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BR-112018073794-B1 - PARTICLE, PARTICLE GENERATING METHOD, VACCINE OR PHARMACEUTICAL COMPOSITION

BR112018073794B1BR 112018073794 B1BR112018073794 B1BR 112018073794B1BR-112018073794-B1

Abstract

This patent application relates to a Pichinde virus with rearrangements of the open reading frames (ORFs) in its genomes. Specifically, the present invention describes a modified genomic segment of the Pichinde virus, wherein the genomic segment of the Pichinde virus is constructed to carry a viral ORF at a position different from the position of the wild-type ORF. Furthermore, trisegmented particles of the Pichinde virus comprising one L segment and two S segments or two L segments and one S segment are also described. The Pichinde virus described herein may be suitable for vaccines and/or the treatment of diseases and/or for use in immunotherapies.

Inventors

  • Weldi Bonilla
  • DANIEL DAVID PINSCHEWER
  • Klaus Orlinger

Assignees

  • UNIVERSITÄT BASEL
  • HOOKIPA BIOTECH GMBH

Dates

Publication Date
20260310
Application Date
20170517
Priority Date
20160518

Claims (9)

  1. 1. Trisegmented particle of Pichinde virus characterized by comprising: one L segment and two S segments, wherein the first S segment comprises an RNA corresponding to SEQ ID NO:8, comprising an open reading frame ("ORF") encoding the Pichinde viral glycoprotein ("GP") at a position under the control of a 3' untranslated genomic region ("UTR") of the Pichinde virus and an ORF encoding a first gene of interest at a position under the control of a 5' genomic UTR of the Pichinde virus, and the second S segment comprises an RNA corresponding to SEQ ID NO:7, comprising an ORF encoding the nucleoprotein ("NP") at a position under the control of a 3' genomic UTR of the Pichinde virus and an ORF encoding a second gene of interest at a position under the control of a 5' genomic UTR of the Pichinde virus, and the L segment comprises an RNA corresponding to SEQ ID NO:2, comprising an ORF encoding RNA-dependent RNA polymerase L. ("L protein") at a position under the control of a 3' genomic UTR of the Pichinde virus and an ORF encoding the Z matrix protein ("Z protein") at a position under the control of a 5' genomic UTR of the Pichinde virus.
  2. 2. Trisegmented particle of Pichinde virus, according to claim 1, characterized in that the genomic 3’ UTR of Pichinde virus is the 3’ UTR of the S segment of Pichinde virus or the L segment of Pichinde virus, and in that the genomic 5’ UTR of Pichinde virus is the 5’ UTR of the S segment of Pichinde virus or the L segment of Pichinde virus.
  3. 3. Pichinde virus trisegmented particle, according to claim 1 or 2, characterized in that the first and/or second gene of interest encodes an antigen derived from an infectious organism, tumor or allergen.
  4. 4. Pichinde virus trisegmented particle, according to claim 3, characterized in that an antigen is selected from human immunodeficiency virus antigens, papillomavirus antigens, hepatitis C virus antigens, hepatitis B virus antigens, varicella-zoster virus antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, tumor-associated antigens and tumor-specific antigens, wherein the tumor-specific antigens comprise tumor neoantigens and tumor neoepitopes.
  5. 5. Trisegmented particle of Pichinde virus, according to any one of claims 1 to 4, characterized in that the trisegmented particle of Pichinde virus is derived from the Munchique CoAn4763 strain isolate P18, or from the P2 strain.
  6. 6. Method for generating the trisegmented Pichinde virus particle, as defined in any one of claims 1 to 5, the method characterized by comprising: (i) transfecting one or more cDNAs from one L segment and two S segments into a host cell; (ii) maintaining the host cell under conditions suitable for virus formation; and (iii) harvesting the Pichinde virus particle.
  7. 7. Method according to claim 6, characterized in that the transcription of said one L segment and two S segments is performed using a bidirectional promoter.
  8. 8. Method according to claim 6 or 7, characterized in that the transcription of the L segment and the two S segments are each under the control of a promoter selected from the group consisting of: (i) an RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) a T7 promoter.
  9. 9. Vaccine or pharmaceutical composition characterized by comprising the Pichinde virus particle as defined in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

