Three-fragment arenavirus as vaccine vector

Abstract

The present application relates to arenavirus having an open reading frame ("ORF") rearrangement in its genome. In particular, described herein are modified arenavirus genomic fragments, where the arenavirus genomic fragments are engineered to carry a viral ORF at a position other than the wild-type position of the ORF. Also described herein are three-fragment arenavirus particles comprising one L fragment and two S fragments, or two L fragments and one S fragment. The arenaviruses described herein may be suitable for vaccines and / or treatment of diseases and / or for immunotherapy.

Description

[0001] This application is a divisional application of patent application No. 201580073235.6, filed on November 12, 2015, entitled "Tri-segment Sand Virus as Vaccine Vector".

[0002] This application claims priority to U.S. Provisional Application No. 62 / 079,493, filed November 13, 2014, the entirety of which is incorporated herein by reference.

[0003] 1. Introduction This application relates to arenaviruses with open reading frame (“ORF”) rearrangements in their genome. Specifically, this application describes modified arenavirus genome fragments, wherein the arenavirus gene fragments are engineered to carry the viral ORF at a location other than the wild-type position of the ORF. This application also describes three-segment arenavirus particles comprising one L segment and two S segments, or two L segments and one S segment. The arenaviruses described herein may be suitable for use in vaccines and / or treatments of diseases and / or for immunotherapy. background 2.1 Research on Lymphocytic Choroid Meningitis Virus and Human Diseases Lymphocytic choriomeningitis virus (LCMV) is a member of the Arenaviridae family and a prototype mouse model virus in viral infection research. Since its isolation in the 1930s (Rivers and McNair Scott, 1935, Science, 81(2105): 439-440), studies using this virus have revealed many key concepts in viral immunology and pathology (summarized in Zinkernagel, 2002, Curr Top Microbiol Immunol, 263:1-5; Oldstone, 2002, Curr Top Microbiol Immunol, 263:83-117). LCMV has been widely used to study viral molecular biology and immune responses, especially in cases of persistent infection. The natural host of LCMV is the mouse; however, several reports suggest that LVMC may also be a neglected human pathogen (Barton, 1996, Clin. Infect. Dis, 22(1):197; Wright). et al.,1997, Pediatrics 100(1): E9). Furthermore, many other members of the Arenaviridae family have been found in rodent populations worldwide. Besides the Old World arenavirus Lassavirus (LASV), which can be found in Africa, several New World arenaviruses such as Junin (JUNV), Guanarito, or Machupo are prevalent in various rodent populations in South America (Johnson). et al., 1966, Am J Trop Med Hyg, 15(1): 103-106; Tesh et al., 1993, Am J Trop Med Hyg 49(2):227-235; Mills et al., 1994, Trop Med Hyg 51(5): 554-562). When transmitted to humans, many of these viruses can cause viral hemorrhagic fever with high mortality (Geisbert and Jahrling, 2004, Nat Med 10(12 Suppl): S110-121).

[0004] 2.2 Genomic structure of lymphocytic choriomeningitis virus Arenaviruses are enveloped viruses. Their genome consists of two negative-sense single-stranded RNA segments (L: 7.2 kb, S: 3.4 kb). Each segment encodes two viral genes in opposite directions. The shorter segment (S segment) encodes the viral glycoprotein (GP) precursor (GP-C; 75 kDa) and the nucleoprotein (NP; 63 kDa) (Salvato... et al., 1988, Virology 164(2):517-522). The long fragment (L fragment) expresses RNA-dependent RNA polymerase (RdRp; L protein; approximately 200 kDa) and matrix protein Z (protein Z), a RING finger protein (11 kDa) (see appendix). Figure 1 A)(Salvato) et al.,1988, Virology 164(2): 517-522). The GP precursor GP-C cleaves post-translationally into GP-1 and GP-2, which remain non-covalently associated (Buchmeier and Oldstone 1979, Virology 99(1): 111-120). The trimers of GP-1 and GP-2 assemble into spikes on the surface of the viral particle, which are crucial for entry into host cells mediated by interaction with cell surface receptors. Viral binding and entry into host cells has long been thought to be mediated by the interaction between LCMV GP and the cell receptor α-dystroglycan, since α-dystroglycan is the only cell receptor for LCMV (Cao). and al., 1998, Science, 282(5396):2079-2081). Until recently, three other human molecules (Axl and Tyro3 from the TAM family and dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin) were inferred to be additional receptors for LCMV and its close relative LASV, which allow LCMV to enter cells independently of α-dystrophic glycoprotein (Shimojima and Kawaoka 2012, J Vet Med, 74(10):1363-1366; Shimojima et al., 2012, J Virol 86(4):2067-2078). NP binds to viral RNA to form a nucleocapsid, which serves as a template for the viral L protein. The nucleocapsid associated with the viral L protein forms the so-called ribonucleoprotein complex, which is active in both replication and transcription and represents the smallest unit of viral infectivity. It has been shown that NP and L proteins are the smallest trans-acting factors (Lee) necessary for viral RNA transcription and replication. et al., 2000, J Virol 74(8): 3470-3477). The two genes on each fragment are separated by an intergenic non-coding region (IGR), flanked by 5' and 3' untranslated regions (UTR). The IGR forms a stable hairpin structure and has been shown to involve structure-dependent termination of viral mRNA transcription (Pinschewer). et al.,2005, J Virol 79(7): 4519-4526). The terminal nucleotides of URT show high complementarity, leading to the formation of secondary structures. These panhandle structures are known to act as viral promoters for transcription and replication, and site-directed mutagenesis analysis has revealed their sequence and structure dependence, making them intolerant of even minor sequence changes (Perez and de la Torre, 2003, Virol 77(2): 1184-1194).

