OA17590A

OA17590A - Malaria Vaccine

Info

Publication number: OA17590A
Application number: OA1201500469
Authority: OA
Inventors: Ryuji Ueno; Wataru Akahata
Original assignee: Vlp Therapeutics, Llc
Priority date: 2013-06-03
Filing date: 2014-06-02
Publication date: 2017-04-28

Abstract

The present invention provides a particle comprising a polypeptide and at least one malaria antigen, and a composition or vaccine comprising thereof, its use in medicine, particularly in the prevention or treatment of malaria infections.

Description

The présent invention relates to a particle comprising a polypeptide and at least one malaria antigen, and a composition or vaccine comprising thereof, its use in medicine, particularly in the prévention or treatment of malaria.

BACKGROUND

Malaria is one of the world’s most prévalent serious infectious diseases, with approximately 250 million cases and 1 million deaths per year (WHO, 2009). Mortality is primarily in children under the âge of five and in prégnant women. Every 45 seconds, an African child dies of malaria. The disease is transmitted from person to person by infected mosquitoes, so past éradication efforts involved massive insecticide campaigns. These were successful in the Southeast U.S. for example, but failed in most poorly developed tropical countries. Current efforts involve distribution of bednets, particularly bednets impregnated with insecticide, to prevent mosquito bites at night. However, résistance to insecticides and to anti-malarial drugs for both prévention and treatment is rapidly rising. Thus, the need for a malaria vaccine is impérative for protection of millions of people from disease (http://www.qlobalvaccines.orq/content/malaria+vaccine+proqram/19614).

Malaria caused by Plasmodium falciparum remains a major public health threat, especially among children and prégnant women in Africa. An effective malaria vaccine would be a valuable tool to reduce the disease burden and could contribute to élimination of malaria in some régions of the world. Current malaria vaccine candidates are directed against human and mosquito stages of the parasite life cycle, but thus far, relatively few proteins hâve been studied for potential vaccine development.

The most advanced vaccine candidate, RTS,S, conferred partial protection against malaria in phase II clinical trials and is currently being evaluated in a phase III trial in Africa. (The Journal of Clinical Investigation 120(12) 4168-4178, 2010).

The CSP is the prédominant surface antigen on sporozoites. CSP is composed of an Nterminal région that binds heparin sulfate proteoglycans (Rl), a central région containing a four-amino-acid (NPNA) repeat, and a GPl-anchored C-terminal région containing a thrombospondin-like domain (RII). The région of the CSP included in the RTS,S vaccine includes the last 16 NPNA repeats and the entire flanking C-terminus. HBsAg particles serve as the matrix carrier for RTS,S, 25% of which is fused to the CSP segment (The Journal of Clinical Investigation 120(12) 4168-4178, 2010).

In a sériés of phase II clinical trials for RTS,S, 30%-50% of malaria-naive adults immunized with RTS,S were protected against challenge by mosquitoes infected with the homologous P. falciparum clone. In phase II field trials in the Gambia and Kenya, RTS,S conferred short-lived protection against malaria infection in approximately 35% of adults, although results from the Kenya triai did not reach statistical significance. Approximately 30%-50% of children and infants immunized with RTS,S in phase II trials conducted in Mozambique, Tanzania, and Kenya were protected from clinical malaria, however, protection was generally short-lived (The Journal of Clinical Investigation 120(12) 4168-4178, 2010). Results from a pivotai, large-scale Phase III trial, published November 9, 2012, online in the New England Journal ofMedicine (NEJM), show that the RTS,S malaria vaccine candidate can help protect African infants against malaria. When compared to immunization with a control vaccine, infants (aged 6-12 weeks at first vaccination) vaccinated with RTS,S had one-third fewer épisodes of both clinical and severe malaria and had similar reactions to the injection.

There are currently no licensed vaccines against malaria. Highly effective malaria vaccine is strongly desired.

Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, potentially yielding safer and cheaper vaccine candidates. A handful of prophylactic VLP-based vaccines is currently commercialized worldwide: GlaxoSmithKIine’s Engerix® (hepatitis B virus) and Cervarix® (human papillomavirus), and Merck and Co., Inc.’s Recombivax HB® (hepatitis B virus) and Gardasil® (human papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical évaluation, such as, influenza virus, parvovirus, Norwalk and various chimeric VLPs. Many others are still restricted to smallscale fundamental research, despite their success in preclinical tests. The implications of large-scale VLP production are discussed in the context of process control, monitorization and optimization. The main up- and down-stream technical challenges are identified and discussed accordingly. Successful VLP-based vaccine blockbusters are briefly presented concomitantly with the latest results from clinical trials and the recent developments in chimeric VLP-based technology for either therapeutic or prophylactic vaccination (Expert Rev. Vaccines 9(10), 1149-1176, 2010).

Chikungunya virus (CHIKV) has infected millions of people in Africa, Europe and Asia since this alphavirus reemerged from Kenya in 2004. The severity of the disease and the spread of this épidémie virus présent a serious public health threat in the absence of vaccines or antiviral thérapies. It is reported that a VLP vaccine for épidémie Chikungunya virus protects non-human primates against infection (Nat Med. 2010 March; 16(3): 334-338). US patent publication No. 2012/0003266 discloses a virus-like particle (VLP) comprising one or more Chikungunya virus structural polypeptides which is useful for formulating a vaccine or antigenic composition for Chikungunya that induces immunity to an infection or at least one symptom thereof. WO2012/106356 discloses modified alphavirus or flavivirus virus-like particles (VLPs) and methods for enhancing production of modified VLPs for use in the prévention or treatment of alphavirus and flavivirus-mediated diseases. (these cited references are herein incorporated by reference).

SUMMARY OF THE INVENTION

In the first aspect, the présent invention provides a particle which is capable of being self-assembled, comprising a polypeptide and at least one malaria antigen, wherein said polypeptide comprises at least one first attachment site and said at least one malaria antigen comprises at least one second attachment site, and wherein said polypeptide and said malaria antigen are linked through said at least one first and said at least one second attachment site.

In the second aspect, the présent invention provides a nucleic acid molécule which is designed for expression of a particle provided in the first aspect of the présent invention.

In the third aspect, the présent invention provides a composition or vaccine comprising the particle provided in the first aspect of the présent invention and/or the nucleic acid molécule provided in the second aspect of the présent invention.

In the fourth aspect, the présent invention provides a method of producing an antibody, comprising contacting the particle provided in the first aspect of the présent invention and/or the nucleic acid molécule provided in the second aspect of the présent invention to a mammal.

In the fifth aspect, the présent invention provides a method of immunomodulation, a method of treating malaria , a method of inducing and/or enhancing immune response against a malaria antigen in a mammal, comprising administering the composition provided in the third aspect of the présent invention to a mammal.

In sixth aspect, the présent invention provides a method of passive immunization against a malaria-causing pathogen, comprising administering the antibody provided in the fourth aspect of the présent invention to a mammal.

In seventh aspect, the présent invention provides a method of presenting an antigen on macrophage, comprising contacting the particle provided in the first aspect of the présent invention and/or the nucleic acid molécule provided in the second aspect of the présent invention to a mammal.

In eighth aspect, the présent invention provides a method for producing the particle provided in the first aspect of the présent invention, comprising preparing a vector which is designed for expression of said particle; culturing a cell which is transfected with said vector to express said particle; and recovering said particle.

BRIEF DESCRIPTION OF THE INVENTION

Fig 1 shows pVLP74_15 (VLP_CHI 532 NPNAx6) vector.

Fig 2 shows pVLP78_15 (VLP_CHI 532 NPNAx25) vector.

Fig 3 shows pVLP74_25 (VLP_VEEV 519 ΝΡΝΑχδ) vector.

Fig 4 shows that the sérum from individual monkeys immunized with Malaria VLPs after 2 weeks induced high titer of antibodies against CSP.

Fig 5 shows mean value and SD of the data shown in Fig 4.

Fig 6 shows effects of combined immunization of CHIKV VLP and VEEV VLP on induction of antibodies against CSP. In the figure, Adj indicates adjuvant.

Fig 7 shows effects of administered VLP fused with no malaria antigen on induction of antibodies against CSP. In the figure, 4w, 6w, 10w and 14w indicate 4 weeks after immunization, 6 weeks after immunization, 10 weeks after immunization and 14 weeks after immunization, respectively.

Fig 8 shows effects of administered VLP fused with malaria antigen on induction of antibodies against CSP. In the figure, 4w, 6w, 10w and 14w indicate 4 weeks after immunization, 6 weeks after immunization, 10 weeks after immunization and 14 weeks after immunization, respectively.

Fig 9 shows effects of administered VLP fused with malaria antigen together with adjuvant on induction of antibodies against CSP. In the figure, 4w, 6w, 10w and 14w indicate 4 weeks after immunization, 6 weeks after immunization, 10 weeks after immunization and 14 weeks after immunization, respectively.

Fig 10 shows schedule of the experiment.

Fig 11 shows détection of 18S malaria DNA by means of PCR.

DETAILED DESCRIPTION OF THE INVENTION

A particle comprising a polypeptide and at least one malaria antigen

In the first aspect, the présent invention provides a particle which is capable of being self-assembled, comprising a polypeptide and at least one malaria antigen, wherein said polypeptide comprises at least one first attachment site and said at least one antigen comprises at least one second attachment site, and wherein said polypeptide and said malaria antigen are linked through said at least one first and said at least one second attachment site.

As used herein, a particle which is capable of being self-assembled” refers to a particle formed by at least one constituent which is spontaneously assembled. The constituent may be a polypeptide or non-peptide chemical compound. In one embodiment, a particle which is capable of being self-assembled may be a particle comprising or consisting of at least one polypeptide. The at least one polypeptide consists of one or more kinds of peptide. In one embodiment, said particle has a diameter of at least 10nm, for example, at least 20nm, preferably at least 50nm. In one embodiment, molecular weight of said particle is from 100 kDa to 100,000 kDa, preferably from 400kDa to 30,000kDa.

A polypeptide used for the présent invention may be spontaneously assembled. The polypeptide may be a virus structural polypeptide. Thus, the particle provided by the présent invention may be a virus like particle.

A virus structural polypeptide may be a naturally occurring viral polypeptide or modified polypeptide thereof. In one embodiment, the modified polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to a naturally occurring viral structural polypeptide including capsid and envelope protein. In one embodiment, the modified polypeptide is a mutant where at most 10% of the amino acids are deleted, substituted, and/or added to a naturally occurring viral structural polypeptide including capsid and envelope protein.

In one embodiment, virus structural polypeptide used for the présent invention consists of or comprises capsid and/or envelope protein or fragment thereof. For example, virus structural polypeptide used for the présent invention consists of or comprises capsid and E2 and E1.

