WO2018076001A1

WO2018076001A1 - Synthetic polypeptide, vaccine composition comprising the same, and uses thereof

Info

Publication number: WO2018076001A1
Application number: PCT/US2017/057796
Authority: WO
Inventors: Trai-Ming Yeh; Yen-chung LAI
Original assignee: National Cheng Kung University; Dcb-Usa Llc
Priority date: 2016-10-21
Filing date: 2017-10-23
Publication date: 2018-04-26
Also published as: TW201828980A; TWI653049B

Abstract

Disclosed herein is a polypeptide comprising the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. Also disclosed is a vaccine composition that comprises the present polypeptide and an adjuvant. The vaccine composition is useful in preventing the DENV infection and/or alleviating the DENV-associated illness in the subject.

Description

SYNTHETIC POLYPEPTIDE, VACCINE COMPOSITION COMPRISING THE

SAME, AND USES THEREOF

CROSS REFERENCES TO RELATED APPLICATIONS

[0001 ] This application relates to and claims the benefit of U.S. Provisional Application No. 62/411,493, filed October 21, 2016; the content of the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. FIELD OF THE INVENTION

[0003] The present disclosure in general relates to the field of preventing viral infection. More particularly, the present disclosure relates to a synthetic polypeptide and its applications in the prophylaxis and/or treatment of dengue virus (DENV) infection.

[0004] 2. DESCRIPTION OF RELATED ART

[0005] Dengue virus (DENV) is a mosquito-borne single positive-stranded RNA virus of the family Flaviviridae and genus Flavivirus. Dengue is an enveloped virus, 40-60 nm in size, having an isometric nucleocapsid of 25-30 nm and an about 10.7 kb, linear, positive-sense RNA genome. Dengue virus exists as five serotypes (Dengue 1-5, the fifth is reported in 2013) and is genetically related to other flaviviruses such as yellow fever and tick-borne encephalitis viruses. DENV infection may cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Although there are several vaccine candidates in clinical trials, the phenomenon, antibody-dependent effect (ADE), may induce more sever disease that limits the development of dengue vaccine. Recently, one dengue vaccine has been licensed which was developed by the company Sanofi Pasteur, while the efficacy against all serotypes of DENVs is still arguable. DENV nonstructural protein 1 (NS1) has been proposed to enhance vascular permeability and contribute to development of DHF. Owing to the benefit of absence of ADE in anti-NSl Abs, active immunization with NS1 protein was suggested to protect against dengue disease. However, several autoantibodies elicited from molecular mimicry of NS1 to host proteins hinders the NS1 -based vaccine development.

[0006] In view of the foregoing, there exists in the related art a need for a novel vaccine for efficiently protecting a subject against the infection caused by different serotypes of DENVs.

SUMMARY [0007] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0008] As embodied and broadly described herein, one aspect of the disclosure is directed to a synthetic polypeptide for preventing or treating a DENV infection in a subject. According to the embodiments of the present disclosure, the synthetic polypeptide comprises a first polypeptide, which has the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. According to one specific example, the first polypeptide has the amino acid sequence of SEQ ID NO: 1. Optionally, for stability purpose, the N-terminus of the synthetic polypeptide is acetylated and/or the C-terminus of the synthetic polypeptide is amidated. According to one specific embodiment, the synthetic polypeptide further comprises a second polypeptide that is disposed at the N- or C- terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of, ovalbumin (OVA), bovine serum albumin (BSA), keyhole limpet haemocyanin (KLH), β-galactosidase, thyroglobulin (TGB), heat shock protein (HSP) and a combination thereof.

[0009] The second aspect of the present disclosure pertains to a vaccine composition for preventing a DENV infection in a subject. According to the embodiments of the present disclosure, the vaccine composition comprises the present synthetic polypeptide and a pharmaceutically acceptable adjuvant. According to one embodiment, the pharmaceutically acceptable adjuvant is selected from the group consisting of, Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant (IFA), complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide (SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP). As mentioned above, the synthetic polypeptide of the present composition comprises a first polypeptide having the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1, and optionally, a second polypeptide disposed at the N- or C- terminus of the first polypeptide. Depending on desired purposes, the second polypeptide may be any of OVA, BSA, KLH, β-galactosidase, TGB, HSP or a combination thereof.

[0010] The third aspect of the present disclosure is directed to a method of preventing a subject from being infected by a DENV. The method comprises administering to the subject an effective amount of the present vaccine composition so as to vaccinate the subject against the DENY According to one embodiment, the vaccine composition may give rise to about 0.8 μg to 80 mg of the polypeptide per kilogram (Kg) of body weight per dose (0.8 μg to 80 mg/Kg/dose); preferably, 8 to 8 mg/Kg/dose; more preferably, 80 to 800 μg/Kg/dose. According to another embodiment, the vaccine composition is administered to the subject at least 2 times in the course of vaccination. Preferably, the vaccine composition is administered to the subject 3 to 5 times in the course of vaccination. The present vaccine composition can be administered by a route selected from the group consisting of transmucosal, subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal injection.

