Tri-segmented pichinde viruses as vaccine vectors

Abstract

To provide Pichinde viruses with rearrangements of their open reading frames ("ORF") in their genomes.SOLUTION: Described herein is a modified Pichinde virus genomic segment, in which the Pichinde virus genomic segment is engineered to carry a viral ORF in a position other than the wild-type position of the ORF. Also described herein are trisegmented Pichinde virus particles comprising one L segment and two S segments or two L segments and one S segment. The Pichinde virus, described herein, may be suitable for vaccines and / or treatment of diseases and / or for the use in immunotherapies.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62 / 338,400, filed May 18, 2016. No. 6,393,633, the entire contents of which are hereby incorporated by reference.

[0002] Reference to Electronically Submitted Sequence Listing The application was created on May 16, 2017, has a size of 61,423 bytes, and is based on the Sequence_Listin The sequence information submitted with this application as a text file entitled "SEQ ID NO: 13194-020-228.TXT" The Tables are incorporated by reference.

[0003] 1. Introduction This application relates to a recombinant human ovarian genome having a rearrangement of its open reading frame ("ORF") within the genome. In particular, the present specification relates to a modified Pichinde virus. A viral genome segment, the Pichinde virus genome segment being a virus The ORF has been engineered to carry an ORF in a position other than the wild-type position of that ORF. The document provides one L segment and two S segments, or two L segments and The present invention relates to a three-segmented Pichinde virus particle, which comprises one S segment and one H segment. The Pichinde virus described herein can be used in vaccines and / or or may be suitable for the treatment of disease and / or for use in immunotherapy. [Background technology]

[0004] 2. Background 2.1 General background and genomic organization of Pichinde virus Pichinde virus was isolated from Oryzomys albigularis (rice rat) in Colombia. (McLay et al., 2014, Journal of General Virology, 95: 1 Pichinde viruses are non-pathogenic and generally do not cause disease in humans. Serological evidence indicates that seroprevalence is high even in native populations. (Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-6 41) The Arenaviridae family includes the Lassa fever virus (LASV) and lymphocytic venous Old World (OW) arenaviruses such as cholangiocarcinoma meningitis virus (LCMV), and Pichinde virus and Junin virus-like New World (NW) arenaviruses are classified into two groups (Buchmeier 2001, "Arenaviridae: The Viruses and Their Replication" Viruses and Their Replication), Fields Virology Vol 2, 1635-1668). Its genome consists of two strands of negative-sense single-stranded RNA. It consists of four segments (Figure 1A). (McLay et al., 2014, Journal of General Virology, 95: 1-15). Each segment encodes two viral genes in opposite orientations. The S segment encodes the viral glycoprotein (GP) and nucleoprotein (NP). The long segment (L segment) encodes RNA-dependent RNA polymerase (RdRp; L protein) and matrix protein Z (protein Z), a RING finger protein The two genes on each segment are separated by a non-coding intergenic region (IGR). The IGR is flanked by 5' and 3' untranslated regions (UTRs). It has been shown that the ribosome-specific ribosome-binding domain (RiB) forms a ribosome-specific ...nuclease (RiB) domain and is involved in the structure-dependent termination of viral mRNA transcription (Pinschewe (R. et al., 2005, J Virol 79(7): 4519-4526). The terminal nucleotides of the UTRs are highly homologous. These panhandle structures are essential for transcription and replication. It is known to function as a viral promoter for Their analysis by gene expression reveals sequence- and structure-dependent structures that cannot tolerate even minor sequence changes. (Perez and de la Torre, 2003, Virol 77(2): 1184-1194).

[0005] 2.2 Reverse genetic systems The isolated and purified RNA of negative-strand viruses such as Pichinde virus is expressed as mRNA. They are not capable of functioning directly, i.e., cannot be translated when introduced into a cell. As a result, transfection of cells with viral RNA results in the generation of infectious viral particles. Conversion of negative-strand RNA virus infection from cDNA to permissive cells in culture To make a viral particle, the viral RNA segment(s) undergoes transcription and replication. The gene must be trans-complemented with the minimum necessary factors. With the help of the viral signaling system, the viral symbionts involved in transcription, replication and formation of viral particles are The gene-acting elements and trans-acting factors could finally be analyzed (Lee et al., 2003). , 2000, J Virol 74(8): 3470-3477; Lee et al., 2002, J Virol 76(12): 6393-6 397; 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 a reverse genetics system successfully rescued Pichinde virus. The method has been developed to successfully demonstrate the i, 1171: E65-E74; Lan et al., 2009, Journal of Virology, 83 (13): 6357-6362 (See ).

[0006] 2.3 Recombinant Pichinde expressing the gene of interest The creation of recombinant negative-strand RNA viruses expressing foreign genes of interest has long been pursued. Various 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): 448 6-4492; Flick and Hobom, 1999, Virology 262(1): 93-103; Machado et al. , 2003, Virology 313(1): 235-249). Live Pichinde virus-based vectors have been published. (Dhanwani et al., 2015, Journal of Virology 90:2551-2560; International Patent Publication No. Publication number WO 2016 / 048949. A three-segmented Pichinde virus has been published (Dhanwa Ni et al., 2015, Journal of Virology 90:2551-2560; International Patent Application Publication No. WO 2015 / 023636 16 / 048949). In a three-segment virus published by Dhanwani in 2015, NP Both the and GP are retained in their native positions within the S segment, and therefore the They were expressed under their native promoters.

[0007] 2.4 Replication-deficient arenaviruses Infectious arenavirus particles have the ability to amplify and express their genetic material in infected cells. Normal cells that contain the genome but are not genetically engineered will generate additional progeny. However, it is possible to engineer an infectious, replication-deficient arenavirus such that it cannot replicate in the host (i.e., infectious replication-deficient arenaviruses). particles) (International Publication No. WO 2009 / 083210 A1 and International Publication No. WO 2014 / 140301 A1). Summary of the Invention

[0008] 3. Summary of the Invention This application relates to Pichinde virus having a rearrangement of its ORFs within its genome. In particular, The present application describes a method for the preparation of a plasmid that has been engineered to carry the Pichinde virus ORF in a location other than its wild-type location. The present application relates to a replicable bi-segmented Pichinde virus genome segment. One L segment and two S segments, or two L segments, that do not recombine into the HIV particle The present invention also provides a three-segmented Pichinde virus particle comprising one S segment and one H segment. The present application provides a method for producing the three-segment Pichinde virus particles with improved genetic stability and sustained viral replication. We demonstrate that the present invention can be engineered to ensure efficient transgene expression.

[0009] In certain embodiments, the viral vectors provided herein are infectious. That is, they are capable of invading a host cell or injecting their genetic material into a host cell. In certain more specific embodiments, the viral vectors provided herein are infectious i.e., by invading or injecting its genetic material into a host cell and The genetic information can then be amplified and expressed in a host cell. In the method, the viral vector amplifies the genetic information in the infected cells and expresses it. In normal, unengineered cells, the genome contains the ability to Infectious replication-deficient pichindels engineered to be unable to produce infectious progeny particles In one embodiment, the infectious Pichinde virus is a viral vector. The viral vectors are capable of replication and induce further infection in normal, non-genetically modified cells. In certain more specific embodiments, such a method can produce dye-activated progeny particles. A replication-competent viral vector is a vector that replicates in a wild-type virus from which it is derived. is comparatively attenuated. 3.1 Non-native open reading frames Thus, in one embodiment, provided herein is a method for the preparation of a Pichinde virus genotype. In one embodiment, the genome segment comprises a viral ORF. In some embodiments, the ORF is engineered to carry the gene at a position other than the wild-type position. The Pichinde virus genome segment comprises: (i) The S segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (ii) The ORF encoding the Z protein is located in the S segment under the control of the 5'UTR of Pichinde virus. to; (iii) The ORF encoding the L protein is located in the S segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the S segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) The ORF encoding the L protein is located in the S segment under the control of the 3'UTR of Pichinde virus. to; (vi) The ORF encoding the Z protein is located in the S segment under the control of the 3'UTR of Pichinde virus. to; (vii) the L segment, in which the ORF encoding GP is under the control of the 5′UTR of Pichinde virus; (viii) the L segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (ix) The ORF encoding the L protein is located in the L segment under the control of the 5'UTR of Pichinde virus. to; (x) L segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (xi) an L segment in which the ORF encoding NP is under the control of the 3'UTR of Pichinde virus; and (xii) The ORF encoding the Z protein is located in the L segment under the control of the 3'UTR of Pichinde virus. nt; is selected from the group consisting of:

[0010] In some embodiments, the 3'UTR of the Pichinde virus is In one embodiment, the 3'UTR of the Pichinde virus L segment is The 5'UTR of the Pichinde virus is a member of the Pichinde virus S segment or the Pichinde virus L segment. The 5'UTR of

[0011] Also provided herein are the Pichinde virus genome segments provided herein. Also provided herein is an isolated cDNA encoding the Pichinde virus. It is a DNA expression vector containing the cDNA of a genomic segment.

[0012] Also provided herein are Pichinde virus genome segments, Pichinde virus Vectors containing cDNA of a segment of the Rus genome or cDNA of a segment of the Pichinde virus genome The host cell comprises a host cell.

[0013] Also provided herein is a method for producing a Pichinde virus particle comprising the steps of: The Pichinde virus genome segment and the second Pichinde virus genome segment are The Pichinde virus particle contains a .mu. segment.

[0014] In certain embodiments, the Pichinde virus particles are infectious and replication competent. In some embodiments, the Pichinde virus particle is attenuated. In a similar manner, the Pichinde virus particles are infectious but do not express further in non-complementing cells. unable to produce infectious progeny.

[0015] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the following is deleted or functionally inactivated:

[0016] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the ORFs has been removed and replaced with a heterologous ORF from an organism other than Pichindevirus. In another embodiment, the GP, NP, Z and L proteins are encoded by Only one of the four ORFs that correspond to the gene has been deleted, indicating that the gene is not derived from any organism other than Pichinde virus. In a more specific embodiment, the ORF encoding the GP is replaced with a species ORF. The sequence of the gene encoding the Pichinde virus has been removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In this manner, the ORF encoding NP was removed, allowing the expression of a heterologous O virus from an organism other than Pichinde virus. In some embodiments, the ORF encoding the Z protein is replaced with an RF. , have been removed and replaced with heterologous ORFs from organisms other than Pichinde virus. In an embodiment, the ORF encoding the L protein is removed, resulting in a virus other than Pichinde virus. The gene has been replaced with a heterologous ORF from the original genome.

[0017] In some embodiments, the heterologous ORF encodes a reporter protein. In an embodiment, the heterologous ORF encodes an antigen derived from an infectious organism, a tumor, or an allergen. In another embodiment, the heterologous ORF encoding the antigen is a human immunodeficiency virus antigen, hepatitis C virus antigen, hepatitis B surface antigen, varicella zoster virus Virus antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, tumor-associated antigens, and tumor neoantigens and tumor specific antigens (such as tumor neoepitopes).

[0018] In one embodiment, the proliferation or infectivity of Pichinde virus particles is determined by the presence of a Pichinde virus It is not affected by heterologous ORFs from organisms other than the host.

[0019] Also provided herein are methods for generating Pichinde virus genome segments. In one embodiment, the method comprises the step of: This includes transcription.

[0020] Also provided herein is a method for producing Pichinde virus particles. In an embodiment, the method for producing Pichinde virus particles comprises: (i) Transfecting a cDNA of a Pichinde virus genome segment into a host cell and; (ii) introducing a plasmid containing the cDNA of a second Pichinde virus genome segment into the host cell; Transfecting into; (iii) maintaining the host cells under conditions favorable for virus formation; and (iv) Collecting Pichinde virus particles Includes.

[0021] In one embodiment, transcription of the L segment and the S segment is controlled by a bidirectional promoter. This is done using

[0022] In one embodiment, the method comprises administering to a subject a subject a nucleic acid sequence encoding one or more nucleic acids encoding the Pichinde virus polymerase. In yet another more specific embodiment, the method further comprises transfecting the nucleic acid of the present invention into a host cell. In another embodiment, the polymerase is an L protein. and further comprising transfecting one or more nucleic acids encoding P into the host cell.

[0023] In one embodiment, transcription of the L segment and the S segment is, respectively: (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter The promoter is under the control of a promoter selected from the group consisting of:

[0024] In another embodiment, provided herein is a vaccine comprising a Pichinde virus particle. The gene is a nucleotide sequence encoding at least four ORFs encoding GP, NP, Z and L proteins. The other one is deleted or functionally inactivated; or the GP, NP, and Z proteins are At least one ORF encoding the L protein is removed, or the GP, NP, Z proteins, and L Only one of the four protein-encoding ORFs was removed, and none of the viruses except Pichinde virus In a more specific embodiment, the gene is replaced with a heterologous ORF from another organism. The cutin further comprises a pharma- ceutical acceptable carrier.

[0025] In another embodiment, provided herein is a pharmaceutical comprising a Pichinde virus particle. The composition comprises at least four ORFs encoding GP, NP, Z and L proteins. one of the GP, NP, and Z proteins has been deleted or functionally inactivated; At least one ORF encoding the L protein and the C protein is removed, and the Pichindewil or the GP, NP, Z proteins, and Only one of the four ORFs encoding the L protein was deleted, and all but Pichinde virus In a more specific embodiment, the medicament and Competently acceptable carriers further include pharma- ceutically acceptable carriers.

[0026] In some embodiments, the Pichinde virus genome segment or the Pichinde virus The virulence particles were derived from the highly pathogenic long-term passage strain Munchique CoAn4763 isolate P18 or the short-term passage strain P2. either from the genus Trapido or from one of several isolates described by Trapido and coworkers. (Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-641).

[0027] 3.2 Three-segmented Pichinde virus In one embodiment, provided herein is a nucleic acid sequence comprising one L segment and two S segments. In some embodiments, the present invention is a three-segmented Pichinde virus particle comprising the The proliferation of three-segmented Pichinde virus particles is regulated by type I interferon receptors and type II interferon receptors. -feron receptor and recombination activating gene 1 (RAG1), 4 PFU three-segment picin After 70 days of persistent infection in mice infected with devirion, two replication-competent segments In one embodiment, instead of two separate segments, In addition, two S segments, one of which contains two Pichinde virus ORFs, are identified. Recombination between the two suppresses viral promoter activity.

[0028] In another aspect, provided herein is a method for the preparation of a medicament for the treatment of a pulmonary artery disease comprising administering to a subject a method ... In one embodiment, the three-segmented Pichinde virus particle comprises The proliferation of the virion is regulated by type I interferon receptors and type II interferon receptors. It lacks the receptor for inflammatory cytokines and recombination activating gene 1 (RAG1), and 4 PFU three-segment picin deuil Replication-competent bisegmented virus after 70 days of persistent infection in mice infected with sarcoplasmic reticulum particles In some embodiments, instead of two separate segments, one The intersegmental fragment of the two L segments that connects the two Pichinde virus ORFs is The recombination suppresses viral promoter activity.

[0029] In one embodiment, one of the two S segments is: (i) The S segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (ii) The ORF encoding the Z protein is located in the S segment under the control of the 5'UTR of Pichinde virus. to; (iii) The ORF encoding the L protein is located in the S segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the S segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) The ORF encoding the L protein is located in the S segment under the control of the 3'UTR of Pichinde virus. T; and (vi) The ORF encoding the Z protein is located in the S segment under the control of the 3'UTR of Pichinde virus. to; is selected from the group consisting of:

[0030] In one embodiment, one of the two L segments is: (i) The L segment, in which the ORF encoding GP is under the control of the 5′UTR of Pichinde virus; (ii) the L segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (iii) The ORF encoding the L protein is located in the L segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the L segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) an L segment in which the ORF encoding NP is under the control of the 3'UTR of Pichinde virus; and (vi) The ORF encoding the Z protein is located in the L segment under the control of the 3'UTR of Pichinde virus. to; is selected from the group consisting of:

[0031] In one embodiment, the 3'UTR of the three segmented Pichinde virus particle is In another embodiment, the 3'UTR is the 3'UTR of the S segment of the virus or the L segment of the Pichinde virus. The 5'UTR of the three-segment Pichinde virus particle is composed of the Pichinde virus S segment or is the 5'UTR of the Pichinde virus L segment.

[0032] In one embodiment, the two S segments are: (i) one from an organism other than Pichinde virus; (ii) one or two overlapping Pichinde virus ORFs; or (iii) one or two overlapping Pichinde virus ORFs. Contains one heterologous ORF from a non-Pichindevirus organism and one overlapping Pichindevirus ORF. .

[0033] In one embodiment, the two L segments are (i) from an organism other than Pichinde virus. (ii) one or two heterologous ORFs; or (iii) one or two overlapping Pichinde virus ORFs; One heterologous ORF from a non-Pitchindevirus organism and one overlapping Pichindevirus ORF were include.

[0034] In certain embodiments, the heterologous ORF is an antigen derived from an infectious organism, a tumor, or an allergen. In another embodiment, the heterologous ORF encoding the antigen encodes a human immunodeficiency virus (HIV) antigen. Virus antigen, Hepatitis C virus antigen, Hepatitis B surface antigen, Varicella zoster (varicella zoster ) viral antigens, cytomegalovirus antigens, tuberculosis antigens, tumor-associated antigens, and (tumor The antigens are selected from tumor-specific antigens (such as tumor antigens and tumor neoepitopes).

[0035] In certain embodiments, at least one heterologous ORF encodes a fluorescent protein. In one embodiment, the fluorescent protein is green fluorescent protein (GFP) or red fluorescent protein (RFT). RFP.

[0036] In one embodiment, the three-segment Pichinde virus particle comprises all four Pichinde segments. In some embodiments, the three-segment Pichinde virus particle comprises a viral ORF. The offspring are infective and capable of replicating.

[0037] In one embodiment, the three-segment Pichinde virus particle comprises four Pichinde virus segments. In another embodiment, the three segmented Pichinde virus lacks one or more of the rus ORFs. The particles are infectious but are incapable of producing further infectious progeny in non-complementing cells.

[0038] In one embodiment, the three-segment Pichinde virus particle comprises four Pichinde virus segments. The three-segmented Pichinde virus particles are infectious and lack one of the gene ORFs. Complementing cells can express the virion vector, but are unable to produce further infectious progeny in non-complementing cells.

[0039] In some embodiments, the three segmented Pichinde virus particle lacks the GP ORF. is.

[0040] In a further aspect, provided herein is a polypeptide comprising one L segment and two S segments. In one embodiment, the first segment of the virus particle is a three-segmented Pichinde virus particle comprising a first segment. The S segment places the GP-encoding ORF under the control of the 3'UTR of Pichinde virus. The ORF encoding the first gene of elephant is placed under the control of the 5'UTR of Pichinde virus. In some embodiments, the second S segment encodes NP. The ORF encoding the second gene of interest was placed under the control of the 3'UTR of Pichinde virus. The gene has been engineered to carry an ORF encoding the 5' UTR of Pichinde virus under the control of the 5' UTR.

[0041] In yet another aspect, provided herein is a polypeptide comprising one L segment and two S segments. In one embodiment, the first segment is a three-segmented Pichinde virus particle. The S segment of the GFP-encoding ORF is located under the control of the 5'UTR of Pichinde virus. The ORF encoding the first gene of interest is placed under the control of the 3'UTR of the Pichinde virus. In some embodiments, the second S segment is engineered to encode NP. The ORF encoding the second gene of interest was placed under the control of the 5'UTR of the Pichinde virus. It has been engineered to carry an ORF encoding the 3′ UTR of Pichinde virus under the control of the 3′ UTR.

[0042] In certain embodiments, the gene of interest is derived from an infectious organism, a tumor, or an allergen. In another embodiment, the gene of interest encodes an antigen that inhibits human immunodeficiency virus (HIV) infection. Gen, Hepatitis C virus antigen, Hepatitis B surface antigen, Varicella zoster virus Virus antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, tumor-associated antigens, and (tumor neoantigens and and tumor-specific antigens (such as tumor neoepitopes). In another embodiment, at least one gene of interest encodes a fluorescent protein. In specific embodiments, the fluorescent protein is GFP or RFP.

[0043] Also provided herein is the isolation of the genome of the three-segment Pichinde virus particle. Also provided herein is a cDNA encoding a three-segment Pichinde virus particle. Also provided herein is a DNA expression vector containing the cDNA of the genome of the One or more DNA expression vectors containing the segments Pichinde virus cDNA individually or as a whole. It is.

[0044] Also provided herein are three-segment Pichinde virus particles, three-segment cDNA of the genome of Pichinde virus particle, or the genome of the three-segment Pichinde virus particle A host cell comprising a vector containing the cDNA of

[0045] In certain embodiments, the three segment Pichinde virus particle is attenuated.

[0046] Also provided herein are methods for producing three-segment Pichinde virus particles. In one embodiment, the method for producing the Pichinde virus particles comprises: (i) transfecting a host cell with one or more cDNAs of one L segment and two S segments; to; (ii) maintaining the host cells under conditions favorable for virus formation; and (iii) Collecting Pichinde virus particles Includes.

[0047] Also provided herein are methods for producing three-segment Pichinde virus particles. In one embodiment, the method for producing the three-segment Pichinde virus particle comprises: , (i) transfecting one or more cDNAs of two L segments and one S segment into a host cell; to; (ii) maintaining the host cells under conditions favorable for virus formation; and (iii) Collecting Pichinde virus particles Includes.

[0048] In one embodiment, transcription of one L segment and two S segments is a bidirectional process. In some embodiments, the promoter is two L segments and one Transcription of the two S segments is driven using a bidirectional promoter.

[0049] In one embodiment, the method comprises administering to a subject a subject a nucleic acid sequence encoding one or more nucleic acids encoding the Pichinde virus polymerase. In yet another more specific embodiment, the method further comprises transfecting the nucleic acid of the present invention into a host cell. In another embodiment, the polymerase is an L protein. and further comprising transfecting a host cell with one or more nucleic acids encoding a P protein. nothing.

[0050] In one embodiment, transcription of one L segment and two S segments is , (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter The promoter is under the control of a promoter selected from the group consisting of:

[0051] In one embodiment, transcription of two L segments and one S segment is , (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter The promoter is under the control of a promoter selected from the group consisting of:

[0052] In one embodiment, the three-segment Pichinde virus particle is a two-segment Pichinde virus particle. In another embodiment, the three-segment peptide has the same tropism as the viral particle. The genovirus particles are replication-deficient.

[0053] In another embodiment, provided herein is a three-segment Pichinde virus particle. and a pharma- ceutical acceptable carrier.

[0054] In another embodiment, provided herein is a three-segment Pichinde virus particle. and a pharma- ceutical composition comprising the compound and a pharma- ceutical acceptable carrier.

[0055] In some embodiments, the Pichinde virus genome segment or the Pichinde virus The particles were derived from the highly pathogenic long-passaged Munchique CoAn4763 isolate P18 or the short-passaged P2 strain. or to one of several isolates described by Trapido and coworkers. (Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-641). 3.3 Conventions and Abbreviations [Table 1] [Brief description of the drawings]

[0056] 4. Brief description of the drawings [Figure 1] Figures 1A-1D: Schematic diagram of the genome organization of bi-segmented and tri-segmented Pichinde virus. The bi-segmented genome of wild-type Pichinde virus consists of one S segment encoding GP and NP and one L segment encoding Z and L proteins. Both segments are flanked by 5'UTR and 3'UTR, respectively. (Figure 1A) Schematic representation of the rPICwt Pichinde virus genome, a cDNA-derived wild-type Pichinde virus with its native genome segments S (SEQ ID NO: 16) and L (SEQ ID NO: 2), which have been modified by silent mutations introduced to suppress the BsmBI and BbsI sites of the respective cDNAs. (Figures 1B-1D) The genome of the recombinant tri-segmented Pichinde virus (r3PIC) consists of one L segment and two S segments, each of which has one position to insert a gene of interest (here a GFP / sP1AGM fusion protein). (Figure 1B) Schematic representation of a tri-segmented Pichinde virus vector genome with an artificial organization. In one of the duplicated S segments, the glycoprotein (GP) ORF is located in place of the nucleoprotein (NP) ORF of the natural S segment, i.e., between the 3'UTR and the IGR. (Figure 1C) r3PIC-GFPart consists of all viral genes in their natural locations, except that the GP ORF is artificially juxtaposed to the 3'UTR and expressed under the control of the 3'UTR (S-GP / GFPart; SEQ ID NO: 13). (Figure 1D) Schematic representation of the three-segment Pichinde virus-based sP1AGM expressing r3PIC-sP1AGMart vector genome.

[0057] [Diagram 2]Figure 2: Three-segment r3PIC-GFPart was attenuated compared to its two-segment wild-type parent virus. Growth kinetics of the indicated viruses in BHK-21 cells infected at a multiplicity of infection (moi) of 0.01 (wild-type Pichinde virus: black squares; r3PIC-GFPart: black circles). Supernatants were harvested at the indicated time points post-infection and virus titers were determined by focus formation assay.

[0058] [Diagram 3] FIG. 3: Schematic representation of the expression cassettes of the plasmids used for the experiments described in FIG. 2 and FIG.

[0059] [Figure 4] Figure 4: Reconstitution of infectious GFP-expressing virus from cDNA in cells using r3PIC-GFPart. Fluorescent images of GFP expression were captured 48 or 168 hours after transfection of BHK-21 cells with combinations of plasmids: S segment minigenome: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-miniS-GFP; L segment minigenome: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L-GFP-Bsm; r3PIC-GFPart: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L, pol-I-PIC-NP-GFP, pol-I-PIC-GP-GFP; rPICwt: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L, pol-I-PIC-S.

[0060] [Diagram 5]Figures 5A-5B: Three-segment Pichinde virus-based viral vectors are highly immunogenic. BALB / c mice were infected intravenously with 10e5 FFU of r3PIC-sP1AGMart. Control mice were not immunized. Eight days later, the frequency of P1A-specific CD8+ T cells in peripheral blood was determined by MHC class I tetramer staining. Exemplary FACS plots (Figure 5A) and the frequency of tetramer-binding cells among CD8+ T cells in peripheral blood (Figure 5B) are shown. Symbols in B represent individual mice.

[0061] [Figure 6] Figure 6: Schematic representation of a three-segment Pichinde virus vector genome designed to express its glycoprotein (GP) and nucleoprotein (NP) genes under the control of the 5' and 3' UTR promoters, respectively, i.e. in their "natural" positions in the context of the artificially duplicated S-segments S-GP / GFPnat (SEQ ID NO: 15) and S-NP / GFP (also known as PIC-NP-GFP; SEQ ID NO: 11). The genome consists of one L segment and two S segments, each of which has one position to insert a gene of interest (here the GFP protein).

[0062] [Figure 7] Figure 7: Early passages of three-segment r3PIC-GFPnat and r3PIC-GFPart were attenuated compared to their two-segment wild-type parent viruses. Growth kinetics of the indicated viruses in cultured BHK-21 cells infected at a multiplicity of infection (moi) of 0.01. Supernatants were harvested 48 hours post-infection and virus titers were determined by focus formation assay. Symbols indicate titers of individual parallel cell culture wells; error bars display the mean + / - SD.

[0063] [Figure 8]Figure 8: Unlike the stably attenuated r3PIC-GFPart, r3PIC-GFPnat reached titers in the range of rPICwt during persistent infection of mice. AGR mice (triple-deficient for type I and type II interferon receptors and RAG1) were infected intravenously with 10e5 FFU of virus as indicated (wild-type Pichinde virus-rPICwt: grey triangles; r3PIC-GFPart: black circles; r3PIC-GFPnat: white squares). Blood was collected on days 7, 14, 21, 28, 35, 42, 56, 77, 98, 120 and 147 and virus infectivity was determined in a focus-forming assay detecting Pichinde virus nucleoprotein (NP FFU).

[0064] [Figure 9] Figure 9: Unlike the stably attenuated r3PIC-GFPart, r3PIC-GFPnat reached titers in the range of rPICwt during persistent infection of mice. AGR mice (triple-deficient for type I and type II interferon receptors and RAG1) were infected intravenously with 10e5 FFU of virus as indicated (wild-type Pichinde virus-rPICwt: grey triangles; r3PIC-GFPart: black circles; r3PIC-GFPnat: white squares). Blood was collected on days 7, 14, 21, 28, 35, 42, 56, 77, 98, 120 and 147 and viral infectivity was determined in a focus-forming assay detecting the viral GFP transgene for r3PIC-GFPnat and r3PIC-GFPart (GFP FFU).

[0065] [Figure 10]Figure 10: Trisegmented Pichinde virus-based viral vectors with artificial genomes are highly immunogenic. AGR mice (triple-deficient for type I and type II interferon receptors and RAG1) were infected intravenously with 10e5 FFU of virus as indicated (r3PIC-GFPart: black circles; r3PIC-GFPnat: white squares). Blood was collected on days 7, 14, 21, 28, 35, 42, 56, 77, 98, 120 and 147 and viral infectivity was determined by focus formation assay as presented in Figures 9 and 10. The obtained values ​​were used to calculate the NP:GFP FFU ratio for each animal and time point.