An antigen may be inserted into E2. In one embodiment, a particle provided by the first aspect of the présent invention can be formed by assembling 240 capsids, 240 E1 proteins and 240 E2 proteins where a malaria antigen is inserted into each of E2 proteins.

[0024] Virus structural polypeptide used for the présent invention may be derived from Alphavirus or Flavivirus. Thus, the particle provided by the présent invention may be a virus like particle derived from Alphavirus or Flavivirus.

Examples of Alphavirus and Flavivirus include, but not limited to, Aura virus, Babanki virus, Barmah Forest virus (BFV), Bebaru virus, Cabassou virus, Chikungunya virus (CHIKV),

Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus (RRV), Salmon pancréas disease virus, Semliki Forest virus, Sindbis virus, Southern éléphant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV).Whataroa virus, West Nile virus, dengue virus, tick-borne encephalitis virus and yellow fever virus.

Malaria is a disease which human or other animal (e.g. mouse) suffers from. Example of malaria include, but are not limited to, a disease caused by Plasmodium (P.) species including P. falciparum, P. malariae, P.ovale, P.vivax, P.knowlesi, P.berghei, P.chabaudi and P.yoelii

As used herein, the term “malaria antigen” refers to any antigen or fragment thereof. The term antigen or fragment thereof, means any peptide-based sequence that can be recognized by the immune system and/or that stimulâtes a cell-mediated immune response and/or stimulâtes the génération of antibodies.

According to Scand. J. Immunol. 56, 327-343, 2002, considering the whole parasite life cycle, there are essentially six targets for a malaria vaccine: (1) sporozoites; (2) liver stages; (3) merozoites; (4) infected RBC; (5) parasite toxins; (6) sexual stages.

Table summarizes the main candidate antigens of each stage identified.

Table 1. Main vaccine candidates from the different phases of Plasmodium life cycle

Targets Candidate antigens

Circumsporozoite protein (CSP)

Sporozoite Thrombospondin-related adhesive protein (TRAP)

Sporozoite and liver-stage antigen (SALSA)

Targets	Candidate antigens
	Sporozoite threonine- and asparagine-rich protein (STARP) CSP Liver-stage antigen (LSA)-1 and -3
Liver stage	SALSA STARP Merozoite surface protein (MSP)-1, -2, -3, -4 and -5 Erythrocyte-binding antigen (EBA)-175 Apical membrane antigen (AMA)-1
Merozoite	Rhoptry-associated protein (RAP)-1 and -2 Acidic-basic repeat antigen (ABRA) Duffy-binding protein (DBP) (Plasmodium vivax) Ring érythrocyte surface antigen (RESA) Serine-rich protein (SERP)
Blood stage	Erythrocyte membrane protein (EMP)-1, -2 and -3 Glutamate-rich protein (GLURP)
Toxins	Glycosilphosphatidylinositol (GPI)
Sexual stages	Ps25, Ps28, Ps48/45 and Ps230

(Scand. J. Immunol. 56, 327-343, 2002)

According to the présent invention, one or more antigens listed above can be used as long as it is formed to a particle. For example, a circumsporozoite protein and a fragment thereof can be used as an antigen. Examples of circumsporozoite protein include, but are 5 not limited to, Plasmodium falciparum circumsporozoite protein consisting of amino acid sequence described below (SEQ ID No.:56):

Mmrklailsvssflfvealfqeyqcygsssntrvlnelnydnagtnlynelemnyygkqenwyslkknsrslgenddgnnnngd ngregkdedkrdgnnedneklrkpkhkklkqpgdgnpdpnanpnvdpnanpnvdpnanpnvdpnanpnanpnanpna npnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnvdpnanpnanpnanpnanpnanpnan pnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnknnqgngqghnmpndpnrn vdenanannavknnnneepsdkhieqylkkiknsistewspcsvtcgngiqvrikpgsankpkdeldyendiekkickmekc ssvfnvvnssiglimvlsflflntr.

In one embodiment, malaria antigen is a Plasmodium falciparum circumsporozoite protein B cell epitope. Example of Plasmodium falciparum circumsporozoite protein B cell epitope may be a repeat sequence of NPNA, including (NPNA)₄.₃₀ (i.e. 4xNPNA, 5xNPNA, 6xNPNA, 7xNPNA, 8xNPNA, 9xNPNA, ΊΟχΝΡΝΑ, 11xNPNA, 12xNPNA, 13xNPNA 14xNPNA, 15xNPNA, 16xNPNA, 17xNPNA, 18xNPNA, 19xNPNA, 20xNPNA, 21xNPNA, 22xNPNA, 23xNPNA, 24xNPNA, 25xNPNA, 26xNPNA, 27xNPNA, 28xNPNA, 29xNPNA or 30xNPNA).

In one embodiment, malaria antigen is a Plasmodium yoelii circumsporozoite protein B cell epitope including (QGPGAP)₃.₁₂.

In one embodiment, malaria antigen is a Plasmodium vivax circumsporozoite protein B cell epitope including (ANGAGNQPG)·!.^.

In one embodiment, malaria antigen is a Plasmodium malariae circumsporozoite protein B cell epitope including (NAAG)4.₃₀.

In one embodiment, malaria antigen is a Plasmodium falciparum circumsporozoite protein T cell epitope. Example of Plasmodium falciparum circumsporozoite protein T cell epitope may be EYLNKIQNSLSTEWSPCSVT (SEQ ID No.:44). (EYLNKIQNSLSTEWSPCSVTjve may be also used as a malaria antigen.

In one embodiment, malaria antigen is a Plasmodium yoelii circumsporozoite protein T cell epitope which is YNRNIVNRLLGDALNGPEEK (SEQ ID No.45). (YNRNIVNRLLGDALNGPEEKjve may be also used as a malaria antigen.

The présent invention addresses one or more of the above needs by providing antigens, vectors encoding the antigens, and antibodies (and antibody-like molécules including aptamers and peptides) that specifically bind to the antigen, together with the uses thereof (either alone or in combination) in the prévention or treatment of malaria infections.

As used herein, the term antibody refers to molécules which are capable of binding an epitope or antigenic déterminant. The term is meant to include whole antibodies and antigen-binding fragments thereof, including single-chain antibodies. Such antibodies include human antigen binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The antibodies can be from any animal origin including birds and mammals. Preferably, the antibodies are mammalian e.g. human, murine, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animais e.g. chicken. As used herein, human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animais transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described, for example, in U.S. Patent No. 5,939,598, the disclosure of which is incorporated herein by reference in its entirety.

The antigen used for the présent invention can be modified polypeptide derived from a naturally occurring protein. The modified polypeptide may be a fragment of the naturally occurring protein. In one embodiment, the modified polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to a polypeptide derived from a naturally occurring protein. In one embodiment, the modified polypeptide derived is a mutant where at most 10% of the amino acids are deleted, substituted, and/or added based on a polypeptide derived from naturally occurring protein.

In the particle as provided by the présent invention, a polypeptide and an antigen may be linked through at least one first attachment site which is présent in the polypeptide and at least one second attachment site which is présent in the antigen.

As used herein, each of a first attachment site and a second attachment site refers to a site where more than one substance is linked each other.

In one embodiment, the polypeptide and the antigen are directly fused. Altematively, one or two linkers may intervene between N-terminal residue of the antigen and the polypeptide and/or between C-terminal residue ofthe antigen and the polypeptide.

The antigen or the polypeptide can be truncated and replaced by short linkers. In some embodiments, the antigen or the polypeptide include one or more peptide linkers. Typically, a linker consists of from 2 to 25 amino acids. Usually, it is from 2 to 15 amino acids in length, although in certain circumstances, it can be only one, such as a single glycine residue.

In one embodiment, a nucleic acid molécule, in which polynucleotide encoding the polypeptide is genetically fused with polynucleotide encoding the antigen, is expressed in a host cell so that the first attachment site and the second attachment site are linked through a peptide bond. In this case, the polypeptide and the antigen are linked through a peptide bond. Relating to this embodiment, the first attachment site and/or the second attachment site may be genetically modified from the original polypeptide or antigen. For example, the first attachment site is modified from the polypeptide so that through a linker peptide including SG, GS, SGG, GGS and SGSG, the polypeptide is conjugated with the antigen.

When the polypeptide are chemically conjugated with the antigen, the first attachment site and the second attachment site may be linked through a chemical cross-linker which is a chemical compound.

Examples of the cross-linker include, but are not limited to, SMPH, Sulfo-MBS, SulfoEMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other crosslinkers available from the Pierce Chemical Company.

In one embodiment, the particle provided by the présent invention comprises a polypeptide linked to an antigen, wherein spatial distance between the N-terminal residue and C-terminal residue of the antigen is 30Â or less when the distance is determined in a crystal of the antigen or a naturally occurring protein containing the antigen or modified protein therefrom.

The antigen used for the présent invention can be designed by a person skilled in the art. For example, the antigen used for the présent invention may be a naturally occurring protein or a fragment thereof. Altematively, the antigen used for the présent invention may be a protein modified from a naturally occurring protein or a fragment thereof. A person skilled in the art can design the antigen so that spatial distance between the N-terminal residue and C-terminal residue of the antigen is 30Â or less when the distance is determined in a crystal of the antigen or a naturally occurring protein containing the antigen or modified protein therefrom. For example, the antigen used for the particle provided by the présent invention can be designed using a free software including PyMOL (e.g. PyMOL v0.99: http:/www.pymol.org). In one embodiment, the spatial distance between the Nterminal residue and C-terminal residue of the antigen is 30Â (angstrom) or less, 20Â or less, or 10Â or less (e.g. from 5 Â to 15 Â , from 5 Â to 12 Â , from 5 Â to 11 Â , from 5 Â to 10 Â , from 5 A to 8 A , from 8 A to 15 A , from 8 Â to 13 A , from 8 Â to 12 Â , from 8 Â to 11 Â , from 9 Â to 12 Â , from 9 A to 11 A, from 9 Â to 10 A or from 10 A to 11 Â ).

Chikunqunya virus like particle or a Venezuelan equine encephalitis virus like particle

In one embodiment, the présent invention provides a Chikungunya virus like particle or a Venezuelan equine encephalitis virus like particle comprising a Chikungunya or Venezuelan equine encephalitis virus structural polypeptide and at least one malaria antigen, wherein said Chikungunya virus structural polypeptide or said Venezuelan equine encephalitis virus structural polypeptide comprises at least one first attachment site and said at least one malaria antigen comprises at least one second attachment site, and wherein said Chikungunya or Venezuelan equine encephalitis virus structural polypeptide and said at least one antigen are linked through said at least one first and said at least one second attachment site.