[001 1 ] According to some embodiments of the present disclosure, the present method is useful in protection the subject against the infection caused by DENV serotype 1, 2, 3 or 4.

[0012] According to the embodiments, the subject is a mammal. Preferably, the subject is a human.

[0013] Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:

[0015] Figure 1A is the data depicting the binding of the modified NS1 wing domain (NSl-WD) immune sera of mice to the surface of different serotypes of DENV-infected HuH-7 cells according to one embodiment of the present disclosure. DENV 1-4 or mock-infected HuH-7 cells were respectively stained the specified immune sera of mice (1 : 100 dilution) and analyzed by flow cytometry: KLH-immune sera (the sera isolated from the mice immunized with KLH control), NS1 -immune sera (the sera isolated from the mice immunized with full length NS1 protein), modified NSl-WD-immune sera (the sera isolated from the mice immunized with the modified NSl-WD polypeptide (SEQ ID NO: 1)), and 2° Ab (stained with secondary antibody only, serving as the negative control in the experiment).

[0016] Figure IB is the line chart depicting the binding affinity of purified pAbs from modified NSl-WD polypeptide-immunized mouse sera to NS1 polypeptide, modified NSl-WD polypeptide-conjugated BSA or BSA according to one embodiment of the present disclosure.

[0017] Figure 2A is the histogram depicting the binding affinity of different doses of purified pAbs from KLH control, wildtype NSl-WD polypeptide (SEQ ID NO: 2), modified NSl-WD polypeptide immune sera to HUVECs according to another embodiment of the present disclosure. The shift ratio (%) represents the percentage of binding compared with the control group (Anti-mouse IgG Alexa 488 only). All data are presented as the mean ± S.D. from at least three independent experiments. *P < 0.05, **P < 0.01.

[0018] Figure 2B is the histogram depicting the binding affinity of different doses of purified pAbs from KLH control, Full length NS1, modified NSl-WD polypeptide immune sera to human platelets according to another embodiment of the present disclosure.

[0019] Figure 3 are histograms respectively depicting antibodies against modified NSl-WD polypeptide caused cytotoxicity only in DENV-infected cells by complement-dependent cytolysis manner according to one embodiment of the present disclosure. HUVECs were infected with specified DENVs (serotypes 1-4 for panels A-D, respectively; multiplicity of infection = 10) or mock infection for 48 hours, and incubated with either phosphate-buffered saline (PBS), purified pAbs from KLH or modified NSl-WD-peptide immune sera (50 μg/ml) for 1 hour at 4°C. The cells were then incubated with or without complement (1 :20) for 4 hours at 37°C. The release of lactate dehydrogenase (LDH) was analyzed as described in the Methods. Anti-NSl Ab 2E8 served as a positive control. **P < 0.01, ***P < 0.001.

[0020] Figures 4A and 4B are histograms respectively depicting antibodies against modified NSl-WD polypeptide inhibit viral propagation by complement-dependent cytolysis of DENV-infected cells and directly reduce viral replication by complement-independent manner respectively according to one embodiment of the present disclosure. HUVECs were infected with DENVs (serotypes 1-4, multiplicity of infection = 10) or mock-infected for 48 hours. After 48 hours of infection, cells were incubated with either PBS, purified pAbs from KLH or modified NSl-WD polypeptide-immunized mouse sera (50 μg/ml) or anti-NSl mAb 2E8 (50 μg/ml) for 1 hour at 4°C and then incubated with (Figure 4A) or without (Figure 4B) complement (C) (1 :20) for 4 hours at 37°C. After refilling with fresh medium for another 24 hours, the infectious virus in supernatants was titrated by fluorescent focus assay (FFA) as described in the Materials and Methods. All data are presented as the mean ± S.D. from at least three independent experiments. *P < 0.05, **P < 0.01, ***p < 0.001, ns indicates no significance.

[0021 ] Figures 5A-5D are dot plots respectively depicting the active immunization of modified NSl-WD polypeptide protects mice against DENV 1, DENV 2, DENV 3 or DENV 4-elicited prolonged bleeding time according to another embodiment of the present disclosure. *P < 0.05, ***P < 0.001.

[0022] Figures 6A-6B are photographs and histograms respectively depicting the active immunization of modified NSl-WD polypeptide protects mice against specified DENV-elicited hemorrhage according to another embodiment of the present disclosure. Figure 6A: representative images of the skins of mice respectively treated with specified treatments. Figure 6B: the quantitative results of hemorrhage of mice challenged with DENV 1 (panel A), DENV 2 (panel B), DENV 3 (panel C) or DENV 4 (panel D). The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity by ImageJ. Arrows indicate hemorrhage in the skin lesions. *P < 0.05, **P < 0.01, ***P < 0.001.

[0023] Figure 7 is the data depicting the active immunization of modified NSl-WD polypeptide protects mice against DENV 2-elicited hemorrhage according to another embodiment of the present disclosure. Hematoxylin and eosin (H&E) staining was performed to analyze local skin hemorrhage in the skin lesions. The arrows indicate the intact blood vessels. The scale bar indicated 40 μπι.

