Tripartite arenaviruses as vaccine vectors

Abstract

Provided arenaviruses having rearrangements of open reading frames (ORFs) in their genomes that are suitable for use as vaccines and / or for treating disease and / or in immunotherapy. The present invention provides tripartite arenavirus particles containing one L segment and two S segments, or two L segments and one S segment.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 079,493, filed November 13, 2014. No. 6,239,693, the entire contents of which are incorporated herein by reference.

[0002] (1. Introduction) This application describes an allele having a rearrangement of an open reading frame ("ORF") in the genome. In particular, the present invention relates to a virus that retains an ORF in a position different from the wild-type position of the ORF. Described herein are modified arenavirus genome segments that have been engineered to One L segment and two S segments, or two L segments and one S segment Also described herein are tripartite arenavirus particles comprising: The arenaviruses are useful for vaccines and / or for the treatment of disease and / or for use in immunotherapy. It may be suitable. [Background technology]

[0003] (2. Background) 2.1 Lymphocytic Choriomeningitis Virus Research and Human Disease Lymphocytic choriomeningitis virus (LCMV), a member of the Arenaviridae family, is a viral infection It is the prototype mouse model virus for the study of infectious diseases. Since its introduction (Rivers and McNair Scott, 1935, Science, 81(2105): 439-440), Studies with viruses have clarified many key concepts in viral immunology and pathogenesis. (Zinkernagel, 2002, Curr Top Microbiol Immunol 263:1-5; Oldst (Reviewed in [End Page 117], 2002, Curr Top Microbiol Immunol, 263:83-117). , particularly to investigate the molecular biology of viruses and immune responses in the context of persistent infection. The natural host of LCMV is the mouse. However, some reports have This finding revealed that LCMV may also be a neglected human pathogen (Barto n, 1996, Clin. Infect. Dis, 22(1):197; Wright et al., 1997, Pediatrics 10 0(1): E9). In addition, many other members of the Arenavirus family are known to infect rodents worldwide. Lassa virus, an Old World arenavirus found in Africa, has been found in clusters of In addition to the (LASV), there are several New World arenas such as Junín (JUNV), Guanarito, or Machupo. Infectious diseases such as rabies, flu, and flu-like viruses are prevalent in diverse rodent populations in South America (Johnson et al., 2014). 1966, Am J Trop Med Hyg, 15(1): 103-106; Tesh et al., 1993, Am J Trop Med Hy g 49(2):227-235; Mills et al., 1994, Trop Med Hyg 51(5):554-562). Many of these viruses can cause viral hemorrhagic fevers with high mortality rates when transmitted to humans. (Geisbert and Jahrling, 2004, Nat Med 10(12 Suppl):S110-121).

[0004] 2.2 Genome organization of lymphocytic choriomeningitis virus Arenaviruses are enveloped viruses. Their genomes consist of a negative-sense It consists of two segments of double-stranded RNA (L: 7.2 kb, S: 3.4 kb). Each segment is in opposite orientation. The shorter segment (S segment) encodes two viral genes. It encodes the glycoprotein (GP) precursor (GP-C; 75 kDa) and nucleoprotein (NP; 63 kDa) of the nucleus. (Salvato et al., 1988, Virology 164(2): 517-522). The longer segment (L segment) The RdRp (L protein; approximately 200 kDa) and matrix protein They express protein Z (protein Z), a RING finger protein (11 kDa) (Figure 1A) (Salvato et al. (1988, Virology 164(2): 517-522). The GP precursor, GP-C, is posttranslationally converted to GP-1 and GP-2. and cleavage, but they remain non-covalently associated (Buchmeier and Oldstone, 1979, Virology 99(1): 111-120). Trimers of GP-1 and GP-2 form spikes on the surface of the virion. and mediates entry into host cells by interacting with cell surface receptors The binding and entry of the virus into the host cell is essential for the Mediated by interaction with the cell receptor α-dystroglycan as the only cell receptor It has long been argued that this is the case (Cao et al., 1998, Science, 282(5396):2079-2081). Recently, three additional human molecules (Axl and Tyro3 of the TAM family, and dendritic cell-specific Intercellular adhesion molecule 3-binding nonintegrin (IL-3) is a further marker of LCMV and its close relative LASV. These are hypothesized receptors that mediate LCMV intracellular entry, independent of α-dystroglycan. allowing invasion (Shimojima and Kawaoka, 2012, J Vet Med, 74(10):1363-1366; S Himojima et al., 2012, J Virol 86(4):2067-2078). NP binds to viral RNA and It forms a nucleocapsid, which serves as a template for the viral L protein. The nucleocapsid associated with the viral L protein is a so-called ribonucleoprotein The complex is active in both replication and transcription and is the minimum infectivity of the virus. The NP and L proteins are the minimum transcription factors required for viral RNA transcription and replication. It has been shown to be a virulence factor (Lee et al., 2000, J Virol 74(8): 3470-3477). The two genes on each segment are separated by a non-coding intergenic region (IGR). The IGR is located on the 5' and 3' untranslated regions (UTRs) and has a stable hairpin structure. It has been shown to be involved in the structure-dependent termination of viral mRNA transcription. (Pinschewer et al., 2005, J Virol 79(7): 4519-4526). These panhandle structures are highly complementary and lead to the formation of secondary structures. It is known to act as a viral promoter for transcription and replication, and These analyses by mutagenesis reveal sequences and sequences that do not tolerate even small sequence changes. Structure dependence has been demonstrated (Perez and de la Torre, 2003, Virol 77(2): 11 84-1194).

[0005] (2.3 Reverse Genetics Systems) Isolated and purified RNA from negative-strand viruses such as LCMV can function directly as mRNA. That is, they cannot be translated when introduced into a cell. As a result, transfection of cells with viral RNA leads to the production of infectious viral particles. Infectivity of negative-strand RNA viruses from cDNA in cultured permissive cells To produce a viral particle, the viral RNA segment(s) must be transcribed and replicated. The minimum factors required for production must be trans-complemented. With the help of the minigenome system published several years ago, transcription, replication, and replication of viral particles can be controlled. The viral cis- and trans-acting elements involved in the formation of (Lee et al., 2000, J Virol 74(8): 3470-3477; Lee et al., 2002, J Virol 76(12): 6393-6397; Perez and de la Torre, 2003, J Virol 77(2): 1184-11 94; Pinschewer et al., 2003, J Virol 77(6): 3882-3887; Pinschewer et al., 2005, J Virol 79(7): 4519-4526). Other arenaviruses, such as LASV and Tacaribe virus, have also been reported. A reverse genetics system has also been established for rus (Lopez et al., 2001, J Virol 75(2) 4): 12241-12251; Hass et al., 2004, J Virol 78(24): 13793-13803). The two publications , completely cDNA-derived infectious using pol-I / -II or T7 / pol-II driven plasmids, respectively. showed recovery of LCMV (termed "viral rescue") (Flatz et al., 2006, Proc N atl Acad Sci USA 103(12): 4663-4668; Sanchez and de la Torre, 2006, Virolo gy 350(2): 370-380).

[0006] 2.4 Recombinant LCMV expressing a gene of interest Generation of recombinant negative-strand RNA viruses expressing exogenous genes of interest is promoted for long periods. Different strategies have been published for different viruses (Garcia-Sastre et al., 2004). Reference, 1994, J Virol 68(10): 6254-6261; Percy et al., 1994, J Virol 68(7): 4486-449 2; Flick and Hobom, 1999, Virology 262(1): 93-103; Machado et al., 2003, Vi rology 313(1): 235-249). To date, additional foreign genes have been incorporated into the genome of bipartite LCMV particles. It has been shown that it is possible to introduce it into the human body (Emonet et al., 2009, PNAS, 106(9) Two foreign genes of interest are inserted into the bipartite genome of LCMV, resulting in two This resulted in a tripartite LCMV particle (r3LCMV) with three S segments and one L segment. In this tripartite virus, published by Emonet et al. (2009), both NP and GP The two sequences are maintained in their natural positions in the S segment and therefore in the adjacent UTRs. The present application, however, is directed to the expression of the ribosomal RNA under their native promoters in the The tripartite LCMV particles disclosed by Emonet et al. are primarily a collection of bipartite particles. (i.e., the arenaviruses enclose one S segment instead of two. (only able to recombine the two S segments), resulting in attenuated growth and the ability to recombine the two S segments. As further shown in this application, this Such recombinations are reproducible and result in phenotypic reversion and transfer to wild-type virus. This results in gene loss.

[0007] (2.5 Replication-Defective Arenaviruses) Recently, infectious arenavirus particles have been developed by amplifying and expressing their genetic material in infected cells. have the ability to produce further offspring, whereas normal, non-genetically engineered cells It has been shown that the virus can be engineered to contain a genome that cannot be transmitted (i.e., infectious Replication-deficient arenavirus particles (International Publication No. WO2009 / 083210 A1 and International Publication No. WO2014 / 140301 A1). Summary of the Invention

[0008] (3. Summary of the Invention) This application relates to arenaviruses having rearrangements of ORFs in their genomes. In particular, this application relates to: , arenaviruses engineered to retain the ORF in a position other than the wild-type position. The present application also relates to replication-competent bipartite arenavirus genome segments. One L segment and two S segments, or two L segments, which do not recombine into a nucleotide particle. The present application provides a tripartite arenavirus particle comprising one S segment and one nucleotide sequence. Segmented arenavirus particles enhance genetic stability and ensure sustained transgene expression Demonstrate that the system can be manipulated to

[0009] In certain embodiments, the viral vectors provided herein are infectious. that is, capable of entering or transferring its genetic material into a host cell. In certain more specific embodiments, the compositions provided herein can be injected into the The viral vectors used are infectious, i.e., capable of entering host cells. Alternatively, the genetic material can be injected into a host cell, followed by intracellular In one embodiment, the viral vector is capable of amplifying and expressing its genetic information. - has the ability to amplify and express its genetic information in infected cells, but does not have the ability to express normal genetic information. In cells that are not engineered, they are unable to produce further infectious progeny particles. Infectious replication-deficient arenavirus viral vectors engineered to contain a non-human genome In one embodiment, the infectious arenavirus viral vector is a can be produced and produce infectious progeny particles in normal, genetically unmodified cells. In a more specific embodiment, such replicable The replication-competent viral vector is a wild-type virus from which the replication-competent viral vector is derived. is attenuated compared to 3.1 Non-Natural Open Reading Frames Thus, in one embodiment, the arenavirus genome segment is In one embodiment, the genome segment comprises a viral ORF in the region of the ORF. In some embodiments, the green mold is engineered to be held in a location separate from that of the green mold. The arenavirus genome segment: (i) The S segment in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (ii) the S segment, in which the ORF encoding the Z protein is under the control of the arenavirus 5′UTR; (iii) The S segment, in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR ; (iv) an S segment in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an S segment in which the ORF encoding the L protein is under the control of the arenavirus 3′UTR; (vi) an S segment in which the ORF encoding the Z protein is under the control of the arenavirus 3′UTR; (vii) the L segment, in which the ORF encoding GP is under the control of the arenavirus 5′UTR; (viii) the L segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (ix) an L segment in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR; (x) L segment in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (xi) an L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR; and (xii) The L segment, in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR , is selected from the group consisting of:

[0010] In some embodiments, the arenavirus 3'UTR comprises an arenavirus S segment. or the 3'UTR of an arenavirus L segment. The 5'UTR is the 5'UTR of an arenavirus S segment or an arenavirus L segment. .

[0011] Also provided herein are isolated cDNAs of arenavirus genome segments. Also provided herein are vectors containing the cDNA of the arenavirus genome segment. Also provided herein are DNA expression vectors containing the vector.

[0012] Also, the arenavirus genome segment, a host cell comprising the cDNA or a vector comprising the cDNA of said arenavirus genome segment; Also provided herein are:

[0013] The arenavirus genome segments may also be modified to include an S segment and an L segment. Also provided herein are arenavirus particles comprising a first and a second arenavirus genome segment. It is provided as follows.

[0014] In certain embodiments, the arenavirus particles are infectious and replication competent. In some embodiments, the arenavirus particles are attenuated. In the present study, the arenavirus particles are infectious but do not infect non-complementing cells. ng cells) cannot give rise to further infectious progeny.

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

[0016] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the ORFs is removed and replaced with a heterologous ORF from an organism other than arenavirus. In another embodiment, the GP, NP, Z protein, and L protein are encoded by Only one of the four ORFs was removed, leaving a heterologous ORF from an organism other than arenaviruses. In a more specific embodiment, the ORF encoding GP is removed. and replaced with a heterologous ORF from an organism other than an arenavirus. In this case, the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than arenavirus. In some embodiments, the ORF encoding the Z protein is removed. and replaced with a heterologous ORF from an organism other than an arenavirus. In this case, the ORF encoding the L protein has been removed to produce a heterologous vector derived from an organism other than an arenavirus. It has been replaced by an ORF.

[0017] In one embodiment, the heterologous ORF encodes a reporter protein. In some embodiments, the heterologous ORF is an antigen derived from an infectious organism, a tumor, or an allergen. In another embodiment, the heterologous ORF encoding an antigen is a human immunodeficiency virus. Whole virus antigen, Hepatitis C virus antigen, Hepatitis B surface antigen, Varicella zoster (varicella zoster) ter) selected from viral antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, and tumor-associated antigens will be done.

[0018] In one embodiment, the proliferation or infectivity of the arenavirus particles is increased by the It is not affected by heterologous ORFs from organisms other than R. rus.

[0019] Also provided herein are methods for generating said arenavirus genome segments. In one embodiment, the method comprises: This includes transcribing the

[0020] Also provided herein are methods for producing said arenavirus particles. In embodiments, the method of producing the arenavirus particles comprises: (i) transfecting a cDNA of the arenavirus genome segment into a host cell; and; (ii) introducing a plasmid containing the cDNA of the second arenavirus genome segment into a host cell; To transfect; (iii) maintaining the host cells under conditions suitable for virus formation; and (iv) recovering the arenavirus particles; Includes.

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

[0022] In certain embodiments, the methods include one or more nucleic acids encoding an arenavirus polymerase. and transfecting the nucleic acid of the present invention into a host cell. In one embodiment, the polymerase is an L protein. The method further comprises transfecting the host cell with one or more nucleic acids encoding NP. include.

[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, a vaccine comprising arenavirus particles, including GP, NP, Z, At least one of the four ORFs encoding the L protein and the L protein has been removed. or functionally inactivated; or GP, NP, Z protein, and L protein At least one ORF encoding a gene has been removed to produce a variant derived from another organism other than an arenavirus. Replaces the species ORF; or encodes GP, NP, Z protein, and L protein Only one of the four ORFs was removed and replaced with a heterologous ORF from an organism other than arenavirus. Provided herein are such vaccines, wherein the In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier.

[0025] In another embodiment, a pharmaceutical composition comprising arenavirus particles, including GP, NP, Z, At least one of the four ORFs encoding the L protein and the L protein has been removed. or functionally inactivated; or GP, NP, Z protein, and L protein At least one ORF encoding a protein has been removed to identify a virus derived from another organism other than an arenavirus. replaced with heterologous ORFs; or encoding GP, NP, Z protein, and L protein Only one of the four ORFs was removed, replacing it with a heterologous ORF from an organism other than arenaviruses. The pharmaceutical compositions are provided herein. In some embodiments, the pharmaceutically acceptable carrier further comprises a pharmaceutically acceptable carrier. include.

[0026] In certain embodiments, the arenavirus genome segment or the arenavirus In some embodiments, the particles are derived from LCMV. The menthos or arenavirus particles were derived from LCMV MP strain, Armstrong strain, or Armstrong clone 13. In another embodiment, the arenavirus genome segment or the arenavirus is derived from a strain. The lenavirus particles are Junin virus vaccine Candid #1 or Junin virus vaccine X. It is derived from J clone 3 strain.

[0027] (3.2 Tripartite arenaviruses) In one embodiment, a tripartite arenavirus comprising one L segment and two S segments. In some embodiments, the tripartite array is provided herein. The proliferation of flu virus particles is controlled by type I interferon receptors, type II interferon receptors, and and lacking recombination activating gene 1 (RAG1), 10 4 Infected with PFU of the tripartite arenavirus particles yielding replication-competent bipartite virus particles after 70 days of persistent infection in mice carrying In one embodiment, the two arenavirus ORFs are located on two separate segments. Intersegmental recombination of the two S segments, resulting in integration onto a single segment rather than suppresses viral promoter activity.

[0028] In another embodiment, a tripartite arenavirus comprising two L segments and one S segment is In one embodiment, the tripartite Arenauer particle is The proliferation of virus particles is regulated by type I interferon receptors, type II interferon receptors, and lacking recombination activating gene 1 (RAG1), 10 4 PFU of the tripartite arenavirus particles did not produce replication-competent bipartite virus particles after 70 days of persistent infection in mice In one embodiment, the two arenavirus ORFs are not located on two separate segments. Intersegmental recombination of the two L segments, which integrates them into a single segment, 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 arenavirus 5′UTR; (ii) the S segment, in which the ORF encoding the Z protein is under the control of the arenavirus 5′UTR; (iii) The S segment, in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR ; (iv) an S segment in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an S segment in which the ORF encoding the L protein is under the control of the arenavirus 3′UTR; and (vi) The S segment in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR , is selected from the group consisting of:

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

[0031] In certain embodiments, the tripartite arenavirus particle 3'UTR is an arenavirus S segment. In another embodiment, the 3'UTR is a 3'UTR of an arenavirus L segment or an arenavirus L segment. The segmented arenavirus particle 5'UTR is composed of the arenavirus S segment or the arenavirus L segment. The 5'UTR of the nucleotide sequence of the 5'-nucleotide fragment.

[0032] In one embodiment, the two S segments are (i) derived from an organism other than an arenavirus. or (ii) one or two replicated arenavirus ORFs; or (iii) one heterologous ORF from an organism other than arenavirus and one replicated arenavirus ORF Contains one.

[0033] In one embodiment, the two L segments are (i) derived from an organism other than an arenavirus. or (ii) one or two replicated arenavirus ORFs; or (iii) one heterologous ORF from an organism other than arenavirus and one replicated arenavirus ORF Contains one.

[0034] In certain embodiments, the heterologous ORF is derived from an infectious organism, a tumor, or an allergen. In another embodiment, the heterologous ORF encoding an antigen is a human immunoglobulin. Incomplete viral antigen, Hepatitis C virus antigen, Hepatitis B surface antigen, Varicella zoster virus antigen , cytomegalovirus antigens, Mycobacterium tuberculosis antigens, and tumor-associated antigens.

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

[0036] In one embodiment, the tripartite arenavirus particle comprises four arenavirus ORFs. In some embodiments, the tripartite arenavirus particles comprise all of the following: It is both reliable and replicable.

[0037] In one embodiment, the tripartite arenavirus particle comprises four arenavirus ORFs. In other embodiments, the tripartite arenavirus particle lacks one or more of the following: Although infectious, they are unable to give rise to further infectious progeny in non-complementing cells.

[0038] In one embodiment, the tripartite arenavirus particle comprises four arenavirus ORFs. wherein the tripartite arenavirus particle is infectious but lacks one of the non-complementing They are unable to give rise to further infectious progeny in the cell.

[0039] In some embodiments, said tripartite arenavirus particle lacks the GP ORF.

[0040] In a further embodiment, a tripartite arena comprising one L segment and two S segments. Viral particles are provided herein. In certain embodiments, arenaviruses The GP-encoding ORF is located under the control of the 3'UTR and is located under the control of the arenavirus 5'UTR. The first S segment is engineered to carry an ORF encoding a first gene of interest in a controlled position. In some embodiments, the arenavirus 3'UTR is under the control of the arenavirus 3'UTR. The ORF encoding NP is placed in a position where it is under the control of the arenavirus 5'UTR. A second S segment is engineered to carry an ORF encoding a second gene of interest. .

[0041] In yet another embodiment, a tripartite allele comprising one L segment and two S segments is In some embodiments, arenavirus particles are provided herein. The GP-encoding ORF is placed under the control of the arenavirus 5'UTR and the arenavirus 3'UTR. The first S segment is engineered to carry an ORF encoding a first gene of interest in a position under the control of In some embodiments, the arenavirus 5'UTR is under the control of the A position that contains an ORF encoding NP at a certain position and is under the control of the arenavirus 3'UTR A second S segment is engineered to carry an ORF encoding a second gene of interest. do.

[0042] In one embodiment, 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 of human immunodeficiency virus. antigen, hepatitis C virus antigen, hepatitis B surface antigen, varicella zoster virus antigen, cytomegalovirus The antigen may encode an antigen selected from a human immunodeficiency virus (HIV) antigen, a Mycobacterium tuberculosis antigen, and a tumor-associated antigen. In another embodiment, at least one gene of interest encodes a fluorescent protein. In a specific embodiment, said fluorescent protein is GFP or RFP.

[0043] The isolated cDNA of the genome of said tripartite arenavirus particle is also referred to herein as Also provided is a DNA expression vector containing the cDNA of the genome of the tripartite arenavirus particle. Also provided herein are the above either individually or in their entirety. Also provided herein are one or more DNA expression vectors containing the cDNA of the tripartite arenavirus. It is served.

[0044] Also, the tripartite arenavirus particle, the genome cD of the tripartite arenavirus particle A host cell containing a vector containing NA or the cDNA of the genome of said tripartite arenavirus particle. Also provided herein are:

[0045] In certain embodiments, the tripartite arenavirus particles are attenuated.

[0046] Also provided herein are methods for producing said tripartite arenavirus particles. In certain embodiments, the method of producing the arenavirus particles comprises: (i) One or more cDNAs of one L segment and two S segments are transfected into a host cell. To act; (ii) maintaining the host cells under conditions suitable for virus formation; and (iii) recovering the arenavirus particles. Includes.

[0047] Also provided herein are methods for producing said tripartite arenavirus particles. In one embodiment, a vector comprising the tripartite arenavirus particle is produced. Here's how: (i) One or more cDNAs of two L segments and one S segment are transfected into a host cell. To act; (ii) maintaining the host cells under conditions suitable for virus formation; and (iii) recovering the arenavirus particles. Includes.

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

[0049] In certain embodiments, the methods include one or more nucleic acids encoding an arenavirus polymerase. and transfecting the nucleic acid of the present invention into a host cell. In one embodiment, the polymerase is an L protein. The method includes transfecting one or more nucleic acids encoding an NP protein into the host cell. It further includes:

[0050] In one embodiment, the transcription of the one L segment and two S segments is Re: (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, the transcription of the two L segments and one S segment is Re: (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 tripartite arenavirus particle is a bipartite arenavirus particle. In another embodiment, the tripartite arenavirus particle has the same tropism as the offspring. are replication-deficient.

[0053] In another embodiment, the tripartite arenavirus particles and a pharmaceutically acceptable carrier A vaccine comprising the antibody is provided herein.

[0054] In another embodiment, the tripartite arenavirus particles and a pharmaceutically acceptable carrier A pharmaceutical composition comprising:

[0055] In one embodiment, the tripartite arenavirus particles are derived from LCMV. In some embodiments, the tripartite arenavirus particles are selected from the group consisting of LCMV MP strain, Armstrong strain, or from Armstrong clone 13. In another embodiment, the tripartite arenavirus The virus particles are Junin virus vaccine Candid #1 or Junin virus vaccine XJ clone. It is derived from strain 3. 3.3 Conventions and Abbreviations [Table 1] [Brief explanation of the drawings]

[0056] (4. Brief description of the drawings) [Figure 1] Recombinant tripartite viruses exhibit impaired growth compared to wild-type LCMV, regardless of the location of the GP ORF in the genome. (A-C) Schematic diagrams of the genome organization of bipartite and tripartite LCMV. The bipartite genome of wild-type LCMV consists of one S segment encoding GP and NP and one L segment encoding Z and L proteins (A). Both segments are flanked by their respective 5' and 3' UTRs. The genome of recombinant tripartite LCMV (r3LCMV) consists of one L segment and two S segments, each of which contains a site for inserting a gene of interest (here, GFP). (B) r3LCMV-GFPnatural (nat) has all viral genes in their natural locations. On the other hand, the GP ORF of r3LCMV-GFPartificial (art) is artificially juxtaposed to the 3' UTR and expressed under the control of the 3' UTR (C). (D) Growth kinetics of the indicated viruses in BHK-21 cells infected at a multiplicity of infection (moi) of 0.01 (wild-type LCMV: gray triangles; r3LCMV-GFPnat: black circles; r3LCMV-GFPart: white squares). Supernatants were harvested at the indicated time points post-infection, and virus titers were determined by focus-forming assay. Symbols and bars represent the mean ± SEM of triplicates per group. Error bars are hidden within the symbol dimensions.

[0057] [Figure 2]Tripartite virus preparations contain the majority of bipartite replication-deficient particles (r2LCMV). (A) r2LCMV (white bars), r3LCMV-GFP / RFPart (black bars, GFP-GP, RFP-NP), and r3LCMV-GFP / RFPnat (gray bars, GP-GFP, RFP-NP) were grown in wild-type BHK-21 cells, and the infectivity of the supernatants was determined in wild-type, non-complementing BHK-21 cells (BHK21), GP-expressing (BHK-GP), or NP-expressing (BHK-NP) BHK-21 cells. Titers in BHK-21 and BHK-GP cells were determined by staining for NP-positive virus foci. Titers in NP-complementing BHK-21 cells were determined by counting GP-positive foci. Titers were normalized to the average titer obtained when assayed in BHK-21 cells and are therefore expressed as a multiple of the average titer. Bars represent the mean ± SEM of six replicates per group. ns.: not statistically significant (p≥0.05); **: p<0.01 (by one-way ANOVA using r2LCMV as a reference followed by Dunnett's post-hoc test). (B) r2LCMV (left plot) or r3LCMV-GFP / RFPart (center and right plots) were grown in wild-type BHK-21 cells (BHK21; left and center plots) or NP-expressing BHK-21 cells (BHK-NP; right plot), and fluorescence was assessed by flow cytometry 12 hours post-infection. r2LCMV-infected cells were used as a gating control. One representative plot per condition is shown. (C) Quantification of GFP+, RFP+, or GFP+RFP+ double-positive cells 12 hours post-infection of BHK-21 or BHK-NP cells with r3LCMV-GFP / RFPart. Bars represent the mean ± SEM of triplicates per group. ns.: not statistically significant (p≧0.05); ***: p<0.001 (by unpaired two-tailed Student's t-test).

[0058] [Figure 3]Design and growth kinetics of recombinant tripartite viruses carrying a partially codon-optimized GP ORF or gene tag in the IGR of the S segment. (A) Schematic diagram of the engineered S segment in which the 255 C-terminal base pairs of GP were codon-optimized and NP replaced GFP (GP ORF designated "WE / WET"). Growth kinetics of tripartite r3LCMV-WEWET / GFPnat, consisting of two S segments and one L segment as detailed in Figure 1B, with modifications of the GP-containing S segment as shown in (A), was performed in BHK-21 cells. Supernatants were harvested at the indicated time points post-infection at moi = 0.01, and virus titers were determined by focus formation assay (B). Symbols and bars represent the mean ± SEM of triplicates per group. Error bars are hidden within the symbol dimensions. (C) Schematic diagram of the NP-encoding S segment in which a single base pair in the IGR was deleted to genetically "tag" the noncoding RNA element. The deleted G residue (indicated by the arrow) is located outside the critical stem-loop structure of the IGR. Comparative growth kinetics of tripartite viruses with or without a gene tag in the IGR of the S segment encoding NP (r3LCMV-GFPnat: black circles; r3LCMV-GFPnatIGR*: white circles) was performed in BHK-21 cells at an moi of 0.01. Supernatants were harvested at the indicated time points postinfection, and virus titers were determined by focus-forming assay. Symbols and bars represent the mean ± SEM of triplicates per group. Representative data from one of two independent experiments are shown.

[0059] [Figure 4]Persistent infection with r3LCMV-GFPnat, but not r3LCMV-GFPart, in immunocompromised mice leads to viremia levels comparable to those of the bipartite wild-type virus and to loss of GFP expression. (A) AGRAG mice were intravenously infected with 1 × 104 PFU of r3LCMV-GFPnat (black circles), r3LCMV-GFPart (white squares), or the control bipartite r2LCMV (gray triangles), and viremia was monitored over time. Symbols represent the mean ± SEM of 3–7 mice per group. (B) LCMV viremia on day 127 after intravenous infection of AGRAG mice with 1 × 104 PFU of r3LCMV-GFPnat or r3LCMV-GFPart is shown. Immunofocus assays were performed to detect either the nucleoprotein NP (gray circles) or GFP (white circles). Symbols represent individual mice. ns.: not statistically significant (p≧0.05); ***: p<0.001 (paired, two-tailed Student's t-test). (C-E) Blood from AGRAG mice infected with r3LCMV-GFPnat, r3LCMV-GFPart, or r2LCMV was analyzed for the presence of GFP+ cells by flow cytometry at day 120 post-infection. Monocytes and macrophages were identified using the gating strategy outlined in (C). One representative FACS plot for each group and one representative histogram overlay of GFP expression are shown in (D). (E) Quantification of the GFP+ population within the CD11b+GR1− monocyte / macrophage population. Symbols indicate individual mice.