In one embodiment, a spatial distance between the N-terminal residue and C-terminal residue of the malaria antigen may be 30 Â or less; 25 Â or less; 20 Â or less; 15 Â or less; 14 Â or less; 13 Â or less; 12 Â or less; 11 Â or less; 10 Â or less; 9 Â or less; or 8 A or less (e.g. from 5 Â to 15 Â , from 5 A to 12 A , from 5 Â to 11 Â , from 5 Â to 10 Â , from 5 Â to 8 Â , from 8 A to 15 Â, from 8 Â to 13 Â , from 8 Â to 12 Â , from 8 Â to 11 Â , from 9 Â to 12 Â , from 9 Â to 11 Â , from 9 Â to 11 Â or from 10 Â to 11 Â ) when the distance is determined in a crystal of the malaria antigen or a naturally occurring protein containing the malaria antigen or modified protein therefrom.

In one embodiment, the malaria antigen is linked to the Chikungunya or Venezuelan equine encephalitis virus structural polypeptide by way of chemical cross-linking or as a fusion protein produced by way of genetic engineering.

A Chikungunya or Venezuelan equine encephalitis virus structural polypeptide used in the présent invention may comprise a Chikungunya or Venezuelan equine encephalitis virus envelope protein and/or a capsid.

Examples of Chikungunya virus include, but are not limited to, strains of 37997 and LR2006 OPY-1.

Examples of Venezuelan equine encephalitis virus include, but are not limited to, TC83.

Chikungunya or Venezuelan equine encephalitis virus structural polypeptide used in the présent invention may naturally occurring virus structural polypeptide or modified polypeptide thereof. The modified polypeptide may be a fragment of the naturally occurring virus structural polypeptide. In one embodiment, the modified polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to a naturally occurring viral capsid and/or envelope protein. In one embodiment, the modified polypeptide is a mutant where at most 10% of the amino acids are deleted, substituted, and/or added based on a naturally occurring viral capsid and/or envelope protein. For example, K64A or K64N mutatation may be introduced into a capsid of Venezuelan equine encephalitis virus structural polypeptide used in the présent invention.

Chikungunya or Venezuelan equine encephalitis virus structural polypeptide may consist of or comprise a capsid, E2 and El.

Examples of Chikungunya virus structural polypeptide include, but are not limited to,

Capsid- E2-E1 of Chikungunya virus Strain 37997, and Capsid- E2-E1 of Chikungunya virus

LR2006 OPY-1.

Examples of Venezuelan equine encephalitis virus structural polypeptide include, but are not limited to, Capsid- E2-E1 of Venezuelan equine encephalitis virus Strain TC-83.

An exemplary Chikungunya virus structural polypeptide sequence is provided at

Genbank Accession No. ABX40006.1, which is described below (SEQ ID No.:1):

mefiptqtfynrryqprpwtprptiqvirprprpqrqagqlaqlisavnkltmravpqq kprrnrknkkqkqkqqapqnntnqkkqppkkkpaqkkkkpgrrermcmkiendcifevk he gkvt gyac1vgdkvmkpahvkgt idnadlaklafkrsskydle c aqipvhmksdask fthekpegyynwhhgavqysggrftiptgagkpgdsgrpifdnkgrwaivlgganega rtalswtwnkdivtkitpegaeewslaipvmcllanttfpcsqppctpccyekepeet lrmlednvmrpgyyqllqasltcsphrqrrstkdnfnvykatrpylahcpdcgeghsch spvalerirneatdgtlkiqvslqigiktddshdwtklrymdnhmpadaeraglfvrts apctitgtmghfilarcpkgetltvgftdsrkishscthpfhhdppvigrekfhsrpqh gkelpcstyvqstaatteeievhmppdtpdrtlmsqqsgnvkitvngqtvrykcncggs negltttdkvinnckvdqchaavtnhkkwqynsplvprnaelgdrkgkihipfplanvt crvpkarnptvtygknqvimllypdhptllsyrnmgeepnyqeewvmhkkewltvpte gle vt wgnnepykywpql s tngt ahghphe i i lyyye lyptmtwws vat f i 11 smvg maagmcmcarrrcitpyeltpgatvpfllsliccirtakaatyqeaaiylwneqqplfw lqaliplaalivlcnclrllpcccktlaflavmsvgahtvsayehvtvipntvgvpykt lvnrpgyspmvlemellsvtleptlsldyitceyktvipspyvkccgtaeckdknlpdy sckvftgvypfmwggaycfcdaentqlseahveksescktefasayrahtasasaklrv lyqgnnitvtayangdhavtvkdakfivgpmssawtpfdnkiwykgdvynmdyppfga grpgqfgdiqsrtpeskdvyantqlvlqrpavgtvhvpysqapsgfkywlkergaslqh tapfgcqiatnpvravncavgnmpisidipeaaftrwdapsltdmscevpacthssdf ggvaiikyaaskkgkcavhsmtnavtireaeievegnsqlqisfstalasaefrvqvcs tqvhcaaechppkdhivnypashttlgvqdisatamswvqkitggvglwavaaliliv vlcvsfsrh

Another exemplary Chikungunya virus structural polypeptide sequence is provided at

Genbank Accession No. ABX40011.1, which is described below (SEQ ID No.:2):

net iptqtf ynrryqprpwaprptiqvirprprpqrqagqlaql ieavnJcXtmravpqcj kprmrknkkqrqkkqapqndpkqkkqppqkkpaqkkkkpgrrermcmkiendcifevk hegkvmgyaclvgdkvmkpahvkgtidnadlaklafkrsskydlecaqipvhMkedask i thekpegyynwhhg avqysggrf t iptg agkpg dsgrpi t dnkgrwaivlgganega rtalswtwnkdivtkltpegaeews1alpvlcllantttpcsqppctpccyekepest Innlednvmrpgyyqllkasltcephrqrrstkdnfnvykatrpylahcpdcgeghech spialerirneatdgtlkiqvslqigiktddshdwtklrymdshtipadaeragllvrts apctitgtmghfilarcpkgetltvgftdsrkightcthpfhheppvigrerfhsrpqh gkelpcstyvqstaataaeievh^pdtpdrtlmcqqsgnvkitvngqtvrykcneggs negltttdkvlnnckidqchaavtnhknwqynsplvprnaelgdrkgkihipfplanvt crvpkarnptvtyg knqvtml lypdhpt 1 Isymmg qepnyheewvthkkevt 1 tvpte glevtwgnnepykyi^qmstngtahghphei i lyyyelyptmtwivsvasf vl lemvg tavgmcvcarrrcitpyeltpgatvpflisllccvrttkaacyyeaaaylwneqqplfw Iqaïiplaalivlcnclkllpcccktlaflavmsigahtvsayehvtvipntvgvpykt Ivnrpgyspmvlemelqsvcleptlsldyitceyktvipspyvkccgtaeckdkslpdy eckvftgvypfnwggaycfcdaentqlseahvekBeecktefaeayrahtasaeaklrv lyqgnn i t vaayangdhavt vkdakf wgpmesawtpf dnk i wykgdvynmdyppC ga grpgqfgdiqsrtpeskdvyantqlvlqrpaagtvhvpysqapsgfkywlkergaslqh tapfgcqiatnpvravncavgnipiaidipdaaftrwdapsvcdmscevpacthssdf ggvai ikytaskkgkcavhsmt navcί readvevegnsqlqi b f stalasaef rvqvcs tqvhcaaachppkdhivnypashttlgvqdisttamswvqkitggvglivavaallliv vlcvsfsrh.

An exemplary Venezuelan equine encephalitis virus structural polypeptide is provided at

Genbank Accession No. L01443.1 (http://www.ncbi.nlm.nih.goV/nuccore/L01443.1), which is described below (SEQ ID No.:3):

mfpfqpmypmqpmpyrnpfaaprrpwfprtdpflamqvqeltrsmanltfkqrrdappe gp s aakpkkeas qkqkgggqgkkkknqgkkkak t gppnpkaqngnkkktnkkpgkrqrm vmklesdktfpimlegkingyacwggklf rpmhvegkidndvlaalktkkaskydley advpqnmradt f kythekpqgyyswhh.gavqyen.gr f tvpkgvgakgdsgrpi ldnqgr waivlggvnegsrtalswmwnekgvtvkytpenceqwslvttmcllanvtfpcaqpp icydrkpaetlamlsvnvdnpgydelleaavkcpgrkrrsteelfneykltrpymarci rcavgschspiaieavksdghdgyvrlqtssqygldssgnlkgrtmrydmhgtikeipl hqvslyt s rpchivdghgyf11arcpagds i tme fkkdsvrhs c svpyevk fnpvgre1 ythppehgveqacqvyahdaqnrgayvemhlpgsevdsslvslsgssvtvtppdgtsal vececggtkisetinktkqfsqctkkeqcrayrlqndkwvynsdklpkaagatlkgklh vpflladgkctvplapepmitfgfrsvslklhpknptylitrqladephythelisepa vrnftvtekgwefvwgnhppkrfwaqetapgnphglphevithyyhrypmstilglsic aaiatvsvaastwlfcrsrvacltpyrltpnaripfclavlccartaraettwesldhl wnnnqqmfwiqlliplaaliwtrllrcvccwpf lvmagaagagayehattmpsqagi syntivnragyaplpisitptkikliptvnleyvtchyktgmdspaikccgsqectpty rpdeqckvftgvypfmwggaycfcdtentqvskayvmksddcladhaeaykahtasvqa flnitvgehsivttvyvngetpvnfngvkitagplstawtpfdrkivqyageiynydfp eygagqpgafgdiqsrtvsssdlyantnlvlqrpkagaihvpytqapsgfeqwkkdkap slkftapfgceiytnpiraencavgsiplafdipdalftrvsetptlsaaectlnecvy ssdfggiatvkysasksgkcavhvpsgtatlkeaavelteqgsatihfstanihpefrl qictsyvtckgdchppkdhivthpqyhaqtftaavsktawtwltsllggsaviiiiglv lativamyvltnqkhn.

In one embodiment, a first attachment site comprises an amino group, preferably an amino group of a lysine residue. In one embodiment, the second attachment site comprises sulfhydryl group, preferably, a sulfhydryl group of a cysteine.

In one embodiment, a conjugation of more than two substances (e.g. antigen and Chikungunya or Venezuelan equine encephalitis virus structural polypeptide) through a first attachment site or a second attachment site is achieved using chemical cross linker. Examples of the cross-linker include, but are not limited to, SMPH, Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available from the Pierce Chemical Company.