[0024] Figures 8A and 8B are photographs and histograms respectively depicting active immunization of modified NSl-WD polypeptide reduces NS3 expression in mice according to still another embodiment of the present disclosure. DENV NS3 expression was detected by IHC staining. Arrows indicate local DENV NS3 expression. The scale bar indicates 40 μπι. ***P < 0.001, ns indicates no significance.

[0025] Figures 9A-9C are dot plots and line chart respectively depicting the protective effect protection of the antibody produced by the modified NSl-WD polypeptide on DENV infection in STATl^_/~ mice according to one embodiment of the present disclosure. Figure 9A: viremia in STATl^_/~ mice 2 days post-infection. Figure 9B: NS1 expression in the sera of STATl^_/~ mice 2 days post-treatment. Figure 9C: survival rate of mice administered with specified treatment. **P < 0.01, ***P < 0.001.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0027] 1. Definition

[0028] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms "a" and "an" include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0029] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0030] The term "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer; thus, "peptides," "oligopeptides", and "proteins" are included within the definition of polypeptide and used interchangeably herein. This term also does not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. Throughout the present disclosure, the positions of any specified amino acid residues within a polypeptide are numbered starting from the N terminus of the polypeptide. When amino acids are not designated as either D-or L-amino acids, the amino acid is either an L-amino acid or could be either a D- or L- amino acid, unless the context requires a particular isomer. Further, the notation used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art.

[0031 ] As used herein, the term "synthetic polypeptide" refers to a polypeptide which does not comprise an entire naturally occurring protein molecule. The polypeptide is "synthetic" in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.

[0032] As used herein, "vaccine" refers to a composition which when inoculated into an animal has the effect of stimulating an immune response (e.g., antigen-specific antibody) in the animal, which serves to fully or partially protect the animal against a disease (e.g., DENV infection) or its symptoms. The term vaccine encompasses prophylactic as well as therapeutic vaccines. A combination vaccine is one which combines two or more vaccines.

[0033] As used herein, the term "adjuvant", unless indicated otherwise, refers to any substance or mixture of substances that enhances, increases, upwardly modulates, diversifies or otherwise facilitates the immune response (e.g., humoral or cellular immune response) to an antigen.

[0034] The term "antigen" or "antigenic agent", unless indicated otherwise, refers to any agent that, when introduced into an immunocompetent human or animal, stimulates a humoral and/or cellular immune response. The antigen may be a pure substance, a mixture of substances, or particulate material (including cells, cell fragments, or cell derived fragments) or a live, usually attenuated, organism or virus. Examples of suitable antigens include, but are not limited to, a protein, glycoprotein, lipoprotein, polypeptide, peptide, carbohydrate/polysaccharide, lipopolysaccharide, toxin, virus, bacterium, fungus, and parasite.

[0035] The term "antibody" as used herein refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies (pAb), monoclonal antibodies (mAb), Fv, Fab and F(ab)₂, as well as single chain antibodies and humanized antibodies.

[0036] As used herein, the term "treat", "treating" and "treatment" are interchangeable, and encompasses partially or completely ameliorating, mitigating and/or managing a symptom, a secondary disorder or a condition associated with DENV infection, in which inducing a DNEV-specific immune response (e.g., DENV-specific antibody) provides a benefit to the subject having or suspected of having such symptom, disorder or condition. The term "treating", "treating" or "treatment" as used herein refers to application or administration of the present polypeptide or vaccine composition to a subject, who has a symptom, a secondary disorder or a condition associated with DENV infection, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms, secondary disorders or features associated with DENV infection. Symptoms, secondary disorders, and/or conditions associated with DENV infection include, but are not limited to, fever, severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands, rash, severe abdominal pain, rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit. Treatment may be administered to a subject who exhibits only early signs of such symptoms, disorder, and/or condition for the purpose of decreasing the risk of developing the symptoms, secondary disorders, and/or conditions associated with DENV infection. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is "effective" if the progression of a symptom, disorder or condition is reduced or halted.

[0037] The term "prevent", "preventing" and "prophylaxis" as used herein are interchangeable, and refers to the prophylactic treatment of a subject who is at risk of developing a symptom, a secondary disorder or a condition associated with DENV infection, so as to decrease the probability that the subject will develop the symptom, secondary disorder or condition. Specifically, the term "prevent", "preventing" or "prophylaxis" refers to inhibit the occurrence of a symptom, a secondary disorder or a condition associated with DENV infection, that is, to reduce the inc

[0038]

[0039] idence or the frequency of occurrence of the symptom, secondary disorder or condition. The term "prevent", "preventing" or "prophylaxis" as used herein referring to a polypeptide, a composition comprising the same and/or a method, does not mean or imply that use of the polypeptide, the composition comprising the same and/or the method will provide a guarantee that the symptom, secondary disorder or condition will never occur, but rather that the polypeptide, the composition comprising the same and/or the method will inhibit the occurrence of the symptom, secondary disorder or condition, and that the incidence and/or frequency of the symptom, secondary disorder or condition will be reduced.

