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WO 2015/101666 Al 9 July 2015 (09.07.2015) W P O P C T

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WO 2015/101666 Al 9 July 2015 (09.07.2015) W P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/101666 Al 9 July 2015 (09.07.2015) W P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 39/12 (2006.01) A61K 39/005 (2006.01) kind of national protection available): AE, AG, AL, AM, C07K 14/005 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/EP20 15/050054 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 5 January 2015 (05.01 .2015) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 14382001 .7 3 January 2014 (03.01 .2014) EP kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicants: FUNDACION BIOFISICA BIZKAIA TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, [ES/ES]; Barrio Sarriena, s/n, E-48940 Leioa- Vizcaya TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (ES). UNIVERSIDAD DEL PAIS VASCO [ES/ES]; Bar DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, rio Sarriena, s/n, E-48940 Leioa- Vizcaya (ES). CON- LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SEJO SUPERIOR DE INVESTIGACIONES SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, CIENTIFICAS [ES/ES]; Serrano, 117, E-28006 Madrid GW, KM, ML, MR, NE, SN, TD, TG). (ES). Published: (72) Inventors: RODRIGUEZ AGUIRRE, Jose Francisco; — with international search report (Art. 21(3)) Albadalejo, 2, E-28037 Madrid (ES). GUERIN AGUILAR, Diego Marcelo; Barrio la venta del Sol, 4, E- — before the expiration of the time limit for amending the 48800 Zalla- Vizcaya (ES). claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (74) Agent: ALCONADA RODRIGUEZ, Agustin; ABG Pat entee, S.L, Avenida de Burgos 16D, Edificio Euromor, E- — with sequence listing part of description (Rule 5.2(a)) 28036 Madrid (ES). o (54) Title: VLPs, METHODS FOR THEIR OBTENTION AND APPLICATIONS THEREOF (57) Abstract: The present invention belongs to the field of virus and virus-like particles (VLPs) as is related to VLPs derived from Triatoma virus(TrV) comprising a heterologous polypeptide, a process for obtaining a Triatoma virus VLP modified to express a heterologous protein of interest, polynucleotides comprising the necessary elements for generation of recombinant TrV virions and the process for obtention of said polynucleotides. The invention relates as well to a process for obtaining infectious TrV particles. VLPs, METHODS FOR THEIR OBTENTION AND APPLICATIONS THEREOF FIELD OF THE INVENTION The present invention belongs to the field of virus and virus-like particles (VLPs) as it relates to VLPs derived from Triatoma virus (TrV) comprising a heterologous polypeptide, a process for obtaining a Triatoma virus VLP modified to express a heterologous protein of interest, polynucleotides comprising the necessary elements for generation of recombinant TrV virions and the process for obtention of said polynucleotides. BACKGROUND OF THE INVENTION The use of vaccines based on attenuated or inactivated forms of the whole pathogens has been the traditional strategy for inducing human and animal immunization. Alternatively, subunits of pathogens such as proteins, sugars and peptides, has been also employed as vaccines, but due to their poor immunogenicity and in order to stimulate the immune response, vaccines based on small portion of an antigen have to be formulated with adjuvants. Unfortunately, current adjuvants cause toxicity and other side effects such as inflammation. A more recent and promising method to produce vaccines is to introduce antigen epitopes in VLPs. Although this approach has some technical limitations, like the size and accessibility of the epitope, and unpredictable modifications of the native viral capsid, some remarkable achievements have shown the potency of this technology to the design and production of vaccines. It is also well established that VLPs are potent inducers of immune response for both themselves and also against any antigen displayed on them. The possibility of producing VLPs recombinantly, achieving high yield in a short time, and at low cost of goods, makes this technology for producing vaccines very promising. Virus-like particles (VLPs) are structures built in an organized and geometrically regular manner from many polypeptide molecules of one or more types. Being comprised of more than one molecule, VLPs can be referred to as being supramolecular. VLPs lack the viral genome and, therefore, are noninfectious. VLPs can be produced in large quantities by heterologous expression and can be easily purified. The geometry of a VLP typically resembles the geometry of the source virus particle. VLPs are being exploited in the area of vaccine production because of their structural properties, their ease in