According to the présent invention, a Chikungunya or Venezuelan equine encephalitis virus like particle comprising a Chikungunya or Venezuelan equine encephalitis virus structural polypeptide and an antigen, wherein said Chikungunya or Venezuelan equine encephalitis virus structural polypeptide and said antigen are expressed as a fusion protein can be provided.

In one embodiment, the antigen can be fused with any site of the Chikungunya or Venezuelan equine encephalitis virus structural polypeptide. For example, the antigen may be directly or indirectly linked to N- or C- terminal of the Chikungunya or Venezuelan equine encephalitis virus structural polypeptide, or the antigen may be inserted into Chikungunya or Venezuelan equine encephalitis virus structural protein.

In one embodiment, at least one antigen is inserted into E2 of Chikungunya or Venezuelan equine encephalitis virus structural protein. For example, regarding Chikungunya virus structural protein, at least one antigen is inserted between residues 519 and 520 of SEQ ID Nos.1 or 2 (i.e. between G at 519-position and Q at 520-position of SEQ ID Nos.1 or 2); between residues 530 and 531 of SEQ ID Nos.1 or 2 (i.e. between G at 530position and S at 531-position of SEQ ID Nos.1 or 2); between residues 531 and 532 of SEQ ID Nos.1 or 2 (i.e. between S at 531-position and N at 532-position of SEQ ID Nos.1 or 2); between residues 529 and 530 of SEQ ID Nos.1 or 2(i.e. between G at 529-position and G at 530-position of SEQ ID Nos.1 or 2); or between residues 510 and 511 of SEQ ID Nos.1 or 2(i.e. between S at 510-position and G at 511-position of SEQ ID Nos.1 or 2); or between residues 511 and 512 of SEQ ID Nos.1 or 2(i.e. between G at 511-position and N at 512position of SEQ ID Nos.1 or 2); or between residues 509 and 510 of SEQ ID Nos.1 or 2(i.e. between Q at 509-position and S at 510-position of SEQ ID Nos.1 or 2).

For example, regarding Venezuelan equine encephalitis virus structural protein, at least one antigen is inserted between residues 517 and 518 of SEQ ID No.3 (i.e. between G at 517-position and S at 518-position of SEQ ID No.3); between residues 518 and 519 of SEQ ID No.3 (i.e. between S at 518-position and S at 519-position of SEQ ID No.3); between residues 519 and 520 of SEQ ID No.3 (i.e. between S at 519-position and V at 520-position of SEQ ID No.3); between residues 515 and 516 of SEQ ID No.3(i.e. between L at 515-position and S at 516-position of SEQ ID No.3); between residues 516 and 517 of SEQ ID No.3(i.e. between S at 516-position and G at 517-position of SEQ ID No.3); between residues 536 and 537 of SEQ ID No.3(i.e. between C at 536-position and G at 537position of SEQ ID No.3) ; between residues 537 and 538 of SEQ ID No.3(i.e. between G at 537-position and G at 538-position of SEQ ID No.3) ; between residues 538 and 539 of SEQ ID No.3(i.e. between G at 538-position and T at 539-position of SEQ ID No.3).

The fusion protein may be expressed using a conventional technique in the art. A variety of expression Systems can be used for the expression of the fusion protein. For example, the fusion protein can be expressed in 293 cells, Sf9 cells or E.coli.

A polypeptide derived from Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may be a naturally occurring viral polypeptide or modified polypeptide thereof. In addition, a polypeptide derived from malaria antigen may be a naturally occurring polypeptide or modified polypeptide of the naturally occurring polypeptide or a fragment of the naturally occurring polypeptide or the modified peptide. The modified polypeptide may be a fragment of the naturally occurring virus structural polypeptide.

In one embodiment, the modified polypeptide derived from malaria antigen has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to a naturally occurring polypeptide. In one embodiment, the modified peptide derived from malaria antigen is a mutant where at most 10% of the amino acids are deleted, substituted, and/or added based on a naturally occurring polypeptide derived from malaria antigen.

When a polypeptide derived from a virus is conjugated with a polypeptide derived from an antigen, a linker peptide including SG, GS, SGG, GGS SGSG and TRGGS may be used. Examples of conjugation of the polypeptide derived from a virus (referred to as “PFV” below) with the polypeptide derived from the antigen (referred to as “PFA” below) include, but not limited to: PFV-SG-PFA-GS-PFV; PFV-SG-PFA-GGS-PFV; PFV-SSG-PFA-GSPFV; PFV-SGG-PFA-GGS-PFV;

PFV-SGSG-PFA-GS-PFV; and PFA-SGG-PFA-TRGGS-PFV.

In one embodiment, the présent invention provides a virus like particle comprising a fusion protein of a polypeptide derived from Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) and a polypeptide derived from malaria antigen, wherein the virus like particle is prepared by transfecting an expression vector comprising a nucleic acid molécule corresponding to the amino acid sequence represented by SEQ ID NO. 28, 31, 34, 37, 39, 41 or 43 into a mammalian cell (e.g. 293F cell). Regarding this embodiment, modified fusion protein can be also used for a virus like particle provided by the présent invention, which can be prepared by transfecting an expression vector comprising a nucleic acid molécule corresponding to the amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO. 28, 31, 34, 37, 39, 41 or 43 into a mammalian cell (e.g. 293F cell).

In one embodiment, the présent invention provides a virus like particle comprising or consisting of:

one or more capsid of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV);

one or more E1 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV); and one or more E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), wherein malaria antigen is inserted into E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV). For example, présent invention provides a virus like particle comprising or consisting of:

240 capsids of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV);

240 E1s of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV); and

240 E2s of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), wherein malaria antigen is inserted into each of E2s of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV).

In this embodiment, the E2 into which the antigen is inserted may consist of an amino acid sequence represented by SEQ ID No.50; the E1 may consist of an amino acid sequence represented by SEQ ID No.51; and the capsid may consist of an amino acid sequence represented by SEQ ID NO.: 52; or the E2 into which the antigen is inserted may consist of an amino acid sequence represented by SEQ ID NO.53; the E1 may consist of an amino acid sequence represented by SEQ ID NO.54; and the capsid may consist of an amino acid sequence represented by SEQ ID NO.: 55.

Further, regarding this embodiment, modified capsid of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), modified E1 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) and modified E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may be used for the virus like particle. For example, the modified capsid of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may hâve at least 70%, 75%, 80%, 85%, 90%, 95% or

98% amino acid sequence identity to the amino acid sequence represented by SEQ ID NO.:

or SEQ ID No.:55; the modified E1 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may hâve at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to the amino acid sequence represented by SEQ ID NO.: 51 or SEQ ID No.:54; and/or the modified E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may hâve at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to the amino acid sequence represented by SEQ ID NO.: 50 or SEQ ID No.:53.

Also, the modified capsid, E1 or E2 may be a mutant where at most 10% of the amino acids are deleted, substituted, and/or added based on the capsid consisting of an amino acid sequence represented by SEQ ID NO.: 52 or SEQ ID No.:55; E1 consisting of an amino acid sequence represented by SEQ ID NO.: 51 or SEQ ID No.:54; and/or E2 consisting of consisting of an amino acid sequence represented by SEQ ID NO.: 50 or SEQ ID No.:53.

(2) Nucléotide, Vector, Host cell

In the second aspect, the présent invention provides a nucleic acid molécule which is designed for expression of a particle as provided in the first aspect of the présent invention.

In one embodiment, the présent invention provides a nucleic acid molécule comprising a nucléotide sequence that encodes the Chikungunya or Venezuelan equine encephalitis virus like particle as described above.

Examples of the nucléotide sequence that encodes the Chikungunya or Venezuelan equine encephalitis virus like particle include, but are not limited to, a nucléotide sequence encoding envelope of Chikungunya virus Strain 37997, a nucléotide sequence encoding Capsid-envelope of Chikungunya virus Strain 37997, a nucléotide sequence encoding envelope of Chikungunya virus Strain LR2006 OPY-1, a nucléotide sequence encoding Capsid-envelope of Chikungunya virus LR2006 OPY-1, a nucléotide sequence encoding envelope of Venezuelan equine encephalitis virus Strain TC-83 and a nucléotide sequence encoding Capsid-envelope of Venezuelan equine encephalitis virus TC-83.

Regarding Chikungunya virus, an exemplary nucléotide sequence that encodes envelope is described below (SEQ ID No.:4):

A!gagc<tcgc«ctcttgg’tt!gtgcc<gMggcaaacaetac»tlcacîgcicteagccgccttgc3«ca:tgc!gctacg»a3gjjaaccgg *»gcaccitgcgca<gcHg»ggacaacgtgatgag#cccggJHactaccagcta€maagcaicgctt»€ttgctcic«cca«gccaa3gac gcaftactaag,gacaatmâaigictaiaaagcca€aagaccauiciagctca«gicctgactgcggagaagggcancgtgcc*ca.gcccutc cccgttccai«igaaccacagtg3taggtagggagagglicc»acicpœacasc»!ggtaaagaguficc«gc®gcacgtacglfc^gag c»cçgctgccactgctpggagaiagaggtgca«ecixccagat»c(cctt»ccgcacg«faieacgc*geagtctggc»acgtg#agatca caaiaaigggvafeseagtaca.iaacaagtgcaactac«aRecteaaacgagggactgacaacc»cagafcaaâtft8icaauufctgca»aatî ^eg^^«ipt®^€*«MtesKMpMtg^aat»K^ea«*f:eœgc^s»grt^mc^^^cfUM^aag atccacatixeaitcccattggcaaacgtgacHgcapgtgccaaaagcaagaaaccctacagtaacKaçgpaaaaaccaagteaccatgctg ct^atcctgaccatcc^caciatgtcitaccgiaaangggacaggaascaaattaccacgaggagtgggtgacacacaapaggaggKacci tpœgîgcctactgagggfctggaggtcacHggg^aacaacgaaccatacaegtactggccgcagatgtclacgaacggtacîgclcatg^c acccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttct^cgatggtgggcaca gcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatalgaattaacaccaggagccactgttcccttcctgcteagcctgctatgcig c^cagiacgaccaaggcggccacatattacgaggctgcggcatatcfatggaacgaacagcagcccctgttctggttgcaggctcttatcccgct ggccgcctigaicgtccigtgcaaagtctgaaactcttgccatgctgc(giaagaccctggc«ttttagccgtaatgagcatcggigcccacactgtg agcgcgtacgaacacg'vaacagt&aÎcccgaacacRgtKggagtaccgtataagacicttgtcaacagaccgggttacagccccatftgtgUgga gatg.gagctacaaîcagtcaccttggaaccaacacigtcacttgactacatcacgtgcgagtacaaaactgtcaÎcccctccccgiacgtgaagtgct gtggîacagcagagtgcaaggacaagagcctaccagaciacagctgcaaggtcittaciggagictacccatttatgtggggcggcgcciactgctt «^Î^cgccgaaaaucgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagttfgcatcggcctacagagcccacaccg catcggcgtcggcgaagctccgcgtccmaccaaggaaacaacattaccgtagctgccfacgcfaacggtgaccaigccgtcacagtaaaggac gccaagttt^cgtgggcccaat^cctccgcctggacacctttt^caacaaaatcgtggtgtacaaaggcgae^cucaacatggactacccac cttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcaiit^tactacapg gccagcagcaggcacggtacatgtaccatac!ctcag.gcaccatctggct!caagtattggctgaaggaacgaggagcatcgciacagcacacgg cacegttcggttgccagattgcga€aa«cccggtaagagct^aaattgcgctgt®ggaacataccaatttccatcgacatac€ggafgcggcctt lactagg^tgtegatgcaccclct^aacggacatgteatgcgaapaccagccigeactcactcctccgacmgggggcijcgccatcatcaaat acacagctagcaagaaaggiaaatglgcagtacaucgatgaçcaacgccgtlaccattcgagaagccgacgtagaagtagaggggaactccca gcîgcaaaiascctîctcaacagccctggcaagcgccgagtttcgcgtgcaagtgigctccacacaagtacacigcgcagccgcatgccacccicc aaaggaccacatagtcaauacccagcatcacac3ccacccnggggtccagg3’.atatcc3caacggcaatgtcttgggtgcagaagattacggg aggagtaggattaattgttgctgttgagcx^taattttaattgtggtgctatgcgtgtcgÎttagcagf^ac