[0066] [Figure 11] Figure 11: Virus in mouse sera collected 147 days post-infection with r3PIC-GFPart showed attenuated growth when directly passaged in cell culture, whereas virus grown from r3PIC-GFPnat-infected mice reached titers comparable to rPICwt. Serum collected 147 days post-infection in BHK-21 cells was passaged and virus infectivity determined 48 hours later by NP FFU assay. Symbols indicate titers of virus from individual mouse sera; error bars display mean + / - SD.

[0067] [Figure 12] Figure 12: Virus isolated and expanded from mouse sera collected 147 days post-infection with r3PIC-GFPart showed attenuated growth when directly passaged in cell culture, whereas virus isolated and expanded from r3PIC-GFPnat-infected mice reached titers comparable to rPICwt. BHK-21 cells were infected at a standardized multiplicity of infection = 0.01 with virus obtained from sera collected 147 days post-infection and previously passaged 48 hours. Viral titers were determined 48 hours later. Symbols indicate titers of virus from individual mouse sera; error bars represent the mean + / - SD.

[0068] [Figure 13]Figure 13: r3PIC-GFPart did not recombine the two S segments during the 147-day persistent infection period in mice, but S segment RNA species containing both NP and GP sequences were detected in the serum of mice persistently infected with r3PIC-GFPnat for 147 days. RT-PCR was performed on serum samples collected 147 days after virus infection, using primers designed to bind to Pichinde virus NP and GP, respectively, and spanning the intergenic region (IGR) of the Pichinde virus S segment, predicted to yield a PCR amplicon of 357 base pairs on the rPICwt genomic template. Each lane represents RT-PCR products from an individual mouse in the experiment shown in Figures 8-10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description of the Invention 4.1 Pichinde virus with an open reading frame at a non-native position Provided herein is a Pichinde virus having a rearrangement of its ORF. In an embodiment, such a Pichinde virus is replication competent and infectious. Such a genomic sequence of the Pichinde virus is provided herein. Provided herein is a Pichinde virus genome segment, wherein the Pichinde virus The Pichinde virus genome segment contains the Pichinde virus ORF, each gene of which is of the virus strain Munchique CoAn4763 isolate P18 (see SEQ ID NOs: 1 and 2 in the sequence listing, 7. The position of the ORF found in such wild-isolated viruses (referred to as the "wild-type position" in this specification) The amino acid sequence may be modified to carry the amino acid at a position other than the naturally occurring position (i.e., a non-native position) referred to herein.

[0070] Wild-type Pichinde virus genome segments and ORFs are known in the art. The Pichinde virus genome consists of an S segment and an L segment. The L segment carries ORFs encoding GP and NP. The L segment encodes the L protein and the Z protein. Both segments are flanked by 5'UTR and 3'UTR, respectively (see Figure 1A). Illustrative wild-type Pichinde virus genome segments are set forth in SEQ ID NO:1 and SEQ ID NO:2. Provided in Issue 2.

[0071] In one embodiment, the Pichinde virus genome segment is two or more Pichinde virus genome segments. The vector can be engineered to carry the RNA ORF in a position other than the wild-type position. The Pichinde virus genome segment comprises two Pichinde virus ORFs, or three carrying one or four Pichinde virus ORFs at positions other than the wild-type position. It can be operated to hold.

[0072] In one embodiment, the Pichinde virus genome segment provided herein is (i) The ORF encoding NP is under the control of the 5'UTR of Pichinde virus S segment; (ii) Pichinde virus, in which the ORF encoding the Z protein is under the control of the 5'UTR of Pichinde virus Viral S segment; (iii) Pichinde virus, in which the ORF encoding the L protein is under the control of the 5'UTR of Pichinde virus Viral S segment; (iv) Pichinde virus S, in which the ORF encoding GP is under the control of the 3'UTR of Pichinde virus segment; (v) The ORF encoding the L protein is under the control of the 3'UTR of the Pichinde virus. Irus S segment; (vi) Pichinde virus, in which the ORF encoding the Z protein is under the control of the 3'UTR of Pichinde virus Viral S segment; (vii) Pichinde virus L, in which the ORF encoding GP is under the control of the 5'UTR of Pichinde virus segment; (viii) Pichinde virus, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus L segment; (ix) Pichinde virus, in which the ORF encoding the L protein is under the control of the 5'UTR of Pichinde virus Viral L segment; (x) The ORF encoding GP is located in the Pichinde virus L sequence under the control of the 3'UTR of Pichinde virus. segment; (xi) Pichinde virus L, in which the ORF encoding NP is under the control of the 3'UTR of Pichinde virus Segments; and (xii) Pichinde virus, in which the ORF encoding the Z protein is under the control of the 3'UTR of Pichinde virus Viral L segment; It could be.

[0073] In some embodiments, the non-human genomic segments of the Pichinde virus described herein are The ORF in its natural location is under the control of the Pichinde virus 3'UTR or the Pichinde virus 5'UTR. In a more specific embodiment, the 3'UTR of the Pichinde virus can be located at In another specific embodiment, the 3'UTR of the Pichinde virus S segment is The 3'UTR of the Pichinde virus is the 3'UTR of the Pichinde virus L segment. In an embodiment, the 5'UTR of the Pichinde virus is the 5'UTR of the Pichinde virus S segment. In other specific embodiments, the 5'UTR is the 5'UTR of the L segment.

[0074] In other embodiments, non-Pichinde virus genome segments described herein The ORF in its native location is under the control of conserved terminal sequence elements of arenaviruses (5'- and 3'-terminal 19-21 nucleotide regions) (see, e.g., Perez and de la Torre (see, e.g., 2003, J Virol. 77(2):1184-1194).

[0075] In one embodiment, an ORF located in a non-natural location in the Pichinde virus genome segment can be placed under the control of a promoter element in the 5'UTR (e.g., Albarino et al. (See, e.g., 2011, J Virol., 85(8):4020-4). In another embodiment, the peptide The ORFs at non-native locations in the Chinde virus genome segments are expressed in the promoter elements of the 3'UTR. The virion can be placed under the control of the virion (see, for example, Albarino et al., 2011, J Virol., 85(8) In a more specific embodiment, the promoter element of the 5'UTR The promoter element is the 5'UTR promoter element of the S or L segment. In one embodiment, the promoter element of the 3'UTR is an S segment or an L segment. The 3'UTR promoter element of the

[0076] In one embodiment, an ORF located in a non-natural location in the Pichinde virus genome segment is placed under the control of a truncated Pichinde virus 3'UTR or a truncated Pichinde virus 5'UTR (See, e.g., Perez and de la Torre, 2003, J Virol. 77(2): 1184- 1194; see Albarino et al., 2011, J Virol., 85(8):4020-4). In one embodiment, the truncated 3'UTR is a 3'UTR comprising a Pichinde virus S segment or a Pichinde virus L segment. In a more specific embodiment, the truncated 5'UTR is the 3'UTR of the Pichinde virus. The 5'UTR of the S or L segment.

[0077] Also provided herein is a method for producing a Pichinde virus particle comprising the steps of: The first gene was engineered to carry an ORF at a position other than the wild-type position of that ORF, so as to include A Pichinde virus comprising a genomic segment of the first genomic segment and a second Pichinde virus genomic segment. In a specific embodiment, the ORF at a position other than the wild-type position of the ORF is It is one of the Pichinde virus ORFs.

[0078] In certain specific embodiments, the Pichinde virus particle comprises all four Pichinde viruses. In a specific embodiment, the second picindel may comprise a complete complement of the sequence ORF. A viral genome segment may be engineered to carry an ORF at a location other than the wild-type location of that ORF. In another specific embodiment, the second Pichinde virus genome segment is The target can be a wild-type genomic segment (i.e., an ORF is located at the wild-type position on the segment). (included).

[0079] In one embodiment, the first Pichinde virus genome segment is an L segment. and the second Pichinde virus genome segment is an S segment. wherein the first Pichinde virus genome segment is an S segment and the second Pichinde virus genome segment is an S segment. The deviral genome segment is the L segment.

[0080] A genome segment having an ORF at a position other than the wild-type position of that ORF and a second genome segment Non-limiting examples of Pichinde virus particles containing ment are shown in Table 1. Table 1 Pichinde virus particles * Position 1 is under the control of the 5'UTR of the Pichinde virus S segment; position 2 is under the control of the Pichinde virus S segment Position 3 is under the control of the 3'UTR of the Pichinde virus L segment; position 3 is under the control of the 5'UTR of the Pichinde virus L segment; position 4 is under the control of the 3'UTR of the Pichinde virus L segment. [Table 2]

[0081] Also provided herein are vectors that can be used to carry an ORF in a location other than the wild-type location of that ORF. The cDNA of a segment of the Pichinde virus genome that has been engineered to In an embodiment, provided herein is a cDNA or a plasmid of the Pichinde virus genome as set forth in Table 1. is the cDNA set.

[0082] In one embodiment, the Pichinde virus genome segments described herein are co-transfected with The nucleic acid to be downloaded has at least some sequence identity to the nucleic acid sequences disclosed herein. Thus, in some embodiments, the Pichinde virus genome segment can be The nucleic acid encoding the compound may be any of the nucleic acid sequences disclosed herein by SEQ ID NO: or SEQ ID NO: For nucleic acid sequences that hybridize to the nucleic acid sequences disclosed herein by Also 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, At least 92% identical, at least 93% identical, at least 94% identical, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least A nucleic acid sequence that is at least 99% identical to or identical to the nucleic acid sequence of the target gene. The conditions for the amplification can be highly stringent, as described herein, and are well known to those of skill in the art. , moderately stringent, or low stringency hybridization conditions Similarly, the methods for producing the Pichinde virus genome segments described herein include The nucleic acids that can be used are those disclosed herein by SEQ ID NO. or those A certain percentage of nucleic acids that hybridize to the nucleic acid sequences disclosed herein are For example, the sequences used to generate the Pichinde virus genome segments may have sequence identity. The nucleic acids to be used are those having at least 80% identity, at least Also 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, At least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity It can be either equal to or identical to the

[0083] Sequence identity (also known as homology or similarity) is the degree of sequence identity between two nucleic acid molecules or between two polypeptides. This refers to the sequence similarity between peptides. Identity is determined by comparing positions in each sequence. The positions in the sequences being compared can be determined by the sequence alignment. If a position is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences is determined by the number of matching or homologous positions shared by the sequences. Alignment of two sequences to determine percent sequence identity For example, see Ausubel et al., "Current Protocols in Molecular Biology." Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) This can be done using software programs known in the art, such as Otherwise, default parameters are used for the alignment. One alignment program known in the art is set to default parameters. In particular, the program uses the default parameters of the genetic code =standard;filter=none;strand=both;cutoff=60;expect=10;matrix Source = BLOSUM62; Description = 50 sequences; Selection order = HIGH SCORE; Database = non-redundant, GenBank + Using EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR, The BLASTN and BLASTP programs are described in detail in the National Center for Biotechnology Information. can be found in Ecology Information.

[0084] Stringent hybridization is when the hybridized polynucleotides are As known to those skilled in the art, the stability of hybridized polynucleotides is The qualitative analysis was performed by measuring the melting temperature (T m In general, the hybridized polynucleotides The stability of a nucleotide is a function of salt concentration, eg, sodium ion concentration, and temperature. The hybridization reaction is carried out under conditions of lower stringency, followed by Washes of various, higher stringency can be performed. References to stringency refer to such washing conditions. For example, stable hybridization at 65° C. in 0.018 M NaCl is The conditions that allow hybridization of only the nucleic acid sequences that form a polynucleotide are defined as For example, if the hybrid is not stable in 0.018 M NaCl at 65° C., it is Therefore, it is not stable under the high stringency conditions contemplated in The stringency conditions are, for example, 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS. Hybridization at 42°C, followed by incubation at 65°C in 0.1x SSPE and 0.1% SDS. Washing may provide other than highly stringent hybridization conditions. The hybridization conditions are also used to describe the nucleic acid sequences disclosed herein. For example, the phrase moderately stringent hybridization may be used. Hybridization was performed at 42°C in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS. This refers to conditions equivalent to lysis followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. The term stringent hybridization refers to 10% formamide, 5× Dendritic cell suspension, Hybridization in rt solution, 6x SSPE, 0.2% SDS at 22°C, followed by 1x SSPE The term refers to conditions equivalent to washing in 0.2% SDS at 37°C. Denhart's solution is a mixture of 1% Ficoll, 1% polystyrene, and 1% cellulose. It contains 20x SSPE (sodium chloride), 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). sodium, sodium phosphate, ethylenediamidetetraacetic acid (EDTA)) are 3M sodium chloride, 0. Contains 2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, medium and high stripping Concurrency hybridization buffers and conditions are well known to those of skill in the art and can be found, for example, in Sambrook and Russell, Molecular Cloning: A Laboratory Manual. ing: A laboratory manual), 3rd edition, Cold Spring Harbor Laboratory NY (2001) and Ausubel et al., Current Protocols in Molecular Biology. and Physiol. 1999, 14th ed., Leucular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0085] In one embodiment, an ORF is engineered to carry an ORF at a location other than the wild-type location of that ORF. The cDNA of the selected Pichinde virus genome segment is either part of a DNA expression vector or In a specific embodiment, the ORF is incorporated into a DNA expression vector. cD of Pichinde virus genome segments engineered to carry positions other than the wild type position NA is a DNA gene that facilitates the production of the Pichinde virus genome segments described herein. It may be part of the expression vector or may be incorporated into the DNA expression vector. In such a manner, the cDNA described herein can be incorporated into a plasmid. A more detailed description of nucleic acids and expression systems is provided in Section 4.5.1. These techniques are routine and conventional techniques of molecular biology and the manipulation and production of DNA. Any cloning technique known in the art may be used. Such techniques are well known and should be understood to be of skill in the art. The vendor recommends the use of the book "Molecular Cloning: A Laboratory Manual" by Sambrook and Russell. Cloning: A Laboratory Manual), 3rd edition, Cold Spring Harbor Laboratory NY (2 001) and other laboratory manuals.

[0086] In one embodiment, an ORF is engineered to carry an ORF at a location other than the wild-type location of that ORF. The cDNA of the Pichinde virus genome segment that has been transfected (e.g. Thus, in some embodiments provided herein, Pichinde virus that has been engineered to carry an ORF in a position other than its wild-type position A host cell containing a cDNA of a genomic segment (i.e., a cDNA of a genomic segment). In one embodiment, the cDNA described herein is part of a DNA expression vector or Alternatively, it can be incorporated into a DNA expression vector and introduced into a host cell. In some embodiments, provided herein is a method for producing the present invention, comprising the steps of: In another embodiment, the host cell comprises the cDNA described herein. The Pichinde virus genome segment to be transformed is introduced into a host cell.

[0087] In one embodiment, provided herein is a method for the isolation of a Pichinde virus genome segment. a method for producing a gene encoding a Pichinde virus genome segment, the method comprising: In some embodiments, the viral polymerase protein is This may occur during transcription of Pichinde virus genome segments in vitro or in vivo.

[0088] In one embodiment, transcription of the Pichinde virus genome segment is a bidirectional process. In another embodiment, the Pichinde virus genome segment is Transcription of the promoter is achieved using a bidirectional expression cassette (see, e.g., Ortiz-Riano et al., 2003). , 2013, J Gen Virol., 94(Pt 6): 1175-1188). In the present invention, the bidirectional expression cassette is inserted into the Pichinde virus genome sequence. Both polymerase I and polymerase II promoters are read from both ends of the fragment. In an even more specific embodiment, a method is provided comprising the steps of: The bidirectional expression cassette reads from both the L segment and the S segment.

[0089] In other embodiments, the cD of the Pichinde virus genome segment described herein The transcription of NA involves a promoter. An example of a promoter is the RNA polymerase I promoter. Motor, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter Examples of promoters include the SP6 promoter or the T3 promoter.

[0090] In one embodiment, the method for generating the Pichinde virus genome segment comprises the steps of: The method can further include introducing a cDNA of the genomical segment of the genomical segment into the host cell. In one embodiment, the method for producing the Pichinde virus genome segment comprises The cDNA of the Chinde virus genome segment was used to generate the Chinde virus genome segment. and extracting the pi from the supernatant of the host cells. The method may further include purifying the Chindevirus genome segment. The methods are well known to those skilled in the art.

[0091] Provided herein are methods for infecting cells with the nucleic acids, vectors, and compositions provided herein. The nucleic acids, vectors and methods of culturing cells described herein are also provided. A more detailed description of the systems and cell lines is provided in Section 4.5.

[0092] In certain embodiments, the Pichinde virus particles described herein are infectious and replicative. In a specific embodiment, the method of the present invention provides a method for producing a Pichinde virus particle capable of producing a Pichinde virus particle as described herein. The Pichinde virus particles produced are attenuated. In certain embodiments, the Pichinde virus particles are The particles allow the virus to replicate in vivo while still being at least partially able to spread. However, they can produce only low viral loads that result in subclinical levels of non-pathogenic infection. Such attenuated viruses can be used as immunogenic compositions. Provided herein are amino acid sequences that can be used at non-native positions, as described in Section 4.7. An immunogenic composition comprising a Pichinde virus having an ORF.

[0093] 4.1.1 Replication-defective Pichinde viruses with open reading frames in non-native locations particle In certain embodiments, provided herein are methods for isolating an ORF, comprising: (i) isolating an ORF other than at the wild-type location of the ORF; and (ii) the resulting virus produces additional infectious progeny virus particles. The ORFs encoding the GP, NP, Z, and L proteins were removed so that the Pichinde virus particles that have been modified or functionally inactivated. One or more ORFs are deleted. Pichinde virus particles containing a genetically modified genome that has been modified or functionally inactivated are capable of inducing complementation cells (i.e., cells expressing a deleted or functionally inactivated Pichinde virus ORF). The resulting genetic material of the Pichinde virus particles can be produced by infection of host cells. Sometimes the genetic material is transferred to a host cell in which it can be expressed and amplified. The genome of the genetically modified Pichinde virus particle described herein is It is possible to encode a heterologous ORF from an organism other than the particle.

[0094] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the sequences was removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In another embodiment, at least one gene encoding the GP, NP, Z and L proteins is At least one ORF, at least two ORFs, at least three ORFs, or at least four ORFs The gene is removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In an embodiment, one of the four ORFs encoding the GP, NP, Z protein, and L protein Only the nucleotide sequence of the gene encoding the nucleotide sequence of ... In a specific embodiment, the ORF encoding the GP of the Pichinde virus genome segment is In another specific embodiment, the NP of the Pichinde virus genome segment is deleted. In a more specific embodiment, the ORF encoding the Pichinde virus genome is deleted. The ORF encoding the Z protein of the segment is deleted. In this case, the ORF encoding the L protein is deleted.

[0095] Thus, in some embodiments, the Pichinde virus particles provided herein comprise ) engineered to carry an ORF in a non-native position; (ii) a GP, NP, Z protein, or L protein (iii) the ORFs encoding the target gene are removed; and The genome segment contains a heterologous ORF replaced by a heterologous ORF.

[0096] In certain embodiments, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length , 25-100 nucleotides long, 50-200 nucleotides long, 50-400 nucleotides long, 200-500 nucleotides, or 400 to 600 nucleotides, or 500 to 800 nucleotides. In an embodiment, the heterologous ORF is 750-900 nucleotides in length, 800-100 nucleotides in length, 850 ~1000 nucleotides long, 900~1200 nucleotides long, 1000~1200 nucleotides long, 1000~1 500 nucleotides or 10-1500 nucleotides long, 1500-2000 nucleotides long, 1700-2000 Nucleotide length, 2000-2300 nucleotides, 2200-2500 nucleotides, 2500-3000 nucleotides Nucleotide length, 3000-3200 nucleotides, 3000-3500 nucleotides, 3200-3600 nucleotides Length of nucleotide, 3300-3800 nucleotides, 4000-4400 nucleotides, 42 00-4700 nucleotides long, 4800-5000 nucleotides long, 5000-5200 nucleotides long, 5200 ~5500 nucleotides long, 5500~5800 nucleotides long, 5800~6000 nucleotides long, 6000~ 6400 nucleotides long, 6200-6800 nucleotides long, 6600-7000 nucleotides long, 7000-72 00 nucleotides long, 7200-7500 nucleotides long, or 7500 nucleotides long. In some embodiments, the heterologous ORF is 5 to 10 amino acids in length, 10 to 25 amino acids in length, 25 to 50 amino acids in length, amino acid length, 50-100 amino acid length, 100-150 amino acid length, 150-200 amino acid length, 200-250 amino acid length amino acid length, 250-300 amino acid length, 300-400 amino acid length, 400-500 amino acid length, 500-750 amino acid length amino acid length, 750-1000 amino acid length, 1000-1250 amino acid length, 1250-1500 amino acid length, 1500-1 750 amino acids long, 1750-2000 amino acids long, 2000-2500 amino acids long, or 2500 or more amino acids long In some embodiments, the variant encodes a peptide or polypeptide having a length of about 1000 bp. A species ORF encodes a polypeptide not exceeding 2500 amino acids in length. In some embodiments, the heterologous ORF does not contain a stop codon. In some embodiments, the nucleotide composition, nucleotide pair composition or Both can be optimized. Techniques for such optimization are well known in the art. It is known and can be applied to optimize heterologous ORFs.

[0097] Any heterologous ORF from an organism other than Pichindevirus can be inserted into the Pichindevirus genome segment. In one embodiment, the heterologous ORF can be a reporter protein. A more detailed description of reporter proteins is provided in Section 4.3. In another embodiment, the heterologous ORF is an antigen of an infectious pathogen or an antigen that induces an immune response. In a specific embodiment, the nucleic acid sequence encodes an antigen associated with any disease that can be treated with the nucleic acid sequence. Antigens may be derived from infectious organisms, tumors (i.e., cancer), or allergens. A more detailed explanation is provided in Section 4.3.

[0098] In one embodiment, the proliferation and infectivity of Pichinde virus particles is determined by the presence of a Pichinde virus It is not affected by heterologous ORFs from organisms other than the host.

[0099] Techniques known to those skilled in the art can be used to engineer a gene to carry the Pichinde virus ORF at a location other than the wild-type location. Generate Pichinde virus particles containing engineered Pichinde virus genome segments For example, reverse genetic techniques may be used to identify such Pichinde viruses. In another embodiment, the replication-deficient Pichinde virus particle (i.e. Pichinde virus ORFs engineered to carry the Pichinde virus ORF at positions other than the wild-type position. Viral genome segments encoding GP, NP, Z protein, and L protein F) can be produced in complementing cells.

[0100] In one embodiment, the present application provides a method for the preparation of a vaccine comprising administering to a subject a subject therapies described herein that are suitable for use as a vaccine. Pichinde virus particles and vaccination and treatment or prevention, e.g., for infection or cancer The present invention also relates to methods of using such Pichinde virus particles in A more detailed description of the methods for using Pichinde virus particles is provided in Section 4.6. .

[0101] In certain embodiments, provided herein is one or more cDNAs described herein. In a specific embodiment, the kit comprises one or more containers. In the upper container, a Pichinde virus genome segment or a Pichinde virus genome segment described herein is The kit comprises a Pichinde virus genome segment or a Pichinde virus Suitable host cells for particle rescue, transfecting the host cells with plasmid cDNA Reagents suitable for the preparation of the vector, helper viruses, plasmids encoding viral proteins and / or or modified Pichinde virus genome segments or Pichinde virus particles or their It may further comprise one or more of the one or more primers specific for the cDNA.

[0102] In some embodiments, the present application provides a pharmaceutical composition comprising the composition described herein suitable for use as a pharmaceutical composition. Pichinde virus particles for use in vaccination and treatment or prevention of infection and cancer The present invention relates to methods of using such Pichinde virus particles. A more detailed description of methods for using deviral particles is provided in Section 4.7.

[0103] 4.2 Three-segmented Pichinde virus particles Provided herein is a three-segment Pichinde virus having a rearrangement of its ORF. In one aspect, provided herein is a particle having one L segment and two S Three-segmented picindeui containing two segments or two L segments and one S segment In one embodiment, the three segmented Pichinde virus particle is a replicative virus particle. The virus is not recombined into a bi-segmented Pichinde virus particle that can be produced. More specifically, In an embodiment, two of the genomic segments (e.g., two S segments or two The L segment of the ribosomal RNA (RIRNA) is a single viral segment that can replace the two parent segments. In a specific embodiment, the triplet is incapable of recombining to produce the triplet. Segmented Pichinde virus particles contain an ORF at a location other than the wild-type location of that ORF. In another specific embodiment, the three segmented Pichinde virus particle comprises all four Thus, in one embodiment, the three segment Pichinde virus ORF is The deviral particles are replication competent and infectious. The Pichinde virus particle lacks one of the four Pichinde virus ORFs. In one embodiment, the three-segment Pichinde virus particle is infectious. but are unable to produce further infectious progeny in non-complementing cells.

[0104] In one embodiment, the GP of the three-segment Pichinde virus particle described herein The ORFs encoding the NP, Z protein, or L protein are located in the 3'UTR or In a more specific embodiment, the 5′ UTR of the Pichinde virus can be placed under the control of the 5′ UTR of the Pichinde virus. wherein the 3'UTR of the three-segment Pichinde virus is In another specific embodiment, the 3'UTR of the three-segment Pichinde virus is is the 3'UTR of the tripartite Pichinde virus L segment(s). In an embodiment, the 5'UTR of the three segmented Pichinde virus is In another specific embodiment, the 5'UTR is the 5'UTR of the L segment ( The 5'UTR of one or more

[0105] In other embodiments, the GP of the three-segment Pichinde virus particle described herein The ORFs encoding the NP, Z, or L proteins are conserved in arenaviruses. It can be placed under the control of terminal sequence elements (19-21 nucleotide regions at the 5' and 3' ends). (See, e.g., Perez and de la Torre, 2003, J Virol. 77(2):1184-1194. (I want to be).

[0106] In one embodiment, the GP, NP, and Z proteins of the three-segment Pichinde virus particle Alternatively, the ORF encoding the L protein may be placed under the control of a promoter element in the 5'UTR. (See, e.g., Albarino et al., 2011, J Virol., 85(8):4020-4. In another embodiment, the GP, NP, and Z proteins of the three-segment Pichinde virus particle are Therefore, the ORF encoding the L protein is placed under the control of the promoter element in the 3'UTR. (See, e.g., Albarino et al., 2011, J Virol., 85(8):4020-4. In a more specific embodiment, the promoter element of the 5'UTR is an S segment ( The 5'UTR promoter element of the L segment(s) or the L segment(s). In one embodiment, the promoter element of the 3'UTR comprises an S segment(s) or an L segment. ment(s) and the 3'UTR promoter element.

[0107] In one embodiment, the GP, NP, and Z proteins of the three-segment Pichinde virus particle or the ORF encoding the L protein is a truncated Pichinde virus 3'UTR or a truncated Pichin The gene can be placed under the control of the 5'UTR of the devirus (see, e.g., Perez and de la Torre, 2003, J Virol., 77(2):1184-1194; Albarino et al., 2011, J Virol., 85(8):40 In a more specific embodiment, the truncated 3'UTR is In a more specific embodiment, the 3'UTR of the viral S or L segment. The truncated 5'UTR comprises the 5'UTR of the Pichinde virus S segment(s) or L segment(s). It is TR.

[0108] Also provided herein is the cDNA of the three-segment Pichinde virus particle. In more specific embodiments, provided herein are any of the compounds listed in Table 2 or Table 3. DNA nucleotide sequence encoding the three-segment Pichinde virus particle It is a set of dot arrays.

[0109] In one embodiment, the three-segment Pichinde virus genome sequence described herein Nucleic acids encoding the fragments include those having at least certain sequences in the nucleic acid sequences disclosed herein. Thus, in some embodiments, a three-segment peptide can have the same structure. Nucleic acids encoding the genomic segments of the genotype virus are disclosed herein by SEQ ID NOs. or hybridizes to the nucleic acid sequences disclosed herein by SEQ ID NO. At least 80% identity, at least 85% identity, at least 90% identity to the nucleic acid sequence , at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical % identity, at least 95% identity, at least 96% identity, at least 97% identity, at least have a nucleic acid sequence with at least 98% identity, or at least 99% identity, or be identical thereto Hybridization conditions may be as described herein, and may be performed under high temperature conditions well known to those skilled in the art. Moderately stringent, moderately stringent, or low stringency hybridization Similarly, the three-segment Nucleic acids that can be used to generate Pichinde virus genome segments are represented by SEQ ID NO: The nucleic acid sequences disclosed herein or the SEQ ID NOs are used to identify the nucleic acid sequences disclosed herein. Hybridizing nucleic acid sequences may have a certain percentage of sequence identity. For example, the nucleic acids used to generate the three-segment Pichinde virus genome segment are at least 80% identical, at least 85% identical to the nucleic acid sequences described herein. Identity, at least 90% identity, at least 91% identity, at least 92% identity, at least Also 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity; or can be identical to it.