[0060] [Figure 5]Persistent infection of mice with r3LCMV-GFPnat leads to S segment recombination and loss of a functional, full-length transgene. Viral RNA was isolated from the serum of AGRAG mice 127 days after intravenous infection with 1 × 104 PFU of r3LCMV-GFPnat or r3LCMV-GFPart. Viral RNA was reverse transcribed, and cDNA containing both the NP and GP sequences was PCR-amplified using appropriate gene-specific primers. (A) DNA electrophoresis of PCR products obtained with (+RT, lanes 1–8) or without (−RT, negative control, lanes 9–12) prior reverse transcription of the RNA template. Serum from an untreated animal served as another negative control (n, lane 8), and plasmid DNA encoding the wild-type LCMV S segment served as a positive control (p, lane 17). The amplified products in lanes 1–3 were subjected to Sanger sequencing. (B) A representative cDNA sequence (r3LCMV-GFPnat #3) from animal #3 reveals a recombinant S segment (SEQ ID NO: 17) combining NP and GP sequences, two IGRs (bold), and a C-terminal GFP portion (gray highlight). (C) Schematic representation of the S segment sequences of three recombinant viruses isolated at day 127 post-infection (each of which is dominant in the viral population in one representative AGRAG mouse). The tagged IGR from the S segment carrying NP is marked with a star (*). The sequenced section is indicated by a double arrow. [ka] The base pair (bp) length designations are for the GFP remnants and truncated (shortened) IGR elements. Indicates the

[0061] [Figure 6]The growth kinetics of recombinant viruses carrying two IGRs in the S segment are similar to those of bipartite viruses. BHK-21 cells were infected at an moi of 0.01 with either bipartite LCMV (gray triangles) carrying a wild-type S segment, tripartite r3LCMV-GFPnat (black circles), or r2LCMV_2IGRs (white diamonds) carrying one S segment corresponding to the recombinant product recovered from infected AGRAG mice (Fig. 5). Supernatants were harvested at the indicated time points, and virus titers were determined by focus-forming assay. Symbols and bars represent the mean ± SEM of triplicates per group. Error bars are hidden within the symbol dimensions. ns.: not statistically significant (p ≥ 0.05); ***: p < 0.001 (one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons).

[0062] [Figure 7]A model of recombination events that could explain the loss of the r3LCMV-GPnat transgene and a hypothesized mechanism for r3LCMV-GPart genetic stability is presented. This model itself is based on sequence data for LCMV transcription termination (Meyer and Southern, 1993, J Virol, 67(5):2621-2627) combined with reverse genetic evidence for the IGR as a transcription termination signal (Pinschewer et al., 2005, J Virol, 79(7):4519-4526). Collectively, these findings suggested structure-dependent polymerase pausing upon completion of the IGR hairpin structure. The GFP remnant between the two IGRs in the recombined S segments was found to originate from either or both S segments, supporting a model in which polymerase template switching (also known as copy selection) occurred when the polymerase paused during either genome or antigenome synthesis (Scenarios A and B, respectively, below). (A) During antigenome synthesis, RNA-dependent RNA polymerase (RdRp) initiates at the 3' UTR of the genomic S-segment template and then reads through the NP ORF and IGR. At the end of the IGR, the polymerase pauses due to a secondary structure ("structure-dependent polymerase pausing"). Polymerase stalling facilitates copy selection and continuation of RNA replication on an alternative template (here, the S-segment genome encoding GP). Template switching must occur upstream of the GP stop codon, most likely targeting a sequence near or proximal to the IGR hairpin. Continuing to read through the C-terminus of GFP on the second template, the polymerase then synthesizes the second IGR, the GP ORF, and the 5' UTR. (B) During genome synthesis, RdRp initiates RNA synthesis at the 3' end of the GP-containing antigenome S-segment template and synthesizes most or all of the 5' UTR, GP, and IGR, followed by structure-dependent polymerase pausing. Copy selection occurs and switches to the C-terminal part of the GFP ORF near the IGR of the NP-containing S segment. Thus, duplication adds a fragment of GFP, followed by the full-length IGR, NP, and 3'UTR.(C-D) Similar to scenarios (A) and (B), template switching can also occur during synthesis of the r3LCMV-GFPart genome or antigenome. This process can also combine the NP and GP ORFs onto a single RNA segment. However, the latter is formed by two 3'UTRs, rather than a 3'UTR and a 5'UTR, which together form only a functional viral promoter. Therefore, such molecules cannot be amplified by RdRp and thus do not form recombinant, replicative viruses.

[0063] [Figure 8] We generated a r3LCMV-OVAart vaccine vector with a similar genomic organization to r3LCMV-GFPart (see Figure 1C), but with two ovalbumin (OVA) genes in place of each GFP gene in the virus. C57BL / 6 mice were immunized intramuscularly (im) with either 104 PFU of r3LCMV-OVAart or 108 particles of a replication-deficient, E1-deleted adenovirus 5-based vector expressing OVA. Eight days later, the animals were euthanized and the T cell responses elicited in response to vaccination were analyzed. A: The frequency of OVA-specific CD8+ T cells in the spleen was [ka] The IL-16 expression level of B220-negative CD8+ lymphocytes was determined using peptide-loaded MHC class I tetramers. The frequency of epitope-specific cells was determined. B: Functionality of OVA-specific CD8+ T cells was evaluated after restimulation. for [ka] Analysis was performed by intracellular cytokine assay using peptides. Bars represent 5 animals per group. The mean values ​​+ / - SEM of the mice are shown. *: p<0.05; **: p>0.01 (paired two-tailed Student's t-test) evening).

[0064] [Figure 9] Tripartite LCMV induces polyfunctional memory CD8+ T cells. C57BL / 6 mice were infected intravenously with 1 × 105 PFU of r3LCMV-OVA or 1 × 108 PFU of rAd-OVA. Spleens were harvested 25 days postinfection, and the functionality of OVA-specific CD8+ T cells was analyzed by intracellular cytokine staining. The cytokine profiles (IFN-γ, TNF-α, and IL-2) of OVA-specific T cells induced by r3LCMV-OVA (black bars) or rAd-OVA (white bars) are shown as the percentage of CD8+ T cells (A) or absolute numbers per spleen (B). Symbols and bars represent the mean ± SEM of five mice per group. An unpaired, two-tailed Student's t-test was used for statistical analysis, and the resulting P values ​​were corrected for multiple comparisons by multiplying by the number of comparisons (n ​​= 7). One representative example of two similar experiments is shown.

[0065] [Figure 10] Antigen-encoding LCMV induces specific T cell responses against foreign and self antigens. C57BL / 6 mice were infected intravenously with 1 × 10 PFU of r3LCMV encoding rat, human, or mouse Her2 peptides (A, B, and C, respectively). Nine days after infection, spleens were harvested and the induction of functional antigen-specific CD8+ T cells was analyzed by intracellular cytokine staining and flow cytometry. The cytokine profile (IFN-γ, TNF-α, and IL-2) of r3LCMV-induced Her2-specific CD8+ T cells is shown as a percentage of CD8+ T cells. Symbols and bars represent the mean ± SEM of three mice per group.

[0066] [Figure 11]Interferon-α is induced upon infection with r3LCMV, but not with recombinant adenovirus or vaccinia virus. C57BL / 6 mice were infected intravenously with 1 × 10 PFU of r3LCMV-OVA, 1 × 10 PFU of rAd-OVA, or 1 × 10 PFU of rVacc-OVA. Blood was collected at the indicated time points postinfection, and serum interferon-α levels were determined by ELISA. Symbols and bars represent the mean ± SEM of four mice per group. ***: p < 0.001 (two-way ANOVA followed by Bonferroni's post-hoc test for multiple comparisons). Representative data from one of two independent experiments are shown.

[0067] [Figure 12] Cell culture growth of r3JUNV-GFPart compared to r3JUNV-GFPnat and r2JUNV-wt. r3JUNV-GFPart and r3JUNV-GFPnat were constructed similarly to the respective r3LCMV vectors outlined in Figure 1. To compare their cell culture growth potential, 293T cells were infected with r2LCMV-wt, r3JUNV-GFPart, and r3JUNV-GFPnat at a multiplicity of infection (MOI) of 0.01, and supernatants were harvested at the indicated time points. Infectious units (FFU) in the supernatants were determined by immunofocus assay. Symbols and bars represent the mean ± SEM of triplicates per group and are hidden within the symbol dimensions.

[0068] [Figure 13]Tripartite JUNV is dramatically attenuated in vivo and only leads to detectable viremia upon loss of GFP. (A) AGRAG mice were infected intravenously with 7 × 10 PFU of r3JUNV-GFPnat (gray squares), r3JUNV-GFPart (white triangles), or the control bipartite r2JUNV strain Candid#1 (black circles), and viremia was monitored over time. Symbols represent individual mice (n = 3–7 per group). (B) JUNV viremia was determined on day 120 after intravenous infection of AGRAG mice with 7 × 10 PFU of r3JUNV-GFPnat or r3JUNV-GFPart. Immunofocus assays were performed to detect either the nucleoprotein NP (gray circles) or GFP (white circles). The virus stock used to inoculate mice served as a staining control in this assay. Symbols represent individual mice and inocula, respectively.

[0069] [Figure 14] Homologous and heterologous prime-boost combinations of tripartite LCMV- and JUNV-based vaccine vectors induce potent P1A autoantigen-specific CD8+ T cell responses. (A) On days 0 and 35 of the experiment, BALB / c mice were immunized intravenously with 8.5 × 104 PFU of r3JUNV-P1Aart (r3JUNV-P1A) and r3LCMV-P1Aart (r3LCMV-P1A) in the homologous or heterologous combinations indicated in the chart. Epitope-specific CD8+ T cells were stained using MHC class I tetramers loaded with the P1A epitope in combination with anti-CD8a antibodies. The frequency of P1A-tetramer-binding cells within the peripheral blood CD8+ T cell compartment (A) and the absolute number of P1A tetramer-binding CD8+ T cells per microliter of peripheral blood (B) were calculated. Symbols represent the mean + / - SEM of 3-5 mice per group and time point. DETAILED DESCRIPTION OF THE INVENTION

[0070] (Detailed Description of the Invention) 4.1 Arenaviruses with Open Reading Frames in Non-Native Locations Provided herein arenaviruses having ORF rearrangements. In this manner, such arenaviruses are replication competent and infectious. The genome sequence of the Arenavirus is provided herein. The ORFs are each a gene of a gene encoding a nucleotide sequence of LCMV-MP (see SEQ ID NOs: 4 and 5), Found in viruses isolated from the wild (referred to herein as the "wild-type position") arenaviruses engineered to hold the nucleotide sequence at a different position (i.e., a non-natural position) In one embodiment, the arena genome segment is The virus particle is LCMV.

[0071] Wild-type arenavirus genome segments and ORFs are known in the art. In particular, the arenavirus genome consists of an S segment and an L segment. The L segment contains ORFs encoding the L protein and Z protein. Both segments are flanked by the respective 5' and 3' UTRs (see Figure 1A). Exemplary wild-type arenavirus genome segments are provided in SEQ ID NOs: 1-10. It is provided.

[0072] In certain embodiments, the arenavirus genome segment is ligated to a position other than the wild-type position. The vector can be engineered to carry more than one arenavirus ORF in one location. wherein the arenavirus genome segment has two loci at positions different from the wild-type position. 1 arenavirus ORF, or 3 arenavirus ORFs, or 4 arenavirus ORFs It can be manipulated to hold.

[0073] In certain embodiments, the arenavirus genome segments provided herein teeth: (i) The ORF encoding NP is located in the arenavirus S segment under the control of the arenavirus 5'UTR. nt; (ii) The ORF encoding the Z protein is an arenavirus that is under the control of the arenavirus 5'UTR. S segment; (iii) Arenavirus L protein ORF is under the control of the arenavirus 5'UTR Russ S segment; (iv) The ORF encoding GP is located in the arenavirus S segment under the control of the arenavirus 3'UTR. nt; (v) an arenavirus in which the ORF encoding the L protein is under the control of the arenavirus 3'UTR; S segment; (vi) an arenavirus in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR; S segment; (vii) The ORF encoding GP is an arenavirus L segment under the control of the arenavirus 5'UTR. ment; (viii) The ORF encoding NP is an arenavirus L segment under the control of the arenavirus 5'UTR. ment; (ix) An ORF encoding the L protein is an arenavirus that is under the control of the arenavirus 5'UTR. S L segment; (x) The ORF encoding GP is located in the arenavirus L segment under the control of the arenavirus 3'UTR. nt; (xi) The ORF encoding NP is located in the arenavirus L segment under the control of the arenavirus 3'UTR. nt; and (xii) Arenavirus Z protein-encoding ORF is under the control of the arenavirus 3'UTR It can be a Luss L segment.

[0074] In certain embodiments, the non-naturally occurring arenavirus genome segments described herein are The ORF in its natural location may be under the control of the arenavirus 3'UTR or the arenavirus 5'UTR. In a more specific embodiment, said arenavirus 3'UTR is In another specific embodiment, the arenavirus 3 is the 3'UTR of the virase S segment. In a more specific embodiment, the 3'UTR is the 3'UTR of an arenavirus L segment. The arenavirus 5'UTR is the 5'UTR of an arenavirus S segment. In one embodiment, the 5'UTR is the 5'UTR of the L segment.

[0075] In other embodiments, the non-naturally occurring arenavirus genome segments described herein are The ORF in its natural position contains conserved terminal sequence elements of arenaviruses (19-20 at the 5'- and 3'-ends). 0 nt region) (see, e.g., Perez and de la Torre, 2003, J Virol. 77 (2): 1184-1194).

[0076] In one embodiment, an ORF in a non-native location in said arenavirus genome segment may be under the control of promoter elements in the 5'UTR (see, e.g., Albarino et al., 2002). 011, J Virol., 85(8):4020-4). In another embodiment, the arenawi ORFs in non-natural locations in the genome segment are regulated by promoter elements 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 promoter. The promoter element of the 5'UTR of the L segment is a 5'UTR promoter element of the L segment. wherein the promoter element of the 3'UTR is a 3'UTR of an S segment or an L segment. TR promoter element.

[0077] In one embodiment, an ORF in a non-native location in said arenavirus genome segment may be under the control of a truncated arenavirus 3'UTR or a truncated arenavirus 5'UTR (e.g., Perez and de la Torre, 2003, J Virol. 77(2): 1184-1194; Albarino (See, e.g., J. Virol., 85(8):4020-4, 2011). In a more specific embodiment, wherein the truncated 3'UTR is the 3'UTR of an arenavirus S segment or L segment. In a more specific embodiment, said truncated 5'UTR is a 5'UTR of an arenavirus S segment. The 5'UTR of the 5' or L segment.

[0078] The ORF is also positioned differently from the wild-type position of the ORF to include the S and L segments. a first genome segment engineered to retain the first genome segment at a position Also provided herein arenavirus particles comprising the genome segments. In one embodiment, the ORF at a location other than the wild-type location of the ORF is an arenavirus ORF. It's one of them.

[0079] In certain specific embodiments, the arenavirus particle contains four arenavirus ORFs It may contain a full complement of all four arenavirus ORFs. In one embodiment, the second arenavirus genome segment comprises an ORF that is a wild-type variant of the ORF. In another specific embodiment, the mold is operated to hold the mold in a separate position. The second arenavirus genome segment may be a wild-type genome segment. (i.e., containing an ORF on the segment at the wild-type position).

[0080] In one embodiment, the first arenavirus genome segment is an L segment. and the second arenavirus genome segment is an S segment. In an embodiment, the first arenavirus genome segment is an S segment. and the second arenavirus genome segment is an L segment.

[0081] A genome segment having an ORF at a location other than the wild-type location of the ORF and a second genome Non-limiting examples of arenavirus particles containing segments are illustrated in Table 1. (Table 1) Arenavirus particles *Position 1 is under the control of the arenavirus S segment 5'UTR; position 2 is under the control of the arenavirus S Position 3 is under the control of the arenavirus L segment 5'UTR position 4 is under the control of the arenavirus L segment 3'UTR. [Table 2]

[0082] Also, the above arenas engineered to retain an ORF in a position other than the wild-type position of the ORF are cDNAs of viral genome segments are also provided herein. In the present specification, a cDNA or a set of cDNAs of the arenavirus genomes listed in Table 1 is It is provided at.

[0083] In one embodiment, an ORF is engineered to maintain it in a position other than the wild-type position of the ORF. Is the cDNA of the arenavirus genome segment being part of a DNA expression vector? In a specific embodiment, the ORF is the arenavirus genome segment engineered to retain the The cDNA of the arenavirus genome segments described herein facilitates the generation of the arenavirus genome segments. It is part of a DNA expression vector that facilitates the expression of the gene, or is incorporated into such a DNA expression vector. In another embodiment, the cDNAs described herein can be incorporated into a plasmid. A more detailed description of the cDNA or nucleic acid and expression system is provided in Section 4.5.1. The techniques for generating said cDNA are routine and are well known in the art of molecular biology and DNA manipulation. Any cloning technique known to those skilled in the art may be used. Such techniques are well known and are described in Sambrook and Russell, "Molecular Cloning: Laboratory Techniques," vol. "Molecular Cloning: A Laboratory Manual," Third Edition, Cold Spring Harbor Laboratory Press, available to those skilled in the art in laboratory manuals such as Harling Harbor Laboratory NY (2001). It is as follows.

[0084] In one embodiment, an ORF is engineered to maintain it in a position other than the wild-type position of the ORF. The cDNA of the arenavirus genome segment is introduced into a host cell (e.g., Thus, in some embodiments, the ORF is transfected into the wild-type The arenavirus genome segment has been engineered to be in a position other than the position of the type. Host cells containing the cDNA (i.e., the cDNA of the genomic segment) are provided herein. In other embodiments, the cDNAs described herein are part of a DNA expression vector. or can be incorporated into a DNA expression vector and introduced into a host cell. In one embodiment, a host cell comprising a cDNA described herein incorporated into a vector is In other embodiments, the arenaviruses described herein are provided. The rus genome segment is introduced into a host cell.

[0085] In one embodiment, the method comprises transcribing a cDNA of an arenavirus genome segment. Described herein are methods for generating arenavirus genome segments. In such a manner, the viral polymerase protein is capable of inhibiting the activity of the enzyme in vitro or in vivo. It may occur during transcription of a virus genome segment.

[0086] In one embodiment, transcription of the arenavirus genome segment is a bidirectional process. In another embodiment, the arenavirus genome segment is transformed using a motor. Transcription of the gene is achieved using a bidirectional expression cassette (see, e.g., Ortiz-Riano et al., 2014). 13, J Gen Virol., 94(Pt 6): 1175-1188). In a more specific embodiment, The bidirectional expression cassette is a vector that expresses two of the inserted arenavirus genome segments. polymerase I and polymerase II promoters, each reading from opposite ends of the promoter. In an even more specific embodiment, the pol-I and pol-II promoters The bidirectional expression cassette with the nucleotide sequence is inserted into the L and S segments from opposite sides. read.

[0087] In other embodiments, the cDNAs of the arenavirus genome segments described herein The transcription of a gene involves a promoter. An example of a promoter is an RNA polymerase I promoter. Motor, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter Examples of promoters include the SP6 promoter, and the T3 promoter.

[0088] In one embodiment, the method for generating arenavirus genome segments comprises: The method may further comprise introducing into the host cell a cDNA of the arenavirus genome segment. In one embodiment, the method for generating arenavirus genome segments comprises: The cDNA of the arenavirus genome segment is then extracted from the arenavirus genome segment. and introducing the arenavirus into a host cell that expresses all other components for synthesis of the arenavirus. The method may further comprise purifying the genome segment from the supernatant of the host cells. The methods are well known to those skilled in the art.

[0089] As used herein, the nucleic acids, vectors, and compositions provided herein are used to infect a mammalian cell. Derived cell lines, cultures, and methods for culturing such cells are provided. A more detailed description of the nucleic acids, vector systems, and cell lines used is provided in Section 4.5. .

[0090] In certain embodiments, the arenavirus particles as described herein result in In a specific embodiment, the present invention provides an arenavirus that is capable of replication and is capable of producing infectious and replicative arenavirus particles. The arenavirus particles described herein are attenuated. In certain embodiments, The arenavirus particles are then dispersed in a manner that leaves the virus at least partially capable of spreading. and is replicable in vivo, but can only produce low viral loads , resulting in a subclinical level of infection that is not pathogenic. Such attenuated viruses can be used as immunogenic compositions. As described above, immunogenic compositions comprising arenaviruses having ORFs in non-natural locations are provided herein. This is provided in the specification.

[0091] 4.1.1 Replication-Defective Arenaviruses with Open Reading Frames in Non-Native Locations particle) In certain embodiments, an arenavirus particle, wherein (i) an ORF is located at the wild-type position of the ORF. and (ii) ORFs encoding GP, NP, Z protein, and L protein. are removed or functionally inactivated, and the resulting virus produces infectious progeny the arenavirus particles are incapable of producing further viral particles. One or more ORFs are deleted or functionally inactivated. Arenavirus particles containing the genetically modified genome are then transferred to complementing cells. ) (i.e., expressing the deleted or functionally inactivated arenavirus ORF The resulting genetic material of the arenavirus particles can be generated in a variety of cells (e.g., cells expressing the virus). can be transferred into a host cell upon infection of the host cell, wherein the genetic material In addition, the genetically modified arenaviruses described herein can be expressed and amplified. The genome of the arenavirus particle can encode heterologous ORFs derived from organisms other than the arenavirus particle.

[0092] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the ORFs is removed and replaced with a heterologous ORF from an organism other than arenavirus. In another embodiment, the GP, NP, Z protein, and L protein are encoded by At least one ORF, at least two ORFs, at least three ORFs, or at least four One ORF can be removed and replaced with a heterologous ORF from an organism other than an arenavirus. In a specific embodiment, four O sequences encoding GP, NP, Z proteins, and L proteins are Only one of the RFs is removed and replaced with a heterologous ORF from an organism other than the arenavirus particle. In a more specific embodiment, said arenavirus genome segments are altered. In another specific embodiment, the ORF encoding the GP of the Arenau gene has been deleted. The ORF encoding the NP of the virus genome segment has been removed. In the arenavirus genome segment, the ORF encoding the Z protein is deleted. In yet another specific embodiment, the ORF encoding the L protein is deleted. It is being done.

[0093] Thus, in certain embodiments, the arenavirus particles provided herein comprise: (i) engineered to retain the ORF in a non-native position; (ii) GP, NP, Z protein, or L protein (iii) the removed ORF is derived from an organism other than an arenavirus; It contains a genomic segment that has been replaced with a heterologous ORF.

[0094] In one embodiment, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length. Length of nucleotides, 25 to 100 nucleotides, 50 to 200 nucleotides, 50 to 400 nucleotides Nucleotide length, 200 to 500 nucleotides in length, or 400 to 600 nucleotides In another embodiment, the heterologous ORF is , 750-900 nucleotides in length, 800-100 nucleotides in length, 850-1000 nucleotides in length Length, 900-1200 nucleotides, 1000-1200 nucleotides, 1000-1500 nucleotides nucleotides or 10-1500 nucleotides in length, 1500-2000 nucleotides in length, 1700 ~2000 nucleotides long, 2000-2300 nucleotides long, 2200-2500 nucleotides long Length, 2500-3000 nucleotides, 3000-3200 nucleotides, 3000-3500 nucleotides Length of nucleotides: 3200-3600 nucleotides; 3300-3800 nucleotides; 4000 Length of 4400 nucleotides, length of 4200 to 4700 nucleotides, length of 4800 to 5000 nucleotides Nucleotide length, 5000–5200 nucleotides, 5200–5500 nucleotides, 55 Length: 00–5800 nucleotides, 5800–6000 nucleotides, 6000–6400 nucleotides Length, 6200-6800 nucleotides, 6600-7000 nucleotides, 7000-7200 nucleotides nucleotide length, 7200-7500 nucleotides in length, or 7500 nucleotides in length In some embodiments, the heterologous ORF is 5 to 10 amino acids in length, 10 to 25 amino acids in length, 25-50 amino acids, 50-100 amino acids, 100-150 amino acids, 150-200 amino acids, 200 ~250 amino acids long, 250-300 amino acids long, 300-400 amino acids long, 400-500 amino acids long, 500 ~750 amino acids, 750-1000 amino acids, 1000-1250 amino acids, 1250-1500 amino acids , 1500 to 1750 amino acids in length, 1750 to 2000 amino acids in length, 2000 to 2500 amino acids in length, or more than 2500 amino acids in length Some In one embodiment, the heterologous ORF encodes a polypeptide not exceeding 2500 amino acids in length. In a specific embodiment, the heterologous ORF does not contain a stop codon. In some embodiments, the heterologous ORF is codon-optimized. The nucleotide composition, the nucleotide pair composition, or both can be optimized. Techniques for such optimization are known in the art and are applicable to the optimization of heterologous ORFs. possible.

[0095] Any heterologous ORF from a non-arenavirus organism may be inserted into an arenavirus genome segment In one embodiment, the heterologous ORF encodes a reporter protein. A more detailed description of reporter proteins is provided in Section 4.3. In an embodiment, the heterologous ORF is a gene encoding an infectious pathogen capable of eliciting an immune response. In a specific embodiment, the antigen encodes an antigen of any disease. The source may be from an infectious organism, a tumor (i.e., cancer), or an allergen. A more detailed explanation is given in Section 4.3.

[0096] In one embodiment, the proliferation and infectivity of the arenavirus particles is enhanced by the It is not affected by heterologous ORFs from organisms other than R. rus.

[0097] Using techniques known to those skilled in the art, the arenavirus ORF can be retained in a position other than the wild-type position. Generating arenavirus particles containing arenavirus genome segments engineered to carry For example, reverse genetic techniques can be used to produce such arenavirus particles. In other embodiments, the replication-deficient arenavirus particles (i.e. , an arenavirus engineered to carry the arenavirus ORF in a position other than the wild-type position. A virus genome segment, an ORF encoding GP, NP, Z protein, and L protein The arenavirus genome segment, which is deleted, is produced in a complementing cell. obtain.

[0098] In certain embodiments, arenavirus genome segments or arenaviruses used in accordance with the present application The virus particle can be an Old World virus, for example, LCMV.

[0099] In some embodiments, 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. arenavirus particles, as well as vaccines and therapeutics, e.g., for the treatment of infectious diseases or cancer. The present invention relates to methods of using such arenavirus particles in prophylaxis. A more detailed description of how to use arenavirus particles is provided in section 4.6. It is being done.

[0100] In one embodiment, a kit containing one or more of the cDNAs described herein in one or more containers is provided. In a specific embodiment, the kit comprises one or two Into the above container, the arenavirus genome segment or arenavirus described herein is added. The kit comprises the following: the arenavirus genome segment or the A suitable host cell for rescuing arenavirus particles is selected, and the plasmid cDNA is transfected into the host cell. suitable reagents for injecting the virus, helper viruses, and vectors encoding viral proteins. mide, and / or modified arenavirus genome segments or arenavirus particles or one or more primers specific for the cDNA thereof. It may also contain.

[0101] In certain embodiments, the present application provides a pharmaceutical composition comprising a compound according to any one of the preceding claims, which is suitable for use as a pharmaceutical composition. arenavirus particles and vaccinations, as well as the treatment or administration of, for example, infectious diseases and cancer. The present invention relates to methods of using such arenavirus particles in prophylaxis. A more detailed description of how to use the arenavirus particles described is provided in section 4.7. It has been done.

[0102] (4.2 Tripartite Arenavirus Particles) Provided herein are tripartite arenavirus particles having ORF rearrangements. In one embodiment, one L segment and two S segments, or two L segments and one Provided herein are tripartite arenavirus particles containing two S segments. In some embodiments, the tripartite arenavirus particles are replication-competent bipartite arenavirus particles. More specifically, in one embodiment, the genome segment does not recombine into a gene particle. Two of the segments (e.g., the two S segments or the two L segments, respectively) ) are recombined to produce a single viral segment that can replace the two parental segments. In a specific embodiment, the tripartite arenavirus particle In yet another specific embodiment, the gene contains an ORF at a location that is different from the wild-type location of the ORF. In this case, the tripartite arenavirus particle contains all four arenavirus ORFs. In certain embodiments, the tripartite arenavirus particles are replication competent and infectious. In another embodiment, the tripartite arenavirus particle comprises four arenaviruses. Thus, in one embodiment, the tripartite arenavirus particle lacks one of the ORFs. The offspring are infectious but are unable to give rise to further infectious progeny in non-complementing cells. I can't come.

[0103] In one embodiment, the GP, NP, Z of the tripartite arenavirus particles described herein The ORF encoding the L protein is located in the arenavirus 3'UTR or In a more specific embodiment, the tripartite gene can be under the control of the 5' UTR of the gene. The arenavirus 3'UTR is the 3'UTR of the arenavirus S segment(s). In a specific embodiment, the tripartite arenavirus 3'UTR is a tripartite arenavirus In a more specific embodiment, the tripartite allele is the 3'UTR of the L segment(s). The arenavirus 5'UTR is the 5'UTR of the arenavirus S segment(s). In certain embodiments, said 5'UTR is the 5'UTR of the L segment(s).

[0104] In other embodiments, the GP, NP, Z of the tripartite arenavirus particles described herein The ORF encoding the L protein is located within the arenavirus conserved terminal sequence element. The nucleotide sequence may be under the control of the nucleotide sequence (19-20 nt regions at the 5'- and 3'-ends) (see, e.g., Perez and de la Torre (See, e.g., 2003, J Virol. 77(2): 1184-1194).

[0105] In certain embodiments, the GP, NP, Z protein of said tripartite arenavirus particle, or The ORF encoding the L protein may be 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 one embodiment, the GP, NP, Z protein, and L protein of the tripartite arenavirus particle are The coding ORF can be under the control of a promoter element in the 3'UTR (e.g., Albari (See, e.g., No et al., 2011, J Virol., 85(8):4020-4). wherein the promoter element of the 5'UTR is an S segment(s) or an L segment In another specific embodiment, the 5'UTR promoter element(s) of The promoter element of the 3'UTR is the 3' end of the S segment(s) or L segment(s). 'UTR promoter element.

[0106] In certain embodiments, the GP, NP, Z protein of said tripartite arenavirus particle, or The ORF encoding the L protein is a truncated arenavirus 3'UTR or a truncated arenavirus It may be under the control of the 5'UTR of the gene (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 a more specific embodiment, said truncated 3'UTR is an arenavirus S segment or is the 3'UTR of the L segment. In a more specific embodiment, the truncated 5'UTR is , the 5'UTR of an arenavirus S segment(s) or L segment(s).

[0107] Also provided herein is a cDNA encoding said tripartite arenavirus particle. In a specific embodiment, a tripartite arenavirus particle encoding a tripartite arenavirus particle as described in Table 2 or Table 3 is A DNA nucleotide sequence or set of DNA nucleotide sequences corresponding to the can be.