Regarding Chikungunya virus, another exemplary nucléotide sequence that encodes envelope is described below (SEQ ID No.:5):

Aigagtci!gccatcccagtiatgtgcctgHggc»aac*c«Kgttc«cifc<cccag«:cccttgcacgc«tgeifctacg*aaafg»ccgg aggi»â€«ctscgca:gcug«ggâcaacgt«ât^iga«agggtactatcagcigciacaagM$cat»catgti«ccccaccgccafCfpc »ggaceaagcigcgttao!gÆ»caaccaaaigcca«caga€«caaagaw?cgg«ct3Ktgm£aaca;cagcaccgigtacaa«M-l£g aacaatgggacacucaîcctggcccgatgtccaaaaggggaaactctgacggtgggaUcactgacagtaggaagauagîcacicatgtacgca cccaîUcaccacgaccctccigigat.aggtcgggaaaaaUccattcccgaccgcagcacggtaaagagctacc'.tgcagcacgiacgîgcagag caccgccgcaactaccgaggagatsgaggtacacatgcccccagacacccctgaicgcacauaalgtcacaacagtccggcaacgtaaagatc acagtcaaiggccagacggtgcggtacaagtgtaaftgcggtggctcaaatgaaggaciaacaactacagacaaagtgattaataactgcaaggtt gatcaatgtcatgccgcggtcaccaatcacaaaaagtggcagtataactcccctctggtcccgcgtaatgctgaacttggggaccgaaaaggaaaa aitcacaîcccgUiccgctggcaaa'gtaacatgcagggîgcctaaagcaaggaaccccaccgtgacgtacgggaaaaaccaaglcaicatgcta cîgîatccigaccacccaacactcclgtcctaccggaataigggagaagaaccaaacîaicaagaagagtgggtgatgcataagaaggaagtcgtg ctaaccgtgccgactgaagggctcgaggtcacgtgggg€aaca®:gagccgtataagtattgg€cgcagttatctacaaacg^acagccœtgg ccaaxgcatgagataattctgtattanaigagctgtaccccacut^ctgtegtagttgtgicagtg^cacgttcatactcctgtcgatggtgggta tggcagcggggatgigcatijtgtgcacgacgcagatgcalcacaccgtatgaactgacaccaggagctaccgtccctttcctgcttagcctaatatg ctgcatcagaacagctaaagcggccacataccaapggctgcgatataactgtggaacgagçagcaac«ng»tttggctacaagccc«attccg ctggcagccctgattgttctatgcaactgtctgagactcltacxatgctgcîgtaaaacgttggcuitttagccgtaatgagcgtc^tgcccacactgt gagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactctagtcaatagacmggctacagccccatggtaMgg agaiggaactacigtcagtcactttggagccaacactatcgcttgattaeatcacgtgcgagtacaaaaccgtcatcccgtctccgtacgtgaagtgct gcggtacagcagagtgcaaggacaaaaacctacctgacucagctgtaaggtcttcaccggcgicucccautatgtggggcggcgcctactgctt ctgcgacgctgaaaacacgcagttgagcgaagcacacgtggagaagtccgaatcatgcaaaacagaamgcatcsgcatacagggcîcataccg catctgcatcagctaagctccgcgtcctttaccaaggaaataacatcactgtaactgccîatgcaaacggcgaccatgccgtcacagttaaggacgc caaatKaMgtfôggccaat^cttcagcctggacacattcgacaMaaaaUgtggt^acaaaggtgacgictataacatggactacccgccctt tggcgcaggaagaccaggacaamggcgatatccaaagtcgcacaccigagagtaaagacgtcutgctaaiacacaactggtactgcagagac cg^tgtgggtacggtacac^gccataacicaggcaccatcig^tttaagtaMggctaaaapacgcggg^^cgctgcagracacagcac caHtggcîgccaaatagcaacaaacccggtaagagcggtgaactgcgccgtagggaacatgcccatctccatcgacataccggaagcggccttc acla^gtcgtcgacgcgccctctttaacggacatgtcgtgcgaggtaccagccigcacccattcctcagactttgggggcgtcgccattattaaata tgcagccagcaagaaaggcaagjgtgcggtgcaitcgatgactaacgccgtcactattcgggaagctgagatagaagttgaagggaaitacagct gcaaatctctuacgaeggccuagccagcgccgaaitccgcgtacaa^ctgttctacacaagtacacigîgcagcc^igtgccacixcccgaag gacca.catagtcaactacccggcgtcacataccaccctcggggtccaggacatctccgctacggcgatgtcatgggtgcagaagaicacgggag gt|pgggactggttg«gagttgœgcac»gattctaatc|^gipgcwgcgtgtcgttcagcaggcac

Regarding Chikungunya virus, an exemplary nucléotide sequence that encodes a

Capsid-envelope is described below (SEQ ID No.:6):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggc cccacgccctacaattcaagtaattagacctagaccacgtccacagaggcaggctgggc aactcgcccagctgatctccgcagtcaacaaattgaccatgcgcgcggtacctcaacag aagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaaaa cgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaac caggccgtagggagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaag catgaaggcaaagtgatgggctacgcatgcctggtgggggataaagtaatgaaaccagc acatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcggtcgt ctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaag tttacccacgagaaacccgaggggtactataactggcatcacggagcagtgcagtattc aggaggccggttcactatcccgacgggtgcaggcaagccgggagacagcggcagaccga tcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaaggtgcc cgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctga gggagccgaagagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacat tcccctgctctcagccgccttgcacaccctgctgctacgaaaaggaaccggaaagcacc ttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaagcatc gctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctata aagccacaagaccatatctagctcattgtcctgactgcggagaagggcattcgtgccac agccctatcgcattggagcgcatcagaaatgaagcaacggacggaacgctgaaaatcca ggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgct atatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttca gcaccgtgcacgatcaccgggaccatgggacactttattctcgcccgatgcccgaaagg agagacgctgacagtgggatttacggacagcagaaagatcagccacacatgcacacacc cgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacat ggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagat agaggtgcatatgcccccagatactcctgaccgcacgctgatgacgcagcagtctggca acgtgaagatcacagttaatgggcagacggtgcggtacaagtgcaactgcggtggctca aacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgcca tgctgcagtcactaatcacaagaattggcaatacaactcccctttagtcccgcgcaacg ctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtgact tgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccat gctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaa attaccacgaggagtgggtgacacacaagaaggaggttaccttgaccgtgcctactgag ggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctac gaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtacc ccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggc acagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatatgaattaac accaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaagg cggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctgg ttgcaggctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactctt gccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgcccacactg tgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagact cttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcac cttggaaccaacactgtcacttgactacatcacgtgcgagtacaaaactgtcatcccct ccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagactac agctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttg cgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaa cagagtttgcatcggcctacagagcccacaccgcatcggcgtcggcgaagctccgcgtc ctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcac agtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgaca acaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgca ggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgttta tgccaacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatact ctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcac acggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgt ggggaacataccaatttccatcgacataccggatgcggcctttactagggttgtcgatg caccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgacttt gggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattc gatgaccaacgccgttaccattcgagaagccgacgtagaagtagaggggaactcccagc tgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgcaagtgtgctcc acacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtcaattaccc agcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgc agaagattacgggaggagtaggattaattgttgctgttgctgccttaattttaattgtg gtgctatgcgtgtcgtttagcaggcactaa.

Regarding Chikungunya virus, another exemplary nucléotide sequence that encodes a Capsid-envelope is described below (SEQ ID No.:7):