[0110] In one embodiment, the nucleic acid encoding the three segmented Pichinde virus genome is Part of or incorporated into one or more DNA expression vectors In a specific embodiment, the genome of the three-segment Pichinde virus particle is encoded by The nucleic acid that promotes the production of the three-segment Pichinde virus particles described herein. It is part of or incorporated into one or more of the above DNA expression vectors. In another embodiment, the cDNAs described herein can be incorporated into a plasmid. A more detailed description of cDNA and expression systems is provided in Section 4.5.1. The techniques for this purpose are routine and conventional techniques of molecular biology and DNA manipulation and production. Any known cloning technique can be used. Such techniques are well known and include Those skilled in the art will be familiar with the techniques described in Sambrook and Russell, "Molecular Cloning: A Laboratory Manual." ular Cloning: A laboratory manual), 3rd edition, Cold Spring Harbor Laboratory NY (2001) and other laboratory manuals.

[0111] In one embodiment, the three-segment Pichinde virus cDNA is Thus, in some embodiments, the present invention provides a method for the preparation of a vector comprising the steps of: Provided herein is the cDNA of the three-segment Pichinde virus particle (i.e., The host cell contains a cDNA of a genome segment of the endothelin virus particle. In embodiments, the cDNAs described herein are part of a DNA expression vector or are DNA It can be incorporated into an expression vector and introduced into a host cell. In an embodiment, provided herein is a method for the preparation of a method for the preparation of a nucleic acid molecule as described herein, which method is incorporated into a vector. In another embodiment, the host cell comprises a cDNA encoding the three-segment Intopichinde virus genome segments (i.e., L segment and / or S segment or or a plurality of such segments) is introduced into a host cell.

[0112] In one embodiment, described herein is the three-segment Pichinde virus. A method for producing particles comprising transcribing the cDNA of the three-segment Pichinde virus particle. In one embodiment, the viral polymerase protein is In vitro or in vivo, the three-segmented Pichinde virus particle is transcribed. In one embodiment, transcription of the Pichinde virus genome segment can be This is done using a directional promoter.

[0113] In another embodiment, the transcription of the Pichinde virus genome segment is bidirectional. This is done using a cassette (see, for example, Ortiz-Riano et al., 2013, J Gen Virol., 94( In a more specific embodiment, the bidirectional expression system The set contains two sets of 5′-terminal fragments, one each flanking the two ends of the Pichinde virus genome segment to be inserted. The primers contain both polymerase I and polymerase II promoters.

[0114] In other embodiments, the cD of the Pichinde virus genome segment described herein The transcription of NA involves a promoter. A specific example of a promoter is an RNA polymerase I promoter. promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter Examples of promoters include the promoter, SP6 promoter, and T3 promoter.

[0115] In one embodiment, the method for producing the three segment Pichinde virus particles further comprises: and introducing the cDNA of the three-segment Pichinde virus particle into a host cell. In one embodiment, the method for producing the three-segment Pichinde virus particles comprises and further expressing all other components for the production of the three-segment Pichinde virus particle. introducing the cDNA of the three-segment Pichinde virus particle into a host cell, and purifying the three-segment Pichinde virus particles from the supernatant of the cells. Such methods are well known to those of skill in the art.

[0116] Provided herein are methods for infecting cells with the nucleic acids, vectors, and compositions provided herein. The nucleic acids, vectors and methods of culturing cells described herein are also provided. A more detailed description of the systems and cell lines is provided in Section 4.5.

[0117] In one embodiment, the three segmented Pichinde virus particle described herein comprises: In a specific embodiment, the present invention provides a method for the production of infectious and replicative Pichinde virus particles. The Pichinde virus particles described herein are attenuated. Segmented Pichinde virus particles show that the virus is still at least partially replicable. A virus that is capable of replication in vivo but produces a subclinical level of non-pathogenic infection. Such attenuated viruses are attenuated so that they can only generate a viral load. It can be used as an immunogenic composition.

[0118] In one embodiment, the three-segment Pichinde virus particle is It has the same tropism as the genovirus particle.

[0119] Also provided herein is a method for producing a method of the present invention comprising the steps of: In a specific embodiment, the kit comprises, in one or more containers, The kit includes a three-segment Pichinde virus particle as described in the specification. Suitable host cells for the rescue of Topichinde virus particles, transfection of the plasmid cDNA into the host cells A suitable reagent for transfecting the virus, a helper virus, a vector encoding a viral protein, A plasmid and / or a modified Pichinde virus genome segment or a Pichinde virus One or more oligonucleotide primers specific for the nucleic acid encoding the particle. The above may further be included.

[0120] Also provided herein are three-segment pitches as described in Sections 4.6 and 4.7. and an immunogenic composition comprising a genovirus particle.

[0121] 4.2.1 Trisegmented Pichinde virus particles containing one L segment and two S segments child In one aspect, provided herein is an L segment and two S segments. In one embodiment, the three-segmented Pichinde virus particle comprises one L segment. The growth of tripartite Pichinde virus particles containing two S segments was replication-competent. In a specific embodiment, the virus particles do not produce bi-segmented virus particles. The proliferation of tripartite virions containing two S segments and two S segments was confirmed by type I interferon. It lacks the interferon receptor, type II interferon receptor, and recombination activating gene (RAG1), 10 4 In mice infected with PFU of three-segmented Pichinde virus particles (see Section 4.8.13), At least 10 days, at least 20 days, at least 30 days, at least 40 days, or At least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or does not produce replication-competent bisegmented virus particles after at least 100 days. In one embodiment, a three-segment picindewi comprising one L segment and two S segments is The propagation of the rRNA particles may be at least 10 passages, at least 20 passages, at least 30 passages, or at least did not yield replication-competent bisegmented virus particles after at least 40 or at least 50 passages. stomach.

[0122] In one embodiment, two on one segment instead of two separate segments. The three-segment picindel provided herein combines arenavirus ORFs. Recombination between the two S segments of the S particle leads to the formation of a non-functional promoter (i.e. a genomic segment of the structure: 5'UTR----5'UTR or 3'UTR----3'UTR, In this case, each UTR that forms one end of a genome is an inverted repeat of the other end of the same genome.

[0123] In one embodiment, a three-segment peptide comprising one L segment and two S segments is Chinde virus particles carry the Pichinde virus ORF at a location other than the wild-type location of the ORF In another embodiment, one L segment and two S segments are engineered to The three-segmented Pichinde virus particles containing two Pichinde virus ORFs or three Pichinde virus ORFs a Chinde virus ORF, or four Pichinde virus ORFs, or five Pichinde virus ORFs, or engineered to carry six Pichinde virus ORFs at positions other than the wild-type positions. In a specific embodiment, a three-segment peptide comprising one L segment and two S segments is The Pichinde virus particle contains the full complement of all four Pichinde virus ORFs. In some embodiments, the three segmented Pichinde virus particles are infectious multiplicities. In a specific embodiment, the three-segmented Pichinde virus particle is The two S segments of the Mentopichinde virus particle share one of their ORFs with the wild type. In a more specific embodiment, the The two S segments comprise a full complement of the ORF of the S segment. and the L segment has been engineered to carry an ORF at a position other than the wild-type position. Or, the L segment can be a wild-type genomic segment.

[0124] In one embodiment, one of the two S segments is: (i) The ORF encoding the Z protein is under the control of the 5'UTR of Pichinde virus. Irus S segment; (ii) Pichinde virus, in which the ORF encoding the L protein is under the control of the 5'UTR of Pichinde virus Viral S segment; (iii) Pichinde virus S, in which the ORF encoding NP is under the control of the 5'UTR of Pichinde virus segment; (iv) Pichinde virus S, in which the ORF encoding GP is under the control of the 3'UTR of Pichinde virus segment; (v) The ORF encoding L is the Pichinde virus S sequence under the control of the Pichinde virus 3'UTR. segment; and (vi) Pichinde virus, in which the ORF encoding the Z protein is under the control of the 3'UTR of Pichinde virus Viral S segment; It could be.

[0125] In one embodiment, a three-segment peptide comprising one L segment and two S segments is Chinde virus particles contain overlapping ORFs (i.e., ORFs of two wild-type S segments, e.g., GP In a specific embodiment, the nucleotide sequence may include one L segment and two S segments. Trisegmented Pichinde virus particles containing segments contain one overlapping ORF (e.g., (GP, GP )) or two overlapping ORFs (e.g., (GP, GP) and (NP, NP)).

[0126] Table 2A below shows a three-segment picin deuterium nucleotide sequence containing one L segment and two S segments. The genome organization of the virus particle is shown, and the two S segments in the three-segment Pichinde virus genome are Recombination between segments of the amplicon does not result in replication-competent bisegmented virus particles, but suppresses renavirus promoter activity (i.e., the resulting recombinant S segment is a 3' It consists of two 3'UTRs instead of a 5'UTR and a 5'UTR). Table 2A Trisegmented Pichinde virus particles contain one L segment and two S segments. Position 1 is under the control of the 5'UTR of the Pichinde virus S segment; position 2 is under the control of the 5'UTR of the Pichinde virus S segment. position 3 is under the control of the 5′UTR of the Pichinde virus S segment; position 4 is under the control of the 3'UTR of the Pichinde virus S segment; Position 5 is under the control of the 5'UTR of the Pichinde virus L segment; position 6 is under the control of the Pichinde virus L segment 5'UTR. It is under the control of the 3'UTR of the lusL segment. *ORF indicates that a heterologous ORF has been inserted. [Table 3] TIFF2024170461000004.tif196170

[0127] In one embodiment, the IGR between position 1 and position 2 is selected from the group consisting of the Pichinde virus S segment and the It may be the IGR of the L segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment or the IGR of the L segment; the IGR between positions 5 and 6 is the IGR of the Pichinde virus L segment. In a specific embodiment, the IGR between positions 1 and 2 can be an IGR of a pichinectin. It may be the IGR of the viral S segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment. the IGR between positions 5 and 6 of the Pichinde virus L segment; In some embodiments, other combinations are possible. For example, a three-segment The threonine virus particle contains one L segment and two S segments. Intersegmental recombination of the two S segments in the Mentopichinde virus genome results in a replication-competent Produces no bisegmented virus particles and suppresses arenavirus promoter activity (i.e., the resulting recombinant S segment consists of two 5'UTRs instead of a 3'UTR and a 5'UTR. (It has been created.)

[0128] In one embodiment, a three-segment peptide comprising one L segment and two S segments is Intersegmental recombination of the S and L segments in the Chinde virus particle results in two A mechanism that carries two viral genes on only one segment instead of separate segments In another embodiment, one L segment and two S segments are restored. Segmentation of the S and L segments in three-segmented Pichinde virus particles containing Recombination between the two segments does not result in replication-competent bisegmented virus particles.

[0129] Table 2B below shows a three-segment picin deu containing one L segment and two S segments. The genome organization of the virus particle is shown, and the S segment in the three-segment Pichinde virus genome is shown. Recombination between the T and L segments produces replication-competent bisegmented virus particles. and suppress arenavirus promoter activity (i.e., the resulting recombinant S segment The ment is composed of two 3'UTRs instead of a 3'UTR and a 5'UTR). Table 2B Trisegmented Pichinde virus particles contain one L segment and two S segments. Position 1 is under the control of the 5'UTR of the Pichinde virus S segment; position 2 is under the control of the 5'UTR of the Pichinde virus S segment. position 3 is under the control of the 5' UTR of the Pichinde virus S segment; position 4 is under the control of the 3'UTR of the Pichinde virus S segment; position 5 is under the control of the 3'UTR of the Pichinde virus S segment; Position 5 is under the control of the 5'UTR of the Pichinde virus L segment; position 6 is under the control of the 5'UTR of the Pichinde virus L segment. It is under the control of the 3'UTR of the L segment. *ORF indicates that a heterologous ORF has been inserted. [Table 4]

[0130] In one embodiment, the IGR between position 1 and position 2 is selected from the group consisting of the Pichinde virus S segment and the It may be the IGR of the L segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment or the IGR of the L segment; the IGR between positions 5 and 6 is the IGR of the Pichinde virus L segment. In a specific embodiment, the IGR between positions 1 and 2 can be an IGR of a pichinectin. It may be the IGR of the viral S segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment. the IGR between positions 5 and 6 of the Pichinde virus L segment; In some embodiments, other combinations are possible. For example, a three-segment The threonine virus particle contains one L segment and two S segments. Intersegmental recombination of the two S segments in the Mentopichinde virus genome results in a replication-competent Produces no bisegmented virus particles and suppresses arenavirus promoter activity (i.e., the resulting recombinant S segment consists of two 5'UTRs instead of a 3'UTR and a 5'UTR. (It has been created.)

[0131] In certain embodiments, one of skill in the art can utilize the structures shown in Table 2A or 2B and described herein. The Pichinde virus genome was then constructed by synthesis and then assayed using the assay described in Section 4.8. In order to determine whether the three-segment Pichinde virus particles are genetically stable, i.e. To determine whether the method produces replication-competent bi-segmented viral particles as discussed herein. obtain.

[0132] 4.2.2 Trisegmented Pichinde virus particles containing two L segments and one S segment child In one aspect, provided herein are two L segments and one S segment. In one embodiment, the three-segmented Pichinde virus particle comprises two L segments. The growth of tripartite Pichinde virus particles containing one α- and one S segment was In a specific embodiment, the two L segments do not result in a bisegmented virus particle. The growth of three-segmented Pichinde virus particles containing one S segment and one T segment was confirmed by type I interferon. It lacks the interferon receptor, type II interferon receptor, and recombination activating gene (RAG1), 10 4 In mice infected with PFU of three-segmented Pichinde virus particles (see Section 4.8.13), At least 10 days, at least 20 days, at least 30 days, at least 40 days, or at least At least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least does not produce replication-competent bi-segmented virus particles after persistence for 100 days. A three-segmented Pichinde virus containing two L segments and one S segment The particles were replicated after at least 10, 20, 30, 40, or 50 passages. It does not result in segmented virus particles.

[0133] In one embodiment, instead of two separate segments, two on one segment The three-segment Pichinde virus ORF provided herein is Recombination between the two L segments of the virus particle results in a non-functional promoter ( That is, a genomic segment of the structure: 5'UTR----5'UTR or 3'UTR----3'UTR, In each UTR, which forms one end of the genome, an inverted repeat of the other end of the same genome is used.

[0134] In one embodiment, a three-segment peptide comprising two L segments and one S segment is Chinde virus particles carry the Pichinde virus ORF at a location other than the wild-type location of the ORF In another embodiment, two L segments and one S segment are engineered. The three-segmented Pichinde virus particles containing two Pichinde virus ORFs or three Pichinde virus ORFs a Chinde virus ORF, or four Pichinde virus ORFs, or five Pichinde virus ORFs, or engineered to carry six Pichinde virus ORFs at positions other than the wild-type positions. In a specific embodiment, a three-segment peptide comprising two L segments and one S segment is The Pichinde virus particle contains the full complement of all four Pichinde virus ORFs. In some embodiments, the three segmented Pichinde virus particles are infectious multiplicities. In a specific embodiment, the three-segmented Pichinde virus particle is The two L segments of the Mentopichinde virus particle share one of their ORFs with the wild type. In a more specific embodiment, the The two L segments comprise a full complement of the ORF of the L segment. In this case, the S segment carries one of these ORFs in a position other than its wild-type position. or the S segment can be a wild-type genomic segment. .

[0135] In one embodiment, one of the two L segments is: (i) The L segment, in which the ORF encoding GP is under the control of the 5′UTR of Pichinde virus; (ii) the L segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (iii) The ORF encoding the L protein is located in the L segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the L segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) an L segment in which the ORF encoding NP is under the control of the 3'UTR of Pichinde virus; and (vi) The ORF encoding the Z protein is located in the L segment under the control of the 3'UTR of Pichinde virus. to; It could be.

[0136] In one embodiment, a three-segment peptide comprising one L segment and two S segments is Chinde virus particles contain overlapping ORFs (i.e., ORFs of two wild-type L segments, e.g., Z-segment In a specific embodiment, the two L segments can include a ribozyme protein (RIP) or an L protein (L protein). The tripartite virion, which contains one S segment and one overlapping ORF (e.g., (Z protein, Z protein)) or two overlapping ORFs (e.g., (Z protein, Z protein) It may contain (protein, L protein) and (L protein).

[0137] Table 3 below shows a three-segment picin deu containing two L segments and one S segment. The genome organization of the virus particle is shown, and the two L segments in the three-segment Pichinde virus genome are Recombination between segments of the amplicon does not result in replication-competent bisegmented virus particles, but Suppress renavirus promoter activity (i.e., the putative resulting recombinant L segment Instead of a 3'UTR and a 5'UTR, the construct may consist of two 3'UTRs or two 5'UTRs. Based on Table 3, instead of a 3'UTR and a 5'UTR, a peptide consisting of two 5'UTRs was used. Similar combinations can be envisaged for creating deviral particles. Table 3 Trisegmented Pichinde virus particles contain two L segments and one S segment. * Position 1 is under the control of the 5'UTR of the Pichinde virus L segment; position 2 is under the control of the 5'UTR of the Pichinde virus L segment. Position 3 is under the control of the 3'UTR of the Pichinde virus L segment; position 4 is under the control of the 3'UTR of the Pichinde virus L segment; Position 5 is under the control of the 5'UTR of the Pichinde virus S segment; position 6 is under the control of the Pichinde virus S segment It is under the control of the 3'UTR of the rusS segment. *ORF indicates that a heterologous ORF has been inserted. [Table 5]

[0138] In one embodiment, the IGR between position 1 and position 2 is selected from the group consisting of the Pichinde virus S segment and the It may be the IGR of the L segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment or the IGR of the L segment; the IGR between positions 5 and 6 may be the IGR of the Pichinde virus S segment. In a specific embodiment, the IGR between position 1 and position 2 may be an IGR of the L segment or an IGR of the L segment. The IGR may be the IGR of the Pichinde virus L segment; the IGR between positions 2 and 3 may be the IGR of the Pichinde virus L segment. the IGR between positions 5 and 6 of the Pichinde virus L segment; It may be an IGR of the S segment. In some embodiments, other combinations are possible.

[0139] In one embodiment, a three-segment peptide comprising two L segments and one S segment is Intersegmental recombination of the L and S segments from the Chinde virus particle occurred in two has two viral genes on only one segment instead of separate segments In another embodiment, two L segments and one S segment are restored to a functional segment. Segmentation of the L and S segments in three-segmented Pichinde virus particles containing mitogens. Recombination between the cleavage sites does not result in replication-competent bisegmented virus particles.

[0140] Table 3B below shows a three-segment picin deu containing two L segments and one S segment. The genome organization of the virus particle is shown, and the L segment in the three-segment Pichinde virus genome is shown. Intersegmental recombination of the T and S segments produces replication-competent bisegmented virus particles. and suppress arenavirus promoter activity (i.e., the resulting recombinant S segment The ment is composed of two 3'UTRs instead of a 3'UTR and a 5'UTR). Table 3B Trisegmented Pichinde virus particles contain two L segments and one S segment. * Position 1 is under the control of the 5'UTR of the Pichinde virus L segment; position 2 is under the control of the 5'UTR of the Pichinde virus L segment. Position 3 is under the control of the 3'UTR of the Pichinde virus L segment; position 4 is under the control of the 3'UTR of the Pichinde virus L segment; Position 5 is under the control of the 5'UTR of the Pichinde virus S segment; position 6 is under the control of the Pichinde virus S segment It is under the control of the 3'UTR of the rusS segment. *ORF indicates that a heterologous ORF has been inserted. [Table 6]

[0141] In one embodiment, the IGR between position 1 and position 2 is selected from the group consisting of the Pichinde virus S segment and the It may be the IGR of the L segment; the IGR between positions 2 and 3 is the IGR of the Pichinde virus S segment or the IGR of the L segment; the IGR between positions 5 and 6 may be the IGR of the Pichinde virus S segment. In a specific embodiment, the IGR between position 1 and position 2 may be an IGR of the L segment or an IGR of the L segment. The IGR may be the IGR of the Pichinde virus L segment; the IGR between positions 2 and 3 may be the IGR of the Pichinde virus L segment. the IGR between positions 5 and 6 of the Pichinde virus L segment; It may be an IGR of the S segment. In some embodiments, other combinations are possible.

[0142] In one embodiment, one of skill in the art can use the compositions shown in Table 3A or 3B and described herein. The Pichinde virus genome was constructed and then analyzed using the assays described in Section 4.8. Whether the three-segment Pichinde virus particles are genetically stable, i.e. It can be determined whether the resulting replication-competent bisegmented viral particles are capable of replication as discussed in.

[0143] 4.2.3 Replication-defective three-segmented Pichinde virus particles In certain embodiments, provided herein are methods for isolating an ORF, comprising: (i) isolating an ORF other than at the wild-type location of the ORF; and (ii) the resulting virus produces additional infectious progeny virus particles. The GP, NP, Z protein, or L protein is not capable of replication (i.e., is replication-deficient). A three-segment gene in which the ORF encoding the protein has been deleted or functionally inactivated. In one embodiment, the third Pichinde virus segment is a Topichinde virus particle. In another embodiment, the third Pichinde virus segment can be an S segment. In a more specific embodiment, the third Pichindewi segment can be an L segment. A gene segment can be engineered to carry an ORF in a position other than its wild-type position. or the third Pichinde virus segment is a wild-type Pichinde virus genomic segment. In an even more specific embodiment, the third Pichinde virus may be a segment. The segments may include Pichinde virus ORs encoding GP, NP, Z, or L proteins. Lacking F.

[0144] In one embodiment, the three-segment genome segment is an S or L segment hybrid. (i.e., a genomic segment that may be a combination of an S segment and an L segment) In other embodiments, the hybrid segment includes an IGR of the L segment. In another embodiment, the hybrid segment is an S segment. In another embodiment, the hybrid segment is an L segment that includes an IGR of the target gene. In another embodiment, the hybridization vector is a UTR of the S segment with an IGR of the L segment. The lid segment is the UTR of the L segment with the IGR of the S segment. In an embodiment, the hybrid segment comprises a 5′ amino acid sequence of the S segment with an IGR of the L segment. The 3'UTR of the S segment is the 3'UTR of the S segment with the IGR of the L segment. In one embodiment, the hybrid segment is an L segment with an IGR of an S segment. the 5'UTR of the L segment with the IGR of the S segment or the 3'UTR of the L segment with the IGR of the S segment.

[0145] Includes genetically modified genomes in which one or more ORFs have been deleted or functionally inactivated The three-segmented Pichinde virus particles are capable of infecting complementation cells (i.e., The virus can be produced in a variety of cells expressing the Pichinde virus ORF, which has been inactivated in the vector. The genetic material of the Pichinde virus particles can be transferred to host cells upon infection. In addition, the genetic material may be expressed and amplified as described herein. The genome of the genetically modified Pichinde virus particles is derived from organisms other than the Pichinde virus particles. The heterologous ORF may encode a heterologous ORF.

[0146] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the sequences is removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In another embodiment, the GP, NP, Z and L proteins are encoded by At least one ORF, at least two ORFs, at least three ORFs, or at least four ORFs. The RF can be removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In a specific embodiment, the GP, NP, Z and L proteins are encoded by Only one of the four ORFs was removed and replaced with a heterologous ORF from an organism other than the Pichinde virion. In a more specific embodiment, the GP of the Pichinde virus genome segment is In another specific embodiment, the coding ORF is deleted. In a more specific embodiment, the ORF encoding NP of the p53 segment is deleted. The ORF encoding the Z protein of the Chinde virus genome segment is deleted. In a specific embodiment, the ORF encoding the L protein is deleted.

[0147] In one embodiment, provided herein is a polypeptide comprising one L segment and two S segments. and (ii) the resulting ORF is in a position other than the wild-type position of the ORF; The ORFs encoding GP or NP are deleted so that the resulting virus is replication-defective and not infectious. The present invention relates to a three-segment Pichinde virus particle which is either transformed or functionally inactivated. In a specific embodiment, one ORF is removed and a variant from an organism other than Pichinde virus is identified. In another specific embodiment, the two ORFs are removed and replaced with the pituitary ORF. In another specific embodiment, the virus is replaced with a heterologous ORF from an organism other than the virus. , three ORFs are removed and replaced with heterologous ORFs from organisms other than Pichindevirus. In a specific embodiment, the ORF encoding GP is removed and a living virus other than Pichinde virus is generated. In another specific embodiment, the ORF encoding NP is replaced with a heterologous ORF from the is deleted and replaced with a heterologous ORF from an organism other than Pichinde virus. In a specific embodiment, the ORF encoding NP and the ORF encoding GP are removed, The Chinde virus particle is replaced with one or two heterologous ORFs from an organism other than the Chinde virus particle. In one embodiment, the three-segmented Pichinde virus particle comprises: (i) one L segment; and two S segments; (ii) an ORF located at a position other than the wild-type position of that ORF; (iii) Pichinde Contains one or more heterologous ORFs from organisms other than viruses.

[0148] In one embodiment, provided herein is a method for producing a fusion protein comprising two L segments and one S segment. and (ii) the resulting ORF is in a position other than the wild-type position of the ORF; The Z protein and / or the L protein are inserted into the chromosome so that the resulting virus is replication-deficient and not infectious. A three-segment peptide having an ORF encoding the In a specific embodiment, one ORF is removed, resulting in a Pichinde virus particle. In another specific embodiment, the two ORFs are replaced with heterologous ORFs from an organism other than the virus. The ORF is removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In one embodiment, the ORF encoding the Z protein is deleted, and the virus is a non-Pichinde virus. In another specific embodiment, the L protein is replaced with a heterologous ORF from the organism. The loading ORF is removed and replaced with a heterologous ORF from an organism other than Pichinde virus. In an even more specific embodiment, the ORF encoding the Z protein and the L protein The coding ORF was removed and replaced with a heterologous ORF from an organism other than the Pichinde virus particle. Thus, in one embodiment, the three-segment Pichinde virus particle comprises: (i) two (ii) an ORF at a position other than the wild-type position of that ORF; iii) contains one or more heterologous ORFs from an organism other than Pichinde virus.

[0149] Thus, in some embodiments, the three-segment picindel provided herein The particles have been engineered to i) carry ORFs in non-native locations; ii) contain GP, ​​NP, and Z proteins; iii) the ORF encoding the P protein or the L protein is deleted; Three segments replaced with one or more heterologous ORFs from an organism other than Chindevirus. Pichinde virus particles (i.e., one L segment and two S segments, or two L segments) segment and one S segment).

[0150] In certain embodiments, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length , 25-100 nucleotides long, 50-200 nucleotides long, 50-400 nucleotides long, 200-500 nucleotides, or 400 to 600 nucleotides, or 500 to 800 nucleotides. In an embodiment, the heterologous ORF is 750-900 nucleotides in length, 800-100 nucleotides in length, 850 ~1000 nucleotides long, 900~1200 nucleotides long, 1000~1200 nucleotides long, 1000~1 500 nucleotides or 10-1500 nucleotides long, 1500-2000 nucleotides long, 1700-2000 Nucleotide length, 2000-2300 nucleotides, 2200-2500 nucleotides, 2500-3000 nucleotides Nucleotide length, 3000-3200 nucleotides, 3000-3500 nucleotides, 3200-3600 nucleotides Length of nucleotide, 3300-3800 nucleotides, 4000-4400 nucleotides, 42 00-4700 nucleotides long, 4800-5000 nucleotides long, 5000-5200 nucleotides long, 5200 ~5500 nucleotides long, 5500~5800 nucleotides long, 5800~6000 nucleotides long, 6000~ 6400 nucleotides long, 6200-6800 nucleotides long, 6600-7000 nucleotides long, 7000-72 00 nucleotides long, 7200-7500 nucleotides long, or 7500 nucleotides long. In some embodiments, the heterologous ORF is 5 to 10 amino acids in length, 10 to 25 amino acids in length, 25 to 50 amino acids in length, amino acid length, 50-100 amino acid length, 100-150 amino acid length, 150-200 amino acid length, 200-250 amino acid length amino acid length, 250-300 amino acid length, 300-400 amino acid length, 400-500 amino acid length, 500-750 amino acid length amino acid length, 750-1000 amino acid length, 1000-1250 amino acid length, 1250-1500 amino acid length, 1500-1 750 amino acids long, 1750-2000 amino acids long, 2000-2500 amino acids long, or 2500 or more amino acids long In some embodiments, the variant encodes a peptide or polypeptide having a length of about 1000 bp. A species ORF encodes a polypeptide not exceeding 2500 amino acids in length. In certain embodiments, the heterologous ORF does not contain a stop codon. In some embodiments, the nucleotide composition, nucleotide pair composition or Both can be optimized. Techniques for such optimization are well known in the art. It is known and can be applied to optimize heterologous ORFs.