[0108] In certain embodiments, the nucleic acid encoding the tripartite arenavirus genome comprises one or more It is part of the above DNA expression vector or is incorporated into one or more DNA expression vectors. In a specific embodiment, the genome of said tripartite arenavirus particle is encoded by The nucleic acid may comprise one or more nucleic acids that facilitate the production of the tripartite arenavirus particles described herein. Is part of a DNA expression vector or is incorporated into one or more such DNA expression vectors In another embodiment, the cDNAs described herein are incorporated into a plasmid. A more detailed description of the cDNAs and expression systems is provided in Section 4.5.1. The techniques for generating said cDNA are routine and are well known in the art of molecular biology and DNA manipulation. Any cloning technique known to those skilled in the art may be used. Such techniques are well known and are described in Sambrook and Russell, "Molecular Cloning: A Review." "Molecular Cloning: A Laboratory Manual," Third Edition, Cold Spring Harbor Laboratory Press, available to those skilled in the art in laboratory manuals such as Spring Harbor Laboratory NY (2001). is.

[0109] In one embodiment, the tripartite arenavirus cDNA is introduced into a host cell ( Thus, in some embodiments, the three-segmented cDNA of arenavirus particles (i.e., the genome segments of the tripartite arenavirus particles) In another embodiment, a host cell comprising the cDNA of the present invention is provided herein. The cDNAs described herein are part of or incorporated into DNA expression vectors. and introduced into a host cell. Host cells containing the cDNA described herein integrated into a vector are provided herein. In other embodiments, the tripartite arenavirus genome sequence described herein is The segment (i.e., the L segment and / or the S segment or segments) , which has been introduced into a host cell.

[0110] In one embodiment, the method of producing tripartite arenavirus particles comprises: the method described herein, comprising transcribing the cDNA of the tripartite arenavirus particle. In some embodiments, the viral polymerase protein is expressed in vitro or It may be present during transcription of the tripartite arenavirus particle in vivo. wherein transcription of the arenavirus genome segment is carried out using a bidirectional promoter. It can be done.

[0111] In another embodiment, transcription of the arenavirus genome segments is by bidirectional expression. This is done using a cassette (see, for example, Ortiz-Riano et al., 2013, J Gen Virol., 94(Pt 6): 1175-1188). In a more specific embodiment, the bidirectional expression cassette The inserted arenavirus genome segment has two terminals, one at each end. It contains both polymerase I and polymerase II promoters reading from opposite sides.

[0112] In other embodiments, the cDNAs of the arenavirus genome segments described herein The transcription of a gene involves a promoter. An example of a promoter is an RNA polymerase I promoter. Motor, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter Examples of promoters include the SP6 promoter, and the T3 promoter.

[0113] In one embodiment, the method for producing tripartite arenavirus particles comprises the step of: The method may further comprise introducing the cDNA of the nodular arenavirus particle into a host cell. In an embodiment, the method for producing tripartite arenavirus particles comprises the step of: The viral particle cDNA and all other components for the generation of the tripartite arenavirus particles and introducing the tripartite arenavirus particles into a host cell expressing the tripartite arenavirus; and It may further comprise purifying it from the supernatant. Such methods are well known to those skilled in the art.

[0114] As used herein, the nucleic acids, vectors, and compositions provided herein are used to infect a mammalian cell. Derived cell lines, cultures, and methods for culturing such cells are provided. A more detailed description of the nucleic acids, vector systems, and cell lines used is provided in Section 4.5. There are.

[0115] In certain embodiments, the tripartite arenavirus particles described herein are The result is infectious and replicative arenavirus particles. Thus, the arenavirus particles described herein are attenuated. wherein the tripartite arenavirus particles are at least partially replicable. remain competent and capable of replication in vivo but may produce low viral loads. and is attenuated to result in a subclinical level of infection that is not pathogenic. Such attenuated viruses can be used in immunogenic compositions.

[0116] In one embodiment, the tripartite arenavirus particle is a bipartite arenavirus particle. It has the same tropism as the child.

[0117] Also included herein are kits containing, in one or more containers, one or more of the cDNAs described herein. In a specific embodiment, the kit comprises, in one or more containers, The kit comprises the following: a tripartite arenavirus particle as described in the specification. A suitable host cell for rescuing segmented arenavirus particles is selected. The plasmid cDNA is transfected into the host cell. suitable reagents for infecting the cells, a helper virus, and a promoter encoding the viral proteins. plasmids, and / or modified arenavirus genome segments or arenaviruses One or more oligonucleotide probes specific for the nucleic acid that encodes the nucleic acid. The polymer may further include one or more of:

[0118] Also, immunoglobulins containing tripartite arenavirus particles, such as those described in Sections 4.6 and 4.7, Immunogenic compositions are also provided herein.

[0119] 4.2.1 Tripartite Arenavirus Particles Containing One L Segment and Two S Segments In one embodiment, a tripartite arenavirus comprising one L segment and two S segments. In one embodiment, the one L segment is a hydroxylase. The propagation of tripartite arenavirus particles containing two S segments is comparable to that of replication-competent bipartite arenaviruses. In a specific embodiment, the one L segment and the two The propagation of tripartite arenavirus particles containing the S segment of the virus is regulated by the type I interferon receptor agonist (IAR). , lacking the type II interferon receptor and recombination activating gene (RAG1), 10 4 PFU's 3 minutes In mice infected with nodal arenavirus particles, 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, At least 70 days, at least 80 days, at least 90 days, or at least 100 days do not produce replication-competent bipartite virus particles after subsequent infection (see section 4.8.13). In another embodiment, the tripartite fragment comprising one L segment and two S segments is The propagation of nodular arenavirus particles is considered to be at least 10 passages, at least 20 passages, or at least 10 passages. Replicative cells after at least 30 passages, at least 40 passages, or at least 50 passages. It does not produce bipartite virus particles.

[0120] Tripartite arenaviruses in which all viral genes are in their respective wild-type positions Such particles are known in the art (see, e.g., Emonet et al., 2011 J. Virol., 8 5(4):1473; Popkin et al., 2011, J. Virol. 85(15):7928). In particular, the tripartite arena The viral genome consists of one L segment and two S segments, in which heterologous ORFs (e.g. For example, a gene encoding a GFP (e.g., GFP) is inserted at one position on each S segment. The other segment, S, encodes GFP and NP, respectively. The L segment encodes the L protein and the Z protein. The segments flank the respective 5' and 3' UTRs.

[0121] In certain embodiments, two of the tripartite arenavirus particles provided herein The two arenavirus ORFs are integrated into one segment rather than into two separate segments. Intersegmental recombination results in the integration of two segments, forming one end of the genome. Each UTR has a non-functional promoter (i.e., an inverted repeat sequence) at the other end of the same genome. Chi, [ka] resulting in the structure of the genome segment.

[0122] In one embodiment, the tripartite antigen comprising one L segment and two S segments is Arenavirus particles carry the arenavirus ORF in a position other than the wild-type position of the ORF. In another embodiment, the one L segment and two S segments are engineered to Tripartite arenavirus particles containing two arenavirus ORFs or three arenavirus ORFs. or four arenavirus ORFs, or five arenavirus ORFs, or six arenavirus ORFs The virus has been engineered to retain the ORF in a position other than that of the wild type. In one embodiment, the tripartite arenavirus comprises one L segment and two S segments. The virus particle contains all four arenavirus ORFs. wherein the tripartite arenavirus particles are infectious and replication-competent tripartite arenavirus particles. In a specific embodiment, the two S segments of said tripartite arenavirus particle are The segments are engineered to retain one of their ORFs in a position other than the wild-type position. In a more specific embodiment, the two S segments are S segment O In a specific embodiment, the L segment comprises the ORF in its entirety. or the L segment has been engineered to be in a different position from the wild-type It can be a genome segment.

[0123] In one embodiment, one of the two S segments is: (i) The ORF encoding the Z protein is under the control of the arenavirus 5'UTR. S segment; (ii) The ORF encoding the L protein is an arenavirus that is under the control of the arenavirus 5'UTR. S segment; (iii) The ORF encoding NP is located in the arenavirus S segment under the control of the arenavirus 5'UTR. ment; (iv) The ORF encoding GP is located in the arenavirus S segment under the control of the arenavirus 3'UTR. nt; (v) The ORF encoding L is located in the arenavirus S segment under the control of the arenavirus 3'UTR. and (vi) an arenavirus in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR; S segment It can be said that:

[0124] In one embodiment, the tripartite antigen comprising one L segment and two S segments is Viral particles contain overlapping ORFs (i.e., two wild-type S segment ORFs, e.g., G In a specific embodiment, the one L segment and two S segments may comprise Tripartite arenavirus particles containing one overlapping ORF (e.g., (GP,GP)) or It may contain two overlapping ORFs (for example, (GP, GP) and (NP, NP)).

[0125] Table 2A below shows tripartite arenaviruses containing one L segment and two S segments. A particle comprising two S segments in the tripartite arenavirus genome. Interrecombination does not result in replication-competent bipartite virus particles and is not a precursor to arenavirus progenitors. suppresses motor activity (i.e., the resulting recombinant S segment does not bind to the 3'UTR and The genome structure of the tripartite arenavirus particle is This is an example of this. (Table 2A) Tripartite arenavirus particles containing one L segment and two S segments. Position 1 is under the control of the arenavirus S segment 5'UTR; position 2 is under the control of the arenavirus S Position 3 is under the control of the arenavirus S segment 5'UTR position 4 is under the control of the arenavirus S segment 3'UTR; position 5 is under the control of the arenavirus S segment 3'UTR position 6 is under the control of the arenavirus L segment 3'UTR; It's under control. *ORF indicates that a heterologous ORF has been inserted. [Table 3]

[0126] In certain embodiments, the IGR between positions 1 and 2 is an arenavirus S segment or The L segment can be an IGR; the IGR between positions 2 and 3 can be an IGR between the arenavirus S segment or the L segment IGR; and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In a specific embodiment, the IGR between Position 1 and Position 2 can be an arenavirus S segment IGR; an IGR between positions 2 and 3 is an Arenavirus and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In some embodiments, other combinations are also possible. For example, a tripartite arenavirus particle contains one L segment and two S segments. Intersegmental recombination of two S segments in a tripartite arenavirus genome However, it does not produce replicative bipartite virus particles and does not induce the arenavirus promoter. - activity (i.e., the resulting recombinant S segment has a 3'UTR and 5'UTR The tripartite arenavirus particle is formed by two 5'UTRs rather than one 5'UTR.

[0127] In one embodiment, a tripartite arena comprising one L segment and two S segments. Intersegmental recombination of the S and L segments in the virus particle results in the separation of two viruses. Functional chromosomes in which the chromosome genes are present on only one segment rather than two separate segments In another embodiment, one L segment and two S segments are restored. S and L segments in tripartite arenavirus particles containing Interrecombination does not result in replication-competent bipartite virus particles.

[0128] Table 2B below shows tripartite arenaviruses containing one L segment and two S segments. A particle comprising a segment of the S segment and a segment of the L segment in a tripartite arenavirus genome. Intersegment recombination does not result in replication-competent bipartite virus particles and is not a cause of arenavirus suppresses the promoter activity of the S segment (i.e., the resulting recombinant S segment the tripartite arenavirus particle is formed by two 3'UTRs rather than a 5'UTR and a 3'UTR), This is an example of the genome composition of the offspring. (Table 2B) Tripartite arenavirus particles containing one L segment and two S segments. Position 1 is under the control of the arenavirus S segment 5'UTR; position 2 is under the control of the arenavirus S Position 3 is under the control of the arenavirus S segment 5'UTR position 4 is under the control of the arenavirus S segment 3'UTR; position 5 is under the control of the arenavirus S segment 3'UTR position 6 is under the control of the arenavirus L segment 3'UTR; It's under control. *ORF indicates that a heterologous ORF has been inserted. [Table 4]

[0129] In certain embodiments, the IGR between positions 1 and 2 is an arenavirus S segment or The L segment can be an IGR; the IGR between positions 2 and 3 can be an IGR between the arenavirus S segment or the L segment IGR; and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In a specific embodiment, the IGR between Position 1 and Position 2 can be an arenavirus S segment IGR; an IGR between positions 2 and 3 is an Arenavirus and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In some embodiments, other combinations are also possible. For example, a tripartite arenavirus particle contains one L segment and two S segments. The intersegmental assembly of two S segments in the tripartite arenavirus genome Recombination does not result in replication-competent bipartite virus particles and does not promote arenavirus suppresses target activity (i.e., the resulting recombinant S segment has a 3'UTR and 5'UTR The tripartite arenavirus particle is formed by two 5'UTRs rather than one 5'UTR.

[0130] In certain embodiments, one of skill in the art can use the methods illustrated in Table 2A or 2B and described herein. Construct arenavirus genomes as described in section 4.8. Using such an assay, it is possible to determine whether the tripartite arenavirus particles are genetically stable. or, as discussed herein, to produce replication-competent bipartite viral particles. It can be determined whether or not

[0131] 4.2.2 Tripartite arenavirus particles containing two L segments and one S segment In one embodiment, a tripartite arenavirus comprising two L segments and one S segment. In one embodiment, the two L segments are The propagation of tripartite arenavirus particles containing one S segment and one S segment is comparable to that of replication-competent bipartite arenaviruses. In a specific embodiment, the two L segments and one The propagation of tripartite arenavirus particles containing the S segment of the virus is regulated by the type I interferon receptor agonist (IAR). , type II interferon receptor, and recombination activating gene (RAG1), and 4 PFU In mice infected with tripartite arenavirus particles, For at least 20 days, at least 30 days, at least 40 days, or at least 50 days, 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days persistence does not result in replication-competent bipartite virus particles (see section 4.8.13). In another embodiment, the two L segments and one S segment The propagation of tripartite arenavirus particles containing the virus has been confirmed by at least 10, 20, or 30 passages. does not produce replication-competent bipartite virus particles after 40, 40, or 50 passages .

[0132] In certain embodiments, the tripartite arenavirus particles provided herein The two arenavirus ORFs of the two L segments are integrated into one segment rather than into two separate segments. Intersegmental recombination, which integrates two segments, results in the formation of one end of the genome. Non-functional promoters, each of which consists of an inverted repeat sequence at the other end of the same genome. - (i.e., [ka] resulting in the structure of the genome segment.

[0133] In one embodiment, the tripartite antigen comprises two L segments and one S segment. Arenavirus particles carry the arenavirus ORF in a position other than the wild-type position of the ORF. In another embodiment, the two L segments and one S segment are engineered to Tripartite arenavirus particles containing two arenavirus ORFs or three arenavirus ORFs. or four arenavirus ORFs, or five arenavirus ORFs, or six arenavirus ORFs The virus ORF has been engineered to be in a different position than the wild-type position. In one embodiment, a tripartite Arenavirus comprising two L segments and one S segment. The virus particle contains all four arenavirus ORFs intact. In such a manner, the tripartite arenavirus particles are infectious and replication-competent tripartite arenavirus particles. In a specific embodiment, two of said tripartite arenavirus particles are The L segment maintains one of these ORFs in a position other than the wild-type position. In a more specific embodiment, the two L segments are In a specific embodiment, the S segment completely contains those ORFs. or the S The segment can be a wild-type genomic segment.

[0134] In some embodiments, one of the two L segments is: (i) The L segment, in which the ORF encoding GP is under the control of the arenavirus 5′UTR; (ii) the L segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (iii) the L segment, in which the ORF encoding the L protein is under the control of the arenavirus 5'UTR; ; (iv) the L segment, in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR; and (vi) The L segment, in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR It can be said that:

[0135] In one embodiment, the tripartite antigen comprising one L segment and two S segments is Lenavirus particles contain overlapping ORFs (i.e., two wild-type L segment ORFs, e.g., Z In a specific embodiment, the two L segments may comprise a Tripartite arenavirus particles containing one S segment and one overlapping ORF (e.g., For example, (Z protein, Z protein)) or two overlapping ORFs (e.g., (Z protein, Z protein) It may contain (protein) and (L protein, L protein)).

[0136] Table 3 below shows a tripartite arenavirus particle containing two L segments and one S segment. intersegmental recombination of two L segments in a tripartite arenavirus genome However, it does not result in replication-competent bipartite virus particles and is not a promoter of an arenavirus. activity (i.e., the putative resulting recombinant L segment (It may be formed by two 3'UTRs or two 5'UTRs rather than one 3'UTR and one 5'UTR), 1 is an illustration of the genome organization of an arenavirus particle. Based on Table 3, similar combinations are To produce arenavirus particles that consist of two 5'UTRs rather than just the R and 5'UTR would be predictable. (Table 3) Tripartite arenavirus particles containing two L segments and one S segment. *Position 1 is under the control of the arenavirus L segment 5'UTR; position 2 is under the control of the arenavirus L Position 3 is under the control of the arenavirus L segment 5'UTR position 4 is under the control of the arenavirus L segment 3'UTR; position 5 is under the control of the arenavirus L segment 3'UTR position 6 is under the control of the arenavirus S segment 3'UTR; It's under control. *ORF indicates that a heterologous ORF has been inserted. [Table 5]

[0137] In certain embodiments, the IGR between positions 1 and 2 is an arenavirus S segment or The L segment can be an IGR; the IGR between positions 2 and 3 can be an IGR between the arenavirus S segment or L segment IGR; and the IGR between positions 5 and 6 can be an arenavirus S segment IGR. In a specific embodiment, positions 1 and 2 may be the L segment or the IGR. The IGR between positions 2 and 3 can be an arenavirus L segment IGR; The IGR can be an arenavirus L segment IGR; and the IGR between positions 5 and 6 can be In some embodiments, other combinations may be used. It is also possible that

[0138] In one embodiment, a tripartite arena comprising two L segments and one S segment. Intersegmental recombination of the L and S segments from the virus particles results in the formation of two viruses. Functional segmentation where the gene is located on only one segment rather than on two separate segments In another embodiment, a gene comprising two L segments and one S segment is used to restore the normal function of the gene. Intersegmental recombination of L and S segments in tripartite arenavirus particles. does not result in replication-competent bipartite virus particles.

[0139] Table 3B below shows tripartite arenaviruses containing two L segments and one S segment. A particle comprising a segment of the L segment and a segment of the S segment in a tripartite arenavirus genome. Intersegment recombination does not result in replication-competent bipartite virus particles and is not a cause of arenavirus suppresses the promoter activity of the S segment (i.e., the resulting recombinant S segment the tripartite arenavirus particle is formed by two 3'UTRs rather than a 5'UTR and a 3'UTR), This is an example of the genome composition of the offspring. (Table 3B) Tripartite arenavirus particles containing two L segments and one S segment. *Position 1 is under the control of the arenavirus L segment 5'UTR; position 2 is under the control of the arenavirus L Position 3 is under the control of the arenavirus L segment 5'UTR position 4 is under the control of the arenavirus L segment 3'UTR; position 5 is under the control of the arenavirus L segment 3'UTR position 6 is under the control of the arenavirus S segment 3'UTR; It's under control. *ORF indicates that a heterologous ORF has been inserted. [Table 6]

[0140] In certain embodiments, the IGR between positions 1 and 2 is an arenavirus S segment or The L segment can be an IGR; the IGR between positions 2 and 3 can be an IGR between the arenavirus S segment or L segment IGR; and the IGR between positions 5 and 6 can be an arenavirus S segment IGR. In a specific embodiment, positions 1 and 2 may be the L segment or the IGR. The IGR between positions 2 and 3 can be an arenavirus L segment IGR; The IGR can be an arenavirus L segment IGR; and the IGR between positions 5 and 6 can be In some embodiments, other combinations may be used. It is also possible that

[0141] In certain embodiments, one of skill in the art can utilize the methods illustrated in Table 3A or 3B and described herein. Construct arenavirus genomes as described in section 4.8. Using such an assay, it is possible to determine whether the tripartite arenavirus particles are genetically stable. or, as discussed herein, to produce replication-competent bipartite viral particles. It can be determined whether or not

[0142] 4.2.3 Replication-deficient trisegmented arenavirus particles In certain embodiments, a tripartite arenavirus particle, wherein (i) the ORF is a wild-type ORF. and (ii) encoding a GP, NP, Z protein, or L protein. The ORF responsible for the replication of the virus has been removed or functionally inactivated, and the resulting virus is are unable to produce further progeny viral particles (i.e., replication-deficient) The tripartite arenavirus particles are provided herein. In embodiments, the third arenavirus segment can be an S segment. In other embodiments, the third arenavirus segment is an L segment. In a more specific embodiment, the third arenavirus segment comprises an ORF , can be engineered to maintain the ORF in a position other than its wild-type position, or a third The arenavirus segment may be a wild-type arenavirus genome segment. In an even more specific embodiment, the third arenavirus segment is GP , lacking arenavirus ORFs encoding NP, Z protein, or L protein.

[0143] In one embodiment, the tripartite genome segment is an S or L segment hybrid ( i.e., a genomic segment that can be a combination of an S segment and an L segment) In another embodiment, the hybrid segment may be an L segment. In another embodiment, the hybrid segment is an S segment containing a nucleotide IGR. is an L segment including an S segment IGR. In another embodiment, the L segment is an S segment UTR followed by an L segment IGR. The hybrid segment is an L-segment UTR with an S-segment IGR. In one embodiment, the hybrid segment comprises an S segment 5 with an L segment IGR. 'UTR or S segment 3'UTR with L segment IGR. In some embodiments, the hybrid segment comprises an L segment 5'UT with an S segment IGR. R or L segment 3'UTR with S segment IGR.

[0144] Contains genetically modified genomes in which one or more ORFs are deleted or functionally inactivated. Tripartite arenavirus particles containing the virion fragments are expressed in complementing cells (i.e., cells lacking the virion fragments or functionally defective). The resulting arenavirus can be generated in a cell expressing an activated arenavirus ORF. The genetic material of the resulting arenavirus particles is transferred into the host cell upon infection of the cell. In addition, the genetic material may be expressed and amplified. The genome of the described genetically modified arenavirus particles may be derived from organisms other than arenavirus particles. The gene may encode a heterologous ORF.

[0145] In one embodiment, four ORFs encoding GP, NP, Z protein, and L protein At least one of the ORFs is removed and replaced with a heterologous ORF from an organism other than arenavirus. In another embodiment, the GP, NP, Z protein, and L protein are encoded by At least one ORF, at least two ORFs, at least three ORFs, or at least four One ORF can be removed and replaced with a heterologous ORF from an organism other than an arenavirus. In a specific embodiment, four ORFs encoding the GP, NP, Z protein, and L protein are Only one of the ORFs is removed and replaced with a heterologous ORF from an organism other than the arenavirus particle. In a more specific embodiment, the GP of the arenavirus genome segment is In another specific embodiment, the ORF encoding the arenavirus genome is deleted. In a more specific embodiment, the ORF encoding NP of the ribosomal segment is removed. The ORF encoding the Z protein of the arenavirus genome segment has been removed. In another specific embodiment, the ORF encoding the L protein is removed.

[0146] In one embodiment, a tripartite arena comprising one L segment and two S segments. A viral particle comprising: (i) an ORF in a location that is different from the wild-type location of the ORF; and (ii) a GP or the ORF encoding NP has been deleted or functionally inactivated, resulting in The tripartite arenavirus particles, in which the virus is replication-deficient and non-infectious, In a specific embodiment, one ORF is removed to create an allele. In another specific embodiment, the ORF is replaced with a heterologous ORF derived from an organism other than a viral vector. In this case, two ORFs were removed and replaced with heterologous ORFs from organisms other than arenaviruses. In another specific embodiment, three ORFs are removed to provide a non-arenavirus. In a specific embodiment, the GP encoding ORF is replaced with a heterologous ORF derived from a different organism. The ORFs that contain the virus have been removed and replaced with heterologous ORFs from organisms other than arenaviruses. In a specific embodiment, the ORF encoding NP is removed to generate a non-arenavirus. In an even more specific embodiment, the NP is replaced with a heterologous ORF derived from an organism. The ORF encoding the GP and the ORF encoding the GP are removed, and the virus particles are derived from organisms other than arenavirus particles. Thus, in one embodiment, the three sequences are replaced with one or two heterologous ORFs. Nodal arenavirus particles consist of (i) one L segment and two S segments; (ii) an ORF. (iii) an ORF derived from an organism other than an arenavirus, wherein the ORF is in a position different from the wild-type position of the ORF; and one or more heterologous ORFs.

[0147] In one embodiment, a tripartite arena comprising two L segments and one S segment. A viral particle comprising: (i) an ORF in a location that is different from the wild-type location of the ORF; and (ii) Z ORFs encoding the L protein and / or the L protein are deleted or functionally inactivated The resulting virus is replication-deficient and non-infectious. In a specific embodiment, a type 1 arenavirus particle is provided herein. One ORF has been removed and replaced with a heterologous ORF from a non-arenavirus organism. In another specific embodiment, two ORFs are removed and the vector is derived from an organism other than an arenavirus. In a specific embodiment, the ORF encoding the Z protein is replaced with a heterologous ORF. The ORFs that contain the virus have been removed and replaced with heterologous ORFs from organisms other than arenaviruses. In a specific embodiment, the ORF encoding the L protein is removed to produce an arenavirus. In a more specific embodiment, the gene is replaced with a heterologous ORF derived from an organism other than the host. The ORF encoding the Z protein and the ORF encoding the L protein are then removed to form the arena. The viral particle is replaced with a heterologous ORF derived from an organism other than the viral particle. wherein the tripartite arenavirus particle comprises (i) two L segments and one S segment. (ii) an ORF in a location that is different from the wild-type location of the ORF; (iii) an arenavirus This includes heterologous ORFs derived from organisms other than the host.

[0148] Thus, in certain embodiments, the tripartite arenaviruses provided herein The particles are: i) engineered to retain ORFs in non-native positions; ii) lacking GP, NP, Z protein, or L protein. iii) the removed ORF encodes a non-arenavirus protein; tripartite arenavirus particles in which one or more heterologous ORFs from the genome have been replaced with those from the genome (i.e. , one L segment and two S segments, or two L segments and one S segment ) is included.

[0149] In one embodiment, the heterologous ORF is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length. Length of nucleotides, 25 to 100 nucleotides, 50 to 200 nucleotides, 50 to 400 nucleotides nucleotide length, 200 to 500 nucleotides in length, or 400 to 600 nucleotides in length; In another embodiment, the heterologous ORF is 750 to 90 nucleotides in length. 0 nucleotides long, 800-100 nucleotides long, 850-1000 nucleotides long, 90 Length: 0-1200 nucleotides, Length: 1000-1200 nucleotides, Length: 1000-1500 nucleotides or 10–1500 nucleotides in length, 1500–2000 nucleotides in length, 1700–2000 nucleotides in length Length of nucleotides: 2000-2300 nucleotides; Length of nucleotides: 2200-2500 nucleotides; Length of nucleotides: 2500 ~3000 nucleotides long, 3000-3200 nucleotides long, 3000-3500 nucleotides long Length, 3200-3600 nucleotides, 3300-3800 nucleotides, 4000 nucleotides Lengths of 4400 to 4400 nucleotides, 4200 to 4700 nucleotides, and 4800 to 5000 nucleotides Length, 5000-5200 nucleotides, 5200-5500 nucleotides, 5500-5800 nucleotides Nucleotide length, 5800–6000 nucleotides long, 6000–6400 nucleotides long, 62 Length: 600–6800 nucleotides, 6600–7000 nucleotides, 7000–7200 nucleotides nucleotides in length, 7200-7500 nucleotides in length, or 7500 nucleotides in length. In this embodiment, 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 longer than 2500 amino acids In some embodiments, the nucleic acid encodes a peptide or polypeptide of 1 or more amino acids in length. In some embodiments, the heterologous ORF encodes a polypeptide not exceeding 2500 amino acids in length. In a specific embodiment, the heterologous ORF does not contain a stop codon. In one embodiment, the heterologous ORF is codon-optimized. The composition, the nucleotide pair composition, or both can be optimized. Techniques for this are known in the art and can be applied to the optimization of heterologous ORFs.

[0150] A heterologous ORF from any non-arenavirus organism may be incorporated into said tripartite arenavirus particle. In one embodiment, the heterologous ORF encodes a reporter protein. A more detailed description of reporter proteins is provided in Section 4.3. In some embodiments, the heterologous ORF is an antigen of an infectious pathogen or an antigen associated with any disease. wherein the antigen is capable of eliciting an immune response. In embodiments, the antigen is an infectious organism, a tumor (i.e., cancer), or an allergen. A more detailed description of heterologous ORFs is provided in Section 4.3.

[0151] In one embodiment, the proliferation and infectivity of the arenavirus particles is enhanced by the It is not affected by heterologous ORFs from organisms other than R. rus.

[0152] Using techniques known to those skilled in the art, the arenavirus ORF can be retained in a position other than the wild-type position. Generating arenavirus particles containing arenavirus genome segments engineered to carry For example, reverse genetic techniques can be used to generate such arenavirus particles. In other embodiments, the replication-deficient arenavirus particles (i.e., Arenaviruses engineered to retain the arenavirus ORF in a position other than the wild-type position The genome segment lacks ORFs encoding GP, NP, Z protein, and L protein. The missing arenavirus genome segment can be generated in a complementing cell. .

[0153] In certain embodiments, the tripartite arenavirus particles used in accordance with the present application are those derived from Old World arenaviruses. The virus may be, for example, LCMV.

[0154] In some embodiments, 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. arenavirus particles and vaccines, as well as therapeutic and / or therapeutic applications for, for example, infectious diseases and cancer. The present invention relates to methods of using such arenavirus particles in prophylaxis. A more detailed description of how to use arenavirus particles is provided in section 4.6. It is being done.

[0155] In certain embodiments, the present application provides a pharmaceutical composition comprising a compound according to any one of the preceding claims, which is suitable for use as a pharmaceutical composition. arenavirus particles and vaccinations, as well as the treatment or administration of, for example, infectious diseases or cancer. The present invention relates to methods of using such arenavirus particles in prophylaxis. A more detailed description of how to use the arenavirus particles described is provided in section 4.6. It has been done.

