Leu Pro Trp Thr Ser Gly Ala Thr Thr Glu Thr Pro Thr Trp

420 425 430

Asn Arg Lys Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys

435 440 445

Gln Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala

450 455 460

Leu Thr Gly Ala Thr Glu Ile Gln Asn Ser Gly Gly Thr Ser Ile Phe

465 470 475 480

Ala Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys

485 490 495

Gly Met Ser Tyr Ala Met Cys Thr Asn Thr Phe Val Leu Lys Lys Glu

500 505 510

Val Ser Glu Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys

515 520 525

Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln

530 535 540

Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr

545 550 555 560

Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu

565 570 575

Ser Asn Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp

580 585 590

Tyr Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg

595 600 605

Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly

610 615 620

Ser Val Gly Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile

625 630 635 640

Phe Gly Ser Ala Tyr Thr Ala Leu Phe Ser Gly Val Ser Trp Ile Met

645 650 655

Lys Ile Gly Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys

660 665 670

Asn Thr Ser Met Ser Phe Ser Cys Ile Ala Ile Gly Ile Ile Thr Leu

675 680 685

Tyr Leu Gly Ala Val Val Gln Ala

690 695

<210> 7

<211> 2100

<212> DNA

<213>artificial sequence

<220>

<223>Dengue 4-prME DNA sequence dna

<400> 7

atggactgga cttggattct gttcctggtc gccgccgcaa ctagggtgca ttcaaacggg 60

agaaaaaggt caactattac tctgctgtgc ctgattccca ccgtcatggc attccacctg 120

agcacaagag acggggagcc actgatgatc gtggccaaac atgaacgggg gagacccctg 180

ctgtttaaaa ccacagaggg aattaacaag tgtacactga tcgccatgga cctgggcgag 240

atgtgcgaag ataccgtcac atacaagtgt cctctgctgg tgaacaccga gccagaagac 300

attgattgct ggtgtaatct gacttccacc tgggtcatgt atggaacatg cactcagtct 360

ggcgagcgga gaagggaaaa acgatccgtg gctctgaccc ctcactctgg gatgggactg 420

gagacccggg cagaaacatg gatgagctcc gagggcgcct ggaagcatgc tcagagagtg 480

gaatcctgga ttctgaggaa ccctgggttc gctctgctgg caggcttcat ggcatacatg 540

attggccaga ctggcatcca gcgcaccgtc ttctttgtgc tgatgatgct ggtggcccca 600

agttatggaa tgcgctgcgt cggcgtgggg aatcgagact tcgtcgaggg cgtgtcaggc 660

ggggcttggg tcgatctggt gctggaacac ggaggctgtg tgactaccat ggcacagggc 720

aagcctactc tggactttga gctgaccaaa acaactgcaa aggaagtggc cctgctgcgc 780

acctactgca ttgaggcctc catttctaac atcaccacag ctactcggtg tccaacccag 840

ggagaaccct acctgaaaga ggaacaggat cagcagtata tctgccgacg agacgtggtc 900

gatcgaggat ggggcaatgg gtgtggactg ttcggcaagg gcggcgtggt cacttgcgcc 960

aagttcagct gttcaggaaa gattaccggc aacctggtgc agatcgagaa tctggaatac 1020

acagtggtcg tgactgtcca caatggcgac acacatgcag tggggaacga tacttctaat 1080

cacggcgtga ccgccacaat cactcctaga agcccatccg tcgaggtgaa gctgcccgac 1140

tatggcgagc tgacactgga ttgcgaacct aggagtggga ttgacttcaa cgagatgatc 1200

ctgatgaaaa tgaagaaaaa gacctggctg gtgcataagc agtggtttct ggacctgcca 1260

ctgccatgga cagcaggagc tgatactagc gaggtgcact ggaattataa ggaaaggatg 1320

gtcacattca aagtgccaca tgccaagcgc caggatgtca ctgtgctggg gagtcaggag 1380

ggagctatgc actcagcact ggcaggagct accgaagtgg acagcggcga tgggaaccac 1440

atgttcgccg gacatctgaa atgcaaggtg cgaatggaga aactgcggat taagggcatg 1500

tcctacacta tgtgttctgg caagttcagc atcgacaagg agatggccga aacccagcac 1560

ggcactaccg tcgtgaaagt gaagtatgag ggagcaggcg ccccctgtaa ggtccctatc 1620

gagattcggg atgtgaacaa ggaaaaggtc gtgggcagaa tcatttctag tacccctctg 1680

gctgagaaca ccaattctgt gacaaacatc gagctggaac cccctttcgg ggactcttac 1740

atcgtcattg gggtgggaaa tagtgccctg acactgcact ggttccggaa aggctcaagc 1800

attgggaaga tgtttgagag cacttatagg ggcgctaaac gcatggcaat cctgggagaa 1860

accgcatggg atttcggcag cgtgggcggg ctgtttacat ccctgggaaa ggctgtccat 1920

caggtgttcg gctcagtcta cacaactatg tttggaggcg tgagctggat gatcagaatt 1980

ctgatcgggt ttctggtgct gtggatcgga accaactcaa ggaatacaag catggctatg 2040

acttgtattg ccgtgggcgg aattacactg tttctgggat tcactgtgca ggcttgataa 2100

<210> 8

<211> 698

<212> PRT

<213>artificial sequence

<220>

<223>Dengue 4-prME protein sequence

<400> 8

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Asn Gly Arg Lys Arg Ser Thr Ile Thr Leu Leu Cys Leu Ile