[0040] The term "effective amount" as referred to herein designate the quantity of a component which is sufficient to yield a desired response. For therapeutic purposes, the effective amount is also one in which any toxic or detrimental effects of the component are outweighed by the therapeutically beneficial effects. The specific effective or sufficient amount will vary with such factors as the particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, in grams, milligrams or micrograms or as milligrams per kilogram of body weight (mg/Kg). Alternatively, the effective amount can be expressed in the concentration of the active component (e.g., the synthetic polypeptide of the present disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction and mixing ratio. Specifically, the term "therapeutically effective amount" used in connection with the synthetic polypeptide described herein refers to the quantity of the synthetic polypeptide, which is sufficient to alleviate or ameliorate the symptoms associated with the cancer in the subject. Persons having ordinary skills could calculate the human equivalent dose (HED) for the medicament (such as the present synthetic polypeptide) based on the doses determined from animal models. For example, one may follow the guidance for industry published by US Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" in estimating a maximum safe dosage for use in human subjects.

[0041 ] The term "subject" refers to a mammal including the human species that is treatable with methods of the present invention. The term "subject" is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0042] 2. Description of the embodiments

[0043] The first aspect of the present disclosure is directed to a polypeptide for preventing or treating DENV infection in a subject, in which the polypeptide comprises an amino acid sequence at least 85% identical to the sequence of "YKDWSEWGKAC" (SEQ ID NO: 1); that is, the amino acid sequence comprised in the present polypeptide may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1. According to one embodiment, the present polypeptide has the amino acid sequence at least 85% identical to SEQ ID NO: 1. According to one working example, the present polypeptide has the amino acid sequence of SEQ ID NO: 1.

[0044] Optionally, the N-terminus of the present polypeptide is acetylated. Additionally or alternatively, the C-terminus of the present polypeptide is amidated.

[0045] Still optionally, the N- or C-terminus of the present polypeptide is conjugated with a carrier molecule so as to increase the immunogenicity of the polypeptide. Exemplary carrier molecules include, but are not limited to, OVA, BSA, KLH, β-galactosidase, TGB, HSP or a combination thereof. According to one working embodiment of the present disclosure, the carrier molecule is KLH.

[0046] The synthetic polypeptides of the invention can be synthesized by commonly used methods such as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the polypeptide. Polypeptides of the invention can also be synthesized by the well-known solid phase peptide synthesis methods. Alternatively, the synthetic polypeptides of the invention can be produced by host cells (e.g., FIEK293 cells), which is transfected with a nucleic acid encoding the polypeptide.

[0047] According to one embodiment of the present disclosure, the present polypeptide possesses an immunogenic function to stimulate an immune response (i.e., the production of antibody) against four serotypes (serotype 1-4) of DENV (hereinafter respectively designated as DENV 1, DENV 2, DENV 3, and DENV 4). Thus, the second aspect of the present disclosure pertains to a vaccine composition that comprises the polypeptide according to any of the above-mentioned aspect and embodiments of the present disclosure, and a pharmaceutically acceptable adjuvant.

[0048] As known by a skilled artisan, the adjuvant is a substance that enhances the immune response against an antigen (e.g., the present polypeptide). Suitable examples of adjuvant for enhancing the present polypeptide include, but are not limited to, Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant (IFA), complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide (SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP). According to one embodiment, the adjuvant is CFA. According to another embodiment, the adjuvant is IFA.

[0049] To produce an antibody (e.g., a mAb or a pAb), a host animal, such as a mouse, a rat, or a rabbit, is immunized with the present polypeptide or the present vaccine composition. The immunization may be performed in accordance with commonly adopted procedures. The immunization interval is not particularly limited. Immunization may be carried out at intervals of several days to several weeks, preferably one week, for 2-10 times, until a desired antibody titer is reached.

[0050] According to certain embodiment, the host animals are vaccinated by subcutaneously or intradermally injecting with the present vaccine composition on weekly basis for 3-5 consecutive weeks. Blood samples are taken regularly after immunization and subject to centrifugation to separate sera. The resultant sera are then subject to measurement of antibody titers by any suitable method, which includes, but is not limited to, ELISA, enzyme immunoassay (EIA), or radio immunoassay (RIA). In one preferred example, antibody titers are measured by ELISA. Then, final immunization is given to those animals showing high antibody titers to the present polypeptide described above.

[0051 ] The blood is collected from the immunized animals, and then separated and purified by an ordinary method, including centrifugation, precipitation using ammonium sulfate or polyethylene glycol, chromatography (such as gel filtration chromatography, ion exchange chromatography, or affinity chromatography) etc., so as to obtain a pAb recognizing the present polypeptide in the form of a polyclonal antiserum.

[0052] According to the embodiments of the present disclosure, the present polypeptide or vaccine composition induces an immune response (e.g., the production of pAb) against DENY According to one embodiment of the present disclosure, the thus produced antibody exhibits binding affinity and specificity to the present polypeptide. According to another embodiment of the present disclosure, the thus produced antibody is capable of binding and/or neutralizing the DENVs (including DENV 1, DENV 2, DENV 3 and DENV 4).