[0151] Any heterologous ORF from an organism other than Pichinde virus can be expressed as a three-segment Pichinde virus In one embodiment, the heterologous ORF can be incorporated into a particle. A more detailed description of reporter proteins is provided in Section 4.3. In another embodiment, the heterologous ORF is associated with an antigen of an infectious pathogen, or any disease, In a specific embodiment, the antigen is capable of eliciting an immune response. The ORF may be derived from an infectious organism, a tumor (i.e., cancer), or an allergen. A more detailed explanation is provided in Section 4.3.

[0152] In one embodiment, the proliferation and infectivity of Pichinde virus particles are It is not affected by heterologous ORFs from organisms other than the host.

[0153] Using techniques known to those skilled in the art, the Pichinde virus ORF can be inserted into a location other than its wild-type location. Pichinde virus particles containing engineered Pichinde virus genome segments For example, reverse genetic techniques can be used to generate such Pichindewi offspring. In another embodiment, the replication-deficient Pichinde virus is capable of producing viral particles. Particles (i.e., ORFs encoding GP, NP, Z protein, and L protein are deleted, P Pichinde virus engineered to carry the Chinde virus ORF at a position other than the wild-type position The complementing genome segment can be produced in a complementing cell.

[0154] In one embodiment, the present application provides a method for the preparation of a vaccine comprising administering to a subject a subject therapies described herein that are suitable for use as a vaccine. Pichinde virus particles, and in vaccination and treatment or prevention of infections and cancers, for example The present invention relates to methods of using such Pichinde virus particles. A more detailed description of methods for using deviral particles is provided in Section 4.6.

[0155] In certain embodiments, the present application provides a pharmaceutical composition comprising any of the compounds described herein suitable for use as a pharmaceutical composition. and for example in vaccination and treatment or prevention of infection or cancer. The present invention also relates to methods of using such Pichinde virus particles in A more detailed description of methods for using the genovirus particles is provided in Section 4.6.

[0156] 4.3 Pichinde virus particles expressing heterologous ORFs or trisegmented Pichinde virus particles In one embodiment, the Pichinde virus genome segment and each Pichinde virus genome segment are The virus particle or trisegment Pichinde virus particle may contain a heterologous ORF. In another embodiment, the Pichinde virus genome segment and each Pichinde virus The virus particle or the three segmented Pichinde virus particle can contain a gene of interest. In a more specific embodiment, the heterologous ORF or gene of interest encodes an antigen. In a specific embodiment, the heterologous ORF or gene of interest comprises a reporter protein or It encodes a fluorescent protein.

[0157] In one embodiment, the Pichinde virus genome segment, the Pichinde virus particle The three segmented Pichinde virus particles may contain one or more heterologous ORFs or one or more genes of interest. In another embodiment, the Pichinde virus genome segment comprises: The Pichinde virus particle or the three-segment Pichinde virus particle comprises at least one Heterologous ORF, at least two heterologous ORFs, at least three heterologous ORFs, or more heterologous ORFs. In another embodiment, the Pichinde virus particle or the three segmented The Topicinde virus particles contain at least one gene of interest, at least two genes of interest, The offspring may contain at least three genes of interest, or more genes of interest.

[0158] Various antigens can be expressed using the Pichinde virus genome segments, Pichinde virus particles or It can be expressed by a three-segment Pichinde virus particle. The heterologous ORF may be an antigen of an infectious agent or any disease capable of eliciting an immune response. In certain embodiments, the heterologous ORF encodes an antigen associated with a virus, a bacterium, It may code for an antigen derived from a fungus, a parasite, or may be used to treat a tumor or tumor-associated disease ( i.e. cancer), autoimmune disease, degenerative disease, genetic disease, substance dependence, obesity, or allergies It can be manifested in inflammatory diseases.

[0159] In some embodiments, the heterologous ORF encodes a viral antigen. Non-limiting examples of the Adenoviridae family include the Adenoviridae (e.g., Mastadenovirus and Trial Adenovirus), Herpesviridae (e.g., Herpes simplex virus type 1, Herpes simplex Herpes simplex virus type 2, herpes simplex virus type 5, herpes simplex virus type 6, Epstein-Barr virus -viruses, HHV6-HHV8 and cytomegalovirus), Leviviridae (e.g., Levivirus rus, bacteriophage MS2, allolevirus), Poxviridae ( For example, Chordopoxvirinae, Parapoxvirus, Avianpoxvirus, Capripox virus, rabbit pox virus, swinepox virus, molluscpox virus and Entomopoxviridae), Papovaviridae (e.g., polioviruses, Viruses and papillomaviruses), Paramyxoviridae (e.g. Influenza virus, parainfluenza virus type 1, mobiliviruses (e.g., measles virus), Viruses (e.g., mumps virus), Pneumovirinae (e.g., pneumovirus, human respiratory syncytial virus), human respiratory syncytial virus and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus), Picornaviridae (e.g., Enterovirus roviruses, rhinoviruses, hepatoviruses (e.g., human hepatitis A virus), Orthoreoviruses, and Aphthoviruses), Reoviridae (e.g., Orthoreoviruses, Orthoreoviruses, Viral viruses, rotaviruses, cypoviruses, physiviruses, phytreoviruses, and Oryzaviruses), Retroviridae (e.g., mammalian B-type retroviruses, mammalian Human C retroviruses, avian C retroviruses, D retrovirus group, BLV-HTLV retroviruses Viruses, lentiviruses (e.g., human immunodeficiency virus (HIV) 1 and HIV-2 (e.g., HIV gp160), Spumaviruses), Flaviviridae (e.g., Hepatitis C virus, Denguine Viruses (e.g., West Nile virus), Hepadnaviridae (e.g., Hepatitis B virus), Gaviridae (e.g., Alphaviruses (e.g., Sindbisvirus) and Rubivirus) (e.g. rubella virus), Rhabdoviridae (e.g. vesiculovirus, Lissaviridae), viruses, ephemeroviruses, cytorhabdoviruses, and nucleorhabdoviruses), Arenaviruses (e.g., Arenaviruses, Lymphocytic Choriomeningitis Virus, IPP-IU) Viruses, and Lassa viruses), and Coronaviridae (e.g., coronaviruses and thromboviruses). In a specific embodiment, the viral antigen is an antigen from HIV gp120, gp41, HIV Nef, RSV F glycoprotein, RSV G glycoprotein, HTLV tax, simplex herpes pesvirus glycoproteins (e.g., gB, gC, gD, and gE) or hepatitis B surface antigen, hepatitis C The viral E protein or coronavirus spike protein. Thus, the viral antigen is not an HIV antigen.

[0160] In other embodiments, the heterologous ORF encodes a bacterial antigen (e.g., a bacterial coat protein). In other embodiments, the heterologous ORF encodes a parasitic antigen (e.g., a protozoan antigen). In yet another embodiment, the heterologous nucleotide sequence encodes a fungal antigen. do.

[0161] Non-limiting examples of bacterial antigens include Aquaspirillaceae, Azospirillaceae, Azotoba Bacteraceae, Bacteroidetes, Bartonella, Bdellovibrio, Campylobacter, Chlamydia (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae (e.g., Citrobacter spp., Edwardsiella spp., Enterobacter aerogenes, Envinia spp., E. coli, Hafnia spp., Klebsiella spp., Morganella spp., Proteus vulgaris, Probiotics genus Dendria, Salmonella, Serratia, and Shigella flexneri), Gardnerellaceae, Influenza Lactobacillus casei, Halobacteriaceae, Helicobacteraceae, Legionellaceae, Listeria genus, Tylococcoccaceae, Mycobacteria (e.g., Mycobacteria tuberculosis), Neisseriaceae, Marine Spirochetes, Pasteurellaceae, Pneumococcus spp., Pseudomonas spp., Rhizobiumaceae, Spirillaceae, Spiroso Staphylococci (e.g., methicillin-resistant Staphylococcus aureus and Staphylococcus pyogenes), Staphylococci Streptococci (e.g., Streptococcus enteritidis, Streptococcus mutans, and Streptococcus pneumoniae) cocci), Vampirovibrio Helicobacteraceae, Yersiniaceae, Bacillus anthracis and Vampirovibrio Examples include antigens from bacteria of the Ophiopogon family.

[0162] Non-limiting examples of parasitic antigens include amoeba, malarial parasites, and Plasmodium. Examples of antigens include those from parasites such as Trypanosoma cruzi and Trypanosoma spp. Typical examples include the genus Abscisicida (e.g., Abscisicida corymbifera and Abscisicida ramosa), Aspergillus spp., (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus Rhus terreus), Basidiobolus ranarum, Blastomyces dermatitidis , Candida spp. (e.g., Candida albicans, Candida glabrata, Candida cane, Candida Candida krusei, Candida parapsilosis, Candida pseudotropicalis, Can Candida quillemondii, Candida rugosa, Candida stellatoidea, and Candida tropicalis), Coccidioides immitis, Conidiobolus spp., Cryptococcus S. neoformis, S. cerevisiae, Dermatophytes, Histoplasma capsulatum, Microsporum gypseum, Mucor pusillus, South American blastomycetes, Pseudoaleshe Rhinosporidii, Rhinosporidium severii, Pneumocystis carinii, Spider nematode Mold genera (e.g., Rhizopus arrhizos, Rhizopus oryzae, and Rhizopus microsporidii) Russ), Saccharomyces sporothrix schenckii, Zygomycetes, and Zygomycetes, Included are antigens from fungi classes such as Saccharomycetes, Basidiomycetes, Fungi Imperfecti, and Oomycetes.

[0163] In some embodiments, the heterologous ORF encodes a tumor antigen or a tumor-associated antigen. In some embodiments, the tumor antigen or tumor-associated antigen is an acute lymphoblastic Leukemia, acute myeloid leukemia, adrenal cortical carcinoma, childhood adrenal cortical carcinoma, AIDS-related cancer, Kaposi's sarcoma, Anal cancer, appendiceal cancer, astrocytoma, atypical teratocarcinoma / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, Extrahepatic (cholangiocarcinoma), bladder cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma brain tumor, ependymoma, medulloblastoma, supratentorial tumor Primary neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, cancer of unknown primary site, central nervous system Lymphoma, primary, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma brain tumor, cervical cancer, Childhood cancer, chronic bronchitis, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, Colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, emphysema, endometrial cancer, ependymoma , esophageal cancer, Ewing sarcoma in Ewing family of tumors, extracranial germ cell tumors, gonadal Extrahepatic germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, carcinoma Id gastrointestinal tumors, gastrointestinal stromal tumors, germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic tumors , brain stem glioma, glioma, pediatric cerebellar astrocytoma, pediatric visual pathway and hypothalamus, gastric carcinoma Id, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, Hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's carcinoma tumor, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphocytic lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lip and oral cavity cancer, liposarcoma, Liver cancer (primary), lung cancer, non-small cell, small cell, AIDS-related lymphoma, Burkitt's lymphoma , cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoma, primary central nervous Myelopathy, Macroglobulinemia, Waldenstrom's, Male breast cancer, Malignant fibroids of bone / osteosarcoma histiocytoma, medulloblastoma, melanoma, intraocular, merkel cell carcinoma, mesothelioma, adult malignant, mesothelioma, genotype Metastatic squamous cell carcinoma of unknown origin, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell Mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, myeloid leukemia, Chronic, myeloid leukemia, adult acute, myeloid leukemia, childhood acute, myeloma, multiple myeloma (cancer of the bone marrow) , myeloproliferative disorders, chronic, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, Oligodendroglioma, oral cancer, oropharynx, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, epithelial ovarian cancer Ovarian cancer (stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell, paranasal sinus and Nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal glioma, pineal germ cell tumor , pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasm / multiple bone tumors Myeloma, pleural embryonal tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer) , Renal pelvis and ureter, Transitional cell carcinoma, Retinoblastoma, Rhabdomyosarcoma, Children, Salivary gland cancer, Sarcoma, Uterine Ing family tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell carcinoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, tonsillitis Squamous cell carcinoma - see Skin cancer (non-melanoma), squamous cell carcinoma of the neck of the body of unknown primary origin, metastatic, gastric cancer, supratentorial primary Primary neuroectodermal tumor, T-cell lymphoma, Cutaneous - see mycosis fungoides and Sézary syndrome, Testicular Cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, childhood transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblast Tumors, unknown primary site, adult cancer of unknown primary site, children, ureter and renal pelvis cancer, transitional cell carcinoma, urethral cancer , uterine cancer, endometrial sarcoma, bronchial tumors, central nervous system embryonal tumors; pediatric chordoma, colon cancer , Craniopharyngioma, Ependymoblastoma, Langerhans cell histiocytosis, Acute lymphoblastic leukemia, Acute Myeloid leukemia (adult / childhood), small cell lung cancer, medulloepithelioma, oral cancer, papillomatosis, intermediate differentiation of the pineal gland Parenchymal tumors, pituitary tumors, respiratory tract cancer involving the NUT gene on chromosome 15, spinal cord tumors, thymus These include antigens from tumor-associated diseases including thyroid cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. can be done.

[0164] Non-limiting examples of tumor or tumor-associated antigens include adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CPSF, cyclin D1, DKK1, ENAH (hMena), EpCAM, Ep hA3, EZH2, FGF5, glypican-3, G250 / MN / CAIX, HER-2 / neu, IDO1, IGF2B3, IL13Rα2 , intestinal carboxylesterase, α-fetoprotein, kallikrein 4, KIF20A, length Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p5 3, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin )1, SOX10, STEAP1, survivin, telomerase, VEGF, or WT1, EGF-R, CE A, CD52, gp100 protein, MELANA / MART1, NY-ESO-1, p53 MAGE1, MAGE3 and CDK4, α- Actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, β-catenin Nin, Cdc27, CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2, elongation factor 2 , ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase AS fusion protein, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras , N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-βRII, Examples of such proteins include lysine phosphate isomerase, Lengsin, M-CSF, MCSP, and mdm-2. can be.

[0165] In some embodiments, the heterologous ORF encodes a respiratory pathogen antigen. In an embodiment, the respiratory pathogen is RSV, coronavirus, human metapneumovirus, influenza virus, parainfluenza virus, Hendra virus, Nipah virus, adenovirus, Viruses such as rhinovirus or PRRSV. Non-limiting examples of respiratory viral antigens include Examples include respiratory syncytial virus F, G and M2 proteins, coronavirus (severe acute respiratory syndrome coronavirus 1 (SRS) (SARS), HuCoV) spike protein (S), human metapneumovirus fusion protein, Parainfluenza virus fusion protein and hemagglutinin protein (F, HN), Hepatitis B virus (HeV) and Nipah virus (NiV) attachment glycoproteins (G and F), adenovirus Capsid proteins, rhinovirus proteins, and PRRSV wild-type or modified GP5 and M Examples include proteins.

[0166] In specific embodiments, the respiratory pathogen is Bacillus anthracis, Mycobacterium tuberculosis, Bordetella pertussis, Streptococcus pneumoniae, or Bacillus subtilis. Streptococcus aureus, Yersinia pestis, Staphylococcus aureus, Francisella tularensis, Legionella pneumophila, Chlamydia Bacteria such as Neisseria pneumoniae, Pseudomonas aeruginosa, Neisseria meningitidis, and Haemophilus influenzae. Respiratory bacterial antigens Non-limiting examples of such antigens include Bacillus anthracis protective antigen PA, Mycobacterium tuberculosis mycobacterial antigen 85A, and HeLa antigen 85B. Hsp65, Bordetella pertussis toxoid (PT) and filamentous hemagglutinin (FHA) FHA), pneumococcal sortase A and surface adhesin A (PsaA), Yersinia pestis F1 and V subunits , Staphylococcus aureus, Francisella tularensis, Legionella pneumophila, Chlamydia pneumoniae, These include proteins from P. aeruginosa, N. meningitidis, and H. influenzae.

[0167] In some embodiments, the heterologous ORF encodes a T cell epitope. In some embodiments, the heterologous ORF encodes a cytokine or growth factor.

[0168] In other embodiments, the heterologous ORF encodes an antigen expressed in an autoimmune disease. In a more specific embodiment, the autoimmune disease is type I diabetes, multiple sclerosis, Non-limiting examples of autoimmune disease antigens include rheumatoid arthritis, lupus erythematosus, and psoriasis. Examples include Ro60, dsDNA, or RNP.

[0169] In other embodiments, the ORF encodes an antigen expressed in an allergic disease. In a more specific embodiment, the allergic disease is seasonal and perennial rhinoconjunctivitis. Non-limiting examples of allergy antigens include rheumatoid arthritis, asthma, and eczema. Examples include Bet v 1 and Fel d 1.

[0170] In other embodiments, the Pichinde virus genome segment, Pichinde virus particle or The three-segment Pichinde virus particle further comprises a reporter protein. The target protein can be expressed simultaneously with the antigens described herein. In some embodiments, expression is visible in normal light or other wavelengths of light. The strength of the effect produced by the protein is similar to that of the Pichinde virus particle or the three-segment protein. It can be used to directly measure and monitor Chinde virus particles.

[0171] Reporter genes will be readily recognized by those of skill in the art. In another embodiment, the reporter gene is a fluorescent protein. The target gene is GFP, which emits a bright green light when exposed to ultraviolet light or blue light. do.

[0172] Non-limiting examples of reporter proteins include β-galactosidase, chloramphenicol, Nicole acetyltransferase, neomycin phosphotransferase, Luciferase Examples of such enzymes include, but are not limited to, ferase or RFP.

[0173] In one embodiment, the Pichinde virus genome segment expressing a heterologous ORF, The Pichinde virus particle or the three-segment Pichinde virus particle is used for vaccination. have desirable properties for use as vectors for In another embodiment, the Pichinde virus genome segment expressing a heterologous ORF, The Pichinde virus particle or the three segment Pichinde virus particle can be used to infect a host (e.g., a mouse). The antibody can induce an immune response in mice (eg, rats, rabbits, goats, donkeys, and humans). In the manner described herein, the Pichinde virus genome segment expressing the heterologous ORF is The Pichinde virus particle or the three-segment Pichinde virus particle inhibits the innate immune response. In another embodiment, the Pichinde virus genomic sequence expressing a heterologous ORF is The Pichinde virus particle or the three-segment Pichinde virus particle is In a more specific embodiment, the Pichindewi expressing a heterologous ORF induces an immune response. rus genome segment, said Pichinde virus particle or said three-segment Pichinde virus particle The child has both innate and adaptive immune responses.

[0174] In another embodiment, the Pichinde virus genome segment expressing a heterologous ORF, Pichinde virus particles or the three-segment Pichinde virus particles induce T cell responses. In an even more specific embodiment, the Pichinde virus genomic vector expressing a heterologous ORF is The Pichinde virus particle or the three-segment Pichinde virus particle is In another embodiment, the antigen-binding domain carrying the exogenous gene of interest is expressed in a 5'-terminal region of the cytoplasmic domain. The deviral particles induce a strong CD8+ T cell response that is high in frequency and functionality. In the above, the virus expresses an antigen derived from an infectious organism, a cancer or an allergen. -segmented genome segment, said Pichinde virus particle or said three-segmented Pichinde virus particle CD8+ T cells specific for one or more epitopes of the corresponding foreign gene of interest. Induce.

[0175] In one embodiment, the Pichinde virus genome segment expressing a heterologous ORF, Pichinde virus particles or the three segmented Pichinde virus particles inhibit T helper 1 differentiation, Can induce memory formation in CD4+ T cells and / or elicit durable antibody responses These antibodies can neutralize, opsonize, and be toxic to tumor cells. or may have other advantageous biological characteristics. The Pichinde virus genome segment expressing a heterologous ORF, The particles or the three-segment Pichinde virus particles have strong tropism for dendritic cells. and activates them during infection, which enhances antigen presentation by antigen-presenting cells. To strengthen.

[0176] In one embodiment, the peptide expresses an antigen derived from an infectious organism, a cancer, or an allergen. Pichinde virus genome segments, said Pichinde virus particles or said three-segment Pichinde virus The virus particles elicited low or undetectable neutralizing antibody titers against Pichinde virus, respectively. In some embodiments, the antibody induces a highly protective neutralizing antibody response against the exogenous transgene. In the present invention, a particle or three segments expressing an antigen derived from an infectious organism, a cancer or an allergen are The Pichinde virus backbone that forms the Pichinde virus particle is composed of Pichinde virus backbone components. It has low ability to induce immunity against the virus.

[0177] 4.4 Preparation of Pichinde virus particles and three-segment Pichinde virus particles In general, Pichinde virus particles are similar to those described for another arenavirus, LCMV. It can be recombinantly generated by standard reverse genetic techniques (Flatz et al., 2006, Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 2006, Virology 350:3 70; see Ortiz-Riano et al., 2013, J Gen Virol. 94:1175-88. (All rights reserved.) The Pichinde virus particles provided herein These techniques can be applied as described below to make The genome of S. rus can be modified as described in Sections 4.1 and 4.2, respectively. .

[0178] 4.4.1 Open reading frames at non-native positions Genomes that have been engineered to carry a viral ORF at a location other than the wild-type location of that ORF The generation of Pichinde virus particles containing the segments can be achieved by any reverse genetics technique known to those skilled in the art. Thus, it can be produced recombinantly.

[0179] (i) Infectious, replication-competent Pichinde virus particles In one embodiment, the method for producing a Pichinde virus particle includes: (i) producing a first Pichinde virus particle; (ii) transfecting a cDNA of a virus genome segment into a host cell; (iii) transfecting a cDNA of a genomic segment of a genotype virus into a host cell; Plasmids expressing the minimal transactivators NP and L of Chinde virus were introduced into host cells. (iv) maintaining said host cells under conditions favorable for virus formation. and (v) harvesting the Pichinde virus particles. In this case, the cDNA is contained in a plasmid.

[0180] When produced from cDNA, Pichinde virus particles (i.e., infectious and replication-competent) can be grown. In certain embodiments, the Pichinde virus particle can be any of the vectors described herein. Any host that allows the virus to grow to a titer that allows the virus to be used for In one embodiment, the host cell is This allows the Pichinde virus particles to grow to titers comparable to those determined by .

[0181] In certain embodiments, the Pichinde virus particles may be propagated in a host cell. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO, or others. In a specific embodiment, the Pichinde virus particles may be propagated in a cell line.

[0182] In certain embodiments, the host cell is maintained in culture and contains one or more plasmid(s). The plasmid(s) is / are suitable for expression in mammalian cells. For example, one or more expression cassettes consisting of the promoter and terminator of polymerase I. The vector expresses the Pichinde virus genome segment(s) produced under the control of the vector.

[0183] Plasmids that can be used to generate Pichinde virus particles include: i) the S genome segment ii) a plasmid encoding a L genomic segment, e.g., pol-I In one embodiment, the viral L and A plasmid encoding the Pichinde virus polymerase directing the intracellular synthesis of the mitochondrial and S segments The codon can be incorporated into the transfection mixture. For example, the L protein can be The plasmids encoding the NP and / or the plasmids encoding the NP (pC-L and pC-NP, respectively) were not present. The L protein and NP are the minimum components required for transcription and replication of viral RNA. Alternatively, the L and S segments of the virus together with the NP and L proteins Intracellular synthesis of menthol is achieved by the L and S segments of two separate plasmids, one on each side. This can be done using an expression cassette with pol-I and pol-II promoters reading into the cDNA. can.

[0184] In one embodiment, the Pichinde virus genome segment is regulated by a promoter. Typically, RNA polymerase I-driven expression cassettes and RNA polymerase II-driven cassettes are used. Alternatively, a T7 bacteriophage RNA polymerase-driven cassette can be used. In one embodiment, a plasmid encoding the Pichinde virus genome segment The (multiple) may be the same, i.e., the genomic sequence and the trans-acting factors are the same as one The transcription can be initiated from the mide by a promoter. Specific examples of promoters include: RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter Examples of the promoter include the T7 promoter, the SP6 promoter, and the T3 promoter.

[0185] Additionally, the plasmid(s) may contain an expression cassette suitable for gene expression in mammalian cells. , e.g., a mammalian selectable marker under the control of a polymerase II expression cassette as described above. - can be characterized, for example, by puromycin resistance or by viral gene transfer After the image(s), there is an internal ribosome entry site, such as the internal ribosome entry site of the encephalomyocarditis virus. The plasmid contains an entry site followed by a mammalian resistance marker. Additionally, the plasmid further features a bacterial selection marker, such as an ampicillin resistance cassette. do.

[0186] Transfection of host cells with the plasmid(s) can be performed using calcium phosphate, lipopolysaccharide, or other suitable plasmid pharmacological agents. Commonly used strategies such as inoculum-based protocols or electroporation After a few days, the appropriate selection agent, e.g., puromycin, Surviving clones are isolated and subcloned according to standard procedures. and Western blot with an antibody against the viral protein(s) of interest. High expressing clones are identified using immunoprecipitation or flow cytometry.

[0187] For the recovery of Pichinde virus particles as described herein, the following procedure is envisaged: Day 1: As above, cells are typically 80% confluent in M6 well plates. The mixture of plasmids is transfected. For this purpose, calcium phosphate, liposomes, Any commonly used strategy such as a genomic-based protocol or electroporation can be used.

[0188] After 3-5 days: Collect the culture supernatant (Pichindane virus vector preparation), aliquot it, and aliquot it before use. Store at 4°C, -20°C, or -80°C depending on how long the Chinde virus vector will be stored. The infectious titer of the Pichinde virus vector preparation was determined by immunofocus assay. Alternatively, transfected cells and supernatants are assayed 3 days after transfection. On day 5, the cells can be passaged in a large vessel (e.g., T75 tissue culture flask) and the culture supernatants Harvest up to 5 days after passaging.

[0189] The present application further provides that the plasmid encoding the genome segment is adapted to incorporate a heterologous ORF. The heterologous ORF is incorporated into a plasmid using restriction enzymes. It can be incorporated.

[0190] (ii) Infectious replication-deficient Pichinde virus particles Infectious replication-defective Pichinde virus particles can be rescued as described above. However, when generated from cDNA, the infectious, replication-defective Pichinde virus provided herein can be propagated in complementing cells, which, due to modifications of their genome, are replication-deficient. These are cells that provide functionality excluded from the Pichimde virus (e.g., GP protein). When the ORF encoding the GP tag is deleted or functionally inactivated, complementing cells (providing protein).

[0191] One or more of the ORFs in the Pichinde virus vector have been deleted or are functionally inactive. The deletion of the glycoprotein GP is used as an example. The target gene is provided in trans to the deleted viral gene(s), e.g., in this example, GP. Such complementing cell lines, hereafter referred to as C cells, can be generated and propagated in BHK-2 1. Express the viral gene(s) of interest in a cell line such as HEK 293, VERO, or other cell line. One or more plasmids for expression (complementary plasmids, called C-plasmids) are inserted into the The C-plasmid(s) are produced by transfecting the C-plasmid into mammalian cells. One or more expression cassettes suitable for expression, e.g., EF1α pro with a polyadenylation signal. Pichindeu is produced under the control of a mammalian polymerase II promoter such as a promoter In addition, the complementation plasmid expresses the viral gene(s) that are deleted in the viral vector. The smid may be an expression cassette suitable for gene expression in mammalian cells, such as a polypeptide as described above. A mammalian selectable marker, e.g., puromycin, under the control of a ribozyme II expression cassette. encephalomyocarditis virus (EMV) characterized by isoform resistance or by the viral gene transcript(s) There is an internal ribosome entry site, such as the internal ribosome entry site of mammalian ribosomes, behind which For production in E. coli, the plasmid is further modified with Ampicillin. It features a bacterial selection marker such as a phosphorus resistance cassette.

[0192] The cells that can be used, for example, BHK-21, HEK 293, MC57G or others, can be grown in culture. Retention, calcium phosphate, liposome-based protocols or electroporation Transfect the complementing plasmid(s) using one of the commonly used strategies such as After several days, a suitable selection agent, e.g., puromycin, is added in increasing concentrations. Surviving clones are isolated and subcloned according to standard procedures. Western blot or flow blotting with antibodies against the viral protein(s) responsible for Identify high expressing C cell clones using a cytometry method. As an alternative to the use of transfected C cells, transient transfection of normal cells can be used to In each of the following steps, the missing viral gene(s) are complemented: In addition, helper viruses can be used to provide the missing functionality in trans. It is possible.