[0156] 4.3 Arenavirus particles or tripartite arenavirus particles expressing heterologous ORFs In one embodiment, the arenavirus genome segments and their respective arenaviruses are The viral particle or tripartite arenavirus particle may comprise a heterologous ORF. wherein the arenavirus genome segments and the respective arenavirus particles or tripartites The nodal arenavirus particle may comprise a gene of interest. The heterologous ORF or the gene of interest encodes an antigen. In a more specific embodiment, The heterologous ORF or the gene of interest encodes a reporter protein or a fluorescent protein. Do it.

[0157] In one embodiment, the arenavirus genome segment, the arenavirus particle, or the tripartite arenavirus particle, wherein the tripartite arenavirus particle contains one or more heterologous ORFs or one or more genes of interest. In another embodiment, the arenavirus genome segment, the arenavirus The viral particle, or said tripartite arenavirus particle, contains at least one heterologous ORF, at least one The heterologous ORF may contain at least two heterologous ORFs, at least three heterologous ORFs, or more heterologous ORFs. In other embodiments, the arenavirus particle or the tripartite arenavirus particle , at least one gene of interest, at least two genes of interest, at least three genes of interest The gene of interest may be a gene encoding a gene encoding a gene encoding a gene encoding a gene encoding a gene for ...

[0158] The arenavirus genome segments, arenavirus particles, or tripartite arenaviruses of the present application A variety of antigens can be expressed by the viral particle. In one embodiment, the heterologous ORF is , an antigen of an infectious pathogen or any disease-associated antigen capable of eliciting an immune response In one embodiment, the heterologous ORF encodes a viral, bacterial, fungal, or parasitic gene. The antigens may encode antigens derived from living organisms or may be used to treat tumors or tumor-related diseases (i.e., cancer), autoimmune diseases, and the like. In autoimmune diseases, degenerative diseases, genetic diseases, substance addiction, obesity, or allergic diseases It can be expressed as

[0159] In some embodiments, the heterologous ORF encodes a viral antigen. Non-limiting examples of antigens include those from the family Adenoviridae (e.g., mastadenovirus, viruses and aviadenoviruses), herpesviridae (e.g., simplex viruses, Herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, herpes simplex virus 6, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), Family Leviviridae (e.g., Levivirus, Enterobacterial phase MS2, Allolevivirus) Poxviridae (e.g., vertebrate poxvirinae), Poxyiridae (e.g., chordates), opoxyirinae), parapoxvirus, avipoxvirus, capripoxvirus , leporipoxvirus, suipoxvirus, morsipoxvirus, and insects Poxviridae (e.g., Entomopoxyirinae), Papovaviridae (e.g., Polyomaviruses virus and papillomavirus), paramyxoviridae (e.g., paramyxoviridae), Myxoviruses, parainfluenza virus 1, mobilliviruses (e.g. , measles virus), rubulaviruses (e.g., mumps virus), pneumonovirus subtypes Family (pneumonovirinae) (e.g., pneumovirus, human respiratory syncytial virus), human respiratory Haustorial syncytial virus and metapneumovirus (e.g., avian pneumovirus and human Metapneumovirus), Picornaviridae (e.g., Enterovirus), viruses, rhinoviruses, hepatoviruses (e.g., human hepatitis A virus), cardioviruses virus, and apthovirus), reoviridae (e.g., Orthovirus, Reovirus, Orbivirus, Rotavirus, Cypovirus, Fijivirus, Fi Threoviruses, and Oryzaviruses), Retroviridae (e.g., mammalian Type B retrovirus, mammalian type C retrovirus, avian type C retrovirus, type D retrovirus virus group, BLV-HTLV retroviruses, lentiviruses (e.g., human immunodeficiency virus (HIV) V)1 and HIV-2 (e.g., HIV gp160), spumaviruses, Flaviviridae e) (e.g., hepatitis C virus, dengue virus, West Nile virus), hepadnavirus Family (hepadnaviridae) (e.g., hepatitis B virus), Family (togaviridae) (e.g., , alphaviruses (e.g., Sindbis virus) and rubiviruses (e.g., rubella virus) rhabdoviridae (e.g., vesiculovirus, lyssavirus) , ephemelovirus, cytorhabdovirus, and necleorhabdovirus bdovirus), Arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus) virus, Ippy virus, and Lassa virus), and Coronaviridae (e.g., For example, antigens derived from coronaviruses and toroviruses. wherein the viral antigen is HIV gp120, gp41, HIV Nef, RSV F glycoprotein, RSV G glycoprotein proteins, HTLV tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and g E), or hepatitis B surface antigen, hepatitis C virus E protein, or coronavirus spike In one embodiment, 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 an antigen of a parasite (e.g., a protozoan). In yet another embodiment, the heterologous nucleotide sequence encodes a fungal antigen. Code the original.

[0161] Non-limiting examples of bacterial antigens include Aquaspirillum family, Azospirillum Azospirillum family, Azotobacteraceae family, Ba Bacteroidaceae family, Bartonella species, Bdello Vibrio family (Bdellovibrio family), Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citronella Citrobacter species, Edwardsiella, Enterobacter Enterobacter aerogenes, Envinia species, Escherichia coli cherichia coli, Hafnia species, Klebsiella species , Morganella species, Proteus vulgaris, Providencia, Salmonella species, Serratia marcescens Serratia marcescens, and Shigella flexneri, Gal Gardenella family, Haemophilus influenzae, Halobacc Halobacteriaceae family, Helicobacter family, Legionallaceae family, Listeria species, Methylococcus Methylococcaceae family, mycobacteria (e.g., Mycobacterium tuberculosis (M Mycobacterium tuberculosis), Neisseriaceae family, Oceanospiria Oceanospirillum family, Pasteurellaceae family, Streptococcus pneumoniae Pneumococcus species, Pseudomonas species, Rhizobium Rhizobiaceae family, Spirillum family, Spiros omaceae family), Staphylococcus (e.g., methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus) and Streptococcus pyogenes (Streptococcus pyogenes) rogenes, Streptococcus (e.g., Streptococcus enteritidis), coccus enteritidis, Streptococcus fasciae, and Streptococcus pneumoniae Streptococcus pneumoniae, Vampirovibr Helicobacteraceae cobacter family), Yersinaceae, Bacillus antracis and Vampirovibrionaceae Examples of antigens derived from bacteria include:

[0162] Non-limiting examples of parasitic antigens include amoeba, malaria parasite, and Plasmodium. Antibodies derived from parasites such as Plasmodium and Trypanosoma cruzi Non-limiting examples of fungal antigens include Absidia species ( For example, Absidia corymbifera and Absidia ramosa Absidia ramosa), Aspergillus species, (e.g., Aspergillus Aspergillus flavus, Aspergillus fumigatus igatus, Aspergillus nidulans, Aspergillus nidulans Aspergillus niger, and Aspergillus terreus, Basidiophora Basidiobolus ranarum, Blastomyces dermatitidis yces dermatitidis, Candida species (e.g., Candida albicans (C andida albicans, Candida glabrata, Candida cologne da kern), Candida krusei, Candida parapsilosis arapsilosis, Candida pseudotropicalis, Candida Candida quillermondii, Candida rugosa, Candida stellatoidea and Candida tropicalis tropicalis), Coccidioides immitis, Conidiobol Conidiobolus species, Cryptococcus neoformus ms), Cunninghamella species, dermatophytes, Histoplasma kaempferi Histoplasma capsulatum, Microsporum gypsum seum), Mucor pusillus, Paracoccidioides brasiliensis Paracoccidioides brasiliensis, Pseudallescheria boisii ia boydii), Rhinosporidium seeberi, Pneumocystis Pneumocystis carinii, Rhizopus species (e.g., Rhizopus Rhizopus arrhizus, Rhizopus oryzae and Rhizopus Rhizopus microsporus, Saccharomyces species , Sporothrix schenckii, Zygomycetes, and Zygomycetes, Included are antigens from fungi such as classes such as Saccharomycetes, Basidiomycetes, Imperfectiycetes, 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 antigen. disease, acute myeloid leukemia, adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancer, Kaposi's sarcoma, anal Cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, Extrahepatic (see cholangiocarcinoma), bladder cancer, osteosarcoma / malignant fibrous histiocytoma of bone, brain Stem glioma, brain cancer, brain tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma brain tumor, ependyma tumors, medulloblastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, Bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid gastroenteroma tumor, carcinoma of unknown primary, central nervous system lymphoma, primary, cerebellar astrocytoma, cerebral astrocytoma / malignant Glioma, cervical cancer, childhood cancer, chronic bronchitis, chronic lymphocytic leukemia, chronic myeloid leukemia disease, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor tumors, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's family of tumors sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, intraocular melanoma, retinoblastoma gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor Germ cell tumors: extracranial, extragonadal, or ovarian gestational trophoblastic tumors, brainstem tumors Glioma, glioma, pediatric cerebral astrocytoma, pediatric visual pathway and hypothalamic carcinoma, gastric carcinoid, Alley cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin's lymphoma melanoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas) , Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphoblastic lymphoma, acute lymphoblastic lymphoma lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lip and Oral cancer, liposarcoma, liver cancer (primary), lung cancer, non-small cell, small cell, AIDS-related lymphoma lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma , lymphoma, primary central nervous system, macroglobulinemia, Waldenstrom's disease, male breast Malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, intraocular (eye), Merkel cell Cancer, mesothelioma, adult malignant, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary, oral cancer, multiple Endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, bone Myelodysplastic / Myeloproliferative Disorders, Myeloid Leukemia, Chronic, Myeloid Leukemia, Adult Acute, Myeloid Leukemia Disease, childhood acute, myeloma, multiple (bone marrow cancer), myeloproliferative disorder, chronic, nasal and paranasal sinuses Cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, Osteosarcoma / malignant fibrous histiocytoma of the bone, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), Ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell, paranasal sinus and nasal cancer, paranasal cancer adenocarcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineal gland Blastoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasia / multiple myeloma, Pleuropulmonary blastoma, 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, -ing family tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin Skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small intestine Soft tissue sarcoma, squamous cell carcinoma - see Skin cancer (non-melanoma), squamous cell carcinoma of the neck of unknown primary Cancer, metastatic, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, cutaneous mycosis See sarcoidosis and Sézary syndrome, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, Pediatric transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, primary site unknown, carcinoma in adults, primary Unknown location, childhood cancer, ureter and renal pelvis, transitional cell carcinoma, rethral cancer, uterus Cancer, uterine sarcoma of the endometrium, bronchial tumors, central nervous system embryonal tumors; pediatric chordoma, colorectal cancer , craniopharyngioma, ependymoblastoma, Langerhans cell histiocytosis, acute lymphoblastic leukemia, acute Myeloid leukemia (adult / childhood), small cell lung cancer, medullary epithelioma, oral cancer, papilloma, moderately differentiated Pineal parenchymal tumors, pituitary tumors, respiratory tract cancers involving the NUT gene on chromosome 15, spinal cord tumors, breast Adenoma, thyroid cancer, vaginal cancer; vulvar cancer, and Wilms' tumor and antigens derived from tumor-associated diseases, including

[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, Eph A3, EZH2, FGF5, glypican-3, G250 / MN / CAIX, HER-2 / neu, IDO1, IGF2B3, IL13Ralpha2 , intestinal carboxylesterase, alpha-fetoprotein, kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5A C, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin (sec ernin)1, SOX10, STEAP1, survivin, telomerase, VEGF, or WT1, EGF-R, CEA, CD5 2, gp 100 protein, MELANA / MART1, NY-ESO-1, p53 MAGE1, MAGE3, and CDK4, Alf Actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, ta-catenin, Cdc27, CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2, elongation Length factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase ferase AS fusion protein, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PT PRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF- Beta RII, triosephosphate isomerase, Lengsin, M-CSF, MCSP, or md Examples include m-2.

[0165] In some embodiments, the heterologous ORF encodes a respiratory pathogen antigen. In specific embodiments, the respiratory pathogen is RSV, coronavirus, human metanuclear virus, or the like. influenza virus, Hendra virus, Nipah virus, adenovirus Respiratory viral antigens include viruses such as flu, rhinovirus, and PRRSV. Non-limiting examples include respiratory syncytial virus F, G, and M2 proteins, coronaviruses (SARS, HuCoV) spike protein (S), human metapneumovirus fusion protein, para Influenza virus fusion and hemagglutinin proteins (F, HN), Hendra virus ( HeV and Nipah virus (NiV) attachment glycoproteins (G and F), adenovirus capsid protein These include proteins, rhinovirus proteins, and PRRSV wild-type or modified GP5 and M proteins. It can be obtained.

[0166] In a specific embodiment, said respiratory pathogen is Bacillus anthracis, Mycobacterium tuberculosis, Bordetella pertussis (Borde tella pertussis, Streptococcus pneumoniae, Yersinia pestis , Staphylococcus aureus, Francisella tularensis, Legionella pneumophila, Chlamydia pneumoniae iae, Pseudomonas aeruginosa, Neisseria meningitides, and Non-limiting examples of respiratory bacterial antigens include those caused by Bacillus anthracis infection. Protective antigen PA, Mycobacterium tuberculosis mycobacterial antigen 85A and heat shock protein (Hsp65), Bordetella pertussis Pertussis toxoid (PT) and filamentous hemagglutinin (FHA), Streptococcus pneumoniae sortase (so rtase A and surface adhesin A (PsaA), Yersinia pestis F1 and V subunits, and Vitis aureus Streptococcus, Francisella tularensis, Legionella pneumophila, Chlamydia pneumoniae, Pseudomonas aeruginosa, Neisseria meningitidis, and and proteins derived from Haemophilus influenzae.

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

[0168] In another embodiment, the heterologous ORF encodes an antigen expressed in an autoimmune disease. In a more specific embodiment, said autoimmune disease is type 1 diabetes, multiple sclerosis The autoimmune diseases may be rheumatoid arthritis, rheumatoid arthritis, lupus erythmatosus, and psoriasis. Non-limiting examples of infectious disease antigens include Ro60, dsDNA, or RNP.

[0169] In other embodiments, the ORF encodes an antigen expressed in an allergic disease. In a more specific embodiment, said allergic disease is seasonal and perennial rhinitis. These may include, but are not limited to, conjunctivitis, asthma, and eczema. Typical examples include Bet v 1 and Fel d 1.

[0170] In another embodiment, the arenavirus genome segment, the arenavirus particle The virus or said tripartite arenavirus particle further comprises a reporter protein. The transporter protein may be co-expressed with the antigen described herein. The reporter is visible under normal light or other wavelengths of light. The strength of the effect caused by the protein is used to measure the arenavirus particle or 3 Segmented arenavirus particles can be measured and monitored directly.

[0171] Reporter genes will be readily recognized by those skilled in the art. The arenavirus particle is a fluorescent protein. The gene is GFP, which emits a bright green light when exposed to UV or blue light.

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

[0173] In certain embodiments, the arenavirus genome segment expressing a heterologous ORF, The arenavirus particle or the tripartite arenavirus particle is a vaccination vector. It has desirable properties for use as a scalar (see, for example, Section 4.6). In another embodiment, the arenavirus genome segment expressing a heterologous ORF, The arenavirus particles, or the tripartite arenavirus particles, can be administered to a host (e.g., mouse, In another embodiment, the antibody can induce an immune response in a mammal (e.g., a goat, a donkey, a human, a heron, a goat, a donkey, a human). the arenavirus genome segment expressing a heterologous ORF as described herein, Arenavirus particles, or the tripartite arenavirus particles, induce an innate immune response In other embodiments, the arenavirus genome segment expressing a heterologous ORF, The arenavirus particle or the tripartite arenavirus particle induces an adaptive immune response. In a more specific embodiment, said arenavirus genome segment expressing a heterologous ORF is The arenavirus particle, or the tripartite arenavirus particle, is a viral vector that inhibits the innate immune response. Induce both immune responses and adaptive immune responses.

[0174] In another embodiment, the arenavirus genome segment expressing a heterologous ORF, The arenavirus particle, or the tripartite arenavirus particle, induces a T cell response. In an even more specific embodiment, said arenavirus genomic sequence expressing a heterologous ORF is The segment, the arenavirus particle, or the tripartite arenavirus particle induces a CD8+ T cell response. In another embodiment, the above-mentioned arenavirus carrying an exogenous gene of interest is induced. The rRNA particles induce a strong CD8+ T cell response with high frequency and functionality. and the arenavirus genome expressing an antigen derived from an infectious organism, a cancer, or an allergen. The arenavirus particle or the tri-segmented arenavirus particle may be a corresponding The target gene induces CD8+ T cells specific to one or more epitopes of the target gene.

[0175] In certain embodiments, the arenavirus genome segment expressing a heterologous ORF, The arenavirus particle or the tripartite arenavirus particle induces T helper 1 differentiation, CD4+ T It can induce cellular memory formation and / or elicit long-lasting antibody responses. These antibodies may be neutralizing, opsonizing, toxic to tumor cells, or other desired In another embodiment, the arenavirus expressing a heterologous ORF has novel biological characteristics. The virus genome segment, the arenavirus particle, or the tripartite arenavirus particle is It has a strong tropism for dendritic cells and activates them upon infection. The presentation of the antigen by antigen-presenting cells is enhanced.

[0176] In some embodiments, prior to expressing an antigen derived from an infectious organism, cancer, or allergen. The arenavirus genome segment, the arenavirus particle, or the tripartite arenavirus The virus particles elicited low or undetectable neutralizing antibody titers against LCMV and the respective exogenous Induce a highly protective neutralizing antibody response against the transgene. and the particles or tripartite antigens expressing antigens derived from infectious organisms, cancers, or allergens. The arenavirus backbone that forms the arenavirus particle is have a low ability to induce immunity against

[0177] 4.4 Production of Arenavirus Particles and Tripartite Arenavirus Particles In general, arenavirus particles can be generated using standard reverse genetic techniques such as those described for LCMV. It can be recombinantly produced by conventional techniques (see, for example, the US Pat. No. 6,229,629, incorporated herein by reference). Flatz et al., 2006, Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 200 6, Virology 350:370; Ortiz-Riano et al., 2013, J Gen Virol. 94:1175-88 In order to produce the arenavirus particles provided herein, these The techniques can be applied as follows: The genome of a virus can be determined as described in Section 4.1 and Section 4.2. Each of these may be modified as described in Section 4.2.

[0178] 4.4.1 Non-Natural Position Open Reading Frames A genome engineered to retain a viral ORF in a position other than the wild-type position of the ORF. The production of arenavirus particles containing genome segments can be achieved by any reverse genetic method known to those skilled in the art. It may be produced recombinantly by technology.

[0179] (i) Infectious and replication-competent arenavirus particles In one embodiment, the method for producing arenavirus particles comprises: (i) the first (ii) transfecting a cDNA of an arenavirus genome segment into a host cell; Transfecting the cDNA of the second arenavirus genome segment into a host cell. (iii) Plasmids expressing the minimal arenavirus transactivators NP and L. (iv) transfecting the host cells with the vector; and (iv) subjecting the host cells to conditions suitable for virus formation. and (v) recovering the arenavirus particles. In one embodiment, the cDNA is contained in a plasmid.

[0180] Once produced from the cDNA, arenavirus particles (i.e., infectious and replicative) In one embodiment, the arenavirus particles are capable of propagation. The virus is propagated to a titer that allows the virus to be used as described in the specification. In one embodiment, the host cell can be propagated in any host cell that allows The primary cells were then incubated with the arenavirus particles at titers similar to those determined for the corresponding wild type. The cells are allowed to grow to a titer of 1000 kJ / ml.

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

[0182] In certain embodiments, the host cells are maintained in culture and express one or more plasmids. The plasmid(s) may contain, for example, a polymerase I promoter. one or more expression vectors suitable for expression in mammalian cells, consisting of a promoter and a terminator the arenavirus genome segment(s) to be produced under the control of the cassette is expressed.

[0183] The plasmids that can be used to produce the arenavirus particles include: i) a plasmid encoding the S genome segment; ii) a plasmid encoding the L genome segment, e.g., pol-I S; For example, it may contain pol-I L. In one embodiment, the L and S segments of the virus are A plasmid encoding an arenavirus polymerase that directs intracellular synthesis was introduced into the For example, a plasmid encoding the L protein and / or Alternatively, plasmids encoding the L protein and NP (pC-L and pC-NP, respectively) may be present. and NP are the minimal trans-acting factors required for viral RNA transcription and replication. Read the cDNA from two separate plasmids into the L and S segments from opposite sides, respectively. Expression cassettes with pol-I and pol-II promoters were used to express the NP and L proteins. Together they may direct the intracellular synthesis of the viral L and S segments.

[0184] In one embodiment, the arenavirus genome segment is under the control of a promoter. Below are the typical RNA polymerase I-driven expression cassettes, RNA polymerase II-driven A T7 bacteriophage RNA polymerase-driven cassette or a T7 bacteriophage RNA polymerase-driven cassette may be used. In one embodiment, the plasmid encoding the arenavirus genome segment ( The sequences (multiple sequences) can be identical, i.e., the genomic sequence and the trans-acting factors. The promoters can be transcribed from one plasmid. RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase Examples of promoters include the enzyme III promoter, the T7 promoter, the SP6 promoter, and the T3 promoter. can be.

[0185] Additionally, the plasmid(s) may contain an expression vector suitable for gene expression in mammalian cells. a set of mammalian selection vectors under the control of a polymerase II expression cassette, such as those described above markers, e.g., puromycin resistance, or viral gene transcripts. The entity(ies) are followed by an internal ribosome entry site, such as that of encephalomyocarditis virus, , followed by a mammalian resistance marker. For production in E. coli, the plasmid contains In addition, it features a bacterial selectable marker such as an ampicillin resistance cassette.

[0186] Transfection of host cells with the plasmid(s) is performed using calcium phosphate, lipopolysaccharide, or Any of the commonly used strategies, such as endothelial cell membrane-based protocols or electroporation, After several days, a suitable selection agent, e.g., puromycin, may be added at a dose of 100 mg / ml. Surviving clones are isolated and subcloned according to standard procedures and then cultured at high Expression clones were incubated with Western blot analysis using antibodies against the viral protein(s) of interest. Identification is performed using a flow blot or flow cytometry procedure.

[0187] The following procedures are envisioned for the recovery of arenavirus particles as described herein: Cells, typically 80% confluent in M6 well plates, were plated as described above. Transfect with a mixture of amides, including calcium phosphate, liposome-based Any commonly used strategy, such as a transfection protocol, or electroporation, may be utilized.

[0188] After 3-5 days: The culture supernatant (arenavirus vector preparation) is collected, aliquoted, and the arenavirus vector is then diluted with water before use. 4°C, -20°C, or -80°C depending on how long the virus vectors should be stored. The infectious titer of the arenavirus vector preparation was determined by immunofocus assay. Alternatively, the transfected cells are evaluated 3 to 5 days after transfection. The collected cells and supernatant were subcultured into a larger vessel (e.g., a T75 tissue culture flask). The culture supernatant is preferably collected up to 5 days after the subculture.

[0189] The present application further relates to the expression of heterologous ORFs, wherein the promoter encoding the genome segment is The smid is modified to incorporate a heterologous ORF, which is then inserted into the smid using restriction enzymes. It can be incorporated into a plasmid.

[0190] (ii) Infectious replication-deficient arenavirus particles Infectious replication-deficient arenavirus particles can be rescued as described above. Once produced from the cDNA, the infectious, replication-deficient arena vectors provided herein can be used to The virus is capable of propagation in complementing cells, which are capable of replicating the virus by modifying their genomes. A cell that provides functionality that has been removed from a replication-deficient arenavirus (e.g., GP If the ORF encoding the protein is deleted or functionally inactivated, Complementary cells provide the very GP protein).

[0191] For the removal or functional inactivation of one or more of the ORFs in an arenavirus vector (Here, deletion of glycoprotein GP is used as an example) arenavirus vectors, The deleted viral gene(s), e.g., GP in this example, is / are inserted in trans. Such complementing cell lines (hereinafter referred to as C-cells) can be produced and propagated in the cells provided. (hereinafter referred to as "transfected cell lines") are transfected with cell lines such as BHK-21, HEK 293, and VERO to express the viral gene(s) of interest. Transform with one or more plasmids for The C-plasmid(s) are prepared by infecting the C-plasmid(s) with the desired C-plasmid for expression in mammalian cells. One or more suitable expression cassettes, e.g., EF1 alpha with a polyadenylation signal and that the gene is produced under the control of a mammalian polymerase II promoter, such as a promoter. and expressing the viral gene(s) deleted in the arenavirus vector. In addition, the complementing plasmid contains an expression cassette suitable for gene expression in mammalian cells. a mammalian selectable marker under the control of, for example, a polymerase II expression cassette as described above; For example, characterized by puromycin resistance or by the viral gene transcript(s). This is followed by an internal ribosome entry site, such as that of encephalomyocarditis virus, which is responsible for the formation of mammalian resistance mutants. For production in E. coli, the plasmid additionally contains an It features a bacterial selectable marker such as a picillin resistance cassette.

[0192] Cells that can be used, such as BHK-21, HEK 293, MC57G, etc., are maintained in culture and Commonly used methods include calcium phosphate, liposome-based protocols, or electroporation. transfected with the complementing plasmid(s) using any of the strategies After several days, a suitable selection agent, such as puromycin, is added at a titrated concentration. Surviving clones were isolated and subcloned according to standard procedures to identify high-expressing C-cell clones. The lines were analyzed by Western blot or immunoblotting using antibodies against the viral protein(s) of interest. are identified using a flow cytometry procedure. Instead of using transient transfection of normal cells, C-cells are used below. At each step, the missing viral gene(s) can be complemented. In addition, helper viruses can be used to provide missing functionality in trans. It can be provided.

[0193] Plasmids can be of two types: i) in C-cells, Two plasmids, called TF-plasmids, were developed for intracellular expression of minimal trans-acting factors of the vector. Plasmids, such as those derived from the NP and L proteins of LCMV in this example. and ii) in the C-cells, arenavirus vector genome segments, e.g., engineered A plasmid called GS-plasmid for intracellular expression of the modified segment. Plasmids contain expression cassettes suitable for protein expression in mammalian cells, typically For example, preferentially, CMV or EF1 alpha, both of which are combined with a polyadenylation signal. Each gene is under the control of a mammalian polymerase II promoter, such as the FA promoter. The GS-plasmid expresses the NP and L proteins of the arenavirus vector. Expresses small (S) and large (L) genome segments. Typically polymerase I driven Using either a T7 bacteriophage RNA polymerase (T7-) driven expression cassette The latter preferentially processes the primary transcript to produce a normal terminal It has a 3'-terminal ribozyme to provide the correct end. In this case, expression of T7 in C-cells is similar to that of the TF-plasmid construct, leading to a recovery process. The T7 gene must be provided either by including an additional expression plasmid that provides the Alternatively, the C-cells may be engineered to additionally stably express T7. In the present invention, the TF and GS plasmids can be identical, i.e., the genomic sequence and trans-acting factors are driven by T7, polI, and polII promoters from a single plasmid. It can be transcribed.

[0194] For the recovery of arenavirus vectors, the following procedure may be used: Day 1: C-cells; Typically, 80% confluent M6 well plates were cultured with the two TF-plasmids. In one embodiment, the cells are transfected with a mixture of the two GS-plasmids. The TF and GS plasmids can be identical, i.e., the genomic sequence and Trans-acting factors are transcribed from a single plasmid by T7, polI, and polII promoters These can include calcium phosphate, liposome-based protocols, or electroporation. Any of the commonly used strategies, such as the hole method, may be utilized.

[0195] After 3 to 5 days: The culture supernatant (arenavirus vector preparation) is collected and divided. Store the vector at 4°C, -20°C, or -80°C depending on how long it should be stored before use. The infectious titer of the arenavirus vector preparation was then determined by incubating in C-cells. Alternatively, the transfected cells and The supernatant was transferred to a larger container (e.g., T75 tissue culture flask) 3–5 days after transfection. The cells may be subcultured (sco) and the culture supernatant collected up to 5 days after subculture.

[0196] The present invention further provides a method for producing a cell infected with an infectious replication-deficient arenavirus that expresses an antigen. It relates to the expression of antigens in culture. When used to express antigens in cultured cells In this case, the following two procedures may be used:

[0197] i) Infecting the target cell type with a gene that results in the production of antigens in all cells already shortly after infection. at a multiplicity of infection (MOI) of 1 or more, e.g., 2, 3, or 4, resulting in the production of The cells are infected with an arenavirus vector preparation.

[0198] ii) Alternatively, a lower MOI can be used and individual cell clones can be transfected with the virus. The vectors can be selected for their level of cytolytic antigen expression. Due to their anaerobic nature, individual clones can then be propagated indefinitely. Then, depending on the nature of the antigen produced, either from the culture supernatant or from the cells themselves, The antigen can be recovered (and purified). However, the present invention is not limited to these two strategies. In addition, infectious, replication-deficient arenaviruses are used as vectors to drive antigen expression. methods may be taken into consideration.

[0199] 4.4.2 Production of Tripartite Arenavirus Particles Tripartite arenavirus particles can be prepared, for example, as described in Emone, et al., J. Immunol. 2002, 10:111-114, which is incorporated herein by reference. t et al., 2008, PNAS, 106(9):3473-3478; Popkin et al., 2011, J. Virol., 85(15) :7928-7932, or by reverse genetic techniques known in the art. The production of the tripartite arenavirus particles provided herein can be carried out recombinantly. , which may be modified as described in Section 4.2.

[0200] (i) Infectious and replication-competent tripartite arenavirus particles In one embodiment, a vector comprising the tripartite arenavirus particle is produced. The method includes (i) one L segment and two S segments, or two L segments and one S segment. (ii) transfecting the cDNA of the segment into a host cell; Plasmids expressing the trans-acting factors NP and L are transfected into host cells. (iii) maintaining the host cells under conditions suitable for virus formation; and (iv) and recovering the virus particles.

[0201] Once produced from the cDNA, the tripartite arenavirus particles (i.e., infectious or In one embodiment, the tripartite arenavirus is capable of propagation. The particles are then cultured to a titer that allows for the virus to be used as described herein. The cells can be propagated in any host cell that allows the cells to grow. The host cell then determines whether the tripartite arenavirus particles are distinct from their wild-type counterparts. The cells are allowed to grow to a titer similar to the titer determined.