atggagttcatcccaacccaaactttttacaataggaggtaccagcctcgaccctggac tccgcgccctactatccaagtcatcaggcccagaccgcgccctcagaggcaagctgggc aacttgcccagctgatctcagcagttaataaactgacaatgcgcgcggtaccacaacag aagccacgcaggaatcggaagaataagaagcaaaagcaaaaacaacaggcgccacaaaa caacacaaatcaaaagaagcagccacctaaaaagaaaccggctcaaaagaaaaagaagc cgggccgcagagagaggatgtgcatgaaaatcgaaaatgattgtattttcgaagtcaag cacgaaggtaaggtaacaggttacgcgtgcctggtgggggacaaagtaatgaaaccagc acacgtaaaggggaccatcgataacgcggacctggccaaactggcctttaagcggtcat ctaagtatgaccttgaatgcgcgcagatacccgtgcacatgaagtccgacgcttcgaag ttcacccatgagaaaccggaggggtactacaactggcaccacggagcagtacagtactc aggaggccggttcaccatccctacaggtgctggcaaaccaggggacagcggcagaccga tcttcgacaacaagggacgcgtggtggccatagtcttaggaggagctaatgaaggagcc cgtacagccctctcggtggtgacctggaataaagacattgtcactaaaatcacccccga gggggccgaagagtggagtcttgccatcccagttatgtgcctgttggcaaacaccacgt tcccctgctcccagcccccttgcacgccctgctgctacgaaaaggaaccggaggaaacc ctacgcatgcttgaggacaacgtcatgagacctgggtactatcagctgctacaagcatc cttaacatgttctccccaccgccagcgacgcagcaccaaggacaacttcaatgtctata aagccacaagaccatacttagctcactgtcccgactgtggagaagggcactcgtgccat agtcccgtagcactagaacgcatcagaaatgaagcgacagacgggacgctgaaaatcca ggtctccttgcaaatcggaataaagacggatgacagccacgattggaccaagctgcgtt atatggacaaccacatgccagcagacgcagagagggcggggctatttgtaagaacatca gcaccgtgtacgattactggaacaatgggacacttcatcctggcccgatgtccaaaagg ggaaactctgacggtgggattcactgacagtaggaagattagtcactcatgtacgcacc catttcaccacgaccctcctgtgataggtcgggaaaaattccattcccgaccgcagcac ggtaaagagctaccttgcagcacgtacgtgcagagcaccgccgcaactaccgaggagat agaggtacacatgcccccagacacccctgatcgcacattaatgtcacaacagtccggca acgtaaagatcacagtcaatggccagacggtgcggtacaagtgtaattgcggtggctca aatgaaggactaacaactacagacaaagtgattaataactgcaaggttgatcaatgtca tgccgcggtcaccaatcacaaaaagtggcagtataactcccctctggtcccgcgtaatg ctgaacttggggaccgaaaaggaaaaattcacatcccgtttccgctggcaaatgtaaca tgcagggtgcctaaagcaaggaaccccaccgtgacgtacgggaaaaaccaagtcatcat gctactgtatcctgaccacccaacactcctgtcctaccggaatatgggagaagaaccaa actatcaagaagagtgggtgatgcataagaaggaagtcgtgctaaccgtgccgactgaa gggctcgaggtcacgtggggcaacaacgagccgtataagtattggccgcagttatctac aaacggtacagcccatggccacccgcatgagataattctgtattattatgagctgtacc ccactatgactgtagtagttgtgtcagtggccacgttcatactcctgtcgatggtgggt atggcagcggggatgtgcatgtgtgcacgacgcagatgcatcacaccgtatgaactgac accaggagctaccgtccctttcctgcttagcctaatatgctgcatcagaacagctaaag cggccacataccaagaggctgcgatatacctgtggaacgagcagcaacctttgttttgg ctacaagcccttattccgctggcagccctgattgttctatgcaactgtctgagactctt accatgctgctgtaaaacgttggcttttttagccgtaatgagcgtcggtgcccacactg tgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagact ctagtcaatagacctggctacagccccatggtattggagatggaactactgtcagtcac tttggagccaacactatcgcttgattacatcacgtgcgagtacaaaaccgtcatcccgt ctccgtacgtgaagtgctgcggtacagcagagtgcaaggacaaaaacctacctgactac agctgtaaggtcttcaccggcgtctacccatttatgtggggcggcgcctactgcttctg cgacgctgaaaacacgcagttgagcgaagcacacgtggagaagtccgaatcatgcaaaa cagaatttgcatcagcatacagggctcataccgcatctgcatcagctaagctccgcgtc ctttaccaaggaaataacatcactgtaactgcctatgcaaacggcgaccatgccgtcac agttaaggacgccaaattcattgtggggccaatgtcttcagcctggacacctttcgaca acaaaattgtggtgtacaaaggtgacgtctataacatggactacccgccctttggcgca ggaagaccaggacaatttggcgatatccaaagtcgcacacctgagagtaaagacgtcta tgctaatacacaactggtactgcagagaccggctgtgggtacggtacacgtgccatact ctcaggcaccatctggctttaagtattggctaaaagaacgcggggcgtcgctgcagcac acagcaccatttggctgccaaatagcaacaaacccggtaagagcggtgaactgcgccgt agggaacatgcccatctccatcgacataccggaagcggccttcactagggtcgtcgacg cgccctctttaacggacatgtcgtgcgaggtaccagcctgcacccattcctcagacttt gggggcgtcgccattattaaatatgcagccagcaagaaaggcaagtgtgcggtgcattc gatgactaacgccgtcactattcgggaagctgagatagaagttgaagggaattctcagc tgcaaatctctttctcgacggccttagccagcgccgaattccgcgtacaagtctgttct acacaagtacactgtgcagccgagtgccaccccccgaaggaccacatagtcaactaccc ggcgtcacataccaccctcggggtccaggacatctccgctacggcgatgtcatgggtgc agaagatcacgggaggtgtgggactggttgttgctgttgccgcactgattctaatcgtg gtgctatgcgtgtcgttcagcaggcactaa.

In one embodiment, the présent invention provides a vector comprising the nucleic acid molécule as described above, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molécule.

Examples of an expression control sequence include, but are not limited to, promoter such as CMV promoter, phage lambda PL promoter, the E. coli lac, phoA and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs.

In this embodiment, the vector comprising an expression control sequence operably linked to the nucleic acid molécule as described above can be used as an expression vector for preparing the particle provided by the first aspect of the présent invention.

The expression vectors can be prepared by a person skilled in the art based on

WO/2012/006180, the entire contents of which are incorporated by reference herein.

Examples of vectors which can be used for expressing a virus like particle comprising a fusion protein of a polypeptide derived from Chikungunya virus (CHIKV) and a polypeptide of antigen include a vector shown in VLP_CHI 512 vector (SEQ ID No.:8) containing CHIKV VLP polynucleotide (SEQ ID No. 13; corresponding amino acid sequence represented by SEQ ID No.:14); and VLP_CHI 532 vector (SEQ ID No.: 9) containing CHIKV VLP polynucleotide (SEQ ID No. 15; corresponding amino acid sequence represented by SEQ ID No.:16).

The expression vectors can be prepared by a person skilled in the art based on

US2012/0003266, the entire contents of which are incorporated by reference herein.

Examples of vectors which can be used for expressing a virus like particle comprising a fusion protein of a polypeptide derived from Venezuelan equine encephalitis virus (VEEV) and a polypeptide of antigen include a vector shown in VLP_VEEV VLP 518 vector (SEQ ID No.:10) containing VEEV VLP polynucleotide (SEQ ID No. 17; corresponding amino acid sequence represented by SEQ ID No.:18); VLP_VEEV VLP 519 vector (SEQ ID No.11) containing VEEV VLP polynucleotide (SEQ ID No. 19; corresponding amino acid sequence represented by SEQ ID No.:20); and VLP_VEEV VLP 538 vector (SEQ ID No.: 12) containing VEEV VLP polynucleotide (SEQ ID No. 21; corresponding amino acid sequence represented by SEQ ID No.:22).

In one embodiment, the présent invention provides a nucleic acid molécule which is designed for expression of a virus like particle comprising a fusion protein of a polypeptide derived from Chikungunya virus (CHIKV) or Venezuela equine encephalitis virus (VEEV) and a polypeptide derived from malaria antigen, which consists of a nucléotide sequence represented by SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40 or 42.

In one embodiment, the présent invention provides a nucleic acid molécule which is modified from the nucleic acid molécule having a nucléotide sequence represented by any one of SEQ ID Nos.26-27, 29-30, 32-33 or 35-36, 38, 40 or 42. The modified nucleic acid molécule may hâve at least 70%, 75%, 80%, 85%, 90%, 95% or 98% nucléotide sequence identity to the nucleic acid molécule having a nucléotide sequence represented by any one of SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40 or 42. Also, the modified nucleic acid molécule may be a mutant where at most 10% of the amino acids are deleted, substituted, and/or added based on the nucleic acid molécule having a nucléotide sequence represented by any one of SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40 or 42.

(3) Composition or vaccine

In one embodiment, the présent invention provides a composition or vaccine comprising the Alphavirus or Flavivirus virus like particle (e.g. Chikungunya virus like particle or Venezuelan equine encephalitis virus like particle) as described above or the nucleic acid molécule as described above. The content of the Alphavirus or Flavivirus virus like particle and the content of the nucleic acid molécule may be 0.00001-1

Dosage amount of the particle provided in the first aspect of the présent invention (e.g. CHIKV VLP or VEEV VLP) may be 1-500pg/day.

One or more malaria antigens may be used for one composition or one vaccine provided by the third aspect ofthe présent invention.

The composition or vaccine may further comprise a pharmaceutical acceptable carrier and/or adjuvant. Examples of adjuvant include, but are not limited to, aluminium salts, sodium hydroxide, Freund's complété adjuva nt, Freund's incomplète adjuvant and Ribi solution (Sigma Adjuvant system, Sigma-Aldrich). The composition or vaccine provided in the third aspect of the présent invention may contain buffering agent such as dibasic sodium phosphate hydrate, sodium dihydrogen phosphate and sodium chloride; and preserving agent such as thimerosal. In one embodiment, the composition or vaccine is an aqueous solution containing 0.001-1 w/w% of the particle provided in the first aspect of the présent invention (e.g. CHIKV VLP or VEEV VLP), 1-10w/w% of buffering agent, 0.01-1w/w% of adjuvant and 0.00001-0.001 w/w% of preserving agent.

A skilled person can préparé the pharmaceutical composition and vaccine using conventional technique. For example, the particle provided in the first aspect of the présent invention is mixed with buffer solution having physiological pH (e.g. pH 5-9, pH7) to préparé the pharmaceutical composition and vaccine provided in the third aspect of the présent invention.

The pharmaceutical composition of the présent invention may contain a single active ingrédient or a combination of two or more active ingrédients, as far as they are not contrary to the objects of the présent invention. For example, cytokines including chemokines, antibody of cytokines such as anti TNF antibody (e.g. infliximab, adalimumab), anti-VEGF antibody (e.g. bevacizumab and ranibizumab), cytokine receptor antagonist such as anti HER2 antibody (e.g. Trastuzumab), anti EGF receptor antibody (e.g. Cetuximab), anti VEGF aptamer (e.g. Pegaptanib) and immunomodulator such as cyclosporine, tacrolimus, ubenimex may be used for the combination therapy.

In a combination of plural active ingrédients, their respective contents may be suitably increased or decreased in considération of their therapeutic effects and safety.

The term “combination used herein means two or more active ingrédient are administered to a patient simultaneously in the form of a single entity or dosage, or are both administered to a patient as separate entities either simultaneously or sequentially with no spécifie time limits, wherein such administration provides therapeutically effective levels of the two components in the body, preferably at the same time.

In one embodiment, the composition is a vaccine composition including a DNA vaccine. In one embodiment, the DNA vaccine provided by the présent invention comprises CpG containing oligonucleotide.