[0193] The plasmids are of two types: i) the minimal transactivator of Pichinde virus; Two plasmids, called TF-plasmids for intracellular expression in C cells, e.g. in this case from the NP and L proteins of the Pichinde virus; and ii) from the Pichinde virus The vector genome segment, e.g., the segment having the engineered modification, is then cultured in a C cell. The TF-plasmid can be a plasmid called GS-plasmid for intracellular expression. The NP and L proteins of each Pichinde virus vector were expressed in mammalian cells. An expression cassette suitable for protein expression, typically a mammalian promoter such as the CMV or EF1α promoter. Either of these can be expressed as polyadenylation under the control of an animal polymerase II promoter. The GS-plasmid is a small ( It expresses the small (S) and large (L) genome segments. It is usually driven by polymerase I. A T7 bacteriophage RNA polymerase (T7-) driven expression cassette or The latter can be used to preferentially catalyze the 3' end ribozyme for processing of the primary transcript. When using a T7-based system, T Expression of T7 was constructed in a similar manner to the TF-plasmid, and an additional expression plasmid providing T7 was used. This must be brought about by including the In one embodiment, the cells are constructed to further express T7 in a stable manner. The plasmid and the GS plasmid can be the same, i.e., the genomic sequence and trans-acting factors are , T7, polI, and polII promoters can be used to transcribe from one plasmid.

[0194] For the recovery of the Pichinde virus vector, the following procedure can be used. Day 1: Incubate C cells, typically at 80% confluence in a 6-well plate, with two TF-plasmids and two In one embodiment, a mixture of TF and GS plasmids is transfected. The plasmids can be the same, i.e. the genomic sequence and trans-acting factors are T7, polI, and polII promoters from one plasmid. Commonly used protocols such as calcium, liposome-based protocols or electroporation Any of the strategies used in the study can be used.

[0195] After 3-5 days: Collect the culture supernatant (Pichindane virus vector preparation), aliquot it, and aliquot it before use. Store at 4°C, -20°C, or -80°C depending on how long the Chinde virus vector will be stored. The infectious titer of the Pichinde virus vector preparation was then determined by immunofluorescence assay for C cells. Alternatively, the transfected cells and supernatants were analyzed by a focus assay. Subculture the cells into a larger container (e.g., T75 tissue culture flask) 3–5 days after transfection. Culture supernatants may be collected up to 5 days after passaging.

[0196] The present invention further relates to a cell culture medium infected with an infectious replication-defective Pichinde virus that expresses an antigen. When used to express antigens in cultured cells, Two procedures can be used:

[0197] i) injecting a Pichinde virus vector preparation described herein into a cell type of interest for 1 For example, when infected with a multiplicity of infection (MOI) of 2, 3, or 4, all of the Antigens are produced in the cells of the

[0198] ii) Alternatively, a lower MOI can be used and individual cell clones can be isolated from their viral load. The Pichinde virus vector can then be used to select for the level of expression of the antigen. Due to the non-lytic nature of the method, individual clones can be multiplied indefinitely. The antigen is then added either to the culture supernatant or to the cells themselves, depending on the nature of the antigen produced. However, the present invention is not limited to these two strategies. The antigen is expressed using an infectious, replication-deficient Pichinde virus as a vector. Other methods of driving the current may be considered.

[0199] 4.4.2 Preparation of three-segment Pichinde virus particles See, e.g., Emonet et al., 2008, PNAS, 106(9), which is incorporated herein by reference. ):3473-3478; Popkin et al., 2011, J. Virol., 85 (15):7928-7932; Dhanwani et al. As described by J. Virology, 2015, Journal of Virology, doi:10.1128 / JVI.02705-15 The three-segment Pichinde virus particles are recombinantly synthesized by reverse genetic techniques known in the art. The three segmented Pichinde virus provided herein can be produced by The production of the particles can be modified as described in Section 4.2.

[0200] (i) Infectious, replication-competent three-segmented Pichinde virus particles In one embodiment, the method for producing the three segment Pichinde virus particles comprises the steps of: (i) cDNA of one L segment and two S segments or two L segments and one S segment (ii) transfecting a host cell with the minimal transfection vector of the Pichinde virus; (iii) transfecting the host cell with plasmids expressing the agents NP and L; maintaining said host cells under conditions favorable for virus formation; and (iv) Pichinde virus Collecting the particles.

[0201] When generated from the cDNA, the three-segment Pichinde virus particles (i.e., infectious, replicative In one embodiment, the three-segment picin dewi The virus particles are then cultured as described herein to a titer that allows for the use of the virus. The virus can be propagated in any host cell that allows the virus to grow. The host cells are cultured to a titer comparable to that determined for the corresponding wild type. Allows the three-segment Pichinde virus particles to propagate.

[0202] In one embodiment, the three segmented Pichinde virus particles are propagated in a host cell. Specific examples of host cells that can be used include BHK-21, HEK 293, or the like. In a specific embodiment, the three segmented Pichinde virus particles are It may be propagated in.

[0203] In certain embodiments, the host cell is maintained in culture and contains one or more plasmid(s). The plasmid(s) is / are suitable for expression in mammalian cells. For example, one or more expression cassettes consisting of a promoter and terminator for polymerase I. The vector expresses the engineered Pichinde virus genome segment(s) under the control of the vector.

[0204] In a specific embodiment, the host cell is maintained in culture and contains one or more plasmids. The plasmid(s) is / are suitable for expression in mammalian cells. Suitable expression vectors, for example those consisting of a promoter and terminator for polymerase I. The viral gene(s) produced are expressed under the control of the cassette.

[0205] To generate a three-segmented Pichinde virus containing one L segment and two S segments The plasmids that can be used include: i) an S genome segment, e.g., pol-I-PIC-S, ii) two plasmids each encoding the L genome segment, e.g., pol-I-PIC-L The plasmid may contain a three-segment plasmid containing two L segments and one S segment. The plasmids required for Mentopichinde virus include: i) the L genome segment, e.g., pol-I-PIC ii) two plasmids each encoding an S genome segment, e.g., pol-I-PIC-S, pol-I-PIC-S, and pol-L, respectively; It is a plasmid encoding

[0206] In one embodiment, a picin devirus directs intracellular synthesis of viral L and S segments. Incorporating a plasmid encoding the spolymerase into the transfection mixture For example, a plasmid encoding the L protein and a plasmid encoding the NP ( The L protein and NP are involved in the transcription and replication of viral RNA. The minimum trans-acting factor required for viral replication, together with the NP and L proteins. The intracellular synthesis of the L and S segments of the plasmid is initiated by the L and S segments of two separate plasmids, respectively, from both sides. The expression cassettes with pol-I and pol-II promoters reading the cDNA of the S segment were used. This can be done.

[0207] Additionally, the plasmid(s) may contain an expression cassette suitable for gene expression in mammalian cells. a mammalian selectable marker, e.g., under the control of a polymerase II expression cassette as described above, For example, by characterizing the vector by puromycin resistance or by cloning the vector after viral gene transcription(s). In particular, there are internal ribosome entry sites, such as the internal ribosome entry site of the encephalomyocarditis virus. , followed by a mammalian resistance marker. For production in E. coli, the plasmid further characterized by a bacterial selection marker such as an ampicillin resistance cassette.

[0208] Transfection of BHK-21 cells with the plasmid(s) was performed using calcium phosphate buffer. Commonly used strategies such as liposome-based protocols or electroporation After a few days, the cells are cultured using a suitable selection agent, e.g., puromycin. Surviving clones are isolated and subcultured according to standard procedures. Cloning and infecting the virus with antibodies against the viral protein(s) of interest High expressing clones are identified using immunoblot or flow cytometry.

[0209] Typically, an RNA polymerase I-driven expression cassette, an RNA polymerase II-driven cassette, or a T 7. Bacteriophage RNA polymerase-driven cassettes can be used, the latter of which is preferentially accompanies the 3' end ribozyme for processing of the secondary transcript to generate accurate ends. In one embodiment, the vector encoding the Pichinde virus genome segment is The sequences can be the same, i.e., the genomic sequence and trans-acting factors can be T7, polI, and polI. It can be transcribed from one plasmid via the lII promoter.

[0210] In order to recover the Pichinde virus and the three-segment Pichinde virus vector, The following steps are envisaged: Day 1: As described above, culture M6 well plates at typically 80% confluency. The cells of the patient are transfected with a mixture of plasmids. commonly used protocols such as liposome-based or electroporation. Any of the strategies available can be used.

[0211] After 3-5 days: Collect the culture supernatant (Pichindane virus vector preparation), aliquot it, and aliquot it before use. Store at 4°C, -20°C, or -80°C depending on how long the Chinde virus vector will be stored. The infectious titer of the Pichinde virus vector preparation was determined by immunofocus assay. Alternatively, transfected cells and The supernatant was passaged into a larger vessel (e.g., a T75 tissue culture flask) and cultured for 5 days after passage. The supernatant may be collected.

[0212] The present application relates to the expression of heterologous ORFs and / or genes of interest, encoding said genomic segments. The plasmid has been modified to incorporate a heterologous ORF and / or gene of interest. The heterologous ORF and / or gene of interest may be conjugated to the plasmid using restriction enzymes. It can be incorporated into it.

[0213] (ii) infectious, replication-deficient, three-segmented Pichinde virus particles Infectious, replication-defective, three-segmented Pichinde virus particles were rescued as described above. However, when made from cDNA, the infectious, replication-defective vectors provided herein can be The genovirus can be propagated in complementation cells, which are capable of adapting to modifications of the genome. Thus, it is a cell that provides functionality that is excluded from the replication-deficient Pichinde virus (e.g., G Complementation when the ORF encoding the P protein is deleted or functionally inactivated cells provide the GP protein).

[0214] Removal or functional inactivation of one or more of the ORFs in the Pichinde virus vector (herein glycoproteins) For example, the Pichinde virus vector contains deleted viral genes. The gene(s), e.g., in this example, GP, can be produced and propagated in cells that provide the gene(s) in trans. Such complementing cell lines, hereafter referred to as C cells, may include BHK-21, HEK 293, VERO, or other The target viral gene (multiple genes) is transfected into a mammalian cell line such as HIV-1 (here, BHK-21 is used as an example). One or more plasmid gene(s) for expression of several genes (complementation plasmids, called C-plasmids) The C-plasmid(s) are generated by transfecting One or more expression cassettes suitable for expression in mammalian cells, e.g., polyadenylation signals Under the control of a mammalian polymerase II promoter such as the CMV or EF1α promoter having The resulting Pichinde virus vector expresses the deleted viral gene(s). In addition, the complementation plasmid contains an expression cassette suitable for gene expression in mammalian cells. , e.g., a mammalian selection marker under the control of a polymerase II expression cassette as described above. Carriers, characterized, for example, by puromycin resistance or by the expression of viral gene transcripts (multiple After the ER site, an internal ribosome entry site such as that of the encephalomyocarditis virus is The plasmid p53 is then inserted into the plasmid p53A, followed by a mammalian resistance marker. The plasmid further features a bacterial selection marker, such as an ampicillin resistance cassette.

[0215] The cells that can be used, for example, BHK-21, HEK 293, MC57G or others, can be grown in culture. Retention, calcium phosphate, liposome-based protocols or electroporation Transfect the complementing plasmid(s) using one of the commonly used strategies such as After several days, a suitable selection agent, e.g., puromycin, is added in increasing concentrations. Surviving clones are isolated and subcloned according to standard procedures. Western blot or flow blotting using antibodies against the viral protein(s) involved Identify high expressing C cell clones using a cytometry method. As an alternative to the use of transfected C cells, transient transfection of normal cells can be used to In each of the following steps, the missing viral gene(s) are complemented: In addition, helper viruses can be used to provide the missing functionality in trans. It is possible.

[0216] Two types of plasmids: i) in this example, the NP and L proteins of Pichinde virus The minimal transactivator of Pichinde virus derived from the cytoplasm was expressed intracellularly in C cells. ii) two plasmids, called TF-plasmids, for the transcription of the mitochondrial endothelial cell line; and ii) the Pichinde virus vector. - Expressing a genomic segment, e.g., a segment with a designed modification, intracellularly in a C cell A plasmid called GS-plasmid can be used to induce the expression of TF-plasmid. The NP and L proteins of each Pichinde virus vector were expressed in mammalian cells. An expression cassette suitable for protein expression, usually a promoter such as the CMV or EF1α promoter. Either one of these can be expressed as poly(A) under the control of a mammalian polymerase II promoter. The GS-plasmid is a small vector that expresses the GS-plasmid in combination with a denylation signal. They express the small (S) and large (L) genome segments. RNA polymerase I-driven expression cassette or T7 bacteriophage RNA polymerase (T7-)-driven expression cassette The latter can be used to encode a 3'-terminal ribozyme for processing of the primary transcript. When using a T7-based system, the C-cell line is preferentially involved, resulting in accurate termination. Expression of T7 in cells was constructed in a similar manner to the TF-plasmid, providing an additional expression vector for T7. Must it be brought about by including a plasmid in the recovery process? Alternatively, the C cells are engineered to further express T7 in a stable manner. Therefore, the TF and GS plasmids can be the same, i.e., the genomic sequence and the transactivation The factors can be transcribed from one plasmid by the T7, polI, and polII promoters. .

[0217] For the recovery of the Pichinde virus vector, the following procedure can be used. Day 1: Incubate C cells, typically at 80% confluence in a 6-well plate, with two TF-plasmids and two In one embodiment, a mixture of TF and GS plasmids is transfected. The plasmids can be the same, i.e. the genomic sequence and trans-acting factors are T7, polI, and polII promoters from one plasmid. Commonly used protocols such as calcium, liposome-based protocols or electroporation Any of the strategies used in the study can be used.

[0218] After 3-5 days: Collect the culture supernatant (Pichindane virus vector preparation), aliquot it, and aliquot it before use. Store at 4°C, -20°C, or -80°C depending on how long the Chinde virus vector will be stored. The infectious titer of the Pichinde virus vector preparation was then determined by immunofluorescence assay for C cells. Alternatively, transfected cells and supernatants were cultured in a 30-well plate using a focus assay. Subculture the cells into a larger container (e.g., T75 tissue culture flask) 3–5 days after transfection. Culture supernatants may be collected up to 5 days after passaging.

[0219] The present invention relates to an infectious, replication-defective, three-segmented Pichinde virus that expresses an antigen in cell culture. The present invention further relates to the expression of antigens in cell cultures transfected with When used to express a CMV antigen, the following two procedures can be used:

[0220] i) injecting a Pichinde virus vector preparation described herein into a cell type of interest for 1 hour. For example, when infected with a multiplicity of infection (MOI) of 2, 3, or 4, all of the The antigen is produced in the cells of the

[0221] ii) Alternatively, a lower MOI can be used and individual cell clones can be cultured using the virus-driven The expression level of the genotype antigen can then be selected. Due to the non-lytic nature of the cells, individual clones can be expanded indefinitely. The antigen is then added either to the culture supernatant or to the cells themselves, depending on the nature of the antigen produced. However, the present invention is not limited to these two strategies. The study uses infectious, replication-deficient Pichinde virus as a vector to deliver CMV antibodies. Other methods of driving expression of the gene may be considered.

[0222] 4.5 Nucleic acids, vector systems and cell lines In certain embodiments, provided herein are the compositions described in Sections 4.1 and 4.2, respectively. Pichinde virus genome segment or three-segment Pichinde virus particle carrying the It is a cDNA comprising or consisting of the same.

[0223] 4.5.1 Open reading frames at non-native positions In one embodiment, provided herein is a method for treating Pichindewi as described in Section 4.1. In a more specific embodiment, the nucleic acid encoding the nucleic acid segment of the present invention is Provided herein are the DNA nucleotide sequences set forth in Table 1 or sequences derived from DNA nucleotide sequences. Host cells containing such nucleic acid are also provided in Section 4.1.

[0224] In a specific embodiment, provided herein is a method for locating an ORF other than at the wild-type location of the ORF. A cDNA of a Pichinde virus genome segment engineered to carry the The Pichinde virus genome segment is a cDNA encoding a heterologous ORF as described in Section 4.1. It is.

[0225] In one embodiment, provided herein is a method for isolating an ORF at a position other than the wild-type position of the ORF. DNA expression vectors encoding Pichinde virus genome segments engineered to carry Specifically provided herein is a vector system, wherein one or more vectors are The two Pichinde virus genome segments of the Pichinde virus particle described in the document, namely That is, a DNA expression vector system encoding an L segment and an S segment. A vector system can encode (one or more separate DNA molecules).

[0226] In another embodiment, provided herein is a method for isolating an ORF at a position other than its wild-type position. It is a cDNA of the S segment of Pichinde virus engineered to be carried by the GFP. In another embodiment, the ORF is located in a region other than its wild-type location. The cDNA of the Pichinde virus L segment engineered to carry the cDNA at the position of In one embodiment, the ORF is a part of a gene encoding the wild-type gene, or is integrated into a DNA expression system. (ii) the GP, NP, Z protein, or The ORF encoding the L protein was removed, and a heterologous ORF from an organism other than Pichindevirus was inserted. This is the cDNA of the Pichinde virus genome segment in which the ribosomal RNA has been replaced by the ribosomal RNA.

[0227] In certain embodiments, the cDNA provided herein is specific to a particular strain of Pichinde virus. The strains of Pichinde virus can be derived from Munchique CoAn4763 isolate P18 and its These and their derivatives, including P2 and their derivatives, or any other work described by Trapido and its co-workers. The strains may be derived from any of several isolates described in Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-641). In a specific embodiment, the cDNA is derived from the Pichinde virus Munc hique Derived from CoAn4763 isolate P18.

[0228] In one embodiment, the Pichinde virus particle or trisegment described herein The vectors engineered to encode the Pichinde virus particles are characterized by the characteristics of the Pichinde virus. The strain of Pichinde virus can be based on the Munchique CoAn4763 isolate P18. and their derivatives, P2 and their derivatives, or by Trapido and collaborators. The strains may be derived from any of several isolates described in (Trapido et al., 1971, Am J. T. rop Med Hyg, 20: 631-641). In some embodiments, the Pichindeu The virus particles or three-segmented Pichinde virus particles are The strain may be based on the 4763 isolate P18. The sequence of the S segment is set forth as SEQ ID NO: 1. The sequence of the S segment of the virus strain Munchique CoAn4763 isolate P18 is set forth in SEQ ID NO:1. The sequence of the L segment of Pichinde virus is listed as SEQ ID NO:2.

[0229] In another embodiment, provided herein is a cDNA or vector as described above in this section. Cells comprising such systems include cell lines derived from such cells, cultures comprising such cells, infections, Methods for culturing such cells are also provided herein. In one embodiment, Provided herein are vectors engineered to carry an ORF at a location other than the wild-type location of the ORF. In some embodiments, the cell contains a cDNA of a Pichinde virus genome segment. In one embodiment, the cell comprises an S segment and / or an L segment.

[0230] 4.5.2 Three-segmented Pichinde virus particles In one embodiment, provided herein is a three-segment peptide comprising the peptides described in Section 4.2. In a more specific embodiment, the nucleic acid encoding the Pichinde virus particle is Provided herein are, for example, DNA nucleotide sequences or DNA nucleotide sequences set forth in Table 2 or Table 3. A host cell containing such a nucleic acid is also provided in Section 4.2. There are.

[0231] In a specific embodiment, provided herein is a method for locating an ORF other than at the wild-type location of the ORF. It consists of a three-segment Pichinde virus particle cDNA engineered to carry the In another embodiment, (i) a Pichinde virus ORF is inserted into the wild-type position of the ORF. (ii) the three-segment Pichinde virus particle is engineered to carry the The offspring are three-segmented Pichinde virus particles encoding the heterologous ORFs described in Section 4.2. It is cDNA.

[0232] In one embodiment, provided herein is a three-segment The present invention relates to a DNA expression vector system that together encodes the Pichinde virus particle. Provided herein is a vector comprising one or more of the three Pichinde virus genome segments, That is, one L segment and one β segment of the three-segment Pichinde virus particle described herein and two S segments or two L segments and one S segment. Such vector systems are capable of encoding (one or more separate DNA molecules) can.

[0233] In another embodiment, provided herein is a method for isolating an ORF at a position other than its wild-type position. and is part of or incorporated into a DNA expression system. In another embodiment, the ORF is a cDNA of the Pichinde virus S segment(s). Pichinde virus L segments engineered to carry the nucleotide sequence at positions other than the wild-type position of The cDNA of the nucleic acid sequence of the present invention is part of or incorporated into a DNA expression system. and (i) has been engineered to carry a GP, ORF, or a nucleotide sequence other than its wild-type position; and ORFs encoding NP, Z protein, or L protein were deleted, and non-Pichinde virus The three-segmented Pichinde virus particle cDNA has been replaced with a heterologous ORF from another organism. be.

[0234] In certain embodiments, the cDNA provided herein is specific to a particular strain of Pichinde virus. The strains of Pichinde virus can be derived from Munchique CoAn4763 isolate P18 and its These and their derivatives, including P2 and their derivatives, or any other work described by Trapido and its co-workers. The strains may be derived from any of several isolates described in Trapido et al., 1971, Am J Trop Med Hyg, 20: 631-641). In a specific embodiment, the cDNA is derived from the Pichinde virus Munc hique Derived from CoAn4763 isolate P18.

[0235] In one embodiment, the Pichinde virus particle or trisegment described herein The vectors engineered to encode the Pichinde virus particles are characterized by the characteristics of the Pichinde virus. The strain of Pichinde virus can be based on the Munchique CoAn4763 isolate P18. and their derivatives, P2 and their derivatives, or by Trapido and collaborators. The strains may be derived from any of several isolates described in (Trapido et al., 1971, Am J. T. rop Med Hyg, 20: 631-641). In some embodiments, the Pichindeu The virus particles or three-segmented Pichinde virus particles are The strain may be based on the 4763 isolate P18. The sequence of the S segment is set forth as SEQ ID NO: 1. The sequence of the S segment of the virus strain Munchique CoAn4763 isolate P18 is set forth in SEQ ID NO:1. The sequence of the L segment of Pichinde virus is listed as SEQ ID NO:2.

[0236] In another embodiment, provided herein is a cDNA or vector as described above in this section. Cells comprising the system. Cell lines derived from such cells, cultures comprising such cells, Also provided herein are methods for culturing such infected cells. Provided herein is a cell containing the cDNA of a three-segment Pichinde virus particle. In some embodiments, the cell is include.

[0237] 4.6 How to use Vaccines are used to prevent and / or treat infectious diseases, such as poliovirus and measles. However, treatment in the setting of established chronic diseases, including cancer and chronic infections, remains limited. Immunotherapy has not been very successful. Pichinde virus particles and / or three-segment Pichinde virus particles The ability to generate deviral particles represents a new and novel vaccine strategy.

[0238] In one embodiment, provided herein is a method for treating an infection and / or cancer in a subject. The method comprises administering one or more types of Pichinde virus particles or administering to the subject a three-segment Pichinde virus particle or a composition thereof; In a specific embodiment, the infection and / or cancer described herein is The method of treating includes administering a therapeutically effective amount of one or more Pichinde virus particles or Administering a three-segmented Pichinde virus particle or composition thereof to a subject in need thereof. The subject may be, but is not limited to, a human, a mouse, a rat, a guinea pig, or However, there are houses for cows, horses, sheep, pigs, goats, cats, dogs, hamsters, donkeys, etc. The subject may be a mammal, such as a livestock animal. In a specific embodiment, the subject is a human. The human subject may be male or female, adult, child or elderly (65 or older), and may be suffering from multiple diseases. In one embodiment, the subject may be a subject with a polymorbid condition (i.e., a polymorbid subject). Subjects are those whose disease has progressed following treatment with chemotherapy, radiation therapy, surgery, and / or biologics. I am the one who did this.

[0239] In another embodiment, provided herein is a method for detecting an infectious organism, a tumor, or a pulmonary edema in a subject. or a method for inducing an immune response to an antigen derived from an allergen, comprising administering to said subject an infectious organism, Pichinde virus particles or trisegments expressing antigens derived from tumors or allergens The method further comprises administering to the subject an intropinde virus particle or a composition thereof. do.

[0240] In another embodiment, the method comprises administering to the subject a cancer cell derived from an infectious organism, tumor, or allergen provided herein. Pichinde virus particles or three-segmented Pichinde virus particles expressing the antigens The subject to which the composition is administered may have an infection, cancer, or allergy, susceptible to, or at risk of, developing cancer or exhibiting precancerous tissue lesions. In certain embodiments, the antibody is derived from an infectious organism, tumor, or allergen described herein. Pichinde virus particles or three-segment Pichinde virus particles or The subject to whom the composition is administered is infected with an infection, cancer, a precancerous tissue lesion, or an allergy. have, are susceptible to, are at risk for, or have been diagnosed with a disorder.

[0241] In another embodiment, the present invention relates to a method for treating a cancer caused by an infectious organism, tumor, or allergen described herein. Pichinde virus particles or three-segmented Pichinde virus particles expressing the antigens The subject to which the composition is administered may have a variety of disorders, including, but not limited to, the pulmonary system, central nervous system, lymphatic system, gastrointestinal system, and the like. or have or are susceptible to circulatory system infection, cancer, precancerous lesions, or allergies In a specific embodiment, the subject is at risk of having any of the disorders described herein. Pichinde virus particles or other particles expressing antigens derived from infectious organisms, tumors, or allergens The subject receiving the three-segmented Pichinde virus particle or a composition thereof has a tumor in the brain, liver, lungs, and the like. any part of the body, including, but not limited to, the eyes, ears, intestines, esophagus, uterus, nasopharynx, or salivary glands Suffering from, susceptible to, or at risk of infection, cancer, or allergies in one or more organs are exposed to

[0242] In another embodiment, the antibody is derived from an infectious organism, cancer, or allergen described herein. Pichinde virus particles or three-segment Pichinde virus particles or The subject receiving the composition may experience symptoms such as fever, night sweats, fatigue, malaise, anxiety, sore throat, swollen glands, , joint pain, muscle pain, loss of appetite, weight loss, diarrhea, gastrointestinal ulcers, gastrointestinal bleeding, shortness of breath, pneumonia, oral Cavity ulcers, vision problems, hepatitis, jaundice, encephalitis, seizures, coma, pruritus, erythema, hyperpigmentation, phosphorus The subject may be suffering from a condition including, but not limited to, changes in nodes, or hearing loss.

[0243] In another embodiment, the antibody is derived from an infectious organism, cancer, or allergen described herein. Pichinde virus or three-segment Pichinde virus particle expressing an antigen that The composition may be used to treat or prevent infection, cancer, or allergies, or to treat or prevent the development of other conditions, such as cancer, allergies, or other conditions that may be caused by the infection, cancer, or allergies. In a specific embodiment, the method of the present invention is administered to a subject of any age group who has been exposed to a pulmonary embolism. Pichinde virus expressing an antigen derived from an infectious organism, cancer or allergen as provided in the document. The present invention relates to a method for administering a virion or trisegment virion or a composition thereof to an immunocompromised subject. , pregnant subjects, subjects who have received an organ or bone marrow transplant, subjects taking immunosuppressants subjects with a chronic disease, subjects undergoing hemodialysis, subjects with cancer, or subjects with a history of infection, cancer, or allergy. It is administered to a subject who is afflicted with, susceptible to, or at risk of developing the disease. In specific embodiments, the antibody is derived from an infectious organism, cancer, or allergen described herein. Pichinde virus particles or three-segment Pichinde virus particles or The composition is for treating or preventing infection, cancer, or allergies in a subject suffering from, susceptible to, or associated with the infection, cancer, or allergies. At risk: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or In yet another specific embodiment, the method of the present invention is administered to a subject who is a 17-year-old child. Pichinde virus expressing an antigen derived from an infectious organism, cancer or allergen described in The particle or three segmented Pichinde virus particle or composition thereof is intended to prevent infection, cancer, or allergy. Subjects who are infants suffering from, susceptible to, or at risk for pulmonary embolism In yet another specific embodiment, the infectious organisms described herein are administered to Pichinde virus particles or trisegment expressing antigens derived from cancer or allergens The Topitinde virus particles or compositions thereof may be used to treat infections, cancer, or allergies, Susceptible or at risk 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 In yet another specific embodiment, the subject is administered to a subject who is a 12-month-old infant. Pichindeu expressing antigens derived from infectious organisms, cancers or allergens as described in the specification. The present invention relates to a method for treating infection, cancer, or elderly subjects who suffer from, are prone to, or are at risk for allergies In a more specific embodiment, the method is administered to an infectious organism, cancer or The present study was carried out to identify the Pichinde virus particles or three-segment Pichinde virus particles expressing antigens derived from allergens. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 , 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 years of age. is administered to the subject.

[0244] In another embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of a cancer or infectious disease, comprising administering to the patient ... Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen The composition is administered to a subject at high risk for disseminated infection, cancer, or allergies. In a preferred embodiment, the antibody is derived from an infectious organism, cancer or allergen described herein. Pichinde virus particles expressing an antigen or three-segment Pichinde virus particles or The composition is administered to a neonatal subject, who is a newborn and therefore has an immature immune system. .