[0202] In one embodiment, the tripartite arenavirus particles are propagated in a host cell. Specific examples of host cells that can be used include BHK-21, HEK 293, and VERO. In a specific embodiment, said tripartite arenavirus particles are The cells may be propagated by the addition of the fermentation agent.

[0203] In certain embodiments, the host cells are maintained in culture and express one or more plasmids. The plasmid(s) may contain, for example, a polymerase I promoter. one or more expression vectors suitable for expression in mammalian cells, consisting of a promoter and a terminator The cassette is used to generate the arenavirus genome segment(s) that will be produced under the control of the cassette. Appear.

[0204] In a specific embodiment, said host cells are maintained in culture and contain one or more plant cells. The plasmid(s) may be transfected with, for example, a polymerase I promoter. one or more promoters and terminators suitable for expression in mammalian cells The viral gene(s) that are produced under the control of the expression cassette are expressed.

[0205] A tripartite arenavirus containing one L segment and two S segments is produced. Plasmids that can be used to construct the S genome include: i) two plasmids each encoding an S genome segment; ii) a plasmid encoding the L genome segment, e.g., pol-I S; For example, pol-I L can be mentioned. The plasmids required for tripartite arenaviruses include: i) a plasmid encoding the L genome segment; ii) two plasmids encoding the S genome segment, e.g., pol-L, and iii) a plasmid encoding the S genome segment. Plasmids such as pol-I S.

[0206] In one embodiment, arenaviruses that direct intracellular synthesis of viral L and S segments are A plasmid encoding the viral polymerase was incorporated into the transfection mixture. For example, a plasmid encoding the L protein and a plasmid encoding NP (or The L protein and NP are required for the transcription and replication of viral RNA. Alternatively, the L and S segments are trans-acting from opposite sides, respectively. It has pol-I and pol-II promoters that read cDNA from two separate plasmids into the Expression cassettes were used to express the L and S segments of the virus together with the NP and L proteins. It can be synthesized intracellularly.

[0207] Additionally, the plasmid(s) may contain an expression vector suitable for gene expression in mammalian cells. a set of mammalian selectable markers under the control of a polymerase II expression cassette, such as those described above Carriers, characterized by, for example, puromycin resistance, or containing viral gene transcripts ( the mammalian ribosome entry site (s) is followed by an internal ribosome entry site, such as that of an encephalomyocarditis virus; For production in E. coli, the plasmid is Additionally, it features a bacterial selectable marker such as an ampicillin resistance cassette.

[0208] Transfection of BHK-21 cells with the plasmid(s) was performed using calcium phosphate buffer. any of the commonly used strategies, such as liposome-based protocols or electroporation. After a few days, a suitable selection agent, e.g., puromycin, may be added at a dose of Surviving clones are isolated and subcloned according to standard procedures. High-expressing clones were screened by lysing with antibodies against the viral protein(s) of interest. Identification is performed using either a tumor blot or flow cytometry procedure.

[0209] Typically, RNA polymerase I-driven expression cassettes, RNA polymerase II-driven cassettes Alternatively, a T7 bacteriophage RNA polymerase-driven cassette may be used. Preferentially, the 3'-terminus is processed to generate normal ends of the primary transcript. In one embodiment, the arenavirus genome segment comprises a ribozyme. The encoding plasmids can be identical, i.e., the genomic sequence and and trans-acting factors are transduced from a single plasmid by T7, pol I, and pol II promoters. It can be photographed.

[0210] In order to recover the arenavirus and the tripartite arenavirus vector, the following steps are carried out: The procedure is as follows: Day 1: Cells, typically 80% confluent in M6 well plates, The cells are transfected with the mixture of plasmids as described above. Any commonly used method, such as calcium, liposome-based protocols, or electroporation strategies that can be utilized.

[0211] After 3-5 days: The culture supernatant (arenavirus vector preparation) is collected, aliquoted, and the arenavirus vector is then diluted with water before use. Depending on how long the virus vectors are to be stored, they can be stored at 4°C, -20°C, or -80°C. The infectious titer of the arenavirus vector preparation was determined by immunofocusing. Alternatively, the transfected cells and supernatants may be used to assess the activity of the transfected cells. Transfer the cells to a larger container (e.g., a T75 tissue culture flask) 3–5 days after transfection. The cells may be subcultured at intervals of 5 days, and the culture supernatant is collected up to 5 days after subculture.

[0212] The present application further relates to the expression of heterologous ORFs and / or genes of interest, wherein the genomic segment The plasmid encoding the vector has been modified to incorporate a heterologous ORF and / or gene of interest. The heterologous ORF and / or gene of interest can be incorporated into the plasmid using restriction enzymes. Cut.

[0213] (ii) Infectious replication-deficient trisegmented arenavirus particles Infectious replication-deficient tripartite arenavirus particles can be rescued as described above. However, once produced from the cDNA, the infectious, replication-defective vectors provided herein Sexual arenaviruses can grow in complementing cells. Complementing cells can replicate by modifying their genomes. a cell that provides functionality that has been removed from the replication-deficient arenavirus by For example, the ORF encoding the GP protein is deleted or functionally inactivated. In this case, the complementing cells provide the very GP protein).

[0214] For the removal or functional inactivation of one or more of the ORFs in an arenavirus vector (Here, deletion of glycoprotein GP is taken as an example) arenavirus vectors A cell line that provides in trans the deleted viral gene(s), e.g., GP in this example. Such complementing cell lines are hereinafter referred to as C-cells and can be produced and propagated in BH cells. Mammalian cell lines such as BHK-21, HEK 293, and VERO (here, BHK-21 is taken as an example) are used. one or more plasmids (complementation plasmids, C-plasmids) for the expression of target viral gene(s) The C-plasmid (multiple C-plasmids) (possibly acceptable) may contain one or more expression cassettes suitable for expression in mammalian cells, e.g., polyadenylation mammalian polymerase II promoters, such as the CMV or EF1 alpha promoters with transcription signals The deletion in the arenavirus vector is generated under the control of the promoter. In addition, the complementing plasmid expresses the viral gene(s) that are present in the mammalian cell. An expression cassette suitable for gene expression in cells, such as a polymerase II expression cassette as described above. A mammalian selectable marker under the control of the cassette, e.g., characterized by puromycin resistance or the gene transcript(s) of said virus contain a sequence such as that of encephalomyocarditis virus An internal ribosome entry site is followed by the mammalian resistance marker. For production, the plasmid may additionally contain a bacterial selectable element such as an ampicillin resistance cassette. It is characterized by a selectable marker.

[0215] Cells that can be used, such as BHK-21, HEK 293, MC57G, etc., are maintained in culture and Commonly used methods include calcium phosphate, liposome-based protocols, or electroporation. transfected with the complementing plasmid(s) using any of the following strategies: After a few days, a suitable selection agent, e.g., puromycin, is added at a titrated concentration. Clones were isolated and subcloned according to standard procedures to obtain high-expressing C-cell clones. by Western blot or immunoblotting using antibodies against the viral protein(s) of interest. are identified using a flow cytometry procedure. Instead of using transient transfection of normal cells, C-cells are used below. At each step, the missing viral gene(s) can be complemented. In addition, helper viruses can be used to provide missing functionality in trans. It can be provided.

[0216] Two types of plasmids: i) C-expressing the minimal trans-acting elements of arenaviruses in cells; Two plasmids, designated TF-plasmids for intracellular expression, e.g. and ii) a plasmid derived from the NP and L proteins of LCMV in C-cells; and Viral vector genome segments, e.g., segments with engineered modifications, are introduced into cells. A plasmid called GS-plasmid can be used for expression in mammals. Expression cassettes suitable for protein expression in mammalian cells, typically e.g., preferentially CMV or EF1 alpha promoter, both in combination with a polyadenylation signal Each arenavirus vector is expressed under the control of a mammalian polymerase II promoter such as The GS-plasmid expresses the small (S) genome of the vector. expresses the large (L) genome segment. Typically, polymerase I-driven Using either a T7 bacteriophage RNA polymerase (T7-) driven expression cassette The latter preferentially processes the primary transcript to produce normal ends. When a T7-based system is used, the C-intracellular Expression of T7 in TF-plasmid provides a T7 construct similar to the TF-plasmid in the recovery process. must be provided either by including an additional expression plasmid to Alternatively, the C-cells are engineered to further stably express T7. The GS plasmid and the GS plasmid can be identical, i.e., the genomic sequence and the The desired elements can be transcribed from a single plasmid by the T7, pol I, and pol II promoters.

[0217] For the recovery of arenavirus vectors, the following procedure may be used: Day 1: C-cells; Typically, 80% confluent M6 well plates were cultured with the two TF-plasmids. In one embodiment, the cells are transfected with a mixture of the two GS-plasmids. The TF and GS plasmids can be identical, i.e., the genomic sequence and Trans-acting factors are transcribed from a single plasmid by T7, polI, and polII promoters These can include calcium phosphate, liposome-based protocols, or electroporation. Any of the commonly used strategies, such as the hole method, may be utilized.

[0218] After 3 to 5 days: The culture supernatant (arenavirus vector preparation) is collected and divided. Store the vector at 4°C, -20°C, or -80°C depending on how long it should be stored before use. The infectious titer of the arenavirus vector preparation was then determined by immunoblotting using C-cells. Alternatively, the transfected cells and Transfer the supernatant into a larger container (e.g., a T75 tissue culture flask) 3–5 days after transfection. ) and the culture supernatant is collected up to 5 days after the subculture.

[0219] The present invention further provides a method for producing a human ovarian tumor cell line comprising: Regarding the expression of antigens in cell cultures containing CMV antigens. If used, the following two procedures may be used:

[0220] i) Infecting the target cell type, resulting in the production of antigen in all cells already shortly after infection. and injecting the arena vectors described herein at a multiplicity of infection (MOI) of 1 or more, e.g., 2, 3, or 4. Infect with viral vector preparation.

[0221] ii) Alternatively, a lower MOI can be used and individual cell clones can be isolated from their respective The level of virus-driven antigen expression can be selected for. Due to their anaerobic nature, individual clones can then be propagated indefinitely. Then, depending on the nature of the antigen produced, either from the culture supernatant or from the cells themselves, The antigen can be recovered (and purified). However, the present invention is not limited to these two strategies. First, we used infectious, replication-deficient arenaviruses as vectors to drive the expression of CMV antigens. Other ways of doing this may also be considered.

[0222] 4.5 Nucleic Acids, Vector Systems, and Cell Lines In some embodiments, the methods described in Sections 4.1 and 4.2, respectively, are containing or consisting of arenavirus genome segments or tripartite arenavirus particles A cDNA comprising the sequence:

[0223] 4.5.1 Non-Natural Position Open Reading Frames In one embodiment, arenavirus genome segments as described in Section 4.1 are used. Nucleic acids encoding the compounds are provided herein. The DNA nucleotide sequences or sets of DNA nucleotide sequences set forth in Table 1 are used herein. Host cells containing such nucleic acids are also provided in Section 4.1. do.

[0224] In a specific embodiment, the ORF is maintained in a position other than the wild-type position of the ORF. cDNA of an engineered arenavirus genome segment, The cDNA fragment encoding a heterologous ORF as described in Section 4.1 is provided in the document.

[0225] In one embodiment, the ORF is engineered to maintain an ORF in a position other than the wild-type position of the ORF. A DNA expression vector system encoding selected arenavirus genome segments is described herein. Specifically, one or more vectors are provided that contain the arenaviruses described herein. The two arenavirus genome segments of the virus particle, namely the L segment and the S segment, DNA expression vector systems encoding such vectors are provided herein. The system may encode (one or more separate DNA molecules).

[0226] In another embodiment, the ORF is engineered to be in a position other than the wild-type position.

[0013] Provided herein arenavirus S segment cDNAs, which can be used in DNA expression In another embodiment, the ORF is part of a system or is incorporated into a DNA expression system. c of arenavirus L segments engineered to be held in a position other than the wild-type position The DNA is part of or incorporated into a DNA expression system. (i) a pre-engineered vector that has been engineered to retain an ORF in a position other than the wild-type position of the ORF; and (ii) a cDNA of the arenavirus genome segment; and The protein-coding ORF is removed and replaced with a heterologous ORF from an organism other than an arenavirus. It has been replaced.

[0227] In certain embodiments, the cDNAs provided herein are derived from specific strains of LCMV. LCMV strains include Clone 13, MP strain, Arm CA 1371, and Room E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Mar Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 2011 2714, Douglas, GR01, SN05, CABN, and their derivatives. In a specific embodiment, the cDNA is derived from LCMV clone 13. wherein the cDNA is derived from the LCMV MP strain.

[0228] In certain embodiments, the arenavirus particles or tripartite arenavirus particles described herein are The vectors that are generated to encode the viral particles may be based on specific strains of LCMV. LCMV strains include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 8103 62, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and their subsidiaries In some embodiments, the arenavirus particles or The tripartite arenavirus particle may be based on LCMV clone 13. The arenavirus particles or tripartite arenavirus particles described herein, The vector constructed to encode the S segment of LCMV clone 13 is the LCMV MP strain. The sequence of LCMV clone 13 is set forth as SEQ ID NO: 2. The sequence of the S segment is the sequence set forth in SEQ ID NO: 1. The L segment of LCMV clone 13 The sequence of the S segment of LCMV strain MP is set forth as SEQ ID NO: 5. The sequence of the L segment of LCMV strain MP is set forth as SEQ ID NO: 53. It is written.

[0229] In another embodiment, a cell comprising the cDNA or vector system described above in this section , provided herein. Cell lines derived from infected such cells, such cells Also provided herein are cultures comprising such cells, and methods for culturing such cells. In one embodiment, the ORF is engineered to maintain the ORF in a position other than the wild-type position. Provided herein are cells containing cDNAs of arenavirus genome segments. In some embodiments, the cells comprise an S segment and / or an L segment. .

[0230] (4.5.2 Tripartite Arenavirus Particles) In one embodiment, a tripartite arenavirus particle as described in Section 4.2 is Nucleic acids encoding the nucleotide sequences are provided herein. For example, a DNA nucleotide sequence or set of DNA nucleotide sequences as set forth in Table 2 or Table 3. are provided herein. Host cells containing such nucleic acids are also described in Section 4. 2 will be provided.

[0231] In a specific embodiment, the ORF is maintained in a position other than the wild-type position of the ORF. The cDNA consisting of the engineered tripartite arenavirus particle cDNA is referred to herein as In other embodiments, (i) an arenavirus ORF is inserted into a region of the wild-type region of the ORF. The cDNA of a tripartite arenavirus particle is engineered to be held in a different position from the i) the tripartite arenavirus particle encodes a heterologous ORF as described in Section 4.2 do.

[0232] In one embodiment, the tripartite arenavirus particle described herein as a whole A DNA expression vector system encoding the vector is provided herein. A vector system comprising one or more vectors of the tripartite arenavirus particles described herein. The vector contains three arenavirus genome segments: one L segment and two or two L segments and one S segment, Vector systems are provided herein. Such vector systems may comprise (one or more separate The DNA molecule may encode a

[0233] In another embodiment, the ORF is engineered to be in a position other than the wild-type position. and arenavirus S cells that are part of or integrated into DNA expression systems. cDNAs of the fragment(s) are provided herein. In other embodiments, the ORF arenavirus L segment members engineered to retain the nucleotide sequence at a position different from that of the wild type. The cDNA(s) of the target gene are part of or incorporated into a DNA expression system. In one embodiment, (i) an ORF that is in a position that is different from the wild-type position of the ORF. (ii) a cDNA encoding the tripartite arenavirus particle, the cDNA being engineered to carry GP ORFs encoding the NP, Z protein, or L protein are removed to produce non-arenaviruses. It has been replaced with a heterologous ORF from another organism.

[0234] In certain embodiments, the cDNAs provided herein are derived from specific strains of LCMV. Examples of LCMV strains include Clone 13, MP strain, Arm CA 1371, and Arm E. -250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN and derivatives thereof. In a specific embodiment, the cDNA is In another specific embodiment, the cDNA is derived from LCMV strain MP. .

[0235] In certain embodiments, the arenavirus particles or tripartite arenavirus particles described herein are The vectors that are generated to encode the viral particles may be based on specific strains of LCMV. LCMV strains include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 8103 62, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and their subsidiaries In some embodiments, the arenavirus particles or The tripartite arenavirus particle may be based on LCMV clone 13. The arenavirus particles or tripartite arenavirus particles described herein, The vector constructed to encode the S segment of LCMV clone 13 is the LCMV MP strain. The sequence of LCMV clone 13 is set forth as SEQ ID NO: 2. The sequence of the S segment is the sequence set forth in SEQ ID NO: 1. The L segment of LCMV clone 13 The sequence of the S segment of LCMV strain MP is set forth as SEQ ID NO: 5. The sequence of the L segment of LCMV strain MP is set forth as SEQ ID NO: 53. It is written.

[0236] In another embodiment, a cell comprising the cDNA or vector system described above in this section , provided herein. Cell lines derived from infected such cells, such cells Cultures comprising such cells, and methods for culturing such cells, are also provided herein. In one embodiment, the cell containing the tripartite arenavirus particle cDNA is In some embodiments, the cells are provided in a manner similar to that described above. Includes segments.

[0237] (4.6 How to use) Vaccines are used to prevent and / or treat infectious diseases, such as those against poliovirus and measles. However, established chronic diseases, including both chronic infections and cancer, Therapeutic immunization in the disease setting has not been very successful. The ability to produce tripartite arenavirus particles is a novel, previously unseen, This means a vaccine strategy.

[0238] In one embodiment, an arenavirus particle or tripartite as described herein administering to a subject one or more types of arenavirus particles, or compositions thereof, to the subject. Provided herein are methods for treating infections and / or cancer in In embodiments, the methods of treating an infectious disease and / or cancer described herein include: an amount of one of the arenavirus particles or tripartite arenavirus particles described herein The method includes administering the above or a composition thereof to a subject in need thereof. These include, but are not limited to, humans, mice, rats, guinea pigs, Sugars from livestock such as cows, horses, sheep, pigs, goats, cats, dogs, hamsters, and donkeys In a specific embodiment, the subject is a human. The elephants were men, women, adults, children, the elderly (65 years and older), and those with multiple illnesses (i.e. In some embodiments, the subject may be a patient receiving chemotherapy, radiation therapy, or other treatments. Those whose disease has progressed after radiation therapy, surgery, and / or biologic treatment .

[0239] In another embodiment, an antigen expressing an antigen from an infectious organism, a tumor, or an allergen is and administering to a subject arenavirus particles or tripartite arenavirus particles, or a composition thereof. and generating an immune response in a subject to an antigen derived from an infectious organism, a tumor, or an allergen, including A method for deriving an answer is provided herein.

[0240] In another embodiment, the method comprises administering to a subject a subject infected with an infectious organism, tumor, or allergen described herein. Arenavirus particles or tripartite arenavirus particles expressing the original antigen, or compositions thereof The subject to whom the substance is administered may have or be at risk of an infection, cancer progression, 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. Arenavirus particles or tripartite arenavirus particles expressing an antigen, or compositions thereof The subject is infected with an infectious disease, cancer, a precancerous tissue lesion, or an allergy. whether you have, are susceptible to, or are at risk for, or have an infection, cancer, or precancerous tissue lesion or diagnosed with allergies.

[0241] In another embodiment, the method comprises administering to a subject a subject infected with an infectious organism, tumor, or allergen described herein. Arenavirus particles or tripartite arenavirus particles expressing the original antigen, or compositions thereof The subject to whom the substance is administered may have a disease, particularly in the pulmonary system, central nervous system, lymphatic system, gastrointestinal system, or circulatory system. have or are susceptible to infections, cancer, precancerous lesions, or allergies in In a specific embodiment, the subject is a subject who has or is at risk of having the disease described herein. Arenavirus particles or particles expressing antigens derived from infectious organisms, tumors, or allergens The subject to which the tripartite arenavirus particles or a composition thereof is administered may have tumors in the brain, liver, lungs, eyes, one of the body's organs, including but not limited to the ear, intestine, esophagus, uterus, nasopharynx, or salivary glands Suffering from or prone to infection, cancer, or allergies in one or more organs or is at risk of being affected.

[0242] In another embodiment, the method comprises administering to a subject a cancer- or infectious organism-derived or allergen-derived cancer described herein. Arenavirus particles or tripartite arenavirus particles expressing the original antigen, or Subjects to whom the composition may be administered include those suffering from 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 Internal ulcers, visual impairment, hepatitis, jaundice, encephalitis, seizures, coma, pruritus, erythema, hyperpigmentation, The subject may be a patient suffering from symptoms including, but not limited to, lymph node changes, or hearing loss. can be done.

[0243] In another embodiment, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. arenaviruses or tripartite arenavirus particles expressing antigens derived from the virus, or The composition of the present invention is intended to be used to treat or prevent the development of diseases such as infections, cancer, or allergies. It is administered to subjects of any age group who are at risk. An arenavirus expressing an antigen derived from an infectious organism, cancer, or allergen as described. The virus particles or tripartite arenavirus particles, or compositions thereof, are useful in treating patients with a compromised immune system. subjects who are pregnant, have undergone organ or bone marrow transplantation, or are taking immunosuppressants subjects with a history of infection, cancer, or allergies, subjects receiving hemodialysis, or subjects with cancer or allergies administered to subjects who are suffering from, susceptible to, or at risk of allergies In more specific embodiments, the present invention is directed to an infectious organism, a cancer, or a combination thereof, as described herein. or arenavirus particles or tripartite arenaviruses expressing antigens derived from allergens The particles, or compositions thereof, may be used to treat or prevent infection, cancer, or allergies. Susceptible or at risk 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 In yet another specific embodiment, the drug is administered to a subject who is a child between the ages of 16, 17, or 18. and expressing an antigen derived from an infectious organism, cancer, or allergen as described herein. Arenavirus particles or tripartite arenavirus particles, or compositions thereof, are useful in the treatment of infectious diseases, Infants who have, are prone to, or are at risk of developing cancer or allergies In yet another specific embodiment, the method described herein is administered to a subject who is a child. Arenavirus particles or viruses expressing antigens derived from infectious organisms, cancers, or allergens The tripartite arenavirus particles or compositions thereof are useful for treating infectious diseases, cancer, or allergies. Have, be susceptible to, or are at risk 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 In yet another specific embodiment, the subject is an infant between the ages of 10, 11, or 12 months. In embodiments, an antigen derived from an infectious organism, cancer, or allergen described herein is The arenavirus particles or tripartite arenavirus particles, or compositions thereof, that express: Do you have, are susceptible to, or are at risk of developing an infection, cancer, or allergy? In a more specific embodiment, the sensitization inhibitors described herein are administered to an elderly subject. Arenavirus particles or 3 expressing antigens derived from infectious organisms, cancer, or allergens Segmented arenavirus particles, or compositions thereof, are also contemplated. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 years of age It is administered to subjects who are elderly subjects.

[0244] In another embodiment, the method comprises administering to a subject a cancer- or infectious organism-derived or allergen-derived cancer described herein. Arenavirus particles or tripartite arenavirus particles expressing the original antigen, or The composition is administered to a subject at increased risk of disseminated infection, cancer, or allergy. In specific embodiments, the method comprises administering to a subject an infectious organism, cancer, or animal described herein. Arenavirus particles or tripartite arenavirus particles expressing allergen-derived antigens or the composition thereof, in a neonatal subject having a neonatal and therefore immature immune system. It is administered.

[0245] In another embodiment, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from the virus, or The composition is administered to a subject with a dormant infection, cancer, or allergy. In specific embodiments, an infectious organism, cancer, or allergen described herein Arenavirus particles or tripartite arenaviruses expressing antigens derived from the virus, or combinations thereof The composition may be used to treat dormant infections, dormant cancers, or other cancers that can reactivate when the immune system is compromised. It is administered to subjects with dormant allergies. Therefore, it is possible to prevent infection, cancer, or allergies. Methods for preventing reactivation of HIV-1 are provided herein.

[0246] In another embodiment, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from the virus, or The composition is administered to a subject with a recurrent infection, cancer, or allergy.

[0247] In another embodiment, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from the virus, or The composition is administered to a subject with a genetic predisposition to an infectious disease, cancer, or allergy. In another embodiment, the present invention relates to an infectious organism, cancer, or allele as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from the virus, or In another embodiment, the composition is administered to a subject. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from allergens and administered to subjects with risk factors. Exemplary risk factors include aging, tobacco, Sun exposure, radiation exposure, chemical exposure, family history, alcohol, poor diet, physical activity Possible causes include lack of exercise or being overweight.

[0248] In another embodiment, an antigen expressing an antigen from an infectious organism, a cancer, or an allergen is Administration of arenavirus particles or tripartite arenavirus particles can improve symptomatic infection, In another embodiment, the method reduces the risk of infection with an infectious organism, cancer, or allergy. Arenavirus particles expressing antigens derived from allergens or tripartite arenavirus particles The administration reduces asymptomatic infections, cancers, or allergies.

[0249] In another embodiment, an allele that expresses an antigen from an infectious organism described herein is provided. arenavirus particles or tripartite arenavirus particles, or compositions thereof, Virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus Rus, infectious laryngotracheitis virus, chicken anemia virus, Marek's disease virus, avian Leukemia virus, avian adenovirus, or avian pneumovirus, the virus that causes SARS virus, human respiratory syncytial virus, human immunodeficiency virus, hepatitis A virus, hepatitis B virus Viruses, Hepatitis C virus, Polio virus, Rabies virus, Hendra virus, Pavirus, human parainfluenza type 3 virus, measles virus, mumps virus, Ebola virus, Marburg virus, West Nile disease virus, Japanese encephalitis virus , Dengue virus, Hantavirus, Rift Valley fever virus, Lassa fever virus, Simple Injected into subjects or animals infected with one or more strains of herpesvirus and yellow fever virus It is given.

[0250] In another embodiment, an arenavirus expressing an antigen derived from a cancer described herein is The virus particles or tripartite arenavirus particles, or compositions thereof, are useful in treating patients suffering from one or more types of cancer. In other embodiments, the vaccines described herein are administered to animals that are sensitive to treatment. In a more specific embodiment, the present invention can target any type of cancer for which the cancer is susceptible. Arenavirus particles or tripartite arenaviruses expressing cancer-derived antigens described in The lus particles or compositions thereof can be used to treat, for example, melanoma, prostate cancer, breast cancer, lung cancer, neuroblastoma, Hepatocellular carcinoma, cervical cancer, and gastric cancer, Burkitt's lymphoma; non-Hodgkin's lymphoma; Hodgkin's lymphoma nasopharyngeal carcinoma (cancer of the upper part of the throat behind the nose), leukemia, mucosa-associated lymphoid tissue lymphoma, etc. The compound is administered to a subject suffering from

[0251] In another embodiment, arenaviruses expressing antigens derived from the allergens described herein are The virus particle or tripartite arenavirus particle, or composition thereof, may contain one or more alleles. In a more specific embodiment, the anti-inflammatory drugs described herein are administered to a subject suffering from hypertension. Arenavirus particles or tripartite arenavirus particles expressing allergen-derived antigens or a composition thereof, for example, for seasonal allergies, perennial allergies, rhinitis, conjunctivitis, asthma, , eczema, and food allergies.

[0252] In another embodiment, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from the virus, or Administering the composition to a subject induces cellular immunity to an infectious disease, cancer, or allergen. Without being bound by theory, in another embodiment, Arenaviruses that express antigens derived from infectious organisms, cancers, and allergens, as described in the literature. The virus particles or tripartite arenavirus particles, or compositions thereof, bind to the major histocompatibility complex ( host (e.g., macrophages, dendritic cells) for direct presentation of antigens on MHC class I and II The cells then infect and express the antigen of interest in antigen-presenting cells (APCs) of the erythrocyte, erythrocyte, or B cell. In an embodiment, the antibody is derived from an infectious organism, cancer, allergen, or the like, as described herein. Antigen-expressing arenavirus particles or tripartite arenavirus particles, or compositions thereof The administration of the compound to the subject is highly important, multifunctional, cytolytic, and IFN-γ and TNF-α. Induce co-produced CMV-specific CD4+ and CD8+ T cell responses to prevent infection, cancer, or allergies Treat or prevent

[0253] In another embodiment, an antigen expressing an antigen from an infectious organism, a cancer, or an allergen is administering arenavirus particles or tripartite arenavirus particles, or compositions thereof; reduces an individual's risk of developing an infection, cancer, or allergy in the absence of such treatment. at least about 10% lower risk of developing an infection, cancer, or allergy compared with the risk of developing an infection, cancer, or allergy in the 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 about 60%, at least about 70%, at least about 80%, A reduction of at least about 90% or more.

[0254] In another embodiment, an antigen expressing an antigen from an infectious organism, a cancer, or an allergen is administering arenavirus particles or tripartite arenavirus particles, or compositions thereof; symptoms of infection, cancer, or allergy in the absence of such treatment At least about 10%, at least about 20%, or less than the manifestation of symptoms of cancer or allergies. 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 reduce it beyond that.

[0255] In one embodiment, the antigen-expressing cell is a cell line that expresses an antigen from an infectious organism, a cancer, or an allergen. The arenavirus particles or tripartite arenavirus particles are preferably administered by multiple injections (e.g. For example, 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 multiple sites (e.g., In some embodiments, the tumor is administered to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 sites. In the method, an arenavirus expressing an antigen derived from the infectious organism, cancer, or allergen is arenavirus particles or tripartite arenavirus particles, given as two or more separate injections, for 6 months, 12 months, or In certain embodiments, the infectious organism, The arenavirus particles or tripartite arenaviruses expressing antigens derived from cancer or allergens. The first dose is administered on the chosen day, the second dose at least two months after the first dose, and and a third dose administered 6 months after the first dose.

[0256] In some instances, dermal injections are administered at multiple sites on the body to reduce the severity of local skin reactions. On a given vaccination day, patients will each receive a dose (e.g., at least 0.4 ml) , 0.2 ml, or 0.1 ml) at least about 5 cm (e.g., at least Each injection was performed 4.5, 5, 6, 7, 8, 9, or 10 cm apart, with 3 to 5 separate injections from a single syringe. On the next vaccination day, each patient will receive the total assigned dose of cells administered by intravenous injection. Rotate between different hands or feet in a clockwise or counterclockwise direction.