20 25 30

Pro Thr Val Met Ala Phe His Leu Ser Thr Arg Asp Gly Glu Pro Leu

35 40 45

Met Ile Val Ala Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr

50 55 60

Thr Glu Gly Ile Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu

65 70 75 80

Met Cys Glu Asp Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr

85 90 95

Glu Pro Glu Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val

100 105 110

Met Tyr Gly Thr Cys Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg

115 120 125

Ser Val Ala Leu Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala

130 135 140

Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val

145 150 155 160

Glu Ser Trp Ile Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe

165 170 175

Met Ala Tyr Met Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe

180 185 190

Val Leu Met Met Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly

195 200 205

Val Gly Asn Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val

210 215 220

Asp Leu Val Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Gln Gly

225 230 235 240

Lys Pro Thr Leu Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val

245 250 255

Ala Leu Leu Arg Thr Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr

260 265 270

Thr Ala Thr Arg Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu

275 280 285

Gln Asp Gln Gln Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp

290 295 300

Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala

305 310 315 320

Lys Phe Ser Cys Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu

325 330 335

Asn Leu Glu Tyr Thr Val Val Val Thr Val His Asn Gly Asp Thr His

340 345 350

Ala Val Gly Asn Asp Thr Ser Asn His Gly Val Thr Ala Thr Ile Thr

355 360 365

Pro Arg Ser Pro Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu

370 375 380

Thr Leu Asp Cys Glu Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile

385 390 395 400

Leu Met Lys Met Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe

405 410 415

Leu Asp Leu Pro Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val

420 425 430

His Trp Asn Tyr Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala

435 440 445

Lys Arg Gln Asp Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His

450 455 460

Ser Ala Leu Ala Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His

465 470 475 480

Met Phe Ala Gly His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg

485 490 495

Ile Lys Gly Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp

500 505 510

Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys

515 520 525

Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp

530 535 540

Val Asn Lys Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu

545 550 555 560

Ala Glu Asn Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe

565 570 575

Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu

580 585 590

His Trp Phe Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr

595 600 605

Tyr Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp

610 615 620

Phe Gly Ser Val Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His

625 630 635 640

Gln Val Phe Gly Ser Val Tyr Thr Thr Met Phe Gly Gly Val Ser Trp

645 650 655

Met Ile Arg Ile Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn

660 665 670

Ser Arg Asn Thr Ser Met Ala Met Thr Cys Ile Ala Val Gly Gly Ile

675 680 685

Thr Leu Phe Leu Gly Phe Thr Val Gln Ala

690 695

<210> 9

<211> 1515

<212> DNA

<213>artificial sequence

<220>

<223>general Dengue antigen dna

<400> 9

aagggcacca gctacgtgat gtgcaccggc agcttcaagc tggaaaaaga ggtggccgag 60

actcagcacg gcactgtgct cgtccaggtc aagtacgagg gcaccgacgc cccctgcaag 120

atccccttca gcacccaaga cgagaagggc gtgacacaga acggccggct gatcaccgcc 180

aaccccatcg tgaccgacaa agaaaagccc gtgaacatcg agaccgagcc ccccttcggc 240

gagagctaca tcgtggtggg agccggcgag aaggccctca agctgagttg gttcaaaaag 300

ggcagcagca tcggcaagat gttcgaggcc accgccaggg gcgctcgcag gatggctatt 360

ctccggggca ggaagcggcg gagcaagggc atgtcctaca gcatgtgtac tggcaagttc 420

aaggtcgtca aagagatcgc cgaaacacaa cacgggacca tcgtgatccg ggtgcagtat 480

gagggcgacg gcagcccttg taagatccct ttcgagatca tggacctgga aaagcggcac 540

gtgctgggcc gcctgatcac agtgaatcct atcgtgacag agaaggacag ccctgtgaat 600

attgaggcag agccaccatt tggcgactcc tacatcatca tcggcgtgga gcccggccag 660

ctgaagctga attggtttaa gaaggggtcc tccattgggc agatgtttga gactactatg 720

agaggcgcca agagaatggc tattctcaga ggccggaaga gaaggtccaa gggcatgagt 780

tacgcaatgt gtctgaacac cttcgtgctg aagaaagaag tgagcgagac acagcatggc 840

acaatcctga ttaaggtgga gtacaagggc gaggatgccc cttgcaagat tccattctcc 900

accgaggacg gccagggcaa ggctcacaac ggcagactga ttacagccaa ccctgtggtg 960

accaagaaag aggaaccagt caatatcgaa gccgaaccac cattcggcga gtccaacatt 1020

gtgatcggca ttggcgataa agccctgaaa atcaactggt ataagaaggg ctcaagcata 1080

gggaaaatgt ttgaggcaac tgcccgcgga gcaagaagaa tggctatctt gcgtgggaga 1140

aagcgccggt caaagggcat gtcttacact atgtgctctg gaaagttcag catcgacaaa 1200

gagatggctg aaacccagca tggaaccacc gtggtgaagg tgaaatatga aggcgctggg 1260

gctccctgta aggtgcccat cgagatcagg gacgtgaaca aagaaaaagt ggtgggccgg 1320

atcatcagca gcaccccttt cgccgagaac accaacagcg tgaccaacat cgagctggaa 1380

ccccctttcg gcgattctta tatcgtgatt ggcgtgggcg actccgccct gaccctgcac 1440

tggttccgga agggctcctc tataggaaag atgtttgaaa gcacctaccg gggagccaaa 1500