[0245] In another embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of a cancer or infectious disease, comprising administering to the patient ... Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen The composition is administered to a subject with a dormant infection, cancer, or allergy. In some embodiments, the antigens derived from infectious organisms, cancers, or allergens described herein are Pichinde virus particles expressing or three-segmented Pichinde virus particles or compositions thereof The substance may be a dormant infection, a dormant cancer, or a dormant allergy that may be reactivated upon failure of the immune system. Thus, provided herein are methods for treating infection, cancer, or allergies. This is a method to prevent the reactivation of energy.

[0246] In another embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of a cancer or infectious disease, comprising administering to the patient ... Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen The composition is administered to a subject with a recurrent infection, cancer, or allergy.

[0247] In another embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of a cancer or infectious disease, comprising administering to the patient ... Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen The composition is administered to a subject having a genetic predisposition to infection, cancer, or allergy. In another embodiment, the antibody is derived from an infectious organism, cancer or allergen described herein. Pichinde virus particles or three-segment Pichinde virus particles or The composition is administered to a subject. Pichinde virus particles or three-segment Pichinde virus expressing antigens derived from gen The particles are administered to a subject having a risk factor. Exemplary risk factors include aging, tumor Baco, sun exposure, radiation exposure, chemical exposure, family medical history, alcohol, poor diet These include lack of activity, lack of exercise, or being overweight.

[0248] In another embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is Administration of the mitochondrial virus particles or the three-segment mitochondrial virus particles may be used to treat symptomatic infections, cancer, or In another embodiment, the antibody is derived from an infectious organism, cancer, or an allergen. Administration of Pichinde virus particles expressing an antigen related to the infection or three-segment Pichinde virus particles reduces asymptomatic infections, cancer, or allergies.

[0249] In another embodiment, a plasmid expressing an antigen derived from an infectious organism described herein is provided. The Chinde virus particle or the three-segmented Chinde virus particle or the composition thereof is Influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis Viruses such as infectious laryngotracheitis, avian anemia, Marek's disease, and avian Leukemia virus, avian adenovirus, avian pneumovirus, SARS virus , Human respiratory syncytial virus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus, C Hepatitis virus, polio virus, rabies virus, Hendra virus, Nipah virus, Human parainfluenza type 3 virus, measles virus, mumps virus, Ebola Viruses, Marburg virus, West Nile virus, Japanese encephalitis virus, Dengue virus Viruses, Hantavirus, Rift Valley fever virus, Lassa fever virus, Herpes simplex The therapeutic agent is administered to a subject or animal infected with one or more strains of the influenza virus and the yellow fever virus.

[0250] In another embodiment, a Pichindeu expressing an antigen derived from a cancer described herein is provided. The virus particle or three-segment Pichinde virus particle or composition thereof may be one or more types In another embodiment, the vaccine described herein is administered to a subject suffering from a cancer of the present invention. Any type of cancer that is susceptible to treatment with ribozymes may be targeted. In the present invention, a Pichinde virus particle or particles expressing an antigen derived from a cancer described herein are The three segmented Pichinde virus particles or compositions thereof can be used to treat, for example, melanoma, prostate cancer, breast cancer, Cancer, lung cancer, neuroblastoma, hepatocellular carcinoma, cervical cancer, and gastric cancer, Burkitt's lymphoma; non-Hodgkin's lymphoma Hodgkin's lymphoma; nasopharyngeal cancer (cancer of the upper part of the throat behind the nose), leukemia, mucosal The subject is administered with membrane-associated lymphoid tissue lymphoma.

[0251] In another embodiment, a plasmid expressing an antigen derived from an allergen as described herein is provided. The Chinde virus particle or the three-segmented Chinde virus particle or the composition thereof may comprise one or more In a more specific embodiment, the method described herein is administered to a subject suffering from an allergy. Pichinde virus particles expressing antigens derived from allergens that are considered to be allergenic or three-segment picin The deviral particles or compositions thereof can be used to treat, for example, seasonal allergies, perennial allergies, nasal allergies, It is administered to subjects suffering from conjunctivitis, asthma, eczema, and food allergies.

[0252] In another embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of a cancer or infectious disease, comprising administering to the patient ... Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen Administration of the composition to a subject results in cell-mediated immunity (CMI) to infection, cancer, or an allergen. Without wishing to be bound by theory, in another embodiment, Pichinde virus particles expressing antigens derived from infectious organisms, cancers, and allergens that are The three-segment Pichinde virus particle or composition thereof may be administered to a host (e.g., a macrophage) or a mammalian cell. The virus infects host cells (cells, dendritic cells, or B cells) and binds to major histocompatibility complex (MHC) class I and II antigens. Expressing the antigen of interest in the host's antigen-presenting cells (APCs) for direct presentation of the antigen In another embodiment, the antibody is derived from an infectious organism, cancer, allergen, or the like as described herein. Pichinde virus particles or three-segment Pichinde virus particles or administering the composition to a subject to treat or prevent an infection, cancer, or allergy; plurifunctional Cytolytic and IFN-γ and TNF-α co-producing CMV-specific CD4+ and highly induces CD8+ T cell responses.

[0253] In another embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is The administration of the Pichinde virus particle or the three segment Pichinde virus particle or the composition thereof comprises: The risk of an individual developing an infection, cancer, or allergy in the absence of such treatment is , at least about 10%, at least about 20%, or less than the risk of developing an allergy. at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or is further reduced.

[0254] In another embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is The administration of the Pichinde virus particle or the three segment Pichinde virus particle or the composition thereof comprises: Symptoms of infection, cancer, or allergy are reduced by the infection, cancer, allergy, or other conditions in the absence of such treatment. At least about 10%, at least about 20%, at least about 25% %, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more Reduce.

[0255] In one embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is The HIV virus particles or three-segment HIV particles are preferably administered by multiple injections. (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40 , 45, or 50 injections) or by continuous infusion (e.g., using a pump) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 sites In one embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is The 12-month period was used for the 6-month period and the 12-month period for the 12-month period. The drug is administered as two or more separate injections over a 3-, 24-, or 48-month period. In one embodiment, a plant expressing an antigen derived from an infectious organism, a cancer, or an allergen is The first dose of the HIV-1 virus or the three-segment HIV-1 virus was administered on the selected day. a second dose at least 2 months after the first dose, and a second dose at 6 months after the first dose. A third dose is administered at this point.

[0256] In one example, dermal injections are administered at multiple body sites to reduce the severity of local skin reactions. On a given vaccination day, patients will receive their total allocated dose of cells in one Dispense 3-5 separate doses (e.g., at least 0.4 ml, 0.2 ml, or 0.1 ml) from the syringe. Intradermal injections were divided into two groups, each with at least one injection site adjacent to the most proximal injection site on the limb. Administer at needle insertion sites spaced approximately 5 cm apart (e.g., at least 4.5, 5, 6, 7, 8, or 9 cm apart). On subsequent vaccination days, the injection sites will be in different limbs in a clockwise or counterclockwise direction. is rotated to.

[0257] In another embodiment, the present invention provides a method for the presentation of CMV antigens to a newborn, and thus a subject with an immature immune system. The infectious, replication-deficient Pichinde virus or a composition thereof that exhibits the same is capable of inhibiting the proliferation, proliferation, and / or proliferation of the virus in the absence of such treatment. at least about 10%, at least about 20%, At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more cell-mediated immune (CMI) responses to infection, cancer, or allergy. To induce.

[0258] In certain embodiments, the antigens derived from the infectious organisms, cancers, or allergens described herein are Pichinde virus particles expressing an antigen or three-segment Pichinde virus particles to a subject. Administration of the vaccine induces detectable antibody titers for a minimum of at least four weeks. In an embodiment, the present invention relates to a method for producing an antigen derived from an infectious organism, a cancer, or an allergen as described herein. Administration of the Pichinde virus particle or three-segment Pichinde virus particle to a subject Increasing the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, An increase of at least 500%, or at least 1000%.

[0259] In one embodiment, the primary antigen exposure is a minimum of 10% of the mean control serum from infected immune human subjects. at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, Induce functional (neutralizing) and minimal antibody titers of at least 500% or at least 1000% In a more specific embodiment, the primary neutralizing geometry is The mean antibody titer is at least 1:50, at least 1:100, at least 1:200, or less. In another embodiment, the sensitization assay described herein is performed in a manner similar to that described herein, but with a peak value of 1:1000. Pichinde virus particles or trisodium falciparum expressing antigens derived from infectious diseases, cancer, or allergies Immunization with Gumentopichinde virus particles can be performed after a single dose of the vaccine or after two or more sequential doses. After the second immunization, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or less Both vaccines produce high titers of antibodies that persist for up to five years.

[0260] In yet another embodiment, the secondary antigen exposure is at least 100%, at least 200%, At least 300%, at least 400%, at least 500%, or at least 1000% antibody titer In another embodiment, the secondary antigen exposure increases the mean control IgG from infected immune human subjects. At least 50%, at least 100%, at least 200%, at least 300%, or less of the serum Functional (neutralizing) and minimum antibody titers of at least 400%, at least 500%, or at least 1000%. In a more specific embodiment, the secondary antibody induces a secondary titer within at least four weeks after immunization. Neutralizing geometric mean antibody titers of at least 1:50, at least 1:100, at least 1:200, or to a peak value of at least 1:1000. Pichinde virus particles expressing antigens derived from infectious organisms, cancer, or allergies carried by the virus Secondary immunization with immunized mice or three-segmented Pichinde virus particles should be performed at least 4 weeks after immunization. At least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 Produce high titers of antibodies that remain for at least 3 years, at least 4 years, or at least 5 years. do.

[0261] In yet another embodiment, the third booster is at least 100%, at least 200% , at least 300%, at least 400%, at least 500%, or at least 1000%, antibody In another embodiment, the booster boosts the mean antibody titer from infected immune human subjects. At least 50%, at least 100%, at least 200%, at least 300%, or at least at least 400%, at least 500%, or at least 1000% functional (neutralizing) and minimal antibodies In a more specific embodiment, the third booster immunization induces a virulent antibody titer in an infected immune human control. At least 50%, at least 100%, at least 200%, at least At least 300%, at least 400%, at least 500%, or at least 1000% functional (neutralizing) ) and induces a minimum antibody titer. In another embodiment, the third boost is administered at least 2 days after immunization. At least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months , antibody titers for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. Extend.

[0262] In one embodiment, the antibody is a polypeptide that expresses an antigen derived from an infectious organism, cancer, or allergy. The Chinde virus particles or trisegmented PiChinde virus particles are T cell-independent or T cell In another embodiment, the antibody induces an addictive response due to an infectious organism, cancer, or allergy. Pichinde virus particles expressing antigens that stimulate T cell proliferation and differentiation, or three-segment Pichinde virus particles In other embodiments, the method comprises administering to the subject an effective amount of the antibody or antibody that induces a cellular response against an infectious organism, cancer, or Pichinde virus particles expressing antigens derived from allergies or three-segment Pichinde virus particles In another embodiment, the virus particles described herein induce a T helper response. Pichinde virus particles expressing antigens derived from infectious organisms, cancer, or allergies The three-segmented Pichinde virus particles induce either a Th1-oriented or a Th2-oriented response. (Th2-oriented) response.

[0263] In a more specific embodiment, the Th1 adaptive response is characterized by a predominance of IgG2 antibodies over IgG1. In other embodiments, the ratio of IgG2:IgG1 is greater than 1:1 and greater than 2:1. In another embodiment, the ratio of the β-amino acid residues described herein is 0.01 to 0.01. Pichinde virus particles expressing antigens derived from infectious organisms, cancer, or allergies The three-segmented Pichinde virus particles can be expressed as IgG1, IgG2, IgG3, IgG4, IgM, IgA or IgC. This is indicated by the predominance of E antibodies.

[0264] In some embodiments, an infectious replication-deficient pituitary gland expressing a CMV antigen or a fragment thereof is In another embodiment, the devirus induces a CD8+ T cell response. Pichinde virus particles expressing antigens derived from allergies or three-segment Pichinde virus particles The viral particles, with or without antibody combination, are transduced into CD4+ and CD8+ T cells. It elicits both responses.

[0265] In some embodiments, the method comprises administering to the patient a therapeutically effective amount of the therapeutic agent derived from an infectious organism, cancer, or allergy as described herein. Pichinde virus particles or three-segment Pichinde virus particles expressing antigens that In another embodiment, the infectious organisms described herein induce a high titer of neutralizing antibodies. Pichinde virus particles or three-segment pichinde virus particles expressing antigens derived from cancer or allergy Chinde virus particles elicit high titers of neutralizing antibodies through expression of individual protein complex components do.

[0266] In another embodiment, one, two, three, four, five or more of the chromosomes are derived from an infectious organism, cancer, or allergy. Pichinde virus particles expressing more than 100 antigens or three-segment Pichinde virus particles The child is a Pichinde virus expressing one antigen derived from an infectious organism, cancer or allergen. particles or three-segment Pichinde virus particles induce higher titers of neutralizing antibodies.

[0267] In one embodiment, the method comprises administering to the patient a viral particle comprising the Pichinde virus or a three-segment Pichinde virus. In some embodiments, the method further comprises co-administration of the viral particles and at least one additional therapeutic agent. In another embodiment, the co-administration is simultaneous. The Gumentopichinde virus particles are administered prior to administration of the additional therapeutic agent. The Pichinde virus particle or the three-segment Pichinde virus particle further comprises In one embodiment, the Pichinde virus particle or the three-segmented The administration of the Mentopichinde virus particles and the additional therapeutic agent is performed within about 1 hour, about 2 hours, about 3 hours, , about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or In one embodiment, the Pichinde virus particle or the three segment Pichinde virus particle is The interval between administration of the virus particles and the additional treatment is about 1 day, about 1 week, about 2 weeks, or about 3 weeks. , about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks In one embodiment, the Pichinde virus particle or the three segmented The interval between administration of the Topitinde virus particles and the additional therapy is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

[0268] In one embodiment, a pup expressing an antigen derived from an infectious organism, a cancer, or an allergen is Administration of influenza virus particles or compositions thereof results in increased antibody titers detected in patient blood or serum samples. In one embodiment, the antibody produced by the antibody is derived from an infectious organism, a cancer, or an allergen. Pichinde virus particles expressing the gene, the composition can be administered by collection into urine, saliva, blood, tears, semen, etc. , exfoliated cell samples, or milk to reduce the amount of infectious organisms, cancer, or allergies detected .

[0269] In another embodiment, the inflammatory cytokines derived from the infectious organisms, cancers, or allergens described herein are Pichinde virus particles or three-segment Pichinde virus particles expressing an antigen The composition may further comprise a reporter protein. Pichinde expressing antigens and reporter proteins derived from organisms, cancers, or allergens The viral particles or trisegment Pichinde viral particles or compositions are intended to prevent infection, cancer, or The compound is administered to a subject to treat and / or prevent an allergy. In embodiments, the reporter protein is used to monitor gene expression, protein localization, and vaccine Delivery can be monitored in vivo, in situ, and in real time. .

[0270] In another embodiment, the inflammatory cytokines derived from the infectious organisms, cancers, or allergens described herein are Pichinde virus particles or three-segment Pichinde virus particles, or compositions expressing an antigen The composition may further comprise a fluorescent protein. Pichinde virus expressing antigens derived from infectious organisms, cancers, or allergens described in the document. The composition comprises a reporter protein, a three-segment virion, or a three-segment virion. The subject may be administered the compound to treat and / or prevent a disease, infection, cancer, or allergy. In another specific embodiment, the fluorescent protein is the reporter protein. This allows for the analysis of gene expression, protein localization, and vaccine delivery in vivo, in situ, and in vivo. It can be used to monitor the current status of a system and in real time.

[0271] Pichinde virus particles expressing antigens derived from infectious organisms, cancers, allergens or Induced in a subject by administration of a three-segment Pichinde virus particle or a composition thereof Changes in CMI response function to infection, cancer, or allergy can be monitored by flow cytometry (e.g. See, e.g., Perfetto SP et al., 2004, Nat Rev Immun., 4(8):648-55), Lymphocyte proliferation assay (see, e.g., Bonilla FA et al., 2008, Ann Allergy Asthma Immunology, 2010). Nol, 101:101-4; and Hicks MJ et al., 1983, Am J Clin Pathol., 80:159-63. (see ), determining changes in surface marker expression after activation of T lymphocytes by measuring cytokines Assays for measuring lymphocyte activation, including (e.g., Caruso A. et al., Cytometry Assays, 2011, 144: 1111-1115, 2011) 1997;27:71-6), ELISPOT assays (see, e.g., Czerkinsky CC et al. , 1983, J Immunol Methods, 65:109-121; and Hutchings PR et al., 1989, J Immunol Methods, 65:109-121. Nol Methods, 120:1-8), or natural killer cell cytotoxicity assays. (For example, see Bonilla FA et al., 2006, Ann Allergy Asthma Immunol., 94(5 Suppl 1 Any assay known to those skilled in the art, including, but not limited to, the following: It can be measured by (i).

[0272] Successful treatment of cancer patients is defined as an extension of expected survival, induction of an antitumor immune response, or the progression of cancer. Examples of cancer characteristics that may be improved include: The tumor size (e.g., T0, T1-4), metastatic status (e.g., M0, , M1), number of visible tumors, lymph node involvement (e.g., N0, N1-4, Nx), grade (i.e., i.e., grade 1, 2, 3, or 4), stage (e.g., 0, I, II, III, or IV), cell Certain markers on the blood or in body fluids (e.g., AFP, B2M, β-HCG, BTA, CA 15-3, CA 27.29) , CA 125, CA 72.4, CA 19-9, calcitonin, CEA, chromogranin A, EGFR, hormone receptor Condition, HER2, HCG, immunoglobulin, NSE, NMP22, PSA, PAP, PSMA, S-100, TA-90, and thyroglobulin) and / or associated medical conditions (e.g., ascites or edema) Improvements include symptoms such as swelling, swelling, and / or pain. At least 5, 10, 15, 20, 25, 30, 40, 50, 60, 7 It may be 0, 80, or 90% (eg, tumor survival, or volume or linear dimension).

[0273] In another embodiment, described herein is a method for treating an infectious disease comprising administering to a subject ... and the use of Pichinde virus particles expressing antigens derived from cancer or allergens. and at least one of the ORFs encoding the GP, NP, Z protein, and L protein. However, nucleotide sequences encoding infectious agents, antigens derived from infectious organisms, cancers, and allergens or antigenic fragments thereof. This is the method.

[0274] 4.7 Composition, Administration, and Dosage The present application relates to a Pichinde virus particle or a three-segment Pichinde virus as described herein. Further, vaccines, immunogenic compositions (e.g., vaccine formulations), and pharmaceutical compositions comprising the particles are Such vaccines, immunogenic compositions and pharmaceutical compositions are disclosed in accordance with the standards of the art. The formulation can be prepared according to standard procedures.

[0275] Suitable modifications and adaptations of the methods and applications described herein will be apparent and are within the scope of the present invention. It will be understood by those skilled in the relevant art that the present invention may be practiced without departing from the scope of the present invention or any of its embodiments. will be readily apparent.

[0276] In another embodiment, provided herein is a method for treating a pulmonary edema as described herein. The present invention relates to a composition comprising a Pichinde virus particle or a three-segment Pichinde virus particle. The products can be used in methods for treating and preventing diseases. In accordance with the present invention, the compositions described herein are useful in treating subjects with an infection or susceptible to an infection. In other embodiments, the compositions described herein are used in the treatment of cancer or tumor formation. A subject who is susceptible to or exhibits symptoms characteristic of cancer, or a subject who is diagnosed with cancer. In another specific embodiment, the immunosuppressants provided herein are used in the treatment of The prophylactic compositions can be used to induce an immune response in a host to which the composition is administered. The immunogenic compositions described herein can be used as vaccines, In a specific embodiment, the compound can be formulated as a pharmaceutical composition as described herein. The immunogenic compositions described herein can be used to prevent infection or cancer in a subject (e.g., a human subject). In another embodiment, the vaccine, immunogenic composition or pharmaceutical composition is administered to an animal and and / or suitable for administration to humans.

[0277] In some embodiments, provided herein is a method for treating a pulmonary circulation disorder comprising administering to a patient a therapeutically effective amount of the pulmonary circulation disorder described herein. In one embodiment, such an immunogenic composition comprises a virus vector. The pharmaceutical composition further comprises a pharma- ceutically acceptable excipient. Such immunogenic compositions may further comprise an adjuvant. The adjuvant for co-administration may be administered before, simultaneously with, or after the administration of the composition. In some embodiments, the term "adjuvant" refers to in combination with or as part of the compositions described herein When administered as a single dose, Pichinde virus particles or three-segment Pichinde virus particles, and most importantly, enhances the immune response to the gene products it vectorises. When the compound is administered alone, it enhances and / or boosts virions or three-segmented Pichinde virions, and the ve ctorised) refers to a compound that does not elicit an immune response against the gene product. In an embodiment, the adjuvant is a Pichinde virus particle or a three segment Pichinde virus. The immune response to the vectorised gene products of the latter. Adjuvants are, for example, phosphorus These include leukocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages or dendritic cells. The immune response can be enhanced by several mechanisms. Alternatively, the immunogenic composition may contain an adjuvant or may be administered together with one or more adjuvants. When used, the adjuvant that can be used is an inorganic salt adjuvant or an inorganic salt adjuvant. Salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immune stimulating adjuvants. Examples of adjuvants include, but are not limited to, Examples of suitable salts include aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, etc.). and aluminum sulfate), 3De-O-acylated monophosphoryl lipid A (MPL) (GB 2220211 (see references), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), Resorbate 80 (Tween 80; ICL Americas), imidazopyridine compounds (International Publication No. WO (see International Application PCT / US2007 / 064857, published as US 2007 / 109812), Noxaline compounds (International Application PCT / US2007 / 064, published under International Publication Number WO2007 / 109813) 858), and saponins such as QS21 (Kensil et al., Vaccine Design: Vaccine Design: The Subunit and Adjuvant Approach "The Ultimate Approach" (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540 (See, but not limited to, US Pat. No. 5,393,962). In some embodiments, The adjuvant is Freund's adjuvant (complete or incomplete). The agent is an oil-in-water solution, optionally in combination with an immunostimulant such as monophosphoryl lipid A. type emulsions (such as squalene or peanut oil) (Stoute et al., N. Engl. JM ed. 336, 86-91 (1997).

[0278] The compositions may comprise any of the peptides described herein, alone or in combination with a pharma- ceutical acceptable carrier. Pichinde virus particles or three-segmented Pichinde virus particles. Suspensions or dispersions of small or three-segmented Pichinde virus particles, especially isotonic aqueous suspensions Alternatively, a dispersion may be used. The pharmaceutical composition may be sterile and / or or excipients, such as preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, osmotic pressure agents, The composition may contain salts and / or buffers for adjusting the pH, which may be added in a manner known per se, e.g. For example, it is prepared by conventional dispersion and suspension processes. The dispersion or suspension may contain a viscosity modifier. The suspension or dispersion may be stored at about 2° C. to 8° C. temperature or, preferentially, for long-term storage, frozen and then thawed immediately prior to use. For injection, the vaccine or immunization The original formulation is in an aqueous solution, preferably Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may be formulated in a physiologically compatible buffer, such as a suspending agent, a stabilizing agent, or the like. and / or formulation agents such as dispersants.

[0279] In certain embodiments, the compositions described herein further contain a preservative, e.g., mercury. In a specific embodiment, the pharmaceutical compositions described herein include the derivative thimerosal. In another embodiment, the composition comprises 0.001% to 0.01% thimerosal. The pharmaceutical compositions described herein are preservative-free.

[0280] The pharmaceutical composition comprises about 10 3 ~about 10 11 Focus-forming units of Pichinde virus particles or triplicates Contains Gumentopichinde virus particles.

[0281] In one embodiment, administration of the pharmaceutical composition is parenteral. Parenteral administration can be by intravenous administration. or subcutaneously. Thus, the unit dosage form for parenteral administration may be, for example, For example, an ampoule or a vial, for example, about 10 3 ~10 10 Focus formation unit or 10 5 ~10 15 Contains physical particles of Pichinde virus particles or three-segment Pichinde virus particles In one embodiment, the term "10eX" means 10 to the power of X. do.

[0282] In another embodiment, the vaccine composition or immunogenic composition provided herein comprises: These include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal and By routes including inhalation routes, as well as scarification (e.g., using a bifurcated needle, The drug is administered to the subject via a skin incision (by creating a wound in the top layer of the skin). Either the intravenous or intravenous route can be used.

[0283] For administration intranasally or by inhalation, the preparations for use according to the invention may be prepared in a suitable Propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane Use trifluoroethane, carbon dioxide, or other suitable gas to pack in a pressurized pack or nebulizer. The composition may be conveniently delivered in the form of an aerosol spray presentation from a pressurized aerosol dispenser. In some cases, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator are The compound may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch. That's fine.

[0284] The dosage of the active ingredient depends on the type of vaccination and on the subject and its age, weight, It depends on the individual condition, the individual pharmacokinetic data, and the mode of administration. In vitro assays can be used to help identify optimal dosage ranges. Doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0285] In one embodiment, the Pichinde virus particle or the three-segment Pichinde virus particle The vaccine, immunogenic composition or pharmaceutical composition comprising the compound may be used as a live vaccine. An exemplary dose of live Pichinde virus particles is 10-100 PFU per dose, or less. In some embodiments, the Pichinde virus particle or The preferred dosage of the three-segmented Pichinde virus particles is 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1 ×10 10 , 5×10 10 , 1×10 11 , 5×10 11 or 10 12 pfu, once at intervals as frequently as necessary In another embodiment, the raw vitamin C can be administered to a subject two, three or more times. The virus is administered in a 0.2 mL dose of 10 6.5 ~10 7.5 Live Pichinde virus particles in fluorescent focus units In another embodiment, the inactivated vaccine is formulated to contain about 15μg to about 100μg, about 15μg to about 75μg, about 15μg to about 50μg, or about 15μg to about 30μg of Pichi It is formulated to contain the influenza virus.

[0286] In one embodiment, for administration to a child, two doses of the peptides described herein are administered. The present invention relates to a method for producing a 3-segment Pichinde virus particle or a composition thereof, comprising the steps of: In a specific embodiment, the doses are administered to adults one month apart. and administering a single dose of the Pichinde virus particles or the three-segment Pichinde virus particles described herein. In another embodiment, two doses of the present invention are administered. Pichinde virus particles or three-segment Pichinde virus particles as described in the specification, or The compositions are administered to an adult at least one month apart. Young children (6 months to 9 years) were vaccinated with the Pichinde virus particles or the three-segment virus described herein. The intropinde virus particles or compositions thereof may be administered initially in two doses, one month apart. In certain embodiments, children who received only one dose in the first year of vaccination will receive the next dose. In some embodiments, two doses are administered four weeks apart. Two doses are for children aged 2 to 8 years receiving the immunogenic composition described herein for the first time. In one embodiment, for children aged 6 to 35 months, subjects over 3 years of age are A half dose (0.25 ml) may be preferred, as opposed to 0.5 ml, which may be preferred There is.

[0287] In one embodiment, the composition comprises a Pichinde virus particle or a three segment Pichinde virus. A single dose containing a therapeutically effective amount of the viral particles can be administered to the patient. In an embodiment, the Pichinde virus particle or the three segment Pichinde virus particle comprises: A therapeutically effective amount of Pichinde virus particles or three-segment Pichin, respectively. The virus particles may be administered to a patient in a single dose and in one or more pharmaceutical compositions. Cut.

[0288] In certain embodiments, the composition is administered to a patient as a single dose, followed by administration over a period of 3 to 6 weeks. In these embodiments, the booster vaccination is administered after the second vaccination. The booster may be administered to the subject at intervals of 6 to 12 months after inoculation. Inoculation may utilize different Pichinde viruses or compositions thereof. In some embodiments, administration of the same composition described herein may be repeated, at least once. days, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least The interval may be six months.

[0289] Also provided herein is a method for producing a Pichinde virus particle or a three-segmented virus as an active ingredient. Process for the production of a vaccine in the form of a pharmaceutical preparation containing thiamine-containing virus particles and The present application relates to the use of said Pichinde virus particle or said three-segment Pichinde virus particle. The pharmaceutical compositions can be prepared in a manner known per se, for example by conventional mixing and / or dispersing processes. It is prepared by

[0290] 4.8 Assay 4.8.1 Pichinde virus detection assay Those skilled in the art can easily elucidate the picin deuil described herein using techniques known in the art. It is possible to detect three genome segments or three segmented Pichinde virus particles. For example, RT-PCR can be used with primers specific for Pichinde virus to identify the ORF. Pichinde virus genome segments engineered to carry positions other than the wild type position , or three-segment Pichinde virus particles can be detected and quantified. Combined with blot, ELISA, radioimmunoassay, immunoprecipitation, immunocytochemistry, or FACS Using combined immunocytochemistry, we identified the Pichinde virus genome segments or the three-segment Pichinde virus genome segments. The gene products of the virus particles can be quantified.