[0257] In another embodiment, a CMV antigen is expressed in a subject having a neonatal immune system. Administering an infectious, replication-deficient arenavirus or composition thereof can be used to treat such infections. Reduces cell-mediated immune (CMI) responses to infection, cancer, or allergies in the absence of 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 about 60%, at least about 70%, at least At least about 80%, at least about 90%, or more, of the patients are at high risk of infection, cancer, or allergies. It elicits a CMI response to

[0258] In certain embodiments, the method comprises administering to a subject an infectious organism, cancer, or allele as described herein. Targeting arenavirus particles expressing antigens derived from genes or tripartite arenavirus particles The administration induces detectable antibody titers for a minimum of at least 4 weeks. In a method of treating a cancer, an infectious disease, or an allergen, the method comprising administering to a patient a therapeutically effective amount of an antigen derived from an infectious organism, a cancer, or an allergen as described herein. Administering to a subject arenavirus particles or tripartite arenavirus particles expressing the At least 100%, at least 200%, at least 300%, at least 400%, at least 50 0%, or increase antibody titers by at least 1000%.

[0259] In one embodiment, exposure to the primary antigen is measured using an average control population derived from an infection-immune human subject. At least 50%, at least 100%, at least 200%, at least 300%, or less of the serum Induces a minimum functional (neutralizing) antibody titer of at least 400%, at least 500%, or at least 1000% In a more specific embodiment, the primary neutralizing geometric mean antibody titer is at least at least 1:50, at least 1:100, at least 1:200, or at least 1:100 within 4 weeks In another embodiment, the sensitization of the sensitizer as described herein increases to a peak value of 1000. Arenavirus particles or tripartites expressing infectious, cancer, or allergy-derived antigens Immunization with type 1 arenavirus particles can be administered after a single dose of vaccine or after two or more consecutive doses. at least 4 weeks, at least 8 weeks, at least 12 weeks, at least at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 It produces high antibody titers that persist for years or at least 5 years.

[0260] In yet another embodiment, exposure to a second antigen increases antibody titers by at least 100%, at least At least 200%, at least 300%, at least 400%, at least 500%, or at least 1000 In another embodiment, exposure to a secondary antigen increases the HIV-1 response in a human subject immune to the infection by 100%. 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) activity In a more specific embodiment, the secondary neutralizing geometric mean antibody titer is: at least 1:50, at least 1:100, at least 1:200 within at least 4 weeks after immunization; or to a peak value of at least 1:1000. Arenaviruses that express antigens derived from infectious organisms, cancer, or allergies, such as a second immunization with the virus particles or tripartite arenavirus particles at least 4 weeks after the first immunization; At least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least High resistance lasting for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. Produces body titers.

[0261] In yet another embodiment, the third boosting immunization is At least 100%, at least 200%, at least 300%, at least 400%, In another embodiment, the boost immunization increases the immune response by at least 500%, or by at least 1000%. The antibody titer should be at least 50% of the mean control serum from human subjects immune to the infectious disease, at least 10% of the mean control serum from human subjects immune to the infectious disease. 0%, at least 200%, at least 300%, at least 400%, at least 500%, or less In a more specific embodiment, the antibody induces a minimum functional (neutralizing) antibody titer of at least 1000%. The third boost immunization is administered using a small amount of the average control serum from a human subject immune to the infection. 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 another embodiment, the third boost immunization increases antibody titers by at least 100 mg / kg post-immunization. 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months , extend for at least two years, at least three years, at least four years, or at least five years .

[0262] In one embodiment, the antigen expressing the infectious organism, cancer, or allergy is Arenavirus particles or tripartite arenavirus particles can be T cell-independent or T cell-dependent. In other embodiments, the antibody induces a virulent response due to an infectious organism, cancer, or allergy. Arenavirus particles or tripartite arenavirus particles expressing the antigen induce T cell responses. In other embodiments, the present invention is directed against an infectious organism, cancer, or Arenavirus particles or tripartite arenavirus particles expressing allergens are In another embodiment, the infection as described herein induces a T helper response. Arenavirus particles or tripartites expressing antigens derived from sexual organisms, cancer, or allergies Arenavirus particles induce either a Th1- or a Th2-oriented response. induces.

[0263] In a more specific embodiment, said Th1 adaptive response is characterized by a predominance of IgG1 antibodies over IgG2. In other embodiments, the ratio of IgG1:IgG2 is greater than 1:1, greater than 2:1, greater than 3:1, or greater than 4: In another embodiment, the antibody is selected from the group consisting of an infectious organism, a cancer, a leukemia, a leukemia virus ... or infectious arenavirus particles or tripartite arenaviruses expressing antigens of allergy origin The virus particles are displayed by predominantly IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, or IgE antibodies. can be.

[0264] In some embodiments, an infectious, replication-deficient antigen expressing a CMV antigen or a fragment thereof is In another embodiment, the infectious organism is a leukemia virus that induces a CD8+ T cell response. Arenavirus particles or tripartite arenaviruses expressing antigens derived from cancer or allergies The virus particles induce both CD4+ and CD8+ T cell responses, either in combination with antibodies or in combination with antibodies. It induces without any warning.

[0265] In certain embodiments, the method comprises administering to a subject an infectious organism, cancer, or animal as described herein. Arenavirus particles or tripartite arenavirus particles expressing antigens derived from allergies are In another embodiment, the antibody is a human antibody as described hereinabove, which induces high titers of neutralizing antibodies. Arenavirus particles or 3 expressing antigens derived from various infectious organisms, cancer, or allergies Segmented arenavirus particles produce higher titers of neutralizing antibodies than expression of individual protein complex components. Trigger the body.

[0266] In another embodiment, one, two, three, four, five, or more infectious organisms, cancer, or Arenavirus particles or tripartite arenavirus particles expressing allergens are , arenavirus particles or particles expressing one antigen from an infectious organism, cancer, or allergen induces higher titers of neutralizing antibodies than tripartite arenavirus particles.

[0267] In certain embodiments, the method comprises administering to the patient a virus particle or a tripartite arenavirus. In some embodiments, the method further comprises co-administering the particles and at least one additional therapy. The administration is simultaneous. In another embodiment, the allele is administered prior to administration of the additional therapy. The arenavirus particles or tripartite arenavirus particles are administered after the administration of the boost therapy. In some embodiments, arenavirus particles or tripartite arenavirus particles and additional therapy The administration of the present invention is carried out for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours. In certain embodiments, the incubation time is about 9 hours, about 10 hours, about 11 hours, or about 12 hours. The interval between administration of the viral particles or tripartite arenavirus particles and the additional therapy is about 1 day, 1 weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about In some embodiments, the arenavirus particles or 3 The interval between administration of segmented arenavirus particles and additional therapy is approximately 1 month, approximately 2 months, approximately 3 months, About 4 months, about 5 months, or about 6 months.

[0268] In some embodiments, the antigen expressing the antigen from an infectious organism, cancer, or allergen is Administering viral particles or compositions thereof may be performed using a patient's blood sample, or serum sample. In one embodiment, the number of antibodies detected in the sample is reduced. administration of arenavirus particles expressing antigens derived from cancer or its allergen composition means the antigen detected in urine, saliva, blood, tears, semen, exfoliated cell samples, or breast milk. Reduces the amount of infectious organisms, cancer, or allergies.

[0269] In another embodiment, an infectious organism, cancer, or allergen as described herein. Arenavirus particles or tripartite arenavirus particles, or compositions expressing antigens derived from The composition may further comprise a reporter protein. and antigens and reporters derived from infectious organisms, cancers, or allergens as described herein. Arenavirus particles or tripartite arenavirus particles expressing target proteins, or The composition may be administered to a subject to treat and / or prevent an infection, cancer, or allergy. In yet another specific embodiment, said reporter protein is administered Protein expression, protein localization, and vaccine delivery can be monitored in vivo, in situ, and in vivo. It can be used for real-time monitoring.

[0270] In another embodiment, an infectious organism, cancer, or allergen as described herein. Arenavirus particles or tripartite arenavirus particles, or compositions expressing antigens derived from The composition may further comprise a fluorescent protein. Antigens and reporters derived from infectious organisms, cancers, or allergens as described herein Arenavirus particles or tripartite arenavirus particles expressing the protein, or compositions The composition is administered to a subject to treat and / or prevent an infection, cancer, or allergy. In yet another specific embodiment, the fluorescent protein is a reporter protein. The quality of the gene expression, protein localization, and vaccine delivery can be determined in vivo. It can be used for in situ and real-time monitoring.

[0271] Arenaviruses expressing antigens derived from infectious organisms, cancers, or allergens in a subject arenavirus particles or tripartite arenavirus particles, or a composition thereof, Changes in CMI response function to infectious diseases, cancer, or allergies were observed using flow cytometry. cytometry (see, e.g., Perfetto SP et al., 2004, Nat Rev Immun., 4(8):648-55) ), lymphocyte proliferation assays (see, e.g., Bonilla FA et al., 2008, Ann Allergy Asthma Immunol, 101:101-4; and Hicks MJ et al., 1983, Am J Clin Pathol., 80:15 9-63), measurement of surface marker expression after activation of T lymphocytes by measuring cytokines. Assays measuring lymphocyte activation, including determining changes in lymphocyte activity (e.g., Caruso A. et al., 2014). Cytometry. 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., 1 989, J Immunol Methods 120:1-8), or natural killer cell cytotoxicity toxicity assay (e.g., Bonilla FA et al., 2006, Ann Allergy Asthma Immunol., 94 (5 Suppl 1):S1-63) known to those skilled in the art. It can be measured by any assay.

[0272] Successful treatment of cancer patients is defined as extending expected survival time, inducing an antitumor immune response, or Examples of cancer characteristics that can be improved include tumor size, size (e.g., T0, T1, or T4), metastatic status (e.g., M0, M1), and number of observable tumors , nodal involvement (e.g., N0, N1–4, Nx), grade (i.e., grade 1, 2, 3, or 4), stage (e.g., 0, I, II, III, or IV), the presence of certain markers on cells or in body fluids, Presence or concentration (e.g., AFP, B2M, beta-HCG, BTA, CA 15-3, CA 27.29, CA 125, CA 72.4) , CA 19-9, calcitonin, CEA, chromgrainin A, EGFR, hormone receptor Condition, HER2, HCG, immunoglobulin, NSE, NMP22, PSA, PAP, PSMA, S-100, TA-90, and thyroglobulin), and / or associated pathologies (e.g., ascites or edema) or symptoms ( For example, cachexia, fever, loss of appetite, or pain. In such cases, the improvement is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90%. The time to survival, or tumor volume or linear dimension, can be measured.

[0273] In another embodiment, the ORFs encoding the GP, NP, Z protein, and L protein are At least one of these is an antigen derived from an infectious organism, cancer, or allergen, or an antigenic fragment thereof. The infectious nucleotide sequence encoding , expressing antigens derived from infectious organisms, cancers, or allergens as described herein. Methods of use using arenavirus particles (eg, LCMV) are described herein.

[0274] 4.7 Compositions, Administration, and Dosages The present application further provides an arenavirus particle or tripartite arenavirus described herein. and vaccines, immunogenic compositions (e.g., vaccine formulations), and pharmaceutical compositions comprising the particles. Such vaccines, immunogenic compositions, and pharmaceutical compositions are well known in the art. The formulation may be carried out according to conventional procedures.

[0275] Suitable modifications and adaptations to the methods and applications described herein may be apparent, and It will be readily apparent to one skilled in the art that other embodiments may be made without departing from the scope thereof or any of its embodiments. It will become clear.

[0276] In another embodiment, the arenavirus particles or tripartite arenavirus particles described herein are Compositions comprising viral particles are provided herein. Such compositions are useful for preventing disease. In a specific embodiment, the compounds described herein may be used in methods for the treatment and prevention of The compositions are used in the treatment of subjects infected with or susceptible to infectious diseases. In other embodiments, the compositions described herein are used to treat cancer or tumor formation. susceptible to, or exhibiting symptoms characteristic of, cancer or tumor formation, or diagnosed with cancer In another specific embodiment, the compounds described herein are used in the treatment of subjects suffering from The immunogenic compositions provided herein can be used to induce an immune response in a host to which the compositions are administered. The immunogenic compositions described herein can be used as vaccines. In a specific embodiment, the present invention can be used in combination with other compounds and formulated into pharmaceutical compositions accordingly. The immunogenic compositions described herein are useful in preventing infectious diseases or cancer in subjects (e.g., human subjects). In another embodiment, the vaccine, immunogenic composition, or pharmaceutical composition The products are suitable for veterinary administration and / or human administration.

[0277] In certain embodiments, an immune response comprising an arenavirus vector as described herein is provided. Immunogenic compositions are provided herein. In certain embodiments, such immunization compositions The composition further comprises a pharmaceutically acceptable excipient. Such immunogenic compositions further comprise an adjuvant. Adjuvants for administration in combination may be administered prior to, simultaneously with, or in combination with the administration of the composition. In some embodiments, the term "adjuvant" is used herein to refer to an adjuvant. arenavirus particles when administered in combination with or as part of a composition described in and / or enhance the immune response to avian or trisegmented arenavirus particles. It refers to a compound that expresses a gene product, and most importantly, the gene product that it vectorizes. When administered alone, arenavirus particles or tripartite arenavirus particles, and In some embodiments, the vector does not generate an immune response against the gene product delivered by the recipient. In embodiments, the adjuvant is an arenavirus particle or a tripartite arenavirus particle. The latter generates an immune response against the vectorized gene product and Adjuvants have the potential to, for example, mobilize lymphocytes, B Several functions, such as stimulation of cells and / or T cells, and stimulation of macrophages or dendritic cells The vaccine or immunogenic composition of the present invention may contain an adjuvant, thereby enhancing the immune response. If included or administered with one or more adjuvants, the adjuvants that may be used Examples of adjuvants include mineral salt adjuvants, mineral salt gel adjuvants, and particulate adjuvants. These include microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum aluminum hydroxide, aluminum phosphate, and aluminum sulfate, etc.), Sylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas) , Inc.), imidazopyridine compounds (published in International Publication No. WO2007 / 109812 See International Application No. PCT / US2007 / 064857), imidazoquinoxaline compounds (International Publication No. See International Application No. PCT / US2007 / 064858, published as WO2007 / 109813. ), and saponins such as QS21 (Kensil et al., "Vaccine Design" , 1995: "The Subunit and Adjuvant Approach" h)" (Powell & Newman, eds., Plenum Press, NY; see U.S. Patent No. 5,057,540) In some embodiments, the adjuvant includes, but is not limited to, , Freund's adjuvant (complete or incomplete). Other adjuvants are optionally oil-in-water emulsions (squalene or peanut oil) (see Stoute et al., 1997, N. Engl. J. Med. 336, 86-91). (I want to be).

[0278] The composition may comprise an arenavirus particle or a tripartite arenavirus as described herein. particles alone or together with a pharmaceutically acceptable carrier. Use of a suspension or dispersion of segmented arenavirus particles, especially an isotonic aqueous suspension or dispersion. The pharmaceutical composition may be sterilized and / or may contain excipients, e.g., preservatives. , stabilizers, wetting agents, and / or emulsifiers, solubilizers, salts for adjusting the osmotic pressure, and / or may contain buffers and may be prepared in a manner known per se, for example by conventional dispersion and suspension processes. In one embodiment, such a dispersion or suspension is prepared by a process comprising: The suspension or dispersion may contain a viscosity modifier. The suspension or dispersion is kept at a temperature of about 2°C to 8°C. or, preferentially, for longer storage, frozen and then thawed immediately prior to use. For injection, the vaccine may be lyophilized or lyophilized for storage. Alternatively, the immunogenic preparation may be in aqueous solution, preferably in Hank's solution, Ringer's solution, or saline solution. The solution may be formulated in a physiologically compatible buffer, such as a saline buffer. The composition may also contain a formulatory agent such as a binder and / or a dispersant.

[0279] In certain embodiments, the compositions described herein do not contain preservatives, such as mercury derivatives. In a specific embodiment, the pharmaceutical composition described herein further comprises thimerosal. In another embodiment, the composition described herein comprises 0.001% to 0.01% thimerosal. The pharmaceutical composition is free of preservatives.

[0280] The pharmaceutical composition comprises about 10 3 ~about 10 11 of focus-forming units of arenavirus particles or 3 min Contains nodal arenavirus particles.

[0281] In one embodiment, the administration of the pharmaceutical composition is parenteral. The unit dosage form for parenteral administration may be, for example, For example, ampoules or vials, e.g., about 10 3 ~10 10 Focus formation unit or 10 5 ~10 15 A virus containing one or more physical particles of said arenavirus particles or tripartite arenavirus particles It is Ial.

[0282] In another embodiment, the vaccine or immunogenic composition provided herein comprises: These include oral, intradermal, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal, and inhalation. routes, including, but not limited to, scarification techniques (e.g., using a bifurcated needle to remove the top layer of skin) Specifically, subcutaneous or intravenous routes may be used.

[0283] For administration intranasally or by inhalation, the preparations for use according to the invention are preferably In this case, a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, Use of fluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. Presentation of an aerosol spray from a pressurized pack or nebulizer, In the case of a pressurized aerosol, the dosage unit may be a device that delivers a metered amount. This may be determined by providing a valve that allows the inhaler or insufflator to Capsules and cartridges, e.g., of gelatin, for use in the manufacture of pharmaceuticals containing the compound and a suitable powder base such as lactose or starch. This may also be done.

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

[0285] In certain embodiments, the viral vectors include arenavirus particles or tripartite arenavirus particles. The vaccine, immunogenic composition, or pharmaceutical composition containing the virus may be used as a live vaccination. Typical doses of live arenavirus particles range from 10 to 100 or more live viruses per dose. In some embodiments, the PFU of the arenavirus particles or tripartite arenavirus particles may vary. The appropriate dosage of lenavirus particles is 10 2 , 5×10 2 , 10 3, 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5 x10 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, and should be tested once, twice, or three times at intervals as often as necessary. In another embodiment, the live arenavirus may be administered to a subject in 0.2 mL or more. The dose is 10 6.5 ~10 7.5 Fluorescent focus units of live arenavirus particles were prepared. In another embodiment, the inactivated vaccine is formulated into a dose of about 15 μg to about 100 μg, about 15 μg about 15 μg to about 75 μg, about 15 μg to about 50 μg, or about 15 μg to about 30 μg of arenavirus It is formulated into

[0286] In one embodiment, for pediatric administration, two doses given at least one month apart are used. a dose of arenavirus particles or tri-segmented arenavirus particles described herein or the composition thereof is administered to a child. In a specific embodiment, the composition is administered to an adult. To achieve this, a single dose of the arenavirus particles or tripartite arenavirus described herein is administered. In another embodiment, the particles, or compositions thereof, are administered at least one month apart. two doses of the arenavirus particles or tripartite arenavirus particles described herein given in a single dose; In another embodiment, the viral particles, or compositions thereof, are administered to adults. Children (6 months to 9 years of age) should first receive the 2 doses described herein given 1 month apart. arenavirus particles or trisegmented arenavirus particles, or a composition thereof, In certain embodiments, only one dose is received during the first year of vaccination. Children without steroids should receive two doses every four weeks. The two doses administered at different times are administered to a patient receiving the immunogenic composition described herein for the first time. In some embodiments, it may be preferred for subjects over the age of 3. Unlike the commonly used 0.5 ml, a half dose (0.25 ml) may be preferred for children 6 to 35 months of age. There is a possibility.

[0287] In certain embodiments, the composition comprises a therapeutically effective amount of arenavirus particles or tripartites. In some embodiments, a single dosage containing the type arenavirus particles may be administered to the patient. wherein the arenavirus particles or tripartite arenavirus particles are administered in a therapeutically effective amount arenavirus particles or trisegmented arenavirus particles, each in a therapeutically effective amount. A single dose containing the above pharmaceutical composition may be administered to a patient.

[0288] In one embodiment, the composition comprises a single dose followed by a second dose 3 to 6 weeks later. These embodiments allow for the administration of a second dose at intervals of 6 to 12 months after the first dose. In some embodiments, the booster vaccination may be administered to the subject. In some embodiments, different arenaviruses or compositions thereof may be utilized. In this case, administration of the same composition as described herein may be repeated, and the administration may be continued for at least one day. , 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 They may be spaced apart by months, or at least six months.

[0289] Also, a method for producing a virus containing the arenavirus particles or trisegmented arenavirus particles as an active ingredient is provided. A process for the production of a vaccine in the form of a pharmaceutical preparation and a method for the production of arenavirus particles or trinucleotides Also provided herein are uses of knotted arenavirus particles. They are prepared in a manner known per se, for example by conventional mixing and / or dispersing processes.

[0290] 4.8 Assay 4.8.1 Arenavirus Detection Assays Those skilled in the art will be able to easily implement the methods described herein using techniques known in the art. These arenavirus genome segments or tripartite arenavirus particles can be detected. For example, RT-PCR can be used with arenavirus-specific primers to identify the ORF. Arenavirus genome segments engineered to retain RF at a position other than the wild-type position Western blot, E LISA, radioimmunoassay, immunoprecipitation, immunocytochemistry using immunocytochemistry, or immunocytochemistry combined with FACS. Quantitating arenavirus genome segments or gene products of tripartite arenavirus particles possible.

[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 an arenavirus vector preparation. For example, determining the virus / vector titer can be used to measure the "focus-forming unit activity." Briefly, complementation cells, e.g., MC57 cells, can be used to measure the FFU (Frequency-Fuel-Unit) levels. Plates are then inoculated with different dilutions of the virus / vector sample. After the incubation period, cells are allowed to form a monolayer and the virus is allowed to attach to the cells. The monolayer is then covered with methylcellulose. The plates are further incubated to remove the original infection. The cells release the virus progeny. Due to the methylcellulose overlay, the new virus The spread of the virus is restricted to neighboring cells. As a result, each infectious particle These foci give rise to circular regions of infected cells called foci. or another protein expressed by an arenavirus particle or a tripartite arenavirus particle. It can be visualized using antibodies against proteins and an HRP-based color reaction, The virus / vector titer can be calculated as the number of viruses per milliliter. It can be calculated in focus forming units (FFU / mL).

[0292] 4.8.3 Multiplication of Arenavirus Particles The propagation of arenavirus particles described herein can be carried out using methods known in the art or This can be assessed by any of the methods described herein (e.g., cell culture). Growth can be achieved by injecting the alleles described herein into cell (e.g., Vero or BHK-21) cultures. This may be determined by inoculating serial dilutions of the virus particles. After incubation, the virus is isolated using standard methods.

[0293] (4.8.4 Serum ELISA) The humoral immune response during vaccination of animals (e.g., mice, guinea pigs) is determined by antigen-specific This can be done by differential serum ELISA (enzyme-linked immunosorbent assay). Cover with antigen (e.g., recombinant protein) and block to avoid non-specific binding of antibodies After incubation, the bound Serum-antibodies can be isolated, for example, using enzyme-linked anti-species (e.g., mouse, guinea pig) specific antibodies (enzyme-c coupled anti-species-specific antibody) (detecting total IgG or IgG subclasses), and The antibody titer can be determined, for example, by measuring the end-point geometric mean. It can be determined as an average titer.

[0294] 4.8.5 Assays for Measuring Neutralizing Activity of Induced Antibodies Determination of neutralizing antibodies in serum was performed using ATCC-derived ARPE-19 cells and GFP-tagged virus. In addition, additional guinea pigs as a source of exogenous complement were used in the following cell assays: This assay is performed in 384-well plates one or two days before use for neutralization. 6.5 x 10 3 Start by seeding cells / well (50 μl / well). Neutralization is performed in a 96-well plate. Neutralization incubation is performed in a sterile tissue culture plate without cells at 37°C for 1 hour. After the incubation step, the mixture was added to the cells and incubated for another 4 days for GFP detection in a plate reader. On each plate, positive neutralizing human serum was added. sera) is used as a positive control in the assay to check the reliability of all results. C50) is determined using a four-parameter logistic curve fit. The wells are then checked under a fluorescent microscope.

[0295] 4.8.6 Plaque Reduction Assay Briefly, the plaque reduction (neutralization) assay for LCMV was performed using a green fluorescent protein-tagged LCMV vector. This can be done using replicated replication-competent or replication-deficient LCMV, and 5% rabbit serum can be used as exogenous The plaques can be enumerated by fluorescence microscopy. Neutralizing titers are defined as the number of plaques that are neutralized compared to those in control (pre-immune) serum samples. It may be defined as the highest dilution of serum that results in a 75%, 90%, or 95% reduction.

[0296] QIAamp viral RNA mini kit (QIAGEN) was used according to the protocol provided by the manufacturer. The LCMV RNA genome is isolated using SuperSc ript® III Platinum® One-Step qRT-PCR Kit (Invitrogen), and Primers and a portion of the LCMV NP coding region or the arenavirus particle or tripartite arenavirus Probes specific for different genomic segments of the nasal virus particle (FAM reporter and NFQ-MG) on a StepOnePlus real-time PCR system (Applied Biosystems) using a quencher The reaction was detected by quantitative PCR. The temperature profile was: 60°C for 30 minutes, 95°C for 1 minute, Alternatively, the incubation may be at 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds and 56°C for 30 seconds. The results of the assay were compared with fragments of the LCMV NP coding sequence or with alleles containing primer and probe binding sites. Spectrophotometric analysis of the genomic segments of the arenavirus or tripartite arenavirus particles. Comparison with a standard curve generated from a log10 dilution series of in vitro transcribed RNA fragments quantified by the method This can be quantified by

[0297] 4.8.7 Western Blotting Infected cells grown in tissue culture flasks or in suspension were incubated at the indicated time points post-infection. Lyse using RIPA buffer (Thermo Scientific) or use directly without cell lysis. The samples were incubated at 99°C for 10 min with a reducing agent and NuPage LDS sample buffer (NOVEX). The mixture was heated for 1 min, cooled to room temperature, and then loaded onto a 4-12% SDS-gel for electrophoresis. Proteins were blotted onto membranes using an Invitrogens iBlot gel transfer device. Finally, the preparations were stained with primary antibodies against the protein of interest and visualized by Ponceau staining. Alkaline phosphatase-conjugated secondary antibody followed by a one-step NBT / BCIP solution (INVITR The tissue is examined using staining with OXYGEN.

[0298] 4.8.8 MHC-Peptide Multimer Staining Assay for Detection of Antigen-Specific CD8+ T Cell Proliferation Any assay known to one of skill in the art can be used to test antigen-specific CD8+ T cell responses. For example, an MHC-peptide tetramer staining assay may be used (see, e.g., Altman JD et al., Science. 1996;274:94-96; and Murali-Krishna K. et al., Immunity. 1998;8:177-187 (See, e.g., tetramer assay). Briefly, this assay involves the following steps: This is used to detect the presence of antigen-specific T-cells. T-cells detect the peptides for which they are specific. To detect the peptide, T cells are exposed to the peptide and T cells (typically fluorescently labeled). Custom-made MHC molecules for a defined antigen specificity and MHC haplotype The tetramer must then be recognized via the fluorescent label. Detected by flow cytometry.

[0299] 4.8.9 ELISPOT Assay for Detection of Antigen-Specific CD4+ T Cell Proliferation Any assay known to one of skill in the art can be used to test antigen-specific CD4+ T cell responses. For example, an ELISPOT assay may be used (see, e.g., Czerkinsky CC et al., J Immunol M Methods. 1983;65:109-121; and Hutchings PR et al., J Immunol Methods. 1989;12 0:1-8). Briefly, the assay involves the following steps: The spot plate is coated with anti-cytokine antibodies. The cells are then immunospotted in the immunospot plate. The cells secrete cytokines and are then washed away. The sample was then coated with a second biotinylated anti-cytokine antibody and visualized with an avidin-HRP system. To visualize.

[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 CD8+ and CD4+ T cell responses. For example, using an intracellular cytokine assay in combination with flow cytometry (See, e.g., Suni MA et al., J Immunol Methods. 1998;212:89-98; Nomura LE Cytometry. 2000;40:60-68; and Ghanekar SA et al., Clinical and Diagnosis. (See, for example, Nostic Laboratory Immunology. 2001;8:628-63). The assay comprises the following steps: activation of cells via specific peptides or proteins; Protein transport inhibitors (e.g., brefeldin A) can be added to preserve cytokines within the cells. After a defined period of incubation, typically 5 hours, a washing step follows, followed by other Antibodies against cellular markers can be added to the cells. The cells are then fixed. Flurochrome-conjugated anti-cytokine antibodies are added, and the cells are then permeabilized. Cells can be analyzed by flow cytometry.

[0301] 4.8.11 Assays to Confirm Replication Defect of Viral Vectors Any assay known to those skilled in the art that determines the concentration of infectious and replicative viral particles may also be used. can be used to measure replication-deficient viral particles in a sample. An FFU assay such as that described above may be used for this purpose.

[0302] Furthermore, plaque-based assays measure the number of plaque-forming units (PFU) in a virus sample. This is the standard method used to determine viral concentrations in non-complementing hosts. Confluent monolayers of cells were infected with the virus at various dilutions, and the cells were then screened for viruses that were not infectious. The virus plaque is covered with a semi-solid medium such as agar to prevent it from spreading. The virus successfully infects, replicates itself in the cells within the fixed cell monolayer, and spreads to surrounding cells. (See, for example, Kaufmann, SH; Kabelitz, D. (2002) "Microbiology "Methods in Microbiology," Vol. 32: "Immunology of Infection" (See Academic Press. ISBN 0-12-521532-0). Plaque formation is This can take 2-14 days depending on the virus being analyzed. Plaques are generally not extracted manually. The results are used in combination with the dilution factor used to prepare the plate. Calculate the number of plaque-forming units per unit volume of sample (PFU / mL). PFU / mL result indicates the number of infectious, replicative particles in the sample. Using the antibody, titration of replication-deficient or tripartite arenavirus particles was performed. obtain.