[0053] The third aspect of the present disclosure is directed to a method for protecting a subject from being infected by a DENV. The present method comprises administering to the subject an effective amount of the present vaccine composition so as to vaccinate the subject against the DENV (i.e., stimulate an immune response against DENV in the subject).

[0054] In general, the effective amount of the active component (i.e., the present polypeptide) comprised in the vaccine composition may vary with various factors, such as the physical condition of the patient (e.g., the patient's body mass, age, or gender), the vaccinated subject, and the immunogenicity of the active component.

[0055] In one embodiment, the subject is a mouse. To elicit a vaccine effect on mice, the present polypeptide is administered to the subject in the amount of about 0.01 to 1,000 mg/Kg body weight per dose; for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 mg/Kg body weight per dose. Preferably, 0.1 to 100 mg/Kg body weight per dose. More preferably, 1 to 20 mg/ Kg body weight per dose. According to one working example, 2-2.5 mg/Kg of the present polypeptide per dose is sufficient to induce an immune response (e.g., the production of antibody) in the subject.

[0056] A skilled artisan could calculate the human equivalent dose (HED) of the present polypeptide, based on the doses determined from animal models. Accordingly, the effective HED of the present polypeptide is about 0.8 μg/Kg to 80 mg/Kg body weight per dose for human; in other words, the effective HED of the present polypeptide may be any of, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 μg/Kg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 mg/Kg body weight per dose for human. Preferably, 8 μg/Kg to 8 mg/Kg body weight per dose. More preferably, 80 to 800 μg/Kg body weight per dose. In one preferred example, the effective HED of the present polypeptide is about 160 to 200 μg/Kg per dose.

[0057] According to the embodiments of the present disclosure, the present polypeptide or vaccine composition is administered to the subject at least 2 times, such as, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times, in the course of vaccination. For the desired purpose, the vaccine composition may be administered to the subject for 2-10 times with an interval from several days to several years.

[0058] According to one example, the subject is a mouse, and the present vaccine composition is administered to the subject 4-5 times within one month so as to induce the polypeptide-specific immune response in the subject. According to the embodiment, the subject is primed with the vaccine composition comprising an effective amount of the present polypeptide and CFA adjuvant, and then boosted 3-4 times with the vaccine composition comprising an effective amount of the present polypeptide and IFA adjuvant.

[0059] The present vaccine composition can be administered to the subject by an appropriate route, such as transmucosal, subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal injection. According to one specific example, the present vaccine composition is administered to the subject via intraperitoneal injection. [0060] As would be appreciated, the present method can be applied to the subject, alone or in combination with additional therapies that have some beneficial effects on the treatment of DENV infection. Depending on the intended/therapeutic purpose, the present method can be applied to the subject before, during, or after the administration of the additional therapies.

[0061 ] According to one embodiment of the present disclosure, the present method is useful in protecting the subject against DENV (including DENV 1, DENV 2, DENV 3 and DENV 4) infection. In the embodiment, the DENV-associated illness/disorder, such as viremia, local hemorrhage, red blood cell (RBC) extravasation and prolonged bleeding time, is alleviated in the vaccinated subject. According to another embodiment, the antibody/polyclonal antiserum produced by the present method is capable of inducing a complement dependent cytolysis (CDC) response to DENV, and accordingly, decreasing the viral titer in the vaccinated subject. According to still another embodiment, the antibody/polyclonal antiserum produced by the present method dose not cross react to the endothelial cell and the platelet of the subject.

[0062] According to one working example of the present disclosure, the present method is useful in prolonging the survival of DENV-infected subject.

[0063] Basically, the subject treatable by the present method is a mammal, for example, a human, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a cat, a cow, a goat, a sheep, a monkey, and a horse. Preferably, the subject is a human.

[0064] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

EXAMPLE

[0065] Cell lines and viral stocks

[0066] Human hepatoma cell line Huh-7, Baby hamster kidney cell line BHK-21 and Aedes albopictus cell line C6/36, purchased from the Japanese Collection of Research Bioresources (Japan) and the American Type Culture Collection (ATCC, Manassas, Virginia), were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT). Human umbilical vein endothelial cells (HTJVECs) were purchased from the Bioresource Collection and Research Center (BCRC) of Taiwan and maintained in endothelial Basal Medium-2 (EMB-2, Lonza, Walkersville, MD) supplemented with 10% FBS, and SingleQuots™ Kit (Lonza, Walkersville, MD). All cells were cultured at 37°C in a 5% C0₂ atmosphere, except for C6/36 which were cultured at 28°C.

[0067] Four serotypes of dengue viruses, DENV 1 (local Taiwan strain 8700828), DENV 2 (16681 and local Taiwan strain 454009 A), DENV 3 (local Taiwan strain 8700829), and DENV 4 (local Taiwan strain 59201818) were propagated in C6/36 cells. To prepare high titers of DENV, cell-free supernatants were concentrated by Macrosep^® Advance Centrifugal Devices (molecular weight cutoff of 30 kDa; Pall Corp., Port Washington, NY) at 6000 g at 4°C and stored below -70 °C until use.