[0291] 4.8.2 Assays for measuring infectivity Any assay known to one of skill in the art can be used to measure the infectivity of a Pichinde virus vector preparation. For example, the determination of virus / vector titers can be used in This can be done by a complementary cell type forming unit assay (FFU assay). For example, plate MC57 cells and inoculate different dilutions of the virus / vector sample. After an incubation period, the cells are allowed to form a monolayer and the virus is allowed to attach to the cells. To achieve this, the monolayer is covered with methylcellulose. The infected cells release the virus progeny. The methylcellulose overlay allows the new The spread of the virus is restricted to neighboring cells. As a result, each infectious particle This results in circular zones of infected cells called foci. Pichinde virus particles or three-segment Pichinde virus particles expressing Pichinde virus The NPs can be visualized using antibodies against the NPs or another protein and an HRP-based color reaction. The virus / vector titer is 1 / 2 the amount of the virus / vector that can be counted. It can be calculated in focus forming units per liter (FFU / mL).

[0292] 4.8.3 Multiplication of Pichinde virus particles The propagation of the Pichinde virus particles described herein may be carried out using techniques known in the art or as described herein. The virus can be assessed by any method described in the literature (e.g., cell culture). Growth can be achieved by injecting the Pichinde virus vector described herein into cell culture (e.g., BHK-21 cells). The viral load can be determined by inoculating serial dilutions of the particles. After incubation of the cultures, the virus is isolated using standard methods.

[0293] 4.8.4 Serum ELISA The determination of humoral immune responses during vaccination of animals (e.g., mice, guinea pigs) is based on antigen-specific This can be done by a specific serum ELISA (enzyme-linked immunosorbent assay). Coat the plate with an antigen (e.g., recombinant protein) to avoid non-specific binding of the antibody. Block and incubate with serial dilutions of serum to remove sera. After purification, the bound serum-antibody is then purified using, for example, an enzyme-linked (total IgG or IgG subclass detection) antibody. using anti-species (e.g., mouse, guinea pig) specific antibodies and subsequent color reaction Antibody titers can be determined, for example, as endpoint geometric mean titers. Cut.

[0294] 4.8.5 Assays for Measuring Neutralizing Activity of Induced Antibodies Determination of neutralizing antibodies in serum using ARPE-19 cells and GFP-tagged virus from ATCC The following cellular assays were used: In addition, supplemented serum (e.g. The assay is performed using 384 U / mL guinea pig serum one or two days before use in neutralization. 6.5 x 10 on well plate 3Start by seeding cells / well (50 μl / well). Neutralization is performed in a cell-free 96-well sterile tissue culture plate at 37°C for 1 hour. After the incubation step, the mixture was added to the cells and incubated for GFP detection by a plate reader. The plates were then incubated for an additional 4 days. Positive neutralizing human sera were assayed on each plate. Used as a positive control to ensure the reliability of all results. 4-parameter logistic Curve fitting is used to determine potency (EC50). As an additional test, wells are fluorescently labeled. Check with a light microscope.

[0295] 4.8.6 Plaque Reduction Assay Briefly, a plaque reduction (neutralization) assay for Pichinde virus was performed using green fluorescent protein. The present study was performed using replication-competent or replication-deficient Pichinde viruses tagged with a photoprotein. 5% rabbit serum can be used as a source of exogenous complement, and plaques can be visualized by fluorescence microscopy. Neutralization titers can be counted by observation. This is defined as the highest dilution of serum that results in a 50%, 75%, 90% or 95% reduction in plaques. This can be done.

[0296] qPCR: Pichinde virus RNA genome was analyzed according to the protocol provided by the manufacturer. The RNA was isolated using the QIAamp Viral RNA Mini Kit (QIAGEN). The genome equivalent was then synthesized using the SuperScript® III Platinum® One-Step qRT-PCR Kit. (Invitrogen) and the Pichinde NP coding region or the Pichinde virus particle or the three segments Primers and promoters specific to parts of different genomic stretches of the threonine virion were used. The StepOnePlus real-time PCR system was used with the FAM reporter and NFQ-MGB quencher. Detection was performed by quantitative PCR performed on a 350-μL PCR system (Applied Biosystems). The file may be: 60°C for 30 minutes, 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds and 56°C for 30 seconds. The RNA was analyzed using the results of the samples and the Pichinde virus particles containing the primer and probe binding sites. Fragmentation of the NP coding sequence or another genomic stretch of the three-segment Pichinde virus particle Generated from a log10 dilution series of spectrophotometrically quantified in vitro transcribed RNA fragments corresponding to the Quantitation can be achieved by comparison to a standard curve established.

[0297] 4.8.7 Western blotting Infected cells grown in tissue culture flasks or in suspension were subjected to RIPA at the indicated time points postinfection. Lyse using buffer (Thermo Scientific) or use directly without cell lysis The samples were heated with reducing agent and NuPage LDS sample buffer (NOVEX) at 99°C for 10 min and then cooled to room temperature. After cooling to RT, the mixture is loaded onto a 4-12% SDS-gel for electrophoresis. Proteins are purified using Invitrogen The samples were blotted onto a membrane using the ns iBlot Gel transfer apparatus and visualized by Ponceau staining. Finally, the preparation is incubated with a primary antibody against the protein of interest and alkaline phosphatase. Probe with a nucleotide-conjugated secondary antibody, followed by 1-Step NBT / BCIP solution (INVITROGEN Stain with EN.

[0298] 4.8.8 MHC-Peptide Multimer Staining Assay for Detection of Antigen-Specific CD8+ T Cell Proliferation Testing antigen-specific CD8+ T cell responses using any assay known to one of skill in the art For example, an MHC-peptide tetramer staining assay can be used (e.g., A Iltman JD et al., Science 1996; 274:94-96; and Murali-Krishna K. et al., (See Immunity 1998;8:177-187). Briefly, the assay comprises the following steps: The method includes the steps of: detecting the presence of antigen-specific T cells using a tetramer assay; To detect peptides specific for the peptide, T cells are co-immunoprecipitated with the peptide (usually fluorescently labeled Tetramers of custom-tailored MHC molecules for T cells of defined antigen specificity and MHC haplotype The tetramers are then detected by flow cytometry via fluorescent labeling. The detection is performed by ELISA.

[0299] 4.8.9 ELISPOT Assay for Detection of Antigen-Specific CD4+ T Cell Proliferation Testing antigen-specific CD4+ T cell responses using any assay known to one of skill in the art For example, an ELISPOT assay can be used (see, for example, Czerkinsky CC 1983; 65:109-121; and Hutchings PR et al., J Immunol Methods. Immunol Methods. 1989; 120:1-8). Briefly, the assay , which involves the steps of: coating an immunospot plate with anti-cytokine antibodies. Cells are incubated in immunospot plates. The cells secrete cytokines, which The plate is then coated with a second biotinylated anti-cytokine antibody. The antibodies are labeled with IgG and visualized with the avidin-HRP system.

[0300] 4.8.10 Intracellular Cytokine Assays for Detection of Functionality of CD8+ and CD4+ T Cell Responses Any assay known to one of skill in the art can be used to test the functionality of the CD8+ and CD4+ T cell responses. For example, intracellular cytokine assays combined with flow cytometry can be used. The method can be used (e.g., Suni MA et al., J Immunol Methods. 1998; 212:89-98; Nomura LE et al., Cytometry 2000; 40:60-68; and Ghanekar SA See, for example, . et al., Clinical and Diagnostic Laboratory Immunology. 2001; 8:628-63. Briefly, the assay involves the following steps: inhibits protein-mediated cell activation and protein transport (e.g., brefeldin A) In addition, the cytokines are retained intracellularly. After a defined period of incubation, typically Typically, after 5 hours, a washing step is followed by the addition of antibodies against other cell markers to the cells. The cells are then fixed and permeabilized. and cells can be analyzed by flow cytometry.

[0301] 4.8.11 Assays to confirm replication deficiency of viral vectors Any assay known to those of skill in the art that determines the concentration of infectious and replicative viral particles. can also be used to measure replication-defective viral particles in a sample. A cell-based FFU assay can be used for this purpose.

[0302] Additionally, plaque-based assays provide information regarding plaque-forming units (PFU) in a virus sample. This is the standard method used to determine virus concentration using non-complementary Confluent monolayers of host cells are infected with various dilutions of virus and plated on a semi-solid medium such as agar. Cover with medium to prevent the virus from spreading indefinitely. When a virus successfully replicates itself in cells within the cell monolayer and spreads to surrounding cells, Plaque forms (see, e.g., Kaufmann, S.H.; Kabelitz, D. (2002), "Microbial Methods in Microbiology, Vol. 32: Immunology of Infection (See, "On the Plaque Formation in the Fibroblasts," Academic Press, ISBN 0-12-521532-0). This can take 2-14 days depending on the virus being tested. Count and use the result in conjunction with the dilution factor used to prepare the plate. The number of plaque forming units per unit volume of sample (PFU / mL) is calculated by: It represents the number of infectious, replicative particles in a sample. When C cells are used, the same assay is used. By titrating replication-deficient or three-segment Pichinde virus particles, This can be done.

[0303] 4.8.12 Assays for Expression of Viral Antigens Any assay known to one of skill in the art may be used to measure the expression of viral antigens. For example, an FFU assay can be performed. Use monoclonal or polyclonal antibody preparation(s) against viral antigens ( transgene-specific FFU).

[0304] 4.8.13 Animal models To examine the recombination and infectivity of the Pichinde virus particles described herein, In vivo animal models can be used. In some embodiments, the three-segment picindel Animal models that can be used to examine viral particle recombination and infectivity include mouse In a preferred embodiment, the Pichindewi Animal models that can be used to examine recombination and infectivity of the virus include mice. In a more specific embodiment, mice are recombinantly infected with Pichinde virus particles. This mouse expresses type I interferon receptors, type II interferon receptors, and can be used to examine the potency of It lacks three genes: type 1 interferon receptor and recombination activating gene 1 (RAG1).

[0305] In one embodiment, an animal model is used to characterize the infectivity and transgene expression of Pichinde virus. In some embodiments, the stability of the viral RNA can be determined. The antibody can be isolated from the serum of a living model by techniques readily known to those skilled in the art. The viral RNA can be reverse transcribed, and cDNA carrying the Pichinde virus ORF is generated using the gene Specific primers can be used for PCR amplification. Flow cytometry can also be used to The infectivity of Pichinde virus and the stability of the transgene can be examined.

[0306] 5. Working Example These examples describe the Pichinde virus-based vector technology by: (1) translating a viral ORF into the ORF; Pichinde virus genome segments having F at positions other than the wild-type position, and (2) capable of replication Successfully produced three-segmented Pichinde virus particles without yielding two-segmented virus particles Demonstrate that it can be used to develop

[0307] 5.1 Materials and Methods 5.1.1 Cells BHK-21 cells were cultured in 10% heat-inactivated fetal calf serum (FCS; Biochrom), 10 mM HEPES (Gibco), 1 1 mM sodium pyruvate (Gibco) and 1× tryptose phosphate broth The cells were cultured in high glucose Dulbecco's Eagle medium (DMEM Sigma) supplemented with 100% glucose broth. The cells were cultured at 37 °C in a humidified 5% CO2 incubator. 293-T cells were cultured in 10% heat-resistant Dulbecco's Eagle medium (DMEM with Glutamax; Sigma) supplemented with activated fetal calf serum (FCS). was cultivated in.

[0308] 5.1.2 Transgenes (1) Green fluorescent protein (GFP) is flanked by BsmBI sites for seamless cloning. (2) i) Vesicular stomatitis virus glycoprotein ) signal peptide of the VSVG protein; ii) the P1A antigen of the mouse P815 mastocytoma tumor cell line; iii) GSG linker, iv) enterovirus 2A peptide, and v) a fusion protein consisting of mouse GM-CSF. This fusion protein is called sP1AGM. We have synthesized it by seamless cloning. (3) The wild type was synthesized with flanking BsmBI sites as sP1AGM-Bsm (SEQ ID NO: 10). Pichinde virus S segment expression plasmid (S segment without BbsI site) (SEQ ID NO: 8) to reconstitute a pip with flanking BsmBI sites for seamless cloning. Chindevirus GP.

[0309] 5.1.3 Plasmids We introduced a noncoding mutation to delete the BsmBI restriction site in Pichinde virus Modification of the L ORF of strain Munchique CoAn4763 isolate P18 (Genbank accession number EF529747.1) cDNA was synthesized. This synthetic ORF was appropriately flanked by BsmBI and EcoRI and NheI restriction sites. F(LΔBsmBI; SEQ ID NO:3) was introduced into the polymerase II (pol-II) expression vector pCAGGS, This resulted in pC-PIC-L-Bsm (Figure 3) for expression of Pichinde L protein in eukaryotic cells.

[0310] We deleted the L ORF and replaced it with the GFP ORF flanked by BsmBI sites. Chindé virus strain Munchique CoAn4763 isolate P18 (Genbank accession number EF529747 . The modified L segment of 1) (PIC-L-GFP-Bsm; SEQ ID NO: 4) was synthesized. This synthetic cDNA was transformed into Polymerase I (pol-I) expression cassette (Pinschewer et al., J Virol. 2003 Mar;77(6) :3882-7) to generate pol-I-PIC-L-GFP-Bsm (Figure 3).

[0311] We digested PIC-L-Bsm with BsmBI and cloned the BsmBI-mutated L ORF into the similarly digested pol-I-PIC -L-GFP-Bsm backbone, thereby replacing the GFP ORF with the L ORF for cloning purposes Pichinde virus L segment cDNA with all restriction sites removed for The resulting pol-I-PIC-L plasmid (Figure 3) was used to express the full-length picin deoxyribonucleic acid in eukaryotic cells. It was designed for intracellular expression of the virus L segment (PIC-L-seg; SEQ ID NO:2).

[0312] We have named it PIC-miniS-GFP (SEQ ID NO: 5), in which the GP ORF has been replaced with two BsmBI restriction sites. and the NP ORF was replaced by GFP with two flanking BbsI restriction sites. Modification of the virus strain Munchique CoAn4763 isolate P18 (Genbank accession number EF529746.1) A mutant S segment cDNA was synthesized. This synthetic cDNA was used to express mouse polymerase I (pol-I). (Pinschewer et al., J Virol. 2003 Mar;77(6):3882-7) and pol-I-PIC-mi This resulted in niS-GFP (Figure 3).

[0313] We introduced noncoding mutations to delete both BbsI restriction sites in Pichinde virus Modification of the NP ORF of strain Munchique CoAn4763 isolate P18 (Genbank accession number EF529747.1) A mutant cDNA was synthesized. This synthetic ODNA was appropriately flanked by BbsI and EcoRI and NheI restriction sites. RF (NPΔBbsI; SEQ ID NO: 6) was introduced into the polymerase II (pol-II) expression vector pCAGGS, and the eukaryotic This resulted in pC-PIC-NP-Bbs (Figure 3) for the expression of Pichinde NP protein in cells.

[0314] We digested NPΔBbsI with BbsI and cloned the BbsI-mutated NP ORF into the pol-I-PIC-mi It was inserted into the niS-GFP backbone, thereby replacing the GFP ORF with the NP ORF for cloning purposes. The 3'UTR-NP-IGR portion of the Pichinde virus S segment cDNA with all restriction sites removed. The resulting plasmid expresses PIC-NP-Bsm (SEQ ID NO: 7) under the control of pol-I. The resulting pol-I-PIC-NP-Bsm plasmid (Figure 3) was used to express the resulting recombinant Pichinde virus S segment. For expression in eukaryotic cells, the 5'U is inserted by seamlessly replacing the BsmBI site. It is designed to accommodate a transgene of interest to be inserted between the TR and the IGR.

[0315] We introduced non-coding mutations to delete both BbsI restriction sites in a Pichinde virus strain. Modification of the GP ORF of Munchique CoAn4763 isolate P18 (Genbank accession number EF529747.1) DNA was synthesized. As with NPΔBbsI, this synthetic ORF was introduced into the pol-I-PIC-miniS-GFP backbone. thereby replacing the GFP ORF with the GP ORF and providing all restriction sites for cloning purposes. Seamless reconstruction of the 3'UTR-GP-IGR portion of the Pichinde virus S segment cDNA with deleted nucleotides The resulting pol-I-PIC-GP-Bsm plasmid expressing PIC-GP-Bsm (SEQ ID NO: 8) (Figure 3) For the expression of recombinant Pichinde virus S segment in eukaryotic cells, a BsmBI site was inserted. The transgene of interest to be introduced between the 5'UTR and IGR by seamless replacement Designed to accommodate children.

[0316] We then inserted the following genes and transgenes into pol-I-PIC-NP-Bsm: The plasmids containing the following genes were inserted: 1. GFP, 2. sP1AGM, and 3. PichindeGP. The vectors were pol-I-PIC-NP-GFP (expressing PIC-NP-GFP, also known as S-NP / GFP; SEQ ID NO: 11) and and pol-I-PIC-NP-sP1AGM (expressing PIC-NP-sP1AGM; SEQ ID NO: 12) and pol-I-PIC-S (expressing PIC-S; SEQ ID NO: 13). Similarly, we used pol-IP to express either GFP or sP1AGM. Inserted into IC-GP-Bsm and expressing pol-I-PIC-GP-GFP (PIC-GP-GFP, also known as S-GP / GFPart) SEQ ID NO:13) and pol-I-PIC-GP-sP1AGM (expressing PIC-GP-sP1AGM; SEQ ID NO:14). .

[0317] 5.1.4 DNA transfection of cells and rescue of recombinant viruses 3. Stably Transfected to Express Lymphocytic Choriomeningitis Virus Glycoprotein The BHK-21 cells (BHK-GP cells, Flatz et al. Nat Med. 2010 Mar;16(3):339-45) 5 x 10 cells in a 6-well plate 5 Cells were seeded at a density of 100 cells / well and incubated for 24 hours as described by the manufacturer. We used Lipofectamine (approximately 3 μl / μg DNA; Invitrogen) according to the instructions provided with the kit. The recombinant two-segment virus was transfected from plasmid DNA in different amounts. For total rescue of viral vectors, two minimal viral transactivators, NP and L from pol-II-driven plasmids (0.8 μg pC-PIC-NP-Bbs, 1.4 μg pC-PIC-L-Bsm). The cells were delivered to the cells and co-transfected with 1 μg of pol-I-PIC-L and 0.8 μg of pol-I-PIC-S. In the case of rescue of a three-segment r3PIC consisting of an L segment and two S segments, the μg of both pol-I driven S segments were included in the transfection mix. 72 hours after transfection, the cells and supernatant were transferred to a 75 cm2 tissue culture flask and Supernatants were collected 48–96 h after incubation. Viral infectivity was assessed in a focus-forming assay. The virus was passaged 48 times in normal BHK-21 cells for further amplification (infectivity at 48 hours). The viral titer of the virus stock thus obtained was determined by focusing This was again determined by formation assay.

[0318] 5.1.5 Viruses and viral growth kinetics Wild-type and recombinant virus stocks were transfected into BHK-21 or 293-T cells at a multiplicity of infection (moi) of 0.01. The viruses were generated by infecting either 100 or 1500 cells with 100% ethanol and the supernatants were collected 48 hours after infection. The proliferation curve of BHK021 cells was performed in vitro in a T75 cell culture flask format. The cells are 5 × 10 6 The cells were seeded at a density of 100 cells / flask and after 24 hours, the cells were inoculated into 5 ml of a 0.01 moi culture. Incubate with the virus inoculum for 90 min on a rocker plate at 37°C and 5% CO2. Fresh medium was added and the cells were incubated at 37°C / 5% CO2. At given time points (usually 24, 48, and 72 hours), supernatants were harvested and assayed for focus formation. The virus titer was analyzed.

[0319] 5.1.6 Focus formation assay The titer of Pichinde virus was then determined by focus-forming assay. If not, 293-T cells or 3T3 cells were used for focus formation assays. 3 × 10 4 Cells were seeded in 96-well plates at a density of 100 cells / well and incubated with 100 mM NaCl, 0.5% CO2, and 0.5% ethanol. After 2–4 h of incubation at 37°C, the 100 μl of 3.17-fold serial dilutions of the virus were mixed with the Then, 80 μl of viscous medium (2% methylcellulose in 2× supplemented DMEM) was added per well. After 48 hours at 37°C, the supernatant was The cells were flicked off and lysed by adding 100 μl of methanol for 20 min at room temperature. The cells were then fixed (all subsequent steps were performed at room temperature). The cells were then plated in 100 μl / well of BSS / 1% Tris The cells were permeabilized with ton X-100 (Merck Millipore) for 20 min, followed by blocking with PBS / 5% FCS for 60 min. For anti-NP staining, rat anti-pichin de NP monoclonal antibody was used as the primary staining antibody. The plates were washed three times with tap water and the secondary HRP -Add goat anti-rat IgG at a dilution of 1:100 in PBS / 2.5% FCS and incubate for 1 hour. The plate was washed again three times with tap water. Color reaction (0.5 g / l DAB (Sigma D-5637), 0.5 g / l nickel ammonium sulfate in PBS / 0.015% H2O2) was added and the reaction was incubated for 10 min before dilution with water. The stained foci were counted and the final titer was calculated according to the dilution factor.

[0320] 5.1.7 Mouse BALB / c mice were purchased from Charles River Laboratories and were specific pathogen-free for the experiments. All animal experiments were performed under specific pathogen-free (SPF) conditions at the University of Basel. The infections were carried out in accordance with the Swiss legislation and with the permission of the respective responsible cantonal authorities. , 1 × 10 per mouse 5 The treatment was performed intravenously at a dose of FFU.

[0321] 5.1.8 Flow cytometry The blood was immunodominantly purified from P1A-derived H-2L in combination with anti-CD8a and anti-B220 antibodies. d -Restricted Episode Group [ka] Epitope-specific CD8+ T cell frequencies were measured using MHC class I tetramers loaded with BD LSRForte The data were analyzed using a ssa flow cytometer and the data were analyzed using FlowJo software (Tree Star, Ashley). and, OR).

[0322] 5.1.9 Statistical analysis Statistical significance was determined by two-tailed unpaired t-test using Graphpad Prism software (version 6.0d). This was determined by regulation.

[0323] 5.2 Results 5.2.1 Trisegmented Pichinde virus-based vectors with artificial genome organization design The genome of wild-type Pichinde virus contains two negative polarity single-stranded RNA segments (one The 5' untranslated region (5'UTR) and Allows seamless insertion of a transgene of choice between the intergenic regions (IGRs) of duplicated S segments Based on the cassette system that enables the generation of replicable three-segment peptides with artificial genome organization, Polymerase-I / II-driven for r3PIC-art virus vectors (Figures 1B, 1C, and 1D) A cDNA rescue system was designed to completely remove the transgene upon insertion, thus obtaining Insertion of a gene into the arenavirus S segment using a BsmBI site that is not present in the recombinant virus Seamless insertion of the gene (i.e., the remaining nucleotide sequence derived from molecular cloning) A molecular cloning strategy for See Pinschewer et al., Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7895-90 0 and in supporting FIG. 4. The BbsI enzyme is described in Flick et al., J Vir For seamless cloning, as reviewed in ol. 2001 Feb;75(4):1643-55, These Pichinde virus-based r3PIC-art genomes were used in the same way to identify human It consists of the wild-type Pichinde virus L segment with a randomly duplicated S segment, and the 3'UTR Under the control of the nucleoprotein (NP) or glycoprotein (GP), i.e., between the 3'UTR and the IGR, This allows for the insertion of a transgene into each S segment. i.e., each transgene is inserted into two S segments, leaving one position for each S segment. The vectors could be inserted between the 5'UTR and IGR of each of the vectors.

[0324] 5.2.2 Rescue from three-segment recombinant virus vectors with artificial genome configuration Isolated infectious GFP-expressing virus To generate the three-segment recombinant Pichinde virus, we used the We synthesized multiple plasmids as described above. We synthesized the following combinations of plasmids: (A) S segment minigenome: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-miniS-GFP ; (B) L segment minigenome: pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L-GFP-Bsm ; (C)r3PIC-GFP art : pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L, pol-I-PIC-NP-GF P, pol-I-PIC-GP-GFP; (D)rPIC wt : pC-PIC-L-Bsm, pC-PIC-NP-Bbs, pol-I-PIC-L, pol-I-PIC-S was used to transfect BHK-21 cells.

[0325] We observed GFP expression 48 hours after transfection of the S and L segment minigenomes. Heading (Figure 4, Plasmid combinations A and B outlined above), active in gene expression Pichinde virus S and L segment analytes as functional ribonucleoproteins (RNPs) We documented the intracellular reconstruction of the log. Similarly, r3PIC-GFP art The purpose of the production of Transfection C demonstrated GFP-positive cells 48 hours after transfection, whereas , rPIC wt The plasmid combination D for constructing the vector showed green fluorescence as expected. At 168 hours after transfection, GFP-positive cells were found to be S and L segment myoglobin-dependent. In the two genome transfection, most of the genes disappeared, but in the r3PIC-GFP art have Infectious GFP-expressing viruses were reconstituted from the cDNA and expressed in the cells. It was shown that it spread during cultivation.

[0326] 5.2.3 Recombinant three-segment viruses grow to lower titers than wild-type Pichinde virus rPIC wt and r3PIC-GFP art Comparative growth curves were performed with the viruses obtained using the above methods (Figure 2). Supernatants from transfections C and D in Section 5.2.2 were pooled and serially replicated in parallel in BHK-21 cells. For both viruses, peak infectivity was reached after 48 h. However, r3PIC-GFP art The latter is essentially rPIC wt This was lower than that of the three segments. r3PIC-GFP art showed that the two-segmented wild-type parent virus was attenuated compared to its parent virus. Ta.

[0327] 5.2.4 Recombinant r3PIC expressing sP1AGM induces rapid, potent, and versatile P1A-specific CD8+ T cell responses Derive the answer. r3PIC for vaccination purposes art To test the utility of vector delivery technology, we , r3PIC-GFP art r3PIC-sP1AGM, which has a similar genomic organization to (Fig. 1C). art Vaccine Vectors We created r3PIC-GFP (Figure 1D). art Similar to that outlined above for procedure, but instead of the plasmids pol-I-PIC-NP-GFP and pol-I-PIC-GP-GFP, Virus expressing sP1AGM using pol-I-PIC-NP-sP1AGM and pol-I-PIC-GP-sP1AGM (r3PIC-sP1AGM art We created a BALB / c mouse lineage that was injected intravenously with 10e5 focus-forming monoclonal antibodies. r3PIC-sP1AGM (FFU) art After immunization with 100 μg / mL of ... Pitopes [ka] CD8+ T cell responses to IgG were analyzed by flow cytometry using MHC class I tetramers. Measured. r3PIC-sP1AGM art Mice immunized with IgG4 showed a significantly increased expression of P1A35-43-specific CD8 T cells. A distinct population of IgG1 expression was observed in the peripheral blood of mice that was not present in the blood of non-immunized mice (Figure 5A). and 5B). These observations suggest that r3PIC-art-based viral vectors are highly immunogenic and , have demonstrated to be a promising tool for immunotherapy and vaccination.

[0328] 5.2.5 When tested at early passages after rescue from cDNA, A recombinant three-segment construct designed to express certain glycoprotein and nucleoprotein genes. and expressing its glycoprotein under the control of the 3' untranslated region (UTR) promoter. Both recombinant three-segment viruses engineered to inhibit wild-type Pichindewi It grows to lower titers than We expressed the GFP gene under the control of the 5' and 3' UTR promoters, respectively, i.e., S-GP / GFPnat (sequence No. 15) and an artificial duplicate of S-NP / GFP (also known as PIC-NP-GFP; SEQ ID NO: 11). The glycoprotein (GP) and We generated a three-segmented Pichinde virus expressing the ribosomal and nucleoprotein (NP) genes (Figure 6). This r3PIC-GFP nat The virus is a three-segment r3PIC-GFP art The virus is outlined above. was created by a procedure similar to that shown. nat The outline is shown in Fig. 6. When grown in culture in BHK-21 cells (multiplicity of infection = 0.01 , collected at 48 hours), r3PIC-GFP nat is effectively rPIC wt Lower titer, r3PIC-GFP art About The low titers reached were similar to those observed with the 100% HO cells cultured in the same wells (Figure 7; symbols represent individual parallel cell culture wells). (Titers for 300-fold increase in 300-fold increase in 1 ... C-GFP nat was shown to be attenuated compared to its bisegmented wild-type parent virus.