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

[0304] (4.8.13 Animal Models) To investigate the recombination and infectivity of the arenavirus particles described herein, In vivo animal models can be used. In certain embodiments, a combination of tripartite arenavirus particles is used. Animal models that can be used to study recombination and infectivity include mice, guinea pigs, In a preferred embodiment, recombinant arenaviruses and Animal models that can be used to investigate the infection and infectivity include mice. In a specific embodiment, it is used to investigate the recombination and infectivity of arenavirus particles. Obtain mice expressing type I interferon receptors, type II interferon receptors, and recombinant It is a triple deletion type of activating gene 1 (RAG1).

[0305] In certain embodiments, the animal model is used to assess arenavirus infectivity and transduction. In some embodiments, viral RNA can be used to determine the stability of genes in animal models. Viral RNA can be isolated from the serum of infected individuals by reverse transcription, a technique well known to those skilled in the art. cDNA carrying the arenavirus ORF can be extracted using gene-specific primers. PCR amplification can be performed using flow cytometry. Arenaviruses can be detected using PCR. The infectivity and stability of the transgene can also be investigated. [Example]

[0306] 5. Working Example These examples use LCMV virus-based vector technology to: (1) transfect the viral ORF an arenavirus genome segment having a sequence similar to that of the wild-type sequence of the ORF, and 2) Development of tripartite arenavirus particles that do not yield replication-competent bipartite virus particles. Demonstrate that it can be successful.

[0307] 5.1 Materials and Methods (5.1.1 Cell) BHK-21 cells were cultured in 10% heat-inactivated fetal calf serum (FCS; Biochrom), 10 mM HEPES (Gibco ), supplemented with 1 mM sodium pyruvate (Gibco), and 1× tryptose phosphate broth MC57 cells were cultured in high-glucose Dulbecco's Eagle's medium (DMEM; Sigma) supplemented with 5% of heat-inactivated FCS, 2 mM L-glutamine (Gibco), and penicillin-streptomycin (1 Minimum essential medium supplemented with 0.000 U / ml penicillin and 50 mg / l streptomycin (Gibco) Both cell lines were maintained in MEM medium (Sigma). Both cell lines were incubated at 37°C in a humidified 5% CO2 incubator. The cells were cultured in a hood.

[0308] NP-expressing BHK-21 cells were transfected with eukaryotic EF1-alpha promoter according to the manufacturer's protocol. A plasmid expressing NP under the control of a promoter and encoding a puromycin resistance gene. BHK-21 cells were transfected with IgG. 48 hours after transfection After 48 hours, the cells were cultured in a T150 flask. When individual clones became visible, the cells were harvested and plated in a 96-well plate. The wells were serially diluted into 100 ml of medium to obtain single clones. Once a confluent monolayer was formed, each cell was plated in 6 wells. The NP-expressing BHK-21 cells were subcultured in well plates. The strain was cultured in the presence of .

[0309] GP-expressing BHK-21 cells have been previously described. Briefly, BHK-21 cells express GP at the codon A plasmid expressing an optimized LCMV-GP cDNA and a puromycin resistance cassette was used to The GP-expressing clones were transfected at constant temperature in the presence of 4 μg / ml puromycin in the medium. Single clones were selected by adding NP-expressing BHK-21 cells as described previously. The antibodies were obtained by serial dilution.

[0310] (5.1.2 Plasmids) The pol-I L, pC-NP, and pC-L plasmids have been previously described. For the generation of pol-IS plasmids encoding either GFP or RFP, and either NP or GP To this end, we used the pol-I Bbs / Bsm cloning plasmid as a basis (pol-I 5 '-BsmBI_IGR_BbsI_3'). This plasmid contains the viral vector p53 followed by two BsmBI restriction sites. the 5' untranslated region (5'UTR) of the S segment of , the intergenic region flanked by BbsI restriction sites (I GR), NP rest, and CAT open reading frame (ORF), as well as the 3′U of the S segment It encodes TR, GP at its natural 5' position, and GFP in the antisense orientation at the 3' position. The pol-I S plasmid (pol-I 5'-GP_IGR_GfP-3') containing GP was subjected to BsmBI site-specific restriction and The fragment was cloned by inserting it into the pol-I Bbs / Bsm plasmid via ligation. In the second step, GFP was inserted by BbsI digestion and ligation. To obtain the pol-I S plasmid encoding GP in a 3′-GFP vector (pol-I 5′-GFP_IGR_GP-3′), GP was transfected into 3′-GFP vector. The GFP was inserted into the Bbs / Bsm plasmid by digestion with BbsI at the 5' position and GFP was inserted into the Bbs / Bsm plasmid by digestion with BsmBI at the 5' position. The pol-I S (pol-I 5'-G) encoding GFP or RFP and NP was inserted by restriction / ligation. FP_IGR_NP-3' or pol-I 5'RFP_IGR_NP-3') was prepared by digesting NP with BbsI and ligating it. Insert the pol-I into the Bbs / Bsm cloning plasmid, and then insert GFP or RFP into the BsmBI cloning plasmid. The vector was cloned by inserting it into the pol-I Bbs / Bsm cloning plasmid using the following procedure: The pol-I plasmids carrying the GPs of the LCMV strains WE and NP from CMV strain clone 13 (Cl13) were cloned from each gene. The gene was inserted into the pol-I Bbs / Bsm cloning plasmid by Bbs and Bsm site-specific restriction / ligation. The clones were inserted into the respective sites of the plasmid.

[0311] The S segment encoding the WE / WET fusion GP is the last 255 base pairs of the WE ORF, designated "WET." This was achieved by replacing the nucleotide sequence with a codon-optimized sequence. In this step, one WE-specific primer (SEQ ID NO: 11) and an overhang complementary to the WET sequence were used. A fragment of WE GP was PCR amplified with one WE-specific fusion primer (SEQ ID NO: 12) carrying the In parallel, the WET sequence was amplified using a WET-specific primer (SEQ ID NO: 13) and and amplified by PCR using a WET-specific fusion primer (SEQ ID NO: 14) complementary to the WE sequence. In the third PCR reaction, the two PCR products were subjected to PCR fusion using the two fusion primers described above. The resulting WE / WET fusion fragment was digested with BsmBI and then purified with the same restriction enzyme. The fragment was ligated into the pol-I BsmBI_IGR_GFP-3' plasmid, which had been digested with pol-I BsmBI_IGR_GFP-3'.

[0312] In vivo recombinant virus r3LCMV-GFP nat #3 recombinant S segment encoding pol-I promoter The synthesized DNA fragment (gene synthesis by GenScript) was cleaved using SacI and XmaI. Site-specific restriction / ligation ligation catalyzes the expression of wild-type S-seq under the control of the pol-I promoter. The fragment was cloned by inserting it into a plasmid (pol-I GP_IGR_NP) encoding the fragment. This resulted in pol-I GP_IGR_GFPrest_IGR_NP.

[0313] 5.1.3 DNA transfection of cells and rescue of recombinant viruses BHK-21 cells were seeded in 6-well plates at a density of 4 × 105 cells / well, and after 24 hours, Lipofectamine (3 μl / μg of DNA; Invitrogen) or jetPRIME (2 μl / μg) were used according to the manufacturer's instructions. The cells were transfected with various amounts of DNA using either Polyplus (1 μg / μg DNA) or Polyplus (1 μg / μg DNA). For the rescue of recombinant bipartite viruses derived from plasmid DNA, two minimal viral fragments were used. The trans-acting factors NP and L were transduced from the pol-II-driven plasmid (0.8 μg of pC-NP, 1 μg of pC-L). The cells were delivered from the phage and co-transfected with 1.4 μg of pol-I L and 0.8 μg of pol-I S. In the case of rescue of tripartite r3LCMV consisting of two S segments, both pol-I-driven S segments 0.8 μg was included in the transfection mixture. 72 hours after transfection, The supernatant was collected and subcultured in BHK-21 cells for further amplification of the virus. The virus titer in the culture was determined by focus-forming assay.

[0314] 5.1.4 Viruses and viral growth kinetics Wild-type Cl13 LCMV, originally derived from wild-type LCMV Armstrong, has been described. Live and recombinant virus stocks were transfected into BHK-21 cells at a multiplicity of infection (moi) of 0.01. The virus was produced by infecting the infected cells with 1000 μg of ... In vitro, the assay was performed in a 6-well format. BHK-21 cells were seeded at a density of 6 x 10 cells / well. After 24 hours, cells were incubated on a rocker plate at 37°C and 5% CO2 with 500 μl of virus inoculum. Infection was carried out by incubating with the material at an moi of 0.01 for 90 minutes. The cells were then incubated at 37°C / 5% CO2 for 72-96 hours. The supernatant was collected at given time points (usually 1000 μg / mL). The cells were harvested at 18, 24, 48, and 72 hours, and the virus titer was analyzed by focus-forming assay.

[0315] 5.1.5 Focus Formation Assay The titer of LCMV was then determined by focus-forming assay. However, it is a non-cytolytic virus that does not lyse the host and therefore does not form plaques. The units in this study are not the correct term focus-forming units (FFUs), but rather a more appropriate term. Unless otherwise stated, the commonly used term is plaque-forming units (PFU). MC57 cells were used for the focus formation assay. The cells were placed in one well of a 24-well plate. Cells were seeded at a density of 1.6 x 105 cells per well and 10-fold dilutions of virus prepared in MEM / 2% FCS were added. After 2-4 hours of incubation at 37°C, 200 μl of the diluted solution was added per well. Add 1 ml of viscous medium (2% methylcellulose in 2x supplemented DMEM) to allow the virus particles to adhere to the plate. After 48 hours at 37°C, the supernatant was flicked off and the cells were Stabilize the cells for 30 min at room temperature by adding 200 μl of 4% paraformaldehyde (PFA) in PBS. (All of the following steps are performed at room temperature.) The cells were fixed in 200 μl of BSS / 1% T Permeabilization was performed with riton X-100 (Merck Millipore) for 20 min, followed by PBS / 5% FCS for 60 min. For anti-NP staining, rat anti-LCMV-NP monoclonal antibody was added to the cells in PBS / 2.5 ml of PBS. For anti-GFP staining, purified Rat IgG was used as the primary staining antibody at a dilution of 1:30 in 1% FCS for 60 min. Anti-GFP antibody (Biolegend 338002) was used at a dilution of 1:2000 in PBS / 2.5% FCS. The plates were washed three times with tap water and the secondary HRP-goat anti-rat-IgG was added at 1:100 in PBS / 2.5% FCS. The plate was then washed 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) The reaction was stopped after 10 minutes with tap water. The final titer was calculated based on the dilution.

[0316] For anti-GP staining of cells, plates were fixed in 50% MeOH / 50% acetone for 5 min and then resuspended in PBS. The cells were washed and blocked as described above. Anti-GP GP83.4 (hybrid) was used as the primary antibody. (produced from Lysoma) was diluted 1:10 in PBS / 2.5% FCS and incubated for 60 minutes. After washing three times with PBS, the secondary HRP-rabbit anti-mouse IgG antibody was added at a dilution of 1:50 in PBS / 2.5% FCS. After further washing with tap water three times, the color reaction was I added it as I did.

[0317] To determine the viremia of mice in the blood, one drop of blood (corresponding to a 50 μl volume) was added to 950 1 μl of BSS-heparin (Na-heparin, Braun, final 1 IE / ml) and inverted. It was mixed further and stored at -80°C until further use.

[0318] (5.1.6 Mouse) AGRAG mice (IFNα / βR- / -, IFNγR- / -, RAG- / -) have been described and are specific pathogen-resistant. AGRAG mice are bred and housed under specific pathogen-free (SPF) conditions. All animal experiments were carried out in accordance with Swiss animal protection law. and, subject to the authorization of the respective responsible cantonal authorities of Geneva and Basel, the University of Geneva and Mice were infected intravenously with 1 × 104 PFU per mouse. So I went.

[0319] 5.1.7 Viral RNA Preparation and Sequencing Viral RNA was analyzed on cell cultures using the QIAamp viral RNA mini kit (QIAGEN) according to the manufacturer's instructions. Viral RNA was extracted from the supernatant or serum of infected mice. The crypt RT-PCR system (Invitrogen) and a primer specific for LCMV NP (SEQ ID NO: 15) were used. PCR amplification was performed using PCR primers according to the manufacturer's protocol. PCR was performed using 2 μl of DNA and NP- and GP-specific primers (SEQ ID NO: 16). The amplification was performed using high-fidelity DNA polymerase (NEB). The amplification products were then gel-filtered on a 2% agarose gel. The NP was analyzed on a gel, excised, and purified using a QIAquick gel extraction kit (QIAGEN). and sent for DNA Sanger sequencing (Microsynth) using GP-specific primers.

[0320] (5.1.8 Flow Cytometry) Blood was purified using immunofluorescence staining for CD11c (N418), CD11b (M1 / 70), CD19 (6D5), NK1.1 (PK136), CD90.2 (30-H12), and The cells were stained with an antibody against GR-1 (RB6-8C5). The expression of surface molecules stained with specific antibodies and GFP and RFP expression was measured using FlowJo software (Tree Star, Ashland, OR) on BD LSR F Analysis was performed using an Ortessa flow cytometer.

[0321] (5.1.9 Statistical analysis) Statistical significance was determined by two-tailed unpaired t-test using Graphpad Prism software (version 6.0d). or one-way ANOVA followed by Dunnett's or Bonferroni's for multiple comparisons. The significance was determined by post-test. P values ​​of p>0.5 were considered not significant (ns), while A P value of p<0.5 was considered significant (*), where p<0.01 (**) and p<0.001 (***) were considered significant. The floor is highly significant.

[0322] (5.2 Results) 5.2.1 Recombinant tripartite viruses grow to lower titers than wild-type LCMV The genome of wild-type LCMV consists of two single-stranded RNA segments of negative polarity (one L segment and one S segment). Recently, additional foreign genes have been incorporated into the normally bipartite LCMV particle (Fig. 1A). It has been shown that the NP and GP genes can be introduced into the genome of the 2 The gene of interest is isolated onto two S segment analogs, and is inserted into each S segment of the resulting LCMV. The virus is inserted into the host and produces a replicative viral particle containing three RNA segments (two S and one L). A recently published strategy is to target both NP and GP in the S segment. They are kept in their natural position, so that GFP or other transgenes can be inserted into each empty space. (r3LCMV-GFP) nat ) (Fig. 1B). This indicates that the genetic reinsertion of the S segment This minimizes the potential risk of the resulting genome becoming non-viable. However, in this study, we aimed to convert GP into 3'U It can also be juxtaposed to the TR and expressed from the promoter elements that normally drive the NP. We hypothesized that this would be possible (r3LCMV-GFP art ; Figure 1C). Each expression plasmid contains recombinant cDNA All three viral constructs were generated by cloning and rescued entirely from plasmid DNA Comparative growth curves were performed for the three viruses (Fig. 1D). All three viruses showed growth rates of 48 p.i. The peak titer of the tripartite virus was 10-100 times higher than that of the wild-type virus. The wild-type LCMV was 3.4 x 10 6 PFU / ml, r3LCMV-GFP natis 2.7 x 10 4 PFU / ml The peak was r3LCMV-GFP. art is 2.2 x 10 5 The PFU / ml peaked and then decreased. Regardless of the peak titer obtained, r3LCMV-GFP nat At early time points, r3LCMV-GFP art Somewhat more showed high cell-free infectivity.

[0323] (5.2.2) Packaging of tripartite virus particles is more efficient than that of bipartite viruses isn't it) These observations suggest that the addition of a second S segment impairs and slows viral growth. This suggests that the fitness of the virus is reduced by the inclusion of all three RNA segments in the virus particle. This may be due to inefficient packaging of the granules into the granules, resulting in excess bipartite granules. The assumption is that when offspring are formed and infect new cells, they fail to undergo productive replication. For these experiments, two different reporter genes were used: one S-seq r3LCMV with GFP together with GP on one S segment, and NP next to RFP on the second S segment. This was a r3LCMV-GFP / R vector that differed only in the arrangement of GFP and GP on each S segment. FP nat and r3LCMV-GFP / RFP art The virus was then transferred to BHK-21 cells, where it was then transferred to the HIV-1 virus. , r3LCMV-GFP-RFP nat or bipartite r2LCMV, and focus formation assays were performed on normal BH In K-21 cells, or in parallel, stably transfected mice expressing either GP (BHK-GP) or NP (BHK-NP) were cultured. Transfected BHK-21 cells were trans-complemented with α- and β-actin-containing vectors lacking the respective genes. ement) virus genome as a cell substrate. NP complementing cells stained for nucleoprotein-expressing viral foci, whereas GP-positive foci were stained, which revealed that the immunophore on wild-type BHK-21 cells Cas formation detected only tripartite virions. Without being limited by theory, BHK-GP cells The vesicles were tripartite virions, plus the NP-expressing S segment (but lacking the GP-expressing S segment). (L) The two-part type containing the L segment should replicate. Conversely, BHK-NP cells replicate three-part types. Segmented LCMV and, in addition, L and GP-expressing S segments (but not NP-expressing S segments) r3LCMV-GFP / RFP should replicate NP-deficient virions (lacking the NP-deficient virion). nat and r3LCMV-GFP / RFP art The infectious titers of both BHK-GP and BHK-21 cells were significantly higher than those of wild-type BHK-21 cells. Conversely, the titer of r2LCMV was consistently higher when assessed in HK-NP cells. The results were similar regardless of the cell substrate used for the evaluation of the efficacy of each cell line against LCMV. To correct for possible inherent differences in the titers of each virus in BHK-21 cells, the titers were Therefore, the titers were normalized to one, and the BHK-GP and BHK-NP titers were expressed as multiples of that. and cell clone-related differences in viral tolerance that may exist. This reflects the difference in titer between the r3LCMV-GFP / RFP vectors (Figure 2A). nat and r3LCMV-GFP / RFP art Regarding A difference in titer of approximately 5-10 fold was observed in either one of the complete cells, which is due to the r2LC This was significantly higher than that for MV. The majority of virus particles expressed either the NP-only S segment (NP-only particles) or the GP segment. Expressed S segment (GP-only particle) - encodes only one of the two S segments This more than five-fold difference in potency suggests that the NP-only particles and the GP-only particles contained only Both the chiral particles and the trisegmented particles outnumbered the virion particles by about five times. This suggests that the IL-16 expression level accounted for only less than 10 percent of the IL-16 expression levels grown on non-complementing cells. These findings are consistent with the delayed growth and reduced peak viral titers when the virus was administered (Fig. 1D). The findings were further validated by flow cytometry. The cells were transfected with r3LCMV-GFP / RFP art or r2LCMV-infected GFP as a gating control. The fluorescence intensity of RFP and IL-1 was evaluated using a flow cytometer (Figure 2B). Because minimal trans-acting factors are not provided, the L segment along with the NP-expressing S segment Only virions containing at least 1 of these virions are able to initiate the infectious cycle after cell entry. Therefore, when BHK-21 cells are infected with NP-only particles, A population of RFP+GFP- cells was observed, reflecting the phenotype of the RFP+GFP+ double-positive cells. This gating also allowed us to observe RFP-GFP+ cells, and RF RFP-GFP+ cells had a higher RFP MFI than P-GFP- cells, indicating that RFP-GFP+ cells were composed of tripartite particles. This interpretation is supported by the fact that this population and the RFP+GFP+ This is also supported by the continuity with double-positive cells. However, the three-segment r3LCMV-GFP / RF P art was grown in BHK-NP cells, thereby substituting for this minimal trans-acting factor. When complementing BHK-21 cells, we observed over 10-fold higher numbers of RFP-GFP+ Conversely, RFP+GFP- (evidence of NP-only particles) and GFP+RFP+ double-positive cells (3 min) were observed. These results suggest that the chromatin-forming particles (nodal particles) were detected at similar abundances (Fig. 2C). This confirmed the findings obtained by the focus-forming assay and therefore confirmed the efficacy of the tripartite virus. This confirmed that the seropreparation contained a majority of bipartite replication-deficient particles. Findings: r3LCMV-GFP / RFP nat and r3LCMV-GFP / RFP art A more plausible explanation for the attenuated proliferation of provides a new explanation for the random packaging of tripartite viruses, which are apparently very inefficient. This provided insight into the

[0324] 5.2.3 Cloning and Rescue of Recombinant Viruses to Monitor Recombination in Vivo The tripartite viruses exhibit impaired growth kinetics as seen in Figure 1, and thus NP and GP, which has high resistance to viruses by recombining its genetic information into only one S segment. We hypothesized that there must be a strong selective pressure for recombination between arenavirus segments. It is hypothesized that this led to the phylogenetic evolution of the North American clade, thus forming a trifoliate This appears to be a mechanism by which segmented viruses can reconstitute functional bipartite genomes. Without being limited by theory, looking at the genome organization of the two tripartite viruses, r3LCMV-GFP nat Selection pressure against may have favored recombination events in the area of ​​IGR, thereby increasing G We assumed that P and NP were both on the same segment, while GFP was removed. r3LCMV-GFP ar t In these populations, the selective pressure should be equally high, but the reshuffling of GP and the 3'UTR Its location next to the nucleotide sequence allows the virus to combine its two S segments into one functional segment. This would make it very difficult, if not impossible, to combine the two (See Figure 7 below.) Taking into account the specific precautions of RNA recombination and the likelihood of it occurring. To distinguish it from other cDNA contaminations, we used GFP along with the terminal 255 nucleotides. The S segment carrying the recombinant GP ORF was cloned. The resulting GP had a different nucleotide sequence, but was identical to the wild-type WE strain GP. (WE / WET-GP, Figure 3A). However, this recombinant WE / WET GP The ORF does not exist as an infectious bipartite virus, and our laboratory has also constructed a cDNA related to NP. Therefore, there are no known cases where WE / WET is present on the same segment as NP. Any bipartite virus is considered to be clear evidence of intersegment recombination. Such viruses may contaminate cDNA or RNA in the respective assays. A. To test whether the chimeric GP had an effect on viral fitness, To achieve this, a recombinant three-part virus carrying the WE / WET fusion GP (r3LCMV-WEWET / GFP) was developed. nat) cells The culture growth curve was measured using a tripartite virus carrying wild-type WE GP (r3LCMV-WE / GFP). nat ) compared to The growth kinetics and peak titers of the two viruses were comparable (Fig. 3B). r3LCMV-WE / GFP nat :1.7x10 6 PFU / ml, r3LCMV-WEWET / GFP nat :2.3x10 6 PFU / ml). The WEWET glycoprotein of La had no detectable effect on viral growth.

[0325] A possible recombination event occurs between the NP and GP genes of the S segment, which may involve an IGR. To test whether NPs can be obtained, single nucleotide deletions were introduced to act as genetic tags. The nucleotide deletion was introduced into the intergenic region of the S segment, which encodes the nucleotide sequence. This is different from most S-segment IGRs in that neither the sequence nor the length is conserved among strains. In the case of a recombination event, this "tagged" (Fig. The intergenic region (marked as * throughout both the text and the S segment) is This should allow identification of the genetic origin of the missing cytosine (marked by an arrow). A schematic diagram of the S segment carrying the NP and the resulting NP is shown in Figure 3C. To test whether the deletion had any effect on viral growth, the single deletion was Recombinant r3LCMV-GFP with or without nucleotide deletion nat Rescued. Growth curve experiment The results were analyzed in BHK-21 cells (moi = 0.01). V-GFP nat) and its comparison with a mutant IGR (r3LCMV-GFP nat IGR*) is a similar speed The NP-harboring S-segments proliferated at 1000 kJ / s and reached indistinguishable peak titers (Fig. 3D). Tagging the IGR on the nucleotides had no detectable effect on viral fitness. , validating its use in subsequent experiments in vivo.

[0326] 5.2.4 r3LCMV-GFP in mice nat The persistent infection of the virus is equivalent to that of the bipartite wt virus. r3LCMV-GFP art is not) Recombinant r3LCMV-GFP nat The goal of this rescue was to ensure that the tripartite virus was recombined in vivo. To this end, AGRAG mice were transfected with r3LCMV-GFP nat , r3LCMV-GFP art AGRAG mice were infected with the target LCMV or the bipartite r2LCMV as a control. The deletions involved the interferon-α / β receptor, interferon-γ receptor, and RAG1 This gene encodes the α-terminal β-actin, which is responsible for the immunodeficiency phenotype and the ability to infect tripartite LCMV. This leads to the establishment of chronic viremia later on. Blood samples are taken over time and the viral titer is measured. The focus formation assay was performed (Fig. 4A). Carriers of bipartite LCMV showed a rapid increase in the number of infected individuals within 5 days after infection. Within 5×10 5 showed high-titer viremia in the range of PFU / ml blood, followed by a short Tomo 50th day 10 4 ~10 5 The patient showed stable viremia in the range of PFU / ml. Infected mice retained approximately 5 x 10 cells by day 20, consistent with attenuated proliferation in cell culture. 3 The viral load in PFU / ml of blood is shown (compare with Figure 1D). From day 30 onwards, r3LCMV-GFP nat No. Rear showed elevated viral load, which was due to the r3LCMV-GFP art observed in infected animals This resulted in a more than 10-fold difference in viremia at day 50. still carry the GFP reporter gene and therefore lead to GFP expression in infected cells. To determine whether 3LCMV-GFP cells were infected, we harvested 127 days post-infection. nat and r3LCMV-GFP art A viral focus-forming assay was performed using blood samples from carriers to identify nucleopeptides. The cells were stained for either protein or the reporter gene GFP (Figure 4B). art From the career Staining of isolated blood resulted in equal amounts of foci for anti-NP and anti-GFP antibody detection. On the other hand (both evaluations are independent, 10 3 containing virus titers ranging from 100 PFU / ml, expressing GFP. Total (NP+) r3LCMV-GFP foci number at least 100-fold greater than the number of foci present nat The focus is clear It was clear that at least 10 4 The viral titer in PFU / ml was determined based on anti-NP detection. On the other hand, two of the three mice showed no detectable GFP-positive infection and one mouse The mice showed GFP-positive phenotypes in the range of 100 PFU / ml, which corresponds to the lower limit of detection of our assay. GFP expression in infected cells was also assessed by fluorescence microscopy. (Data not shown). r3LCMV-GFPnat GFP fluorescence when blood from carriers was assayed Foci were virtually undetectable. art GFP derived from carrier blood Manual counting of positive foci results in titers obtained in anti-NP focus formation assays The reporter gene expression was further analyzed in infected mice at 120 days post-infection. This was confirmed by flow cytometry analysis of PBMCs. art infected animal In r3LCMV-GFP, more than 10% of CD11b+GR1- monocytes / macrophages were GFP positive. nat Blood samples taken from animals infected with non-fluorescent r2LCMV showed minimal background This finding further supports the finding that GFP-containing nucleotides with GFP in their native position are highly expressed in the GFP-containing nucleotide sequences (Figure 4C-E). The tripartite virus loses reporter gene expression over time, whereas the artificial 3'UTR juxtaposition These results supported the hypothesis that GP transposition in β-actin prevents transgene loss.

[0327] (5.2.5) Tripartite viruses with GP in the natural position recombine their two S segments In this study, a single S-segment with partial or complete IGR duplications flanking the traces of the transgene sequence was generated. (which can bring about Figure 4 shows r3LCMV-GFP nat Elevated viremia and reporter in mice infected with Therefore, we hypothesize that recombination events may explain this experimental result. Intersegment recombination combines GP and NP on the same S segment to form a virus. This should make the second S segment unnecessary in the cell replication cycle. In combination with the loss of gene expression, such events further reduce the level of wild-type virus. To test this possibility, serum samples from infected mice were collected. The original viral RNA was isolated and analyzed using a pair of primers that bind to the NP and GP sequences, respectively. , which carries both the NP and GP ORFs in an ambisense orientation on a single RNA segment Only the putatively recombined RNA molecules were selectively amplified by RT-PCR. The resulting PCR fragments were analyzed by gel electrophoresis (Figure 5A). nat Kya Leah's serum gave rise to RT-dependent PCR products, whereas r3LCMV-GFP art Career and Untreated The control PCR reaction did not show any specific bands. To exclude RNA contamination, this was performed on mock RT-treated RNA samples. -GFP nat The sequencing results for each carrier are shown diagrammatically in Figure 5C. , which contained viral RNA segments with similar patterns but distinct sequences: The C-terminal portions of GP and NP were found in an ambisense orientation on one RNA segment. Among these, both intergenic regions, i.e., those of the NP-expressing segment and the original GP-expressing segment, were identified. The S-segment of the tripartite virus is at least partially retained, and the parental S-segment of the tripartite virus is The fragments were separated by fragments derived from either or both of the GFP reporter genes in the The orientation and length of the GFP fragment varied among the three RNA species recovered from individual mice. This indicated independent recombination events. Further supporting this idea, Recombinant RNA sequences were collected from two samples taken from the same mouse with a sampling interval of more than 3 weeks. Based on the recombinant S segment sequences obtained, the present invention The authors used r3LCMV-GFP, as outlined schematically in Figure 7 and described in the figure legend. n at However, the two S segments are recombined to cause the loss of the transgene and its expression back to the wild-type virus. We proposed a molecular mechanism that leads to the reversion of the present type. r3LCMV-GFP by the mechanism of fragment recombination art The NP ORF and GP ORF are recombined to form a single This explains why the functional S segment cannot be combined.

[0328] (5.2.6) Recombinant r2LCMV with two IGRs on the S segment is viable and (It grows to titers similar to those of bipartite LCMV, which has only one IGR per virus.) The above sequencing data suggest that the (at least partial) overlap of IGRs is particularly noteworthy between them. The genetic elements of the virus in the recombinant S segment were both novel and distinctive. However, there were no intergenic sequences on one S segment. No arenaviruses with repeat regions were known. Murup is found naturally in the Old World arenavirus Mopeia. Therefore, the present inventors have developed r3LCMV-GFP nat The reconfigured S-segment of Carrier #3 was then split into two IGRs. and cloned into a pol-I-driven S segment expression plasmid along with a remnant of GFP. The virus (r2LCMV_2IGRs) was grown in BHK-21 cells. Reproduction kinetics of the three-segmented r3LCMV-GFP nat and bipartite r2LCMV (Fig. 6). The infectious cell-free titer of IGR was already high at early time points, compared with r3LCMV-GFP nat It exceeds that of r2LC MV reached peak titers identical to those of MV (1.7 × 10 7 PFU / ml vs. 1.6 × 10 7 PFU / ml). r2LCMV_2IGR is a 3-segmented strain of its parent, r3LCMV-GFP. nat increased to a peak titer significantly higher than This demonstrates a selective advantage for intersegment recombination despite the overlap of IGRs during this process. It was revealed.