Description

[001] This patent application claims the benefit of priority over U.S. Provisional Patent Application No. 62/338,400, filed May 18, 2016, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO THE LIST OF SEQUENCES SUBMITTED ELECTRONICALLY [002] This patent application incorporates by reference a Sequence Listing, submitted with this patent application as a text file entitled “Sequence_Listing_13194-020-228.TXT”, created on May 16, 2017 and with a size of 61,423 bytes. 1. INTRODUCTION [003] This patent application relates to Pichinde viruses with rearrangements of the open reading frames (“ORFs”) in their genomes. Specifically, a modified genomic segment of the Pichinde virus is described herein, in which the genomic segment of the Pichinde virus is constructed to carry a viral ORF at a position different from the position of the wild-type ORF. In addition, trisegmented particles of the Pichinde virus comprising one L segment and two S segments or two L segments and one S segment are also described. The Pichinde virus described herein may be suitable for vaccines and/or the treatment of diseases and/or for use in immunotherapies. 2. BACKGROUND OF THE INVENTION 2.1 General background and genomic organization of the Pichinde virus [004] Pichinde virus is an arenavirus isolated from Oryzomys albigularis (rice mice) in Colombia (reviewed in McLay et al, 2014, Journal of General Virology, 95: 1-15). Pichinde virus is non-pathogenic and is generally not known to cause disease in humans. Serological evidence suggests a very low seroprevalence even in the local human population (Trapido et al, 1971, Am J Trop Med Hyg, 20: 631-641). The Arenaviridae family is classified into two groups: Old World (OW) arenaviruses, such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV), and New World (NW) arenaviruses, such as Pichinde virus and Junin virus (Buchmeier et al., 2001, Arenaviridae: The Viruses and Their Replication, Fields Virology Vol. 2, 1635-1668). Arenaviruses are enveloped RNA viruses. Their genome consists of two segments of negative-sense single-stranded RNA (Figure 1A) (McLay et al., 2014, Journal of General Virology, 95: 1-15). Each segment encodes two viral genes in opposite orientations. The short segment (S segment) encodes the viral glycoprotein (GP) and nucleoprotein (NP). The long segment (L segment) expresses RNA-dependent RNA polymerase (RdRp; L protein) and matrix Z protein (Z protein), a RING finger protein. The two genes in each segment are separated by a non-coding intergenic region (IGR) and flanked by 5’ and 3’ untranslated regions (UTRs). The IGR forms a stable hairpin-shaped structure and has been shown to be involved in structure-dependent termination of viral mRNA transcription (Pinschewer et al., 2005, J Virol 79(7): 4519-4526). The terminal nucleotides of the UTR show a high degree of complementarity, thus being considered to result in the formation of secondary structures. These panhandle-like structures are known to serve as the viral promoter for transcription and replication, and their analysis by site-directed mutagenesis has revealed sequence and structure dependence, with even minimal sequence alterations not being tolerated (Perez and de la Torre, 2003, Virol 77(2): 1184-1194). 2.2 Reverse Genetics System [005] RNAs isolated and purified from negative-strand viruses, such as Pichinde virus, cannot directly serve as mRNA, i.e., they cannot be translated when introduced into cells. Consequently, transfection of cells with viral RNA does not lead to the production of infectious viral particles. In order to generate infectious viral particles from negative-strand RNA viruses from cDNA in cultured permissive cells, the viral RNA segment(s) must be trans-complemented with the minimum factors required for transcription and replication. With the help of a minigenome system that was published several years ago, cis-acting viral elements and trans-acting factors involved in transcription, replication, and viral particle formation could finally be analyzed (Lee et al., 2000, J Virol 74(8): 3470-3477; Lee et al., 2002, J Virol 76(12): 6393-6397; Perez and de la Torre 2003, J Virol 77(2): 1184-1194; Pinschewer et al., 2003, J Virol 77(6): 3882-3887; Pinschewer et al., 2005, J Virol 79(7): 4519-4526). Such reverse genetics systems have been developed and have been able to demonstrate rescue of the Pichinde virus (See, e.g., Liang et al, 2009, Ann N Y Acad Sci, 1171: E65-E74; Lan et al, 2009, Journal of Virology, 83 (13): 63576362). 2.3 Recombinant Pichinde virus expressing genes of interest [006] The generation of recombinant negative-strand RNA viruses expressing exogenous genes of interest has been pursued for a long time. Different strategies have been published for other viruses (Garcia-Sastre et al., 1994, J Virol 68(10): 6254-6261; Percy et al., 1994, J Virol 68(7): 4486-4492; Flick and Hobom, 1999, Virology 262(1): 93103; Machado