[0329] 5.2.6 Recombinant three-segment virus with artificial genomic organization is expressed during persistent infection of immunodeficient mice. Virus (r3PIC-GFP art ) maintained transgenic GFP expression and expressed wild-type Pichin Deu Illus (rPIC wt ) maintains a consistently lower viral titer in the blood, while its glycoprotein and and a three-segment fusion protein designed to express nuclear protein genes in their respective natural locations. Virus (r3PIC-GFP nat ) eventually lost GFP expression and wt equivalent to animals infected with The blood viral load is reached. We developed a triple-deficient mouse model of type I and type II interferon receptors and RAG1 (AGR mouse). Grob et al., 1999, "Effects of attenuated pseudorabies virus on the control of systemic outbreaks Role of the individual interferon system and specific immunity in mice al interferon systems and specific immunity in mice in controlling systemic diss 'emission of attenuated pseudorabies virus infection)', J Virol, 4748-54). On day 0, 10e5 focus forming units ("FFU") of r3PIC-GFP art , r3PIC-GFP nat , or rPIC wt We infected mice intravenously (iv) with one of the following viruses: Blood was collected on days 56, 77, 98, 120, and 147, and viral infectivity was determined by FFU assay. In these assays, we detected the Pichinde virus nucleoprotein (NP FFU; Fig. 8). or r3PIC-GFP nat and r3PIC-GFP art detected the viral GFP transgene (GFP FFU; Fig. 9 ). From these values, we calculated the NP:GFP FFU ratio for each animal and each time point (Figure 10). .

[0330] r3PIC-GFP for the first 21 days postinfection natand r3PIC-GFP art Total infectivity (NP FFU assay) (as determined by I) persisted at similar levels in the blood of AGR mice and wt infection This was approximately 10-fold lower than in the control (Figure 8). However, from day 28 onwards, nat infection Potency is determined by NP FFU assay, as well as rPIC wt reached a level that was indistinguishable from In contrast, r3PIC-GFP art NP FFU titers were higher for rPICwt than for rPICwt throughout the 147-day observation period. The levels remained approximately 10-fold lower than those observed in the control group (Figure 8).

[0331] In addition to detecting the viral structural protein NP to determine total viral infectivity (Figure 8) We used r3PIC-GFP nat - and r3PIC-GFP art -Infectivity expressed in the blood of infected AGR mice To evaluate the transgene expressing GFP, an FFU assay was performed (GFP FFU, FIG. 9). In striking contrast to the NP FFU titers (Figure 8), r3PIC-GFP nat GFP FFU titers in -infected AGR mice After the 28th day, the levels of r3PIC-GFP decreased, and after the 120th day, the levels were undetectable (Figure 9). a rt - This was in contrast to the almost constant GFP FFU titers in the blood of infected mice (Figure 9). By calculating the “P:GFP FFU ratio” (Figure 10), we found that r3PIC-GFP art -In infected mice It was determined that virtually all of the infectivity (NP FFU) also expressed the GFP transgene. This was supported by a NP:GFP FFU ratio in the range of 1 throughout the 147-day observation period (Figure 10). In striking contrast, r3PIC-GFP nat - The NP:GFP FFU ratio in the blood of infected mice The number of storms started around 1 on the 28th, but by the 28th, it had reached several hundred and more (Figure 10). Populations of virions circulating in the blood of r3PIC-GFPnat-infected mice on day 28 and later Within the 100 or fewer cells, only one in 100 still expressed the GFP transgene, and the GFP-expressing infectivity , which eventually dropped below detectable levels. art A.G. GFP transgene expression was maintained through 147 days of persistent infection in R mice.

[0332] 5.2.7 r3PIC-GFP art -Viruses recovered from the serum of infected mice were r3PIC wt -An infected animal? r3PIC-GFP reached titers similar to those of viruses isolated from nat -Compared to those from infected animals and maintained attenuated To evaluate the growth characteristics of viruses circulating in the serum of persistently infected AGR mice, we Viremic serum collected 147 days after infection of BHK-21 cells was passaged and NP FFU antigen was obtained 48 hours later. Viral infectivity was determined by r3PIC-GFP assay. nat -Grown in serum from infected mice The virus was rPIC wt Reaching IFF titers similar to or higher than those from virus-infected animals (Figure 11; symbols indicate virus titers from individual mouse sera; error bars indicate mean (Mean values ​​are shown + / - SD). In contrast, r3PIC-GFP art-obtained after passage of sera from infected mice Viral titers were substantially lower than in any of the previously described groups (Figure 11).

[0333] From these viruses that were passaged for 48 h, we further analyzed cell culture growth. Four were randomly selected from each group for the experiment shown in Figure 11 (direct measurement of infectivity from serum). Unlike the previous experiment (passages), this experiment (Figure 12) was normalized for input infectivity, as a potential confounding factor in the assessment of viral titers achieved in culture. Different amounts of input infectivity were excluded. Therefore, we standardized the BHK-21 cells. The cells were infected at a multiplicity of infection of 0.01, and the virus titer was determined 48 hours later (Figure 12; symbols indicate Titers from virus derived from individual mouse sera are shown; error bars represent the mean + / - SD). Similar to the differences in titers found after direct ex vivo passaging from serum, r3PIC-GF P nat -The virus was derived from rPIC wt - Reached a titer at least equivalent to that of the source virus. In contrast, in vivo passaged r3PIC-GFP art The titers achieved by the virus derived from was substantially lower than that of the other two groups.

[0334] This is r3PIC-GFP nat - The virus recovered from the serum of infected animals is already attenuated. However, r3PIC-GFP art -The virus circulating in the blood of infected mice was rPIC wt -Origin of Wii These results suggest that the virus was still clearly attenuated compared to the AGR mice. Through experiments in rPIC wtViremia lower than r3PIC-GFP art Viremia (section 5.2.6 ), and also rPICs when re-amplified from blood in cell culture. wt Potency Lower r3PIC-GFP art Judging from the titer, r3PIC-GFP art In vivo duplication in mice The attenuation was maintained throughout the 147-day production period.

[0335] 5.2.8 r3PIC-GFP nat Unlike the above, the recombinant three-segment virus has an artificial genome structure. Rus(r3PIC-GFP art ) did not recombine its two S segments and maintained its transgene. We demonstrated that r3PIC-GFP was expressed during the course of persistent infection in AGR mice. nat But those two S-segments recombining the NP and GP genes onto a single RNA segment, thereby We wanted to determine whether the FP transgene had been eliminated. To test this possibility, We extracted viral RNA from serum samples collected from each animal 147 days after viral infection. We have designed and constructed two NP and GP-binding domains, which bind to the Pichinde virus NP and GP, respectively. rPIC wt PCR on genomic templates is predicted to produce an amplicon of 357 base pairs. Using primers spanning the intergenic region ("IGR") of the Pichinde virus S segment, Such amplicons were indeed rPIC wt or r3PIC-GFP nat Noise When viral RNA from animals infected with either r3PIC-GFP was used, art Feeling When viral RNA from blood of infected mice was used, no results were obtained (Figure 13; each lane is 8–10 represent RT-PCR products from one individual mouse in the experiment shown).

[0336] Taken together, these data suggest that r3PIC-GFP is a novel marker for the pathogenesis of persistent infection in AGR mice. nat The two S segments (S-GP / GFPnat and S-NP / GFP) were recombined to form the NP and GP open reads. This shows that the binding frame of the RNA is recombined into one single segment of RNA. This resulted in loss of GFP transgene expression and re-expansion upon collection from blood and in cell culture. The increased viremia seen in cell cultures and in levels of viremia was evident In mice, the proliferation ability of rPIC wt In contrast, r3PIC-GFP increased the proliferation ability of art teeth , as evidenced by the lack of RT-PCR amplicons spanning the NP and GP genes. The S segment did not recombine.

[0337] 6. Equivalents The viruses, nucleic acids, methods, host cells, and compositions disclosed herein are The scope should not be limited by the specific embodiments described. In addition, various modifications of the viruses, nucleic acids, methods, host cells, and compositions are described above and are not intended to be limiting. Such modifications will become apparent to those skilled in the art from the accompanying drawings and drawings. is intended to be within the scope.

[0338] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated herein by reference. The entirety is incorporated by reference. The present application provides the following aspects of the invention. (Aspect 1) A Pichinde virus genome segment, the genome segment being a viral open reading frame. The gene is engineered to carry an open reading frame ("ORF") at a position other than the wild-type position of the ORF. The Pichinde virus genome segments are: (i) The ORF encoding the nucleoprotein ("NP") is located in the 5' untranslated region of Pichinde virus (" the S segment under the control of the UTR); (ii) The OFR encoding matrix protein Z ("Z protein") is the S segment under the control of the 5′UTR of rus; (iii) The ORF encoding the RNA-dependent RNA polymerase L ("L protein") is the S segment under the control of the 5′UTR of virus; (iv) The ORF encoding the viral glycoprotein ("GP") is located in the 3'UTR of Pichinde virus. S segment under control; (v) The ORF encoding the L protein is located in the S segment under the control of the 3'UTR of Pichinde virus. ment; (vi) The ORF encoding the Z protein is located in the S segment under the control of the 3'UTR of Pichinde virus. ment; (vii) the L segment, in which the ORF encoding GP is under the control of the 5′UTR of Pichinde virus; (viii) L segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus ; (ix) The ORF encoding the L protein is located in the L segment under the control of the 5'UTR of Pichinde virus. ment; (x) L segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (xi) L segment, in which the ORF encoding NP is under the control of the 3′UTR of Pichinde virus; and (xii) The ORF encoding the Z protein is located in the L sequence under the control of the 3'UTR of Pichinde virus. Segment A Pichinde virus genome segment selected from the group consisting of: (Aspect 2) The 3'UTR of the Pichinde virus is the 3'UTR of the viral L segment and the 5'UTR of the Pichinde virus 2. The method according to claim 1, wherein the 5′UTR of said Pichinde virus S segment or said Pichinde virus L segment is Pichinde virus genome segment. (Aspect 3) 2. A cDNA of the Pichinde virus genome segment according to embodiment 1. (Aspect 4) A DNA expression vector comprising the cDNA according to embodiment 3. (Aspect 5) A Pichinde virus genome segment according to embodiment 1, a cDNA according to embodiment 3, or a nucleic acid sequence according to embodiment 4. A host cell containing a vector. (Aspect 6) The Pichinde virus particles according to embodiment 1 are prepared by the method of the present invention, such that the Pichinde virus particles comprise an S segment and an L segment. A Pichinde virus genome segment and a second Pichinde virus genome segment Virus particles. (Aspect 7) The Pichinde virus according to embodiment 6, wherein the Pichinde virus particle is infectious and replicable. Russ particle. (Aspect 8) The Pichinde virus particle of embodiment 6, wherein the Pichinde virus particle is attenuated. . (Aspect 9) The Pichinde virus particles are infectious but produce further infectious particles in non-complementing cells. The Pichinde virus particle of embodiment 6, which is incapable of producing progeny. (Aspect 10) At least one of the four ORFs encoding the GP, NP, Z protein, and L protein is deleted. 10. The Pichinde virus particle of embodiment 9, wherein said viral protein has been removed or functionally inactivated. (Aspect 11) At least one of the four ORFs encoding the GP, NP, Z protein, and L protein is deleted. and replaced with a heterologous ORF from an organism other than Pichinde virus. The Pichinde virus particle described. (Aspect 12) Only one of the four OFRs encoding the GP, NP, Z protein, and L protein was removed. and replaced with a heterologous ORF from an organism other than Pichinde virus. Pichinde virus particles as shown. (Aspect 13) The ORF encoding the GP has been removed, and a heterologous O virus from an organism other than Pichinde virus has been isolated. 10. The Pichinde virus particle of embodiment 9, wherein the RF is replaced by RF. (Aspect 14) The ORF encoding the NP has been removed, and a heterologous O virus from an organism other than Pichinde virus has been identified. 10. The Pichinde virus particle of embodiment 9, wherein the RF is replaced by RF. (Aspect 15) The ORF encoding the Z protein has been removed, and is not from an organism other than Pichinde virus. 10. The Pichinde virus particle of embodiment 9, wherein the ORF is replaced with a heterologous ORF from any of the preceding claims. (Aspect 16) The ORF encoding the L protein has been removed, and it is not derived from an organism other than Pichinde virus. 10. The Pichinde virus particle of embodiment 9, wherein the ORF is replaced with a heterologous ORF from any of the preceding claims. (Aspect 17) 17. The method according to any one of claims 11 to 16, wherein the heterologous ORF encodes a reporter protein. Pichinde virus particles. (Aspect 18) The heterologous ORF encodes an antigen derived from an infectious organism, a tumor, or an allergen. 17. The Pichinde virus particle according to any one of claims 11 to 16. (Aspect 19) The heterologous ORF encoding an antigen is selected from the group consisting of a human immunodeficiency virus antigen, a hepatitis C virus antigen, Varicella zoster virus antigen, cytomegalovirus antigen, tuberculosis antigen From tumor-associated antigens and tumor-specific antigens (such as tumor neoantigens and tumor neoepitopes) 20. The Pichinde virus particle of embodiment 18, which is selected. (Aspect 20) The proliferation or infectivity of the Pichinde virus particles is determined to be due to a prior infection with an organism other than the Pichinde virus. 19. The Pichinde virus particle according to any one of embodiments 11 to 18, which is not affected by the heterologous ORF. . (Aspect 21) 2. A method for producing a Pichinde virus genome segment according to claim 1, said method comprising: 4. A method comprising transcribing the cDNA of embodiment 3. (Aspect 22) 7. A method of making a Pichinde virus particle according to embodiment 6, said method comprising: (i) transfecting a host cell with the cDNA of embodiment 3; (ii) introducing into the host cell a plasmid containing the cDNA of a second Pichinde virus genome segment. transfecting; (iii) maintaining the host cells under conditions favorable for virus formation; and (iv) harvesting the Pichinde virus particles. The method includes: (Aspect 23) The transcription of the L segment and the S segment is carried out using a bidirectional promoter. 23. The method of embodiment 22, (Aspect 24) The method further comprises transfecting a host cell with one or more nucleic acids encoding a Pichinde virus polymerase. 23. The method of embodiment 22, further comprising transfecting. (Aspect 25) 25. The method of embodiment 24, wherein said Pichinde virus polymerase is said L protein. (Aspect 26) The method further comprises transfecting the host cell with one or more nucleic acids encoding the NP protein. 25. The method of embodiment 22 or 24, further comprising injecting the (Aspect 27) Transcription of the L segment and the S segment, respectively: (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter 23. The method of embodiment 22, wherein the promoter is under the control of a promoter selected from the group consisting of: (Aspect 28) The Pichinde virus particle according to any one of aspects 6 to 19 and a medicamentously acceptable carrier. Including vaccines. (Aspect 29) The Pichinde virus particle according to any one of aspects 6 to 19 and a medicamentously acceptable carrier. A pharmaceutical composition comprising: (Aspect 30) The Pichinde virus genome segment or the Pichinde virus particle is 2. The Pichinde virus genome segment according to embodiment 1, derived from the An4763 isolate P18 or P2 strain. Or a Pichinde virus particle according to embodiment 6. (Aspect 31) The tripartite virus particle contains one L segment and two S segments. Therefore, the proliferation of the three-segmented Pichinde virus particles is mediated by the type I interferon receptor, I Lacking type I interferon receptor and recombination activating gene 1 (RAG1) and 4 PFU's After 70 days of persistent infection in mice infected with three-segment Pichinde virus particles, Trisegmented Pichinde virus particles do not yield replication-competent bisegmented virus particles . (Aspect 32) Instead of two separate segments, the two Pichinde virus ORFs were combined into only one Recombination between the two S segments suppresses viral promoter activity. 32. The three segmented Pichinde virus particle according to embodiment 31, (Aspect 33) The tripartite virus particle contains two L segments and one S segment. Therefore, the proliferation of the three-segmented Pichinde virus particles is mediated by the type I interferon receptor, I Lacking type I interferon receptor and recombination activating gene 1 (RAG1) and 4 PFU's After 70 days of persistent infection in mice infected with three-segment Pichinde virus particles, Trisegmented Pichinde virus particles do not yield replication-competent bisegmented virus particles . (Aspect 34) Instead of two separate segments, the two Pichinde virus ORFs were combined into one Recombination between the two L segments suppresses viral promoter activity. 34. The three segmented Pichinde virus particle of embodiment 33. (Aspect 35) One of the two S segments is: (i) The S segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (ii) The ORF encoding the Z protein is located in the S segment under the control of the 5'UTR of Pichinde virus. to; (iii) The ORF encoding the L protein is located in the S segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the S segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) an S segment in which the ORF encoding L is under the control of the 3'UTR of Pichinde virus; and (vi) The ORF encoding the Z protein is located in the S segment under the control of the 3'UTR of Pichinde virus. nt; 32. The three segmented Pichinde virus particle according to embodiment 31, selected from the group consisting of: (Aspect 36) The two L segments are: (i) The L segment, in which the ORF encoding GP is under the control of the 5′UTR of Pichinde virus; (ii) the L segment, in which the ORF encoding NP is under the control of the 5′UTR of Pichinde virus; (iii) The ORF encoding the L protein is located in the L segment under the control of the 5'UTR of Pichinde virus. nt; (iv) the L segment, in which the ORF encoding GP is under the control of the 3′UTR of Pichinde virus; (v) an L segment in which the ORF encoding NP is under the control of the 3'UTR of Pichinde virus; and (vi) The ORF encoding the Z protein is located in the L segment under the control of the 3'UTR of Pichinde virus. to 34. The trisegment Pichinde virus particle according to embodiment 33, selected from the group consisting of: (Aspect 37) The 3'UTR of the Pichinde virus is the 3'UTR of the viral L segment and the 5'UTR of the Pichinde virus 35. The method according to claim 34, wherein the 5′UTR of said Pichinde virus S segment or said Pichinde virus L segment is The three-segmented Pichinde virus particle described in 36. (Aspect 38) The two S segments are each independently selected from the group consisting of (i) one or two heterologous ORFs from an organism other than Pichinde virus; or (ii) one or two overlapping Pichinde virus ORFs; or (iii) one or more 32. The three-segmented vector according to embodiment 31, comprising a heterologous ORF from a foreign organism and one overlapping Pichinde virus ORF. Mentopichinde virus particles. (Aspect 39) The two L segments are each independently selected from the group consisting of (i) one or two heterologous ORFs from an organism other than Pichinde virus; or (ii) two overlapping Pichinde virus ORFs; or (iii) one non-Pichinde virus organism. 34. The three-segment p53 vector according to claim 33, comprising a heterologous ORF from p53 and one overlapping p53 virus ORF. Chinde virus particles. (Aspect 40) The heterologous ORF encodes an antigen derived from an infectious organism, a tumor, or an allergen. 38 or 39. A three segmented Pichinde virus particle according to claim 38 or 39. (Aspect 41) The heterologous ORF encoding the antigen is a human immunodeficiency virus antigen, a hepatitis C virus antigen, Varicella zoster virus antigen, cytomegalovirus antigen, tuberculosis antigen From tumor-associated antigens and tumor-specific antigens (such as tumor neoantigens and tumor neoepitopes) 41. The trisegment Pichinde virus particle according to embodiment 40, which is selected. (Aspect 42) 40. The three-segmented nucleic acid molecule of embodiment 38 or 39, wherein at least one heterologous ORF encodes a fluorescent protein. Topitinde virus particles. (Aspect 43) 43. The method of claim 42, wherein the fluorescent protein is a green fluorescent protein or a red fluorescent protein. of three-segmented Pichinde virus particles. (Aspect 44) The three-segment Pichinde virus particle contains all four Pichinde virus ORFs, 42. Any of aspects 31 to 41, wherein the three-segment Pichinde virus particle is infectious and replicable. 2. The three segmented Pichinde virus particle according to claim 1. (Aspect 45) The three-segment Pichinde virus particle contains one or more of the four Pichinde virus ORFs. The three-segmented Pichinde virus particles are infectious but not expressed in non-complementing cells. 44. The three-segment peptide according to any one of embodiments 31 to 43, which is unable to produce infectious progeny consisting of Devirion. (Aspect 46) The three-segment Pichinde virus particle lacks one of the four Pichinde virus ORFs. The three-segment Pichinde virus particles are infectious but not expressible in non-complementing cells. The three-segment peptide according to any one of embodiments 31 to 43, which is unable to produce infectious progeny comprising Virus particles. (Aspect 47) 46. ​​The three segment Pichinde virus according to embodiment 44 or 45, wherein the Pichinde virus lacks the GP ORF. Russ particle. (Aspect 48) The tripartite virus particle contains one L segment and two S segments. Thus, the first S segment inserts the ORF encoding GP into the 3'UTR of Pichinde virus under its control. and locating an ORF encoding a first gene of interest under the control of the Pichinde virus 5'UTR. and a second S segment is engineered to carry an ORF encoding NP. The ORF encoding the second gene of interest was placed under the control of the 3'UTR of the virus. A three-segment peptide that has been engineered to be placed under the control of the viral 5'UTR. Virus particles. (Aspect 49) The tripartite virus particle contains one L segment and two S segments. Thus, the first S segment inserts the ORF encoding GP into the 5'UTR of Pichinde virus under its control. The ORF encoding the first gene of interest is placed under the control of the 3'UTR of the Pichinde virus. and a second S segment is engineered to carry an ORF encoding NP. The ORF encoding the second gene of interest is placed under the control of the 5'UTR of the genotype virus. A three-segment peptide that has been engineered to carry the gene under the control of the 3'UTR of the virus Virus particles. (Aspect 50) The gene of interest encodes an antigen derived from an infectious organism, a tumor, or an allergen. 50. The three segment Pichinde virus particle according to embodiment 48 or 49. (Aspect 51) The target gene is a human immunodeficiency virus antigen, a hepatitis C virus antigen, a varicella zoster virus antigen, Varicella zoster virus antigen, cytomegalovirus antigen, tuberculosis antigen, tumor-related antigen and tumor-specific antigens (such as tumor neoantigens and tumor neoepitopes). 51. The trisegment Pichinde virus particle according to embodiment 50, encoding an antigen. (Aspect 52) 50. The method according to claim 48 or 49, wherein at least one gene of interest encodes a fluorescent protein. Segmented Pichinde virus particles. (Aspect 53) 53. The method of claim 52, wherein the fluorescent protein is a green fluorescent protein or a red fluorescent protein. of three-segmented Pichinde virus particles. (Aspect 54) The three segmented Pichinde virus particle according to any one of aspects 31, 33, 35, 36, 48 or 49. cDNA of the offspring genome. (Aspect 55) A DNA expression vector comprising the cDNA of embodiment 54. (Aspect 56) The three segment Pichinde virus particle according to embodiment 31 or 33, the cDNA according to embodiment 54, or the 55. A host cell comprising the vector described in 55. (Aspect 57) 50. Any one of aspects 31 to 49, wherein the three-segment Pichinde virus particle is attenuated. 4. The three segmented Pichinde virus particle according to claim 3. (Aspect 58) 32. A method for producing a three segment Pichinde virus particle according to claim 31, said method comprising: : (i) transfecting a host cell with the cDNA of one or more of the L segment and the two S segments; To do; (ii) maintaining the host cells under conditions favorable for virus formation; and (iii) Collecting Pichinde virus particles The method includes: (Aspect 59) 34. A method for producing a three segment Pichinde virus particle according to claim 33, said method comprising the steps of: : (i) transfecting one or more cDNAs of two L segments and one S segment into a host cell; To do; (ii) maintaining the host cells under conditions favorable for virus formation; and (iii) Collecting Pichinde virus particles The method includes: (Aspect 60) Transcription of one L segment and two S segments is carried out using a bidirectional promoter. 59. The method of embodiment 58, wherein (Aspect 61) Transcription of two L segments and one S segment is carried out using a bidirectional promoter. The method of embodiment 59, (Aspect 62) The method further comprises injecting one or more nucleic acids encoding a Pichinde virus polymerase into the host cell. 60. The method of embodiment 58 or 59, further comprising transfecting the (Aspect 63) 63. The method of embodiment 62, wherein said Pichinde virus polymerase is said L protein. (Aspect 64) The method further comprises transferring one or more nucleic acids encoding the NP protein into the host cell. 62. The method of embodiment 58, 59, 60 or 61, further comprising injecting (Aspect 65) Transcription of the L segment and the two S segments is, respectively: (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter 60. The method of embodiment 58, wherein the promoter is under the control of a promoter selected from the group consisting of: (Aspect 66) Transcription of the two L segments and the S segment is, respectively: (i) RNA polymerase I promoter; (ii) an RNA polymerase II promoter; and (iii) T7 promoter 60. The method of embodiment 59, wherein the promoter is under the control of a promoter selected from the group consisting of: (Aspect 67) The three-segment Pichinde virus particle is a two-segment Pichinde virus particle. The three-segment picin deuil according to any one of embodiments 31 to 49, having the same tropism. Particles. (Aspect 68) 50. Any one of aspects 31 to 49, wherein the three-segment Pichinde virus particle is replication-deficient. A three-segmented Pichinde virus particle as described. (Aspect 69) The three segmented Pichinde virus particle according to any one of aspects 31 to 49, 67 and 68, and A vaccine comprising a pharma- ceutical acceptable carrier. (Aspect 70) The three segmented Pichinde virus particle according to any one of aspects 31 to 49, 67 and 68, and A pharmaceutical composition comprising a pharma- ceutical acceptable carrier. (Aspect 71) 31. The Pichinde virus is strain Munchique CoAn4763 isolate P18 or strain P2. 69. The three segmented Pichinde virus particle according to any one of claims 49, 67 or 68.

[0339] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated herein by reference. The entirety is incorporated by reference. 7.Sequence Listing [Table 7] TIFF2024170461000011.tif223170TIFF2024170461000012.tif223170TIFF2024170461000013.tif224170TIFF2024170461000014.tif222170TIFF2024170461000015.tif222170TIFF2024170461000016.tif224170TIFF2024170461000017.tif223170TIFF2024170461000018.tif222170TIFF2024170461000019.tif223170TIFF2024170461000020.tif223170TIFF2024170461000021.tif225170TIFF2024170461000022.tif224170TIFF2024170461000023.tif225170TIFF2024170461000024.tif224170TIFF2024170461000025.tif225170TIFF2024170461000026.tif224170TIFF2024170461000027.tif223170TIFF2024170461000028.tif223170TIFF2024170461000029.tif223170TIFF2024170461000030.tif221170TIFF2024170461000031.tif223170TIFF2024170461000032.tif224170TIFF2024170461000033.tif224170TIFF2024170461000034.tif223170TIFF2024170461000035.tif224170TIFF2024170461000036.tif73170

Claims

1. In the manufacture of a medicament for treating or preventing an infection and / or cancer in a patient. Use of a pharmaceutical composition comprising three-segment Pichinde virus particles, The three-segmented Pichinde virus particle contains one L segment and two S segments. fruit, The first S segment contains an ORF encoding GP under the control of the 3'UTR of the Pichinde virus genome. Position and control of the 5'UTR of the Pichinde virus genome ORF encoding the first gene of interest and is operated to carry it in a lower position, The second S segment transmits the ORF encoding NP under the control of the 3'UTR of the Pichinde virus genome. Position and control of the 5'UTR of the Pichinde virus genome ORF encoding a second gene of interest and The L segment regulates the 3'UTR of the Pichinde virus genome, where it encodes the ORF encoding the L protein. The ORFs encoding the and Z proteins are located under the control of the 5'UTR of the Pichinde virus genome. The use, wherein the device is held in a position.

2. The first and / or second gene of interest are (i) encoding an antigen derived from an infectious organism, a tumor, or an allergen; (ii) Human immunodeficiency virus antigen, papillomavirus antigen, hepatitis C virus antigen, B Hepatitis virus antigen, Varicella zoster virus antigen, Cytomegalovirus antigen, Mycobacterium tuberculosis antigen From tumor-associated antigens, tumor-specific antigens including tumor neoantigens and tumor neoepitopes or (iii) encoding a fluorescent protein; 2. The use according to claim 1.

3. 1. The method of claim 1, wherein the Pichinde virus is strain Munchique CoAn4763 isolate P18 or strain P2. Or the use as described in 2.

4. The two Pichinde virus ORFs were combined into one segment instead of into two separate segments. Recombination between the two S segments enhances viral promoter activity. The use according to any one of claims 1 to 3, which is to inhibit.

5. The proliferation of the three-segment Pichinde virus particles is mediated by type I interferon receptor, type II Lacking interferon receptor and recombination activating gene 1 (RAG1) and 4 PFU's third After 70 days of persistent infection in mice infected with segmented Pichinde virus particles, multiple The use according to any one of claims 1 to 4, which does not result in a producible bi-segmented virus particle. .

6. The three-segment Pichinde virus particle of claims 1 to 5 is infectious and replicable.

2. The use according to any one of claims 1 to 11.

7. Any of claims 1 to 6, wherein the three-segment Pichinde virus particle is attenuated.

10. The use according to claim 1.

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