large scale preparation and purification, and their non-infectious nature. Examples of VLPs include the self-assemblages of capsid or nucleocapsid polypeptides of hepatitis B virus, Sindbis virus, hepatitis C virus, rotavirus, Norwalk virus, retroviruses, retrotransposons and human papilloma virus. Plant-infecting virus capsid polypeptides also self-assemble into VLPs in vitro and in vivo. The structural components of some VLPs have also proven amenable to the insertion or fusion of foreign antigenic sequences, allowing the production of chimeric VLPs exposing the foreign antigen on their surface. Other VLPs have been used as carriers for foreign antigens, including non-protein antigens, via chemical conjugation. Methods for generating VLPs linked to antigens through protein-protein interaction have been described in WO 201 1057134 Al. Similarly, antigen presenting systems based on VLP particles have been described in the art. WO 1996030523 A2 describes an antigen presentation system based on retrovirus-like particles, WO 201 1091279 A2 describes antigen presentation based on calicivirus VLPs, WO 20101 18424 A2 describes a VLP based on papilloma virus LI major capsid protein comprising a neutralizing epitope of a papillomavirus L2 protein, WO 201 1102900 Al describes VLPs that display one or more truncated, re-engineered or remodeled hemagglutinin molecules on their surface. However, the insertion of epitopes into particular VLPs remains fairly unpredictable despite some remarkable successes. Major limitations include accessibility of the epitope, size limitations, and perturbation of the structure of the viral protein by the inserted epitope, and enforcement of a non-native structure on the epitope. In addition, some of the viral carriers face major challenges for regulatory approval due to infectivity or presence of DNA and resistance genes. However, despite the efforts made to date, there still exists a continuing need in the art for novel ways of introducing antigen epitopes in VLPs compounds, as well as developing VLPs useful in eliciting an immune response against particular antigen epitopes. SUMMARY OF THE INVENTION The inventors have developed a system for heterologous antigen presentation based on VLPs derived from the insect viral pathogen Triatoma virus (TrV) (Dicistroviridae:Cripavirus, Dicistroviridae family, Cripavirus genus). This antigen presenting system, called TrV-VLP (or TRWLP), allows epitope exposure on the internal or external surface of the VLPs by insertion or substitution of amino acids, thus facilitating their recognition by the immune system and/or increasing their immunogenicity. Furthermore, production of TrV-VLPs bearing different epitopes may occur through the expression in insect cells by infection of recombinant baculoviruses. In a first aspect, the present invention relates to a VLP comprising (i) the viral structural proteins VPl, VP2, VP3 and VP4 of a virus of the Dicistroviridae family, or the viral structural proteins VPl, VP2, and VPO of a virus of the Dicistroviridae family, or the structural protein precursor (PI) from a virus of the Dicistroviridae family, or a functionally equivalent variant any thereof, and (ii) a heterologous polypeptide, wherein said heterologous polypeptide is provided as a fusion protein with at least one of said viral structural proteins. In a second aspect, the present invention relates to a polynucleotide encoding a fusion protein selected from the group consisting of (i) a fusion protein comprising a structural protein from a virus of the Dicistroviridae family selected from the group consisting of VPl, VP2, VP3, VP4 and VPO, or a functionally equivalent variant thereof, and a heterologous polypeptide, or (ii) a fusion protein comprising a structural protein precursor (PI) from a virus of the Dicistroviridae family, or a functionally equivalent variant thereof, and a heterologous polypeptide. In a further aspect, the invention relates to a vector comprising the polynucleotide encoding a fusion protein as above. In a further aspect, the invention relates to a host cell comprising the polynucleotide encoding a fusion protein or the vector as above. In a further aspect, the present invention relates to a process for obtaining a Dicistroviridae family virus VLP modified to express a heterologous polypeptide of interest comprising the steps of: (i) contacting a host cell with a first polynucleotide which encodes a fusion protein comprising a heterologous polypeptide of interest and the structural protein precursor (PI) of a virus of the Dicistroviridae family or a functionally equivalent variant thereof, and (ii) maintaining the cells under conditions suitable for the expression of the polynucleotide introduced in the cell in step (i).
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