[0068] Virus titration and fluorescent focus assay (FFA)

[0069] For virus titration, the fluorescent focus assay was used to determine the virus titer. In brief, supernatants containing infectious virus were collected and stored below -70°C until use. Supernatant was serially diluted and incubated with BHK-21 cells for 2 hours at 37°C. The monolayers were then overlaid with DMEM containing 2% FBS and 1% methylcellulose and incubated at 37°C for 2-3 days. Virus foci were stained with anti-NSl antibody followed by Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) and visualized with a fluorescence microscope (Leica Geosystems AG, St. Gallen, Switzerland).

[0070] Enzyme-linked immunosorbent assay (ELISA)

[0071 ] 50 μΐ of NS1, bovine serum albumin (BSA), peptides-conjugated BSA or antibody (2 ^ηύ) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4°C overnight. After blocking for 1 hour with 1% BSA in PBS, mice or human sera (1 :50 dilution) were incubated on wells at 37°C for 1 hour. Next, horseradish peroxidase (HRP)-conjugated goat anti-rabbit, anti-mouse IgG (Leadgene Biomedical, Taiwan), anti-human IgG (Jackson ImmunoRe search Laboratories, West Grove, PA) or mouse anti-M13 (Zymed Laboratories, California, USA) secondary Abs (1 : 10,000 dilution) were incubated on wells at 37°C for another hour.

[0072] Subsequently, plates were washed with PBST, followed by color development and visualization using tetramethylbenzidine (TMB, Clinical Science Products, Mansfield, MA) as the substrate. The absorbance was read after adding stop solution (2 N H₂SO₄) at OD₄₅₀ nm by a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA).

[0073] Complement-mediated cytolysis and LDH release assay

[0074] To analyze complement-dependent cytolysis of infected cells, HUVECs (8 ^χ 10³) were infected with DENV (multiplicity of infection = 10) for 48 hours. Cells were washed with PBS and then incubated with 56°C heat-inactivated antibodies for 1 hour at 4°C. After washing with PBS, cells were incubated with Low-Tox-M rabbit complement (1 :20 dilution) (Cedarlane Laboratories Ltd, Ontario, Canada) containing 2% FBS EMB-2 medium for another 4 hours. Then, 50 μΐ supernatants were collected and mixed with 50 μΐ CytoTox 96® substrate Reagent (Promega, Madison, Wis.) in each well. After 30 minutes incubation in the dark, 50 μΐ of stop solution were added into each well. Finally, the absorbance was read at 490 nm by a VersaMax microplate reader.

[0075] Disease and lethal infection models in mice

[0076] C3H/HeN wildtype mice were used in a DENV-induced disease murine model. For active immunization, the KLH-conjugated modified NS1-WD polypeptide (SEQ ID NO: 1; 50 μg; ChinaPeptides, Shanghai, China) or the control protein (KLH) was emulsified with Complete or Incomplete Freund's Adjuvant (CFA, IF A). 6-week-old mice were intraperitoneally immunized with one dose of the CFA-emulsion; two weeks later, the immunized mice were boosted with 3 doses of IFA-emulsion with an interval of one week between each dose (each dose contained 50 μg of KLH-conjugated modified NS1-WD polypeptide or KLH control protein). Three days following the last immunization, the mice were challenged intradermally with concentrated DENV (2* 10⁸ PFU/mouse) or concentrated C6/36 medium as a control at four sites on the upper back.

[0077] 5- to 6-week-old STAT] ^~'^~ mice were used in a lethal infection murine model. A DENV 2 lethal strain (454009 A) (4 ^χ 10⁷ PFU/mouse) or C6/36 control medium were inoculated intravenously (i.v.) into STAT] ^~ mice. Sera isolated from the immunized mice were injected intraperitoneally one day before the DENV challenge. Two days later, the sera were collected from the DENV-challenged mice to determine the viremia and NS1 secretion by FFA and quantitative NS1 ELISA. The body weight and survival rates of mice were monitored for 14 days.

[0078] Hematoxylin and Eosin staining (H&E stain) and Immunohistochemistry (IHC)

[0079] Fresh mice skins were fixed with 4% formalin and embedded with paraffin. For histopathology analysis, tissue sections were stained with H&E. For immunohistochemistry staining, the slides were blocked following the manufacturer's instructions (BIOTnA biotech) and incubated with anti-NS3 Ab (GTX124252, GeneTex, Inc, Irvine, CA) at 4°C overnight. After washing with PBS, the slides were incubated with HRP -labeled secondary antibody for 30 minutes at room temperature and then loaded onto 3,3'-diaminobenzidine (DAB) or HRP green mixed reagent for 1-5 minutes. Hematoxylin counterstain was applied for 2 minutes. The quantification of DAB staining was determined by ImageJ software.

[0080] Skin hemorrhage quantification

[0081 ] The degree of murine skin hemorrhage was image-processed by Photoshop^® and digitally quantified by ImageJ software. In brief, the samples of mice skin were collected and adjusted to the same image sizes. Then, the hemorrhagic areas were isolated and created as new images in Photoshop 6.0 (Adobe, San Jose, CA). To quantify the hemorrhagic portion, the processed images were loaded into ImageJ software, and converted into 16-bit images. After image type was set to black and white, the total hemorrhage volume was calculated and analyzed by Prism software.