cgcatggcca tcctg 1515

<210> 10

<211> 505

<212> PRT

<213>artificial sequence

<220>

<223>general Dengue antigen

<400> 10

Lys Gly Thr Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys

1 5 10 15

Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr

20 25 30

Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu

35 40 45

Lys Gly Val Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val

50 55 60

Thr Asp Lys Glu Lys Pro Val Asn Ile Glu Thr Glu Pro Pro Phe Gly

65 70 75 80

Glu Ser Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser

85 90 95

Trp Phe Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala

100 105 110

Arg Gly Ala Arg Arg Met Ala Ile Leu Arg Gly Arg Lys Arg Arg Ser

115 120 125

Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys

130 135 140

Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr

145 150 155 160

Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu

165 170 175

Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val

180 185 190

Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly

195 200 205

Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn

210 215 220

Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met

225 230 235 240

Arg Gly Ala Lys Arg Met Ala Ile Leu Arg Gly Arg Lys Arg Arg Ser

245 250 255

Lys Gly Met Ser Tyr Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys

260 265 270

Glu Val Ser Glu Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr

275 280 285

Lys Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly

290 295 300

Gln Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val

305 310 315 320

Thr Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly

325 330 335

Glu Ser Asn Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn

340 345 350

Trp Tyr Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala

355 360 365

Arg Gly Ala Arg Arg Met Ala Ile Leu Arg Gly Arg Lys Arg Arg Ser

370 375 380

Lys Gly Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys

385 390 395 400

Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr

405 410 415

Glu Gly Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val

420 425 430

Asn Lys Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Phe Ala

435 440 445

Glu Asn Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly

450 455 460

Asp Ser Tyr Ile Val Ile Gly Val Gly Asp Ser Ala Leu Thr Leu His

465 470 475 480

Trp Phe Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr

485 490 495

Arg Gly Ala Lys Arg Met Ala Ile Leu

500 505

Claims (10)

1. one kind can express the core for causing the polypeptide of the immune response for more than one subtypes of dengue virus of mammal Acid con-struct, the nucleic acid construct includes:

The coding nucleotide sequence of the polypeptide is expressed, wherein the polypeptide includes sub- from least two different dengue virus The PRM/E structural domain of type,

Regulate and control the expression of the polypeptide in the mammal and is operably connected with the coding nucleotide sequence Promoter.

2. nucleic acid construct as described in claim 1, also comprising being operably connected simultaneously with the N-terminal end of the coded sequence And the IgE leader sequence being operably connected with the promoter.

3. such as nucleic acid construct of any of claims 1-2, also comprising being connected with the C-terminal end of the coded sequence Polyadenylation sequence.

4. nucleic acid construct as claimed in any one of claims 1-3, wherein the nucleic acid construct is codon optimization.

5. such as nucleic acid construct of any of claims 1-4, wherein coding nucleotide sequence coding includes coming From dengue virus-hypotype 1, dengue virus-hypotype 2, dengue virus-hypotype 3 or dengue virus-hypotype 4 or their combination The polypeptide of DIII structural domain.

6. nucleic acid construct according to any one of claims 1 to 5, wherein the coding nucleotide sequence is selected from by following The group of composition: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

7. one kind can generate the DNA plasmid vaccine for the immune response of a variety of subtypes of dengue virus, institute in mammals Stating vaccine includes:

The amount expression that the immune response of the mammal can effectively be caused in the cell of the mammal, which shares, steps on The DNA plasmid of antigen is removed from office, the shared Dengue antigen includes dengue virus-hypotype 1, dengue virus-hypotype 2, dengue virus-Asia The shared DIII structural domain of type 3 or dengue virus-hypotype 4 or their combination, and

Pharmaceutically acceptable excipient；

The DNA plasmid includes the promoter being operably connected with the coded sequence for encoding the shared Dengue antigen.

8. DNA plasmid vaccine as claimed in claim 7, wherein the DNA plasmid also includes and the N-terminal of coded sequence end The IgE leader sequence that end is connected and is operably connected with the promoter.

9. the DNA plasmid vaccine as described in any one of claim 7-8, wherein the DNA plasmid also includes and the coding The connected polyadenylation sequence in the C-terminal end of sequence.

10. DNA plasmid vaccine as claimed in any one of claims 7-9, wherein the DNA plasmid is codon optimization.