[0329] 5.2.7 Recombinant r3LCMV expressing ovalbumin (OVA) is a rapid, potent, and versatile OVA Induce A-specific CD8+ T cell responses) r3LCMV for vaccination purposes art To test the usefulness of vector delivery technology, They used r3LCMV-GFP art (Fig. 1C) but with the respective GFP gene in the virus. Instead of the offspring, r3LCMV-OVA, which has a genome structure with two ovalbumin (OVA) genes, art The inventors have produced a vaccine vector. 4 PFU r3LCMV-OVA art C57BL / 6 mice were immunized with IgG1 and IgG2, and 8 days later, we analyzed T cell responses in the spleen. For comparison with widely used vector platforms, we used C57BL / A second group of 6 mice was8 The particles are also replication-deficient E1-deleted adenoviruses that express OVA. Immunocytosine-5 (rAd5-OVA) was used to induce immunodominant OVA. [ka] The frequency of OVA-specific CD8+ T cells that recognize the epitope was significantly higher in r3LCMV-OVA than in r3LCMV-OVA. art C in the vaccine group The proportion of D8+ T cells ranged from 10% to 10%, which was significantly higher than that in the rAd5-OVA group (Figure 8A). -OVA art Induced CD8+ T cell responses, as determined by intracellular cytokine assays, Not only are they of high importance but also highly functional, and most [ka] Reactive r3LCMV-OVA art Induced CD8+ T cells produced IFN-γ in response to peptide stimulation. , and a significant proportion were found to co-produce TNF-α and / or IL-2. The effect of r3LCMV-OVA on vaccine delivery art The usefulness of vector technology was demonstrated.

[0330] 5.2.8 Tripartite LCMV induces polyfunctional memory CD8+ T cells To address the question of whether the r3LCMV vector induces functional CD8+ T cell memory, To achieve this, we inoculated C57BL / 6 mice with 10e5 PFU of r3LCMV-OVA. art iv immunization with 2 On day 5, OVA-specific ( [ka] The control group of mice was a recombinant E1-deficient mouse expressing OVA. Vaccination was performed by the same route with 10e8 viral particles (vp) of adenoviral vector (rAd). OVA-specific CD8+ T cells that produced IFN-γ, TNF-α, and / or IL-2 upon peptide stimulation were isolated. Cells, [ka] Peptide stimulation was assessed by standard intracellular cytokine assays. The frequency (Fig. 9A) and absolute number (Fig. 9B) of such cytokine-producing cells were determined. art Immunized mice expressed significantly higher frequencies and numbers of polyfunctional IFN-γ / TN antigens than rAd-OVA-immunized mice. F-α and IFN-γ / TNF-α / IL-2 co-producing OVA-specific CD8+ T cells were shown.

[0331] 5.2.9 LCMV-encoded antigens induce specific T cell responses against foreign and self antigens do) r3LCMV art The vector can be used to induce CD8+ T cell responses against tumor-expressed self-antigens To investigate whether this is the case, the inventors used BALB / c mice as rats. [ka] Human [ka] or mouse [ka] r3LCMV expressing any of the Her2-induced CD8+ T cell epitopes art vector (Fig. 1 After 9 days, we measured the activity of each peptide in an intracellular cytokine assay. Specific CD8+ T cells producing IFN-γ, TNF-α, and / or IL-2 upon stimulation with IFN-γ were measured. FIG. 10 shows the frequency of epitope-specific CD8+ T cells upon stimulation with the cognate peptide. The percentage of CD8+ T cells producing the cytokine combinations is shown. The frequency of cytokine-producing CD8+ T cells was very low. art Vector demonstrated the ability to induce a significant frequency of tumor autoantigen-reactive CD8+ T cell responses. do.

[0332] (5.2.10 r3LCMV art Interferon-α is induced during infection, but recombinant adenovirus (It is not induced by infection with the ruse vector or vaccinia virus vector) Type I interferons may have multiple immunostimulatory and antitumor effects. Thus, induction of type I interferon is a favorable candidate for virally mediated vaccines. The present inventors performed ELISA measurements and found that r3LCMV-OVA art , rAd-OV A, or OVA-expressing recombinant vaccinia virus (rVacc) 24, 48, or 72 hours in advance. Interferon-alpha concentrations were determined in the serum of immunized mice (Figure 11). a rt is a detectable and sustained (at least 48 hours) systemic interferon-alpha response r3LCMV was induced, but neither rAd nor rVacc was induced. art Vector's powerful This provides evidence of the ability to induce a potent innate immune response.

[0333] (5.2.11 r3JUNV-GFP nat and r3JUNV-GFP compared with the parental Junin strain Candid#1. art Cell culture expansion cultivation) r3LCMV-GFP carrying the genome outlined in Figure 1B nat and r3LCMV-GFP art Baek In analogy with the previous work, we have engineered a GFP gene into one of the two S segments of each of the 2000 cells. r3JUNV-GFP, consisting of a tripartite Junin vaccine strain Candid#1-based vector carrying the offspring na t and r3JUNV-GFP art We designed the r3JUNV-GFP nat and r3JUNV-GFP art ) The inventors of the present invention We tested their proliferation in 293T cells infected at a multiplicity of infection (MOI) and analyzed the supernatant over time. The present inventors have investigated the effects of r3JUNV-GFP on the phenotype of r3JUNV-GFP. art However, its parent, the two-segmented funin vaccine We found that the strain grew more slowly than the strain Candid#1 (Figure 12). r3JUNV-GFP nat This tripartite Junin virus-based vector grew faster than the vector (Figure 12). The different growth behavior of r3LCMV-GFP nat and r3LCMV-GFP art The growth rate of the vector is similar. (Figure 1D).

[0334] (5.2.12) Tripartite JUNV is dramatically attenuated in vivo and is not capable of long-term in vivo replication. In this case, r3JUNV-GFP nat loses GFP expression, but r3JUNV-GFP art will not be lost) r3JUNV-GFP nat and r3JUNV-GFP art To investigate the genetic stability of AG, we RAG mice (IFNα / βR- / -, IFNγR- / -, RAG- / -) were transfected with 7×10e4 PFU of these GFP-expressing vectors. For comparison purposes, a third group was infected with either wild-type bipartite Cand. The latter virus infected all infected mice by day 20 post-infection. The tripartite virus was readily detected in the blood (Fig. 13A), whereas the tripartite virus remained detectable for at least 40 days. This finding supports the notion that in vivo virulence may be attenuated as a result of genome rearrangements. We demonstrated growth in the r3LCMV-GFP vector and confirmed our findings in Figure 4A using the Junin-based After 40 days, the vector was expanded to r3JUNV-GFP. nat and r3JUNV-GFP art Several animals in each group Importantly, however, r3JUNV-GFP was detected in the r3JUNV-GFP vector (Fig. 13A). nat of infected mice While some reached viral loads in the range of wild-type Candid#1-infected mice, r3JUNV-GFP art Infected mice sustained lower viral loads than Candid#1 infected controls.

[0335] The dominant virus population in these viremic animals was identified as a GFP reporter gene. We then determined whether the GFP expression in infected cells still retains its original GFP-like activity. To investigate this, we used r3JUNV-GFP cells harvested 120 days after infection. nat and r3JUNV-GFP art tree A viral focus-forming assay was performed using blood samples from carriers. The infectious titer of the virus retaining GFP expression (anti-GFP, Figure 13B) and the total Junin virus titer were The staining ability (anti-NP, Figure 13B) of r3JUNV-GFP was compared. art Titers were measured using anti-GFP or anti-NP immunofocus. The majority of the virus population was GFP when determined by either assay. Conversely, the highest viremia (same as wild-type Candid#1) was observed. Four r3JUNV-GFP mice with nat In the blood of infected animals, the anti-GFP infectious titer was determined by NP staining. This was at least 10-fold lower than the total infectious titer determined by r3JUNV-GFP. art Yes Although the GFP transgene was stably maintained in vivo, r3JUNV-GFP nat demonstrated that it did not .

[0336] 5.2.13 Homologous and heterologous prime-boost combination of tripartite LCMV and JUNV-based vaccine vectors The combination induces potent P1A autoantigen-specific CD8+ T cell responses. Next, the present inventors investigated the r3LCMV art and r3JUNV art The tumor self-antigen-specific vector can be used in homologous and heterologous prime-boost combinations to induce targeted CD8+ T cell responses The present inventors investigated whether the autoantigen P1A (SEQ ID NO: 1) derived from P815 mouse mastocytoma was involved in the cytotoxicity of IL-1. No.:24)(r3LCMV-P1A art (SEQ ID NOs: 18, 19, 20) and r3JUNV-P1A art (SEQ ID NO: 21, 22, 23) r3LCMV expressing art and r3JUNV art These vaccine vectors were constructed. The vector was used to infect BALB / c mice in a homologous and heterologous prime-boost combination as outlined in FIG. Mice were immunized i.v. with r3LCMV-P1A art and r3JUNV-P1A art Both of these are homologous primed When administered in a first vaccination, [ka] H-2L loaded with peptide (P1A epitopes 35–43) d -Determined from blood using tetramers P1A epitope-specific CD8+ T cells were induced. The average frequency of cytoplasmic vesicles was 1.2% (r3JUNV-P1A art ) and 3.9% (r3LCMV-P1A art ) was. r3JUNV-P1A in a heterogeneous fashion art Primed with r3LCMV-P1A art Boosted motion The subject developed a higher response with a mean epitope-specific CD8+ T cell frequency of 19.5% at day 63. r3LCMV-P1A art Primed by r3JUNV-P1A art Boosted by The frequency of animals (3.1%) was r3LCMV-P1A art those who received the homologous prime-boost vaccination It was about the same.

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

[0338] Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated herein by reference. 10, No. 10 / 199,297, filed on Oct. 1, 2004, incorporated by reference in its entirety.

[0339] Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated herein by reference. 10, No. 10 / 199,297, filed on Oct. 1, 2004, incorporated by reference in its entirety. (7. Sequence Listing) [Table 7] TIFF0007796680000021.tif239170TIFF0007796680000022.tif240170TIFF0007796680000023.tif239170TIFF0007796680000024.tif240170TIFF0007796680000025.tif240170TIFF0007796680000026.tif240170TIFF0007796680000027.tif239170TIFF0007796680000028.tif239170TIFF0007796680000029.tif240170TIFF0007796680000030.tif239170TIFF0007796680000031.tif239170TIFF0007796680000032.tif239170TIFF0007796680000033.tif238170TIFF0007796680000034.tif240170TIFF0007796680000035.tif239170TIFF0007796680000036.tif240170TIFF0007796680000037.tif239170TIFF0007796680000038.tif240170TIFF0007796680000039.tif240170TIFF0007796680000040.tif239170TIFF0007796680000041.tif240170TIFF0007796680000042.tif240170TIFF0007796680000043.tif240170TIFF0007796680000044.tif239170TIFF0007796680000045.tif240170TIFF0007796680000046.tif240170TIFF0007796680000047.tif240170TIFF0007796680000048.tif240170TIFF0007796680000049.tif239170TIFF0007796680000050.tif239170TIFF0007796680000051.tif240170TIFF0007796680000052.tif239170TIFF0007796680000053.tif240170TIFF0007796680000054.tif239170TIFF0007796680000055.tif233170The present application provides the following invention: (Configuration 1) The viral open reading frame ("ORF") is inserted into a region different from the wild-type location of the ORF. 1. An arenavirus genome segment engineered to hold in position: (i) The ORF encoding the nucleoprotein ("NP") is located within the arenavirus 5' untranslated region ("UT"). R”) under the control of the S segment; (ii) an ORF encoding matrix protein Z ("Z protein") is inserted into the arenavirus 5' S segment under the control of UTR; (iii) The ORF encoding the RNA-dependent RNA polymerase L ("L protein") is S segment under the control of the 5'UTR; (iv) The ORF encoding the viral glycoprotein ("GP") regulates the arenavirus 3'UTR. the underlying S segment; (v) an S segment in which the ORF encoding the L protein is under the control of the arenavirus 3′UTR; (vi) an S segment in which the ORF encoding the Z protein is under the control of the arenavirus 3′UTR; (vii) the L segment, in which the ORF encoding GP is under the control of the arenavirus 5′UTR; (viii) the L segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (ix) an L segment in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR; (x) L segment in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (xi) an L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR; and (xii) The L segment, in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR The arenavirus genome segment selected from the group consisting of: (Configuration 2) The arenavirus 3'UTR is an arenavirus S segment or an arenavirus L segment. and the 3'UTR of an arenavirus 5'UTR is an arenavirus S segment or is the 5'UTR of the arenavirus L segment, nt. (Configuration 3) cDNA of the arenavirus genome segment described in construct 1. (Configuration 4) A DNA expression vector containing the cDNA according to construct 3. (Configuration 5) The arenavirus genome segment according to embodiment 1, the cDNA according to embodiment 3, or the vector according to embodiment 4 A host cell containing a vector. (Configuration 6) 1. The arenavirus genome segment of claim 1, wherein the segment comprises an S segment and an L segment. and a second arenavirus genome segment. (Configuration 7) 7. The arenavirus particle of embodiment 6, which is infectious and replicable. (Configuration 8) The arenavirus particle of embodiment 6, which is attenuated. (Configuration 9) Although infectious, they are unable to give rise to further infectious progeny in non-complementing cells. 10. The arenavirus particle according to claim 6. (Configuration 10) At least one of the four ORFs encoding the GP, NP, Z protein, and L protein 10. The arenavirus of claim 9, wherein the particle. (Configuration 11) At least one of the four ORFs encoding the GP, NP, Z protein, and L protein A construct in which the nucleotide sequence of ... Arenavirus particles. (Configuration 12) Only one of the four ORFs encoding the GP, NP, Z protein, and L protein was removed. 10. The virus of claim 9, wherein the vector is a non-arenavirus-derived heterologous ORF. Lenavirus particles. (Configuration 13) The ORF encoding GP was removed and replaced with a heterologous ORF from an organism other than arenavirus. 10. The arenavirus particle of configuration 9, (Configuration 14) The ORF encoding NP was removed and replaced with a heterologous ORF from an organism other than arenavirus. 10. The arenavirus particle of configuration 9, (Configuration 15) The ORF encoding the Z protein was removed to contain a heterologous ORF from an organism other than an arenavirus. 10. The arenavirus particle of construct 9, wherein the vector is replaced with (Configuration 16) The ORF encoding the L protein was removed to contain a heterologous ORF from an organism other than arenaviruses. 10. The arenavirus particle of construct 9, wherein the vector is replaced with (Configuration 17) 17. Any one of aspects 11 to 16, wherein the heterologous ORF encodes a reporter protein. Arenavirus particles. (Configuration 18) The heterologous ORF encodes an antigen derived from an infectious organism, a tumor, or an allergen. 18. The arenavirus particle according to any one of 1 to 17. (Configuration 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 antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, and tumor-associated antigens 19. The arenavirus particle of embodiment 18, selected from: (Configuration 20) The proliferation or infectivity of the arenavirus particles is determined by a different virus derived from an organism other than the arenavirus. 19. The arenavirus particle of any one of embodiments 11 to 18, which is unaffected by a seed ORF. (Configuration 21) 10. The arenavirus genome segment of claim 1, comprising transcribing the cDNA of claim 3. How to generate it. (Configuration 22) A method for producing arenavirus particles according to aspect 6, comprising: (i) transfecting a host cell with the cDNA described in Configuration 3; (ii) introducing a plasmid containing the cDNA of the second arenavirus genome segment into the host cell transfecting into; (iii) maintaining the host cells under conditions suitable for virus formation; and (iv) recovering the arenavirus particles; The method comprising: (Configuration 23) The transcription of the L segment and the S segment is carried out using a bidirectional promoter. 23. The method of claim 22. (Configuration 24) One or more nucleic acids encoding an arenavirus polymerase are transfected into a host cell. 23. The method of claim 22, further comprising: (Configuration 25) 23. The method of claim 22, wherein the arenavirus polymerase is an L protein. (Configuration 26) transfecting the host cell with one or more nucleic acids encoding an NP protein; 25. The method of claim 22 or 24, further comprising: (Configuration 27) 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 23. The method of claim 22, wherein the promoter is under the control of a promoter selected from the group consisting of: (Configuration 28) A vaccine comprising the arenavirus particles according to any one of claims 10 to 16 and a pharmaceutically acceptable carrier. Kuching. (Configuration 29) A pharmaceutical composition comprising the arenavirus particles according to any one of claims 10 to 16 and a pharmaceutically acceptable carrier. Pharmaceutical composition. (Configuration 30) 1. The arenavirus genome of claim 1, wherein the arenavirus genome is derived from lymphocytic choriomeningitis virus ("LCMV"). 7. The arenavirus particle according to claim 6, wherein the viral segment comprises a virion. (Configuration 31) The LCMV is MP stain, Armstrong strain, or Armstrong clone 13 strain. The arenavirus genome segment or arenavirus particle described above. (Configuration 32) Derived from Junin virus vaccine Candid #1 or Junin virus vaccine XJ clone 3 strain an arenavirus genome segment according to embodiment 1, or an arenavirus particle according to embodiment 6; child. (Configuration 33) A tripartite arenavirus particle comprising one L segment and two S segments, The proliferation of the tripartite arenavirus particles is mediated by type I interferon receptors, type II interferon receptors, and lacking the RON receptor and recombination activating gene 1 (RAG1), and 4The three-segmented arenavirus in PFU After 70 days of persistent infection in mice infected with virulence particles, replication-competent bipartite viruses were generated. The tripartite arenavirus particle does not produce virus particles. (Configuration 34) Two arenavirus ORFs on a single segment rather than on two separate segments Intersegmental recombination of the two S segments, which integrates the promoter activity of the virus, 34. The tripartite arenavirus particle of construct 33, which inhibits sexual activity. (Configuration 35) A tripartite arenavirus particle comprising two L segments and one S segment, The proliferation of the tripartite arenavirus particles is mediated by type I interferon receptors, type II interferon receptors, and lacking the RON receptor and recombination activating gene 1 (RAG1), and 4 The three-segmented arenavirus in PFU After 70 days of persistent infection in mice infected with virulence particles, replication-competent bipartite viruses were generated. The tripartite arenavirus particle does not produce virus particles. (Configuration 36) Two arenavirus ORFs on a single segment rather than on two separate segments Intersegmental recombination of the two L segments, which integrates the promoter activity of the virus, 36. The tripartite arenavirus particle of construct 35, which inhibits sex. (Configuration 37) One of the two S segments is: (i) The S segment in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (ii) the S segment, in which the ORF encoding the Z protein is under the control of the arenavirus 5′UTR; (iii) The S segment, in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR ; (iv) an S segment in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an S segment in which the ORF encoding L is under the control of the arenavirus 3'UTR; and (vi) The S segment in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR , 34. The tripartite arenavirus particle of embodiment 33, selected from the group consisting of: (Configuration 38) One of the two L segments is: (i) The L segment, in which the ORF encoding GP is under the control of the arenavirus 5′UTR; (ii) the L segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (iii) the L segment, in which the ORF encoding the L protein is under the control of the arenavirus 5'UTR; ; (iv) the L segment, in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR; and (vi) The L segment, in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR , 36. The tripartite arenavirus particle of embodiment 35, selected from the group consisting of: (Configuration 39) The arenavirus 3'UTR is an arenavirus S segment or an arenavirus L segment. and the 3'UTR of an arenavirus 5'UTR is an arenavirus S segment or is the 5'UTR of the arenavirus L segment, S particle. (Configuration 40) The two S segments contain (i) one or two heterologous ORFs from an organism other than an arenavirus; or (ii) one or two replicated arenavirus ORFs; or (iii) a live non-arenavirus 33. A three-segmented vector comprising one heterologous ORF derived from a virus and one replicated arenavirus ORF. Type arenavirus particles. (Configuration 41) The two L segments contain (i) one or two heterologous ORFs from an organism other than an arenavirus; or (ii) two replicated arenavirus ORFs; or (iii) from an organism other than an arenavirus. 36. The tripartite arenavirus of claim 35, comprising one heterologous ORF and one replicated arenavirus ORF. navirus particles. (Configuration 42) Configuration 4, wherein the heterologous ORF encodes an antigen derived from an infectious organism, a tumor, or an allergen. 0 or 41. The tripartite arenavirus particle according to claim 1. (Configuration 43) 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 antigens, cytomegalovirus antigens, Mycobacterium tuberculosis antigens, and tumor-associated antigens 43. The tripartite arenavirus particle of embodiment 42, selected from: (Configuration 44) 42. The tripartite construct of claim 40 or 41, wherein at least one heterologous ORF encodes a fluorescent protein. Type arenavirus particles. (Configuration 45) 44. The method according to claim 44, wherein the fluorescent protein is a green fluorescent protein or a red fluorescent protein. The three-segmented arenavirus particles shown. (Configuration 46) Constructs 33-43 contain all four arenavirus ORFs and are infectious and replicative. The tripartite arenavirus particle according to any one of the preceding claims. (Configuration 47) They lack one or more of the four arenavirus ORFs and are infectious but not viable in non-complementing cells. 46. ​​The 3-cell vector according to any one of aspects 33 to 45, wherein the vector is unable to further produce infectious progeny. Nodal arenavirus particles. (Configuration 48) Lacking one of the four arenavirus ORFs, it is infectious but not in non-complementing cells 46. ​​The tripartite vector of any one of aspects 33 to 45, which is incapable of further producing infectious progeny. Arenavirus particles. (Configuration 49) 48. The tripartite arenavirus particle of configuration 46 or 47, wherein said arenavirus lacks a GP ORF. child. (Configuration 50) A tripartite arenavirus particle comprising one L segment and two S segments, The first S segment contains an ORF encoding GP under the control of the arenavirus 3'UTR. and encoding the first gene of interest in a position under the control of the arenavirus 5'UTR. The second S segment is engineered to carry the ORF of the arenavirus 3'UTR. The ORF encoding NP is placed under the control of the arenavirus 5'UTR. the three segments, each engineered to carry an ORF encoding a second gene of interest at a certain location; Type arenavirus particles. (Configuration 51) A tripartite arenavirus particle comprising one L segment and two S segments, The first S segment contains an ORF encoding GP under the control of the arenavirus 5'UTR. and encoding the first gene of interest in a position under the control of the arenavirus 3'UTR. The second S segment is engineered to carry the ORF, which controls the arenavirus 5'UTR. It contains an ORF encoding NP at the underlying position and is under the control of the arenavirus 3'UTR. the tripartite antigen-binding fragment is engineered to carry an ORF encoding a second gene of interest at position 1. Lenavirus particles. (Configuration 52) The gene of interest encodes an antigen derived from an infectious organism, a tumor, or an allergen. 50 or 51. A tripartite arenavirus particle according to claim 50 or 51. (Configuration 53) The target gene is a human immunodeficiency virus antigen, a hepatitis C virus antigen, a varicella zoster antigen, a viral antigen, a cytomegalovirus antigen, a Mycobacterium tuberculosis antigen, and a tumor-associated antigen; 53. The tripartite arenavirus particle of claim 52, encoding an antigen. (Configuration 54) 52. The method of claim 50 or 51, wherein at least one gene of interest encodes a fluorescent protein. Nodal arenavirus particles. (Configuration 55) 54. The method according to claim 54, wherein the fluorescent protein is a green fluorescent protein or a red fluorescent protein. The three-segmented arenavirus particles shown. (Configuration 56) The tripartite arenavirus particle genome of any one of constructs 33, 35, 37, 38, 50, or 51. cDNA of. (Configuration 57) 57. A DNA expression vector comprising the cDNA of construct 56. (Configuration 58) A tripartite arenavirus particle according to any one of claims 33 to 35, a cDNA according to claim 56, or a cDNA according to claim 57. A host cell containing the vector. (Configuration 59) 52. The tripartite arenavirus particle of any one of configurations 33 to 51, which is attenuated. (Configuration 60) 34. A method of producing a tripartite arenavirus particle of claim 33, comprising: (i) transfecting one or more cDNAs of the L segment and two S segments into a host cell; To act; (ii) maintaining the host cells under conditions suitable for virus formation; and (iii) recovering the arenavirus particles; The method comprising: (Configuration 61) 36. A method of producing a tripartite arenavirus particle of embodiment 35, comprising: (i) One or more cDNAs of two L segments and one S segment are transfected into a host cell. To act; (ii) maintaining the host cells under conditions suitable for virus formation; and (iii) recovering the arenavirus particles; The method comprising: (Configuration 62) Transcription of one L segment and two S segments is driven by a bidirectional promoter. The method of claim 60, (Configuration 63) Transcription of two L segments and one S segment is driven by a bidirectional promoter. The method of claim 61, (Configuration 64) One or more nucleic acids encoding an arenavirus polymerase are transferred into the host cell. 62. The method of claim 60 or 61, further comprising: (Configuration 65) 63. The method of claim 62, wherein the arenavirus polymerase is an L protein. (Configuration 66) The method further comprises transfecting a host cell with one or more nucleic acids encoding an NP protein. The method of any one of claims 60, 61, 62, or 63, further comprising: (Configuration 67) 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, 61. The method of claim 60, wherein the promoter is under the control of a promoter selected from the group consisting of: (Configuration 68) 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, 62. The method of claim 61, wherein the promoter is under the control of a promoter selected from the group consisting of: (Configuration 69) 52. The method according to any one of claims 33 to 51, wherein the method has the same tropism as a bipartite arenavirus particle. Trisegmented arenavirus particles. (Configuration 70) 52. The tripartite arenavirus particle of any one of aspects 33 to 51, which is replication-deficient. (Configuration 71) A tripartite arenavirus particle according to any one of configurations 33 to 51, 69, and 70, and a pharmaceutical composition comprising the same. and a carrier acceptable to the animal. (Configuration 72) A tripartite arenavirus particle according to any one of configurations 33 to 51, 69, and 70, and a pharmaceutical composition comprising the same. and a suitable, and optionally acceptable, carrier. (Configuration 73) The tripartite arenavirus particle of any one of compositions 33 to 51, 69, or 70, which is derived from LCMV. child. (Configuration 74) The LCMV is MP stain, Armstrong strain, or Armstrong clone 13 strain. The three-segmented arenavirus particles shown. (Configuration 75) Derived from Junin virus vaccine Candid #1 or Junin virus vaccine XJ clone 3 strain The tripartite arenavirus particle according to any one of configurations 33 to 51, 69, or 70.

Claims

1. 1. A pharmaceutical composition for treating infection and / or cancer in a subject, comprising a tripartite arenavirus particle comprising one L segment and two S segments, wherein one of the two S segments is: (i) the S segment, in which the ORF encoding the nucleoprotein (“NP”) is under the control of the arenavirus genome 5′ untranslated region (“UTR”); (ii) the S segment, in which the ORF encoding matrix protein Z ("Z protein") is under the control of the arenavirus genome 5'UTR; (iii) the S segment, in which the ORF encoding the RNA-dependent RNA polymerase L ("L protein") is under the control of the arenavirus genome 5'UTR; (iv) the S segment, in which the ORF encoding the viral glycoprotein (“GP”) is under the control of the arenavirus genome 3′UTR; (v) an S segment in which the ORF encoding the L protein is under the control of the arenavirus genome 3'UTR; and (vi) the S segment, in which the ORF encoding the Z protein is under the control of the arenavirus genome 3′UTR; The pharmaceutical composition is selected from the group consisting of:

2. 2. The pharmaceutical composition of claim 1, wherein intersegmental recombination of the two S segments, which integrates two arenavirus ORFs onto a single segment rather than onto two separate segments, suppresses viral promoter activity.

3. The propagation of the tripartite arenavirus particles lacks type I interferon receptor, type II interferon receptor, and recombination activating gene 1 (RAG1), and 4 2. The pharmaceutical composition of claim 1, which does not produce replication-competent bipartite virus particles after 70 days of persistent infection in mice infected with PFU of said tripartite arenavirus particles.

4. 4. The pharmaceutical composition of claim 1, wherein the arenavirus genome 3'UTR is the 3'UTR of an arenavirus S segment, and the arenavirus genome 5'UTR is the 5'UTR of an arenavirus S segment.

5. 5. The pharmaceutical composition of claim 1, wherein the two S segments comprise: (i) one or two heterologous ORFs derived from an organism other than an arenavirus; (ii) one or two replicated arenavirus ORFs; or (iii) one heterologous ORF derived from an organism other than an arenavirus and one replicated arenavirus ORF.

6. 6. The pharmaceutical composition of claim 5, wherein at least one heterologous ORF encodes (i) an antigen derived from an infectious organism, a tumor, or an allergen; or (ii) an antigen selected from a human immunodeficiency virus antigen, a hepatitis C virus antigen, a varicella-zoster virus antigen, a cytomegalovirus antigen, a Mycobacterium tuberculosis antigen, and a tumor-associated antigen.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the tripartite arenavirus particle comprises all four arenavirus ORFs and is infectious and replicable.

8. 1. A pharmaceutical composition for treating infection and / or cancer in a subject, comprising a tripartite arenavirus particle comprising one L segment and two S segments, wherein a first S segment is engineered to carry an ORF encoding GP located under the control of the arenavirus genome 3' UTR and an ORF encoding a first gene of interest located under the control of the arenavirus genome 5' UTR, and a second S segment is engineered to carry an ORF encoding NP located under the control of the arenavirus genome 3' UTR and an ORF encoding a second gene of interest located under the control of the arenavirus genome 5' UTR.

9. 9. The pharmaceutical composition of claim 8, wherein the gene of interest encodes an antigen selected from (i) an antigen derived from an infectious organism, a tumor, or an allergen; or (ii) a human immunodeficiency virus antigen, a hepatitis C virus antigen, a varicella-zoster virus antigen, a cytomegalovirus antigen, a Mycobacterium tuberculosis antigen, and a tumor-associated antigen.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the tripartite arenavirus particle is derived from LCMV.

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