The composition or vaccine provided in the third aspect of the présent invention can be administered one or more times. When the composition or vaccine provided in the third aspect of the présent invention is administered more than one time, different particle provided in the first aspect of the présent invention (e.g. CHIKV VLP or VEEV VLP) may be used for each of the administration. In one embodiment, combination of immunization using CHIKV VLP provided in the first aspect of the invention and immunization using VEEV VLP provided in the first aspect of the invention is employed. For example, CHIKV VLP provided in the first aspect of the présent invention may be used for the 1 st immunization and VEEV

VLP provided in the first aspect of the présent invention may be used for the 2nd immunization, or VEEV VLP provided in the first aspect of the présent invention may be used for the 1st immunization and CHIKV VLP provided in the first aspect of the présent invention may be used for the 2nd immunization.

A skilled person can détermine timing of immunization using the composition or vaccine provided in the third aspect of the présent invention. For example, 2nd immunization is performed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks after 1st immunization.

In one embodiment, the présent invention provides a kit comprising a vaccine composition comprising the particle provided in the first aspect of the présent invention; and another vaccine composition comprising the particle provided in the first aspect of the présent invention, wherein the particle contained in (a) is a virus like particle which is different from the particle contained in (b). In this embodiment, the particle contained in (a) may be Chikungunya virus like particle and the particle contained in (b) may be Venezuelan equine encephalitis virus like particle.

In one embodiment, the présent invention provides a kit comprising a vaccine composition comprising the particle provided in the first aspect of the présent invention; and another vaccine composition comprising the particle provided in the first aspect of the présent invention, one or more vaccine composition, each of which comprises the particle provided in the first aspect of the présent invention, wherein (a) is used for priming immunization and (b) and (c) are used for boosting immunization; and the particle contained in (a) is a virus like particle which is different from the particle contained in (b); and the particle contained in (c) is different from the particle contained in (a) and (b), or the same as the particle contained in (a) or (b).

The respective vaccine compositions contained in the above-described kit may be administered simultaneously, separately or sequentially.

The Alphavirus or Flavivirus virus like particle (e.g. Chikungunya virus or Venezuelan equine encephalitis virus) provided in the first aspect of the présent invention or the nucleic acid molécule provided by the second aspect of the invention can be used for the composition and vaccine provided in the third aspect of the présent invention.

For example, Chikungunya or Venezuelan equine encephalitis virus like particle comprising or consisting of:

one or more (e.g. 240) capsid of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV);

one or more (e.g. 240) E1 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV); and one or more (e.g. 240) E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), wherein malaria antigen is inserted into E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) may be used for preparing the composition or vaccine provided in the third aspect of the présent invention. The E2 into which the antigen is inserted may consist of an amino acid sequence represented by SEQ ID No.50; the E1 may consist of an amino acid sequence represented by SEQ ID No.51; and the capsid may consist of an amino acid sequence represented by SEQ ID NO.: 52; or the E2 into which the antigen is inserted may consist of an amino acid sequence represented by SEQ ID NO.53; the E1 may consist of an amino acid sequence represented by SEQ ID NO.54; and the capsid may consist of an amino acid sequence represented by SEQ ID NO.: 55.

The composition or vaccine provided in the third aspect of the présent invention can be administered to a mammal (e.g. human) intramuscularly (i.m.), intracutaneously (i.c.), subcutaneously (s.c.), intradermally (i.d.) or intraperitoneally (i.p.).

The composition or vaccine provided in the third aspect of the présent invention may be used for treating or preventing malaria.

Thus, use of the Alphavirus or Flavivirus (e.g. Chikungunya virus or Venezuelan equine encephalitis virus) virus like particle provided in the first aspect of the présent invention or the nucleic acid molécule provided by the second aspect of the invention for manufacturing a pharmaceutical composition or vaccine for treating or preventing malaria is also provided by the présent invention.

(4) Method of producing an antibody, Method of immunomodulation, Method of treating malaria, Method of inducinq and/or enhancing immune response against a malaria antigen in a mammal, Method of passive immunization, Method of presenting an antigen on macrophage, and Method for producing a particle

The antibody produced in the fourth aspect of the présent invention may be humanized using a conventional technique. Thus, in one embodiment, the method provided in the fourth aspect of the invention further comprises a step of humanizing non-human mammal produced antibody.

The particle provided in the first aspect of the présent invention and/or the nucleic acid molécule provided in the second aspect of the présent invention may be administered directly into the patient, into the affected organ or systemically, or applied ex vivo to cells derived from the patient or a human cell line which are subsequently administered to the patient, or used in vitro to select a subpopulation from immune cells such as B-cell and Tcell derived from the patient, which are then re-administered to the patient.

According to the présent invention, the virus like particle can be applied for the immune therapy.

In the fifth aspect, the présent invention provides a method of immunomodulation, a method of treating malaria, a method of inducing and/or enhancing immune response against a malaria antigen in a mammal comprising administering the composition provided in the third aspect of the présent invention to a mammal.

In seventh aspect, the présent invention provides a method of presenting a malaria antigen on macrophage, comprising contacting the particle provided in the first aspect of the présent invention and/or the nucleic acid molécule provided in the second aspect of the présent invention to a mammal.

In eighth aspect, the présent invention provides a method for producing the particle provided in the first aspect of the présent invention, comprising preparing a vector designed for expression of said particle; culturing a cell which is transfected with said vector to express said particle; and recovering said particle.

Examples of mammal include, but are not limited to, a human.

In one embodiment, the présent invention provides a method of producing an antibody against malaria antigen, comprising contacting the Chikungunya or Venezuelan equine encephalitis virus like particle as described above and/or the nucleic acid molécule as described above to a mammal. The produced antibody may be an antibody which can specifically bind to a malaria antigen comprised in the Chikungunya or Venezuelan equine encephalitis virus like particle or a malaria antigen encoded by the nucleic acid molécule. The method of producing an antibody provided by the présent invention may be a useful for producing a monoclonal or polyclonal antibody against a malaria antigen.

In one embodiment, an antibody against malaria antigen obtained by the method of producing an antibody according to the présent invention is used for passive immunization. The method of passive immunization may comprise administering the obtained antibody to a mammal.

In one preferred embodiment, the immunomodulation provided by the présent invention is inducing and/or enhancing immune response against malaria antigen in a mammal. Thus, in one embodiment, the présent invention provides a method of inducing and/or enhancing immune response against malaria antigen in a mammal, comprising administering an effective amount of the composition as described above to the mammal.

Given the symptom of patients infected with Chikungunya or Venezuelan equine encephalitis together with unusual big molécule of Chikingunya or Venezuelan equine encephalitis, this VLP can act effectively and efficiently to target macrophage and its composition such as cytokines and immunomodulative compounds.

In one aspect, the présent invention provides a method of presenting an antigen on macrophage, comprising administering the Chikungunya or Venezuelan equine encephalitis virus like particle as described above and/or the nucleic acid molécule as described above to a mammal. The Chikungunya or Venezuelan equine encephalitis virus like particle provided by the présent invention is good to target macrophage. In one embodiment, the Chikungunya or Venezuelan equine encephalitis virus like particle provided by the présent invention is a kind of delivery System of the at least one antigen, which is comprised in the Chikungunya or Venezuelan equine encephalitis virus like particle, to macrophage.

In one embodiment, the présent invention provides a method for producing Chikungunya or Venezuelan equine encephalitis virus like particle provided in the first aspect of the présent invention, comprising preparing a vector designed for expression of said particle; culturing a cell which is transfected with said vector to express said particle; and recovering said particle. In this embodiment, transfection can be conducted using a conventional method. Cells using for the transfection may be 293 cells. Recovering VLP may include collecting a conditioned medium after cells are transfected with a vector, and may further include purify VLP from the conditioned medium using ultracentrifugation.

The présent invention will be described in detail with reference to the following example, which, however, is not intended to limit the scope of the présent invention.

EXAMPLES

EXAMPLE 1: Préparation of Chikungunya virus (CHIKV) like particle comprising a virus structural polypeptide and a fragment of malaria antigen

The following polynucleotides of malaria CSP1 proteins are used. N terminal linker is SGG and C terminal linker is GGS.

VLP74 (6 repeat of NPNA amino acid sequence) Sggnpnanpnanpnanpnanpnanpnaggs (SEQ ID No.:46) (Tccggaggaaacccgaatgccaatcccaacgcgaaccccaatgctaacccaaatgccaacccaaacgccaaccccaac gctggtggatcc) (SEQ ID No.:47)

VLP78 (25 repeat of NPNA amino acid sequence)

Sggnpnanpnanpnanpnanpnanpnvdpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanp nanpnanpnanpnanpnanpnanpnanpnaggs (SEQ ID No.:48) (tccggaggaaacccgaatgccaatcccaacgcgaaccccaacgctaaccccaacgccaatccgaatgcaaacccgaacg ttgacccaaacgccaacccgaatgccaatcccaacgcgaaccccaatgctaacccaaatgccaacccaaacgccaacccc aacgctaatccaaacgccaaccctaacgccaatcccaacgcgaatcctaacgctaatcccaacgcaaatcccaatgctaatc cgaacgcgaaccctaatgcaaaccccaacgccaacccgaacgctaacccgaacgctaatcccaacgccggtggatcc) (SEQ ID No.:49)

The respective polynucleotides was inserted between the codons encoding Ser at 531position and Asn at 532-position of SEQ ID Nos. 15 or 16 (SEQ ID Nos.1 or 2) to construct a plasmid (hereinafter referred to as CHIKV-VLP74 or 78) for expressing Chikungunya virus like particle where the modified VLP74 or 78-derived peptide is inserted into E2 of Chikungunya virus structural polypeptide.

293F cells (Lifetechnology) were transfected with the plasmid using PEI (GE Healthcare) or GeneX (ATCC). 4 days after the transfection, the conditioned medium was collected and centrifuged at 3000rpm for 15 minutes to separate it from cells. The supernatant was filtrated using 0.45pm filter to obtain virus like particles. The virus like particles were concentrated using TFF column and purified using QXL column (GE Healthcare) to obtain purified virus like particles. When animais were immunized with virus like particles, the purified virus like particles were further concentrated using spin column (Molecular Weightcutoff: 100kDa) to préparé the virus like particles for the immunization.

The expression of VLP comprising VLP74 or 78 conjugated with Chikungunya virus structural polypeptide was confirmed by Western Blot using an antibody spécifie for CHIVK (ATCC: VR-1241AF) and an antibody spécifie for VLP74 or 78.

EXAMPLE 2: Préparation of Venezuelan equine encephalitis virus (VEEV) like particle comprising a virus structural polypeptide and a fragment of malaria antigen

The same polynucleotides of malaria CSP1 proteins (VLP74 and VLP78) used in EXAMPLE 1 are used. N terminal linker is SGG and C terminal linker is GGS.