[0082] Statistical analysis

[0083] All statistical analyses were performed using Prism software (GraphPad Software Inc., CA). The results were analyzed using the unpaired Student's t-test or one-way ANOVA test to compare two independent groups or more than two comparisons, respectively. For each result, all data are presented as the means ± S.D. from three independent experiments. The survival rate was analyzed by Log-rank Mantel-Cox test. *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significance for 95% two-tail confidence intervals.

[0084] Example 1 Protective effect of modified NS1-WD polypeptide against DENV infection in vitro

[0085] For the purpose of evaluating the bioactivity of the present polypeptide (e.g., the modified NSl -WD polypeptide), the sera isolated from mice respectively immunized with specified antigens (including full length NSl protein, the KLH-conjugated modified NSl -WD polypeptide, and the KLH control protein) were incubated with DENV-infected cells, DENV NSl protein, HUVEC cells and human platelets followed by the analysis of flow cytometry and ELISA assay. The data were respectively depicted in Figs. 1-4.

[0086] 1.1 Modified NS1-WD polypeptide induced DENV-specific immune response

[0087] Owing to the highly conserved homology between DENV NSl -WD and LYRIC protein (SEQ ID NO: 3) on HUVEC cells, a critical pathogenic amino acid, Lysine (K), in KXWG motif was identified. After modification of the amino acid on NSl -WD, the data of flow cytometry and ELISA respectively depicted that the modified NSl -WD induced antibodies exhibiting binding affinity to four-serotype DENV infected cells (Fig. 1A) and DENV NSl protein (Fig. IB).

[0088] It is known that several reported anti-NSl antibodies exhibited molecular mimicry with host proteins, and accordingly, cross-reacting with platelets, endothelial cells and coagulation factors, causing platelet dysfunction, endothelial cell damage and hemorrhage. The binding affinity of the modified NSl -WD induced antibodies to the endothelial cells and platelets was also examined in the present disclosure. The data of Figs. 2A and 2B respectively demonstrated that the antibodies induced by the wildtype NSl -WD polypeptide (Fig. 2 A) and full length NSl protein (Fig. 2B) respectively exhibited binding affinity to the endothelial cells and platelets, while the antibody induced by the modified NSl-WD (hereinafter designated as "modified NSl-WD pAb") would not cross react to human endothelial cells and platelets.

[0089] 1.2 Modified NSl-WD pAb exhibited inhibitory effect on four serotype DENVs

[0090] The cell-protective effect of the modified NSl-WD pAb was determined by LDH release assay. The data of Fig. 3 indicated that the modified NSl-WD pAb activated complement that induced significant LDH release in the DENV-infected HUVECs, but not the uninfected HUVECs, in which the anti-NSl 2E8 monoclonal antibody (mAb) and the KLH polyclonal antibody (hereinafter designated as "KLH pAb") respectively served as the positive and negative control antibodies.

[0091 ] The viral titers in the supernatants from Fig. 3 were further analyzed by fluorescent focus assay (FFA). In agreement with the LDH release assay results, the modified NSl-WD pAb in the presence of the complement significantly reduced the viral titers in all four different serotypes of DENV-infected cells as compared to KLH pAb or PBS control groups (Fig. 4A). In addition to conventional complement-facilitated manner, we also found that modified NSl-WD pAb could directly reduce viral titers without complement in all four serotypes of DENV-infected cells.

[0092] These results indicate that modified NSl-WD pAb could reduce virus replication both by complement-mediated lysis of infected cells and complement independent manner.

[0093] Example 2 Protective effect of modified NSl-WD pAb on DENV infection by active immunization

[0094] Whether the modified NSl-WD pAb was useful in protecting a subject against DENV infection was examined in this example. The data was depicted in Figs. 5-9.

[0095] 2.1 Active immunization of modified NSl-WD polypeptide attenuates DENV-induced hemorrhage and coagulopathy

[0096] After three to four times boosts of the modified NSl-WD polypeptide, DENVs were inoculated intradermally in the back of mice skin. Three days later, the bleeding time of mice tail vein was determined. The results in Figs. 5A-5D respectively indicated that compared to the KLH treatment, the modified NSl-WD polypeptide significantly attenuated the DENV (including DENV 1, DENV 2, DENV 3 and DENV 4)-induced prolonged bleeding time in mice.

[0097] Similarly, the DENV (including DENV 1, DENV 2, DENV 3 and DENV 4)-induced hemorrhage was also significantly attenuated in mice immunized with modified NSl-WD polypeptide as compared with that of KLH treatment (Figs. 6A-6B). [0098] Next, H&E staining of the skin dermis was performed to analyze the red blood cell extravasation. The data of Fig. 7 depicted that the immunization with modified NSl-WD polypeptide reduced the DENV 2-elicited red blood cell extravasation.

[0099] The effect of the modified NSl-WD polypeptide on DENV replication was further evaluated by IHC staining with anti-NS3 antibody (NS3 is a marker of DENV replication). Compared with the KLH treatment, there was at least a two-fold decrease in the NS3 expression in the skin dermis of mice immunized with modified NSl-WD polypeptide (Figs. 8 A and 8B).

[00100] These data indicated that immunization with modified NSl-WD polypeptide inhibited DENV replication, and exhibited therapeutic efficacy on DENV-induced local hemorrhage and RBC extravasation.

[00101 ] 2-2. Modified NSl-WD-induced pAb protected mice from lethal DENV infection

[00102] Since immunodeficient mice are more susceptible to DENV infection, a lethal infection mouse model in STAT 1 -deficient mice (STAT V'- mice) was established so as to test the prophylactic effects of the modified NSl-WD pAb against lethal DENV 2 infection. The non-mouse adapted DENV 2 strain 454009 A actively replicated in STAT] ^~ mice, and the viremia in the sera of these mice could reach 10³— 10⁴ PFU/ml at 2-3 days post infection (data not shown). In addition, mice died within two weeks after the DENV challenge (data not shown). Administration of anti-NSl-WD pAb significantly reduced the DENV-induced viremia, NS1 secretion and lethality in STATl^ mice. (Figs. 9A-9C).

[00103] In conclusion, the present invention demonstrated that a modified NSl-WD polypeptide can stimulate an immune response against DENV infection, including DENV 1, DENV 2, DENV 3 and DENV 4. Accordingly, the present vaccine composition comprising the modified NSl-WD polypeptide and an adjuvant may provide a potential means to efficiently prevent a subject from being infected by DENV.

[00104] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

WHAT IS CLAIMED IS:

1. A synthetic polypeptide for the prophylaxis or treatment of a dengue virus infection in a subject comprising a first polypeptide having the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1.

2. The synthetic polypeptide of claim 1, wherein the first polypeptide has the amino acid sequence of SEQ ID NO: 1.

3. The synthetic polypeptide of claim 1, wherein the N-terminus of the synthetic polypeptide is acetylated and/or the C-terminus of the synthetic polypeptide is amidated.

4. The synthetic polypeptide of claim 1, further comprising a second polypeptide disposed at the N- or C- terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of, ovalbumin, bovine serum albumin, keyhole limpet haemocyanin, β-galactosidase, thyroglobulin, heat shock protein, and a combination thereof.

5. A vaccine composition for preventing a subject from being infected by a dengue virus comprising the synthetic polypeptide of claim 1, and a pharmaceutically acceptable adjuvant.

6. The vaccine composition of claim 5, wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of, Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant, complete Fruend's adjuvant, endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide, calcium phosphate, saponin, and muramyl dipeptide.

7. The vaccine composition of claim 5, wherein the synthetic polypeptide further comprises a second polypeptide disposed at the N- or C- terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of, ovalbumin, bovine serum albumin, keyhole limpet haemocyanin, β-galactosidase, thyroglobulin, heat shock protein, and a combination thereof.

8. A method of preventing a subject from being infected by a dengue virus comprising, administering to the subject an effective amount of the vaccine composition of claim 5 so as to vaccinate the subject against the dengue virus.

9. The method of claim 8, wherein the vaccine composition gives rise to about 0.8 μg to 80 mg the synthetic polypeptide of claim 1 per kilogram of body weight per dose.

10. The method of claim 9, wherein the vaccine composition gives rise to about 8 μg to 8 mg the synthetic polypeptide of claim 1 per kilogram of body weight per dose.

11. The method of claim 10, wherein the vaccine composition gives rise to about 80 to 800 μg the synthetic polypeptide of claim 1 per kilogram of body weight per dose.

12. The method of claim 8, wherein the vaccine composition is administered to the subject at least 2 times in the course of vaccination.

13. The method of claim 12, wherein the vaccine composition is administered to the subject 3 to 5 times in the course of vaccination.

14. The method of claim 8, wherein the dengue virus is dengue virus serotype 1, 2, 3 or 4.

15. The method of claim 8, wherein the subject is a human.

16. Use of the synthetic polypeptide of claim 1 for the preparation of a medicament for the prophylaxis or treatment of a dengue virus infection in a subject.

17. The use of claim 16, wherein the synthetic polypeptide is administered to the subject in an amount of 0.8 μg to 80 mg per kilogram of body weight per dose.

18. The use of claim 17, wherein the synthetic polypeptide is administered to the subject in an amount of 8 μg to 8 mg per kilogram of body weight per dose.

19. The use of claim 18, wherein the synthetic polypeptide is administered to the subject in an amount of 80 to 800 μg per kilogram of body weight per dose.

20. The use of claim 17, wherein the synthetic polypeptide is administered to the subject at least 2 times in the course of vaccination.

21. The use of claim 20, wherein the synthetic polypeptide is administered to the subject 3 times in the course of vaccination.

22. The use of claim 16, wherein the dengue virus is dengue virus serotype 1, 2, 3 or 4.

23. The use of claim 16, wherein the subject is a human.