The respective polynucleotides was inserted between the codons encoding Ser at 518position and Ser at 519-position of SEQ ID Nos. 19 or 20 (SEQ ID No.3) to construct a plasmid (hereinafter referred to as VEEV-VLP74 or 78) for expressing Venezuelan equine encephalitis virus like particle where the modified VLP74 or 78 -derived peptide is inserted into E2 of Venezuelan equine encephalitis virus structural polypeptide.

The expression of VLP comprising VLP 74 or 78 conjugated with Venezuelan equine encephalitis virus structural polypeptide was confirmed by Western Blot using an antibody spécifie for VEEV and an antibody spécifie for VLP74 or 78.

EXAMPLE 3:lmmunogenicity in Non-human Primate (Monkey)

The monkeys were immunized with X25-CHI (80ug) at 0 week and x6-VEE (80ug) at week by intramuscular injection with or without adjuvant (Sigma Adjuvant System,

Sigma, S6322). X25-CHI means 25 times malaria CSP repeat amino acid NPNA on CHIKV VLP partide (VLP78_15). X6-VEE means 6 times malaria CSP repeat amino acid NPNA on VEEV VLP partide (VLP74_25). The blood is taken at 2 and 5 weeks after the first immunization.

well ELISA plate were coated with 50ng of Recombinant Circumsporozoite Protein (rCSP) (Reagent Proteins, ATG-422) in 100ul PBS buffer pre well. The Plates after 2 hours incubation were washed three times TBS buffer containing 0.05% Tween-20 and blocked with TBS buffer containing 0.05% Tween-20 and 5% dry milk. The heat inactivated diluted sérum from monkeys were added in the blocking buffer and incubated for 1 h at room température. After washing three times, peroxidase labeled goat anti-human IgG or antimouse IgG was added at 1:4000 dilution and incubated for 1h at room température. After washing three times, Peroxidase substrate was added for development and incubated for 10 mins and 2N H2SO4 was added to stop the development. The data were analyzed using Gen5 (BioTek) and GraphPad Prism6 (GraphPad software Inc).

The Immunogenicities are shown in Figures 4 to 6.

Induction of antibodies against CSP was found in the sérum of ail monkeys immunized with Malaria VLPs (see Figure 4). The mean OD values indicating titer of antibodies against CSP is shown in Figure 5. Figure 5 shows that the sérum from immunized monkeys induced high titer of antibodies against CSP.

As seen in Figure 6, higher titer of antibodies against CSP was achieved when CHIKV VLP partide comprising NPNA and VEEV VLP partide comprising NPNA were used for the priming immunization and boosting immunization, respectively, compared to when only CHIKV VLP partide comprising NPNA was used for both of the priming immunization and the boosting immunization. In addition, Figure 6 shows that use of adjuvant further enhanced the titer of antibodies against CSP. Further, Figure 6 shows that administration of

25-repeats of NPNA induces higher titer of antibodies against CSP compared to administration of 6-repeats of CSP.

The anti-Pf CSP antibody titer in the sérum from the monkeys immunized with x25-CHI (80ug) at 0 week and x6-VEE (80ug) at 3 week without using adjuvant was measured by

ELISA at Malaria Serology Lab Malaria Vaccine Branch, WRAIR. In the ELISA performed at

Malaria Serology Lab Malaria Vaccine Branch, WRAIR, the plates were coated with CSPrp ((NPNA)6 Peptide) [0.2pg/pL] (Supplier: Eurogentec EP070034) and Goat α-Human IgG (KPL/074-1002 LOT# 120714) was used as 2nd antibody. The final titer was determined by the dilution factor that yields an OD of 1.0 (414nm).

As a resuit, the anti-Pf CSP antibody titer in the sérum from the monkeys was enhanced after 2nd immunization compared to 1st immunization (see Table 2). Compared to the antiPf CSP antibody titer in the sérum from the monkeys immunized with RTS,S (GlaxoSmithKIine), the anti-Pf CSP antibody titer in the sérum from the monkeys immunized with x25-CHI (80ug) and x6-VEE (80ug) in the absence of adjuvant was considered to be higher even though RTS,S (GlaxoSmithKIine) contains adjuvant.

[Table 2]

Animal No.	After 1 st immunization Week 2	After 2nd immunization Week 5
1	8990	29420
2	48210	44100
3	80400	51230
4	16260	19640
Géométrie mean	27359	33801

Example 4: Immunoqenicity in mouse

The mice immunized with 10ug of VLP78_15 at week 0, 10ug of VLP74_25 at week3 and 10ug of VLP78_15 at week 6 with or without adjuvant (Sigma Adjuvant System,

Sigma, S6322) by intramuscular injection.

The anti-Pf CSP antibody titer in the sérum from the immunized mice were measured by

ELISA at Malaria Serology Lab Malaria Vaccine Branch, WRAIR, where plates were coated with CSPrp ((NPNA)6 Peptide) [0.2pg/pL] (Supplier: Eurogentec EP070034) and Goat aMouse IgG (KPL/074-1806 LOT# 100737) is used as 2nd antibody to detect the antibodies in the sérum.

The final titer was determined by the dilution factor that yields an OD of 1.0 (414nm).

The Immunogenicity are shown in Tables 3 and 4.

Tables 3 and 4 show that higher titer of antibodies against CSP was achieved after immunizing virus like particle three times. In addition, Tables 3 and 4 show that use of adjuvant enhanced the titer of antibodies against CSP.

[Table 3]

Mouse Week 3 (after 1^st immunization)

Mouse No.	VLP	VLP+Adjuvant
1	6070	QNS
2	5850	13680
3	9610	7610
4	5440	23370
5	16320	27390
Géométrie Mean	7875	16066

[Table 4]

Mouse Week 9 (after 1 st immunization)

GNS=Quantity not sufficient to test

Mouse No.	VLP	VLP+Adjuvant
1	35510	729000
2	15040	197800
3	41650	106700
4	37250	436000
5	48200	497600
Géométrie Mean	33134	319666

Example 5: Irnmunoqenicity of P. yoelii CSP inserted VLP in mice

QGPGAP seen in the rodent malaria CSP (P.yoelii CSP) was used as an antigen.

6x QGPGAP was inserted into CHIKV VLP. The mice were immunized with the CHIKV VLP 2 times at 0 and 8 week (20ug VLP per mouse) by intramuscle injection with or without Adjuvant Ribi.

Immunogenicity was confirmed at 4, 6, 10 and 14 weeks after the first immunization. The anti- P. yoelii CSP antibody were measured by ELISA. The ELISA was performed in the same way as ELISA described in Example 3 except that the plates were coated with P. Yoelii CSP repeat sequence peptide at 0.1 ng/μΙ. The secondary antibody was anti-mouse IgG HRP (Cell signal, #7076S). The results are shown in Figures 7-9.

Figures 7-9 show that higher titer of antibodies against CSP was achieved by intramuscler administration of CHIKV VLP comprising 6X QGPGAP. In addition, Figures 7-9 show that use of adjuvant enhanced the titer of antibodies against CSP.

Example 6: Protection of mice against malaria by intramuscle injection of CHIKV VLP comprising P. yoelii CSP epitope: 6x QGPGAP

6x QGPGAP was inserted into CHIKV VLP. The mice (n=5) were immunized with the CHIKV VLP 2 times at 0 and 8 week (20ug VLP per mouse) by intramuscle injection with or without Adjuvant Ribi (see Figure 10). Rodent malaria: P. yoeliichallenge (i.v.) was conducted at 17 weeks(see Figure 10).

Malaria infection was confirmed by PCR. Genomic DNA was purified from the mice blood day 6 after challenge. 18S malaria DNA was amplified by PCR. Figure 11 shows results of the PCR, indicating that ail of 5 control mice (PBS injection) were infected with malaria; ail of 5 mice immunized with Control VLP were infected with malaria; among 5 mice immunized with CHIKV VLP comprising 6x QGPGAP, 2 mice were infected with malaria and 3 mice were not infected with malaria; and among 5 mice immunized with CHIKV VLP comprising 6x QGPGAP with adjuvant: Ribi, 1 mouse was infected with malaria and 4 mice were not infected with malaria.

Example 7: Préparation of vaccine composition comprising Chikunqunya virus (CHIKV) like particle comprising NPNA repeat or Venezuelan equine encephalitis virus (VEEV) like particle comprising NPNA repeat

Chikungunya virus (CHIKV) like particle comprising 6x or 25x NPNA was prepared according to Example 1, and Venezuelan equine encephalitis virus (VEEV) like particle comprising 6x NPNA was prepared according to Example 2.

To préparé a vaccine composition, 80pg of each of the prepared particles was mixed with

1ml of Sucrose Phosphate Solution, pH 7.2, Endotoxin Free (Teknova, SP buffer).

Claims

1. A particle comprising a virus structural polypeptide and at least one malaria antigen, wherein said virus structural polypeptide comprises at least one first attachment site and said at least one malaria antigen comprises at least one second attachment site, and wherein said virus structural polypeptide and said malaria antigen are linked through said at least one first and said at least one second attachment site, and wherein said particle is virus like particle.

2. The particle according to Claim 1, wherein said virus structural polypeptide comprises the capsid and/or the envelope proteins E1 and E2.

3. The particle according to Claim 2, wherein said at least one malaria antigen is inserted into E2 of the envelope protein.

4. The particle according to any one of Claims 1-3, wherein said virus structural polypeptide is a polypeptide derived from Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV).

5. The particle according to any one of Claims 1-4, wherein said at least one malaria antigen is a fragment derived from the circumsporozoite protein.

6. The particle according to any one of Claims 1-5, wherein said at least one malaria antigen is an antigen comprising (NPNA)n wherein n is from 4 to 30 and/or an antigen comprising (EYLNKIQSLSTEWSPCSVT)y wherein y is from 1 to 6.

1. An isolated nucleic acid molécule comprising a nucléotide sequence for expressing the particle according to any one of Claims 1-6.

8. A vector comprising the nucleic acid molécule according to Claim 7, wherein the vector

5 optionally comprises an expression control sequence operably linked to the nucleic acid molécule.

9. A pharmaceutical composition comprising:

(a) the particle according to any one of Claims 1-6 and/or the nucleic acid molécule according to

10 Claim 7; and (b) a pharmaceutically acceptable carrier.

10. A vaccine for use in the inducing immunomodulation, inducing and/or enhancing immune response against a malaria antigen, or treating malaria in a mammal, comprising, as separate

15 components, a priming composition comprising the particle according to any one of claims 1-6 which comprises at least one first malaria antigen or at least one polynucleotide encoding at least one first malaria antigen; and a boosting composition comprising the particle according to any one of claims 1-6 which comprises at least one polypeptide comprising at least one second malaria antigen or at least one polynucleotide encoding at least one second malaria antigen.