[0005] 2.3 Reverse Genetic System Isolated and purified RNA from negative-strand viruses such as LCMV cannot directly function as mRNA; that is, it cannot be translated when introduced into cells. Therefore, transfection of cells with viral RNA does not induce the production of infectious viral particles. To generate infectious viral particles of negative-strand RNA viruses from cDNA in cultured competent cells, the viral RNA fragments must be trans-complemented with the minimum factors required for transcription and replication. Using a mini-genome system published several years ago, it is finally possible to analyze viral cis-acting elements and trans-acting factors (Lee) involved in transcription, replication, and viral particle formation. et al., 2000, J Virol 74(8):3470-3477; Lee et al., 2002, J Virol 76(12): 6393-6397; Perez and de la Torre2003, J Virol 77(2): 1184-1194; Pinschewer et al., 2003, J Virol 77(6): 3882-3887; Pinschewer et al., 2005, J Virol 79(7): 4519-4526.). For other arenaviruses such as LASV and Tacarib virus, reverse genetic systems have been established (Lopez). et al., 2001, J Virol 75(24):12241-12251; Hass et al., 2004, J Virol 78(24): 13793-13803). Using plasmids driven by pol-I / -II or T7 / pol-II respectively, two publications showed complete recovery of infectious LCMV from cDNA (also known as “virus rescue”) (Flatz). et al.,2006, Proc Natl Acad Sci USA 103(12): 4663-4668; Sanchezand de la Torre, 2006, Virology 350(2): 370-380).

[0006] 2.4 Recombinant LCMV expressing the target gene The generation of recombinant negative-sense RNA viruses expressing target exogenous genes has been pursued for a long time. Different strategies have been disclosed for other viruses (Garcia-Sastre...). et al., 1994, J Virol 68(10): 6254-6261; Percy et al., 1994, J Virol 68(7): 4486-4492; Flick and Hobom, 1999, Virology262(1): 93-103; Machado et al., 2003, Virology 313(1): 235-249). It has been previously shown that it is possible to introduce other exogenous genes into dual-segment LCMV particles (Emonet...). et al., 2009, PNAS, 106(9):3473-3478). Two target exogenous genes were inserted into the two-segment genome of LCMV, producing a three-segment LCMV particle (r3LCMV) with two S segments and one L segment. In the three-segment virus disclosed by Emonet et al. (2009), NP and GP remain in their respective native positions in the S segment and are thus expressed below their native promoters in the flanking UTR (see appendix). Figure 1 (B). However, this application shows that the three-segment LCMV particles disclosed by Emonet et al. primarily assemble into two-segment particles (i.e., the sand-like virus packages only one, not two, S fragments), resulting in reduced growth and strong selection pressure on the two S fragments for recombination. This application further shows that such recombination is reproducibly present, leading to phenotypic reversion to wild-type virus and transgene loss.

[0007] 2.5 Replication-defective sand virus Recently, it has been shown that infectious arenavirus particles can be engineered to contain a genome, enabling them to amplify and express their genetic material in infected cells, but not to produce further progeny in normal, unengineered cells (i.e., infectious, replication-defective arenavirus particles) (International Publication No. WO 2009 / 083210 A1 and International Publication No. WO 2014 / 140301 A1). Invention Overview This application relates to arenaviruses with rearranged ORFs in their genome. Specifically, this application relates to arenavirus genome fragments engineered to carry arenavirus ORFs at locations other than the wild-type location. This application also provides tri-fragment arenavirus particles comprising one L fragment and two S fragments, or two L fragments and one S fragment, which do not recombine to form replicative two-fragment arenavirus particles. This application demonstrates that tri-fragment arenavirus particles can be engineered to improve genetic stability and ensure sustained transgene expression.

[0008] In some embodiments, the viral vector provided herein is infectious, i.e., capable of entering host cells or injecting its genetic material into host cells. In some more specific embodiments, the viral vector provided herein is infectious, i.e., capable of entering host cells or injecting its genetic material into host cells, and subsequently amplifying and expressing its genetic information within said host cells. In some embodiments, the viral vector is an infectious, replication-defective arenavirus viral vector engineered to contain a genome, possessing the ability to amplify and express its genetic information in infected cells, but unable to produce further infectious progeny particles in normal, unengineered cells. In some embodiments, the infectious arenavirus viral vector is replicative, capable of producing further infectious progeny particles in normal, unengineered cells. In some more specific embodiments, such a replicative viral vector is attenuated relative to the wild-type virus from which the replicative viral vector is derived.

[0009] 3.1 Unnatural open reading frames Therefore, in one respect, this document provides a sand-like virus genome fragment. In some embodiments, the genome fragment is engineered to carry the viral ORF at a location other than the wild-type location of the ORF. In some embodiments, the sand-like virus genome fragment is selected from: S segment, in which the ORF encoding NP is under the control of the sand virus 5' UTR; The S fragment, in which the ORF encoding the Z protein is under the control of the 5' UTR of the isopyrvirus; The S fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; S segment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; The S fragment, in which the ORF encoding the L protein is under the control of the arena virus 3' UTR; The S fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR; The L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; The L segment, in which the ORF encoding NP is under the control of the 5' UTR of the sand virus; The L fragment, in which the ORF encoding the L protein is under the control of the arena virus 5' UTR; The L segment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; The L segment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and The L fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR.

[0010] In some embodiments, the 3' UTR of the isovirus is the 3' UTR of the isovirus S fragment or the isovirus L fragment. In some embodiments, the 5' UTR of the isovirus is the 5' UTR of the isovirus S fragment or the isovirus L fragment.

[0011] This article also provides the isolated cDNA of the arenavirus genome fragment provided herein. Furthermore, this article provides a DNA expression vector containing the cDNA of the arenavirus genome fragment.

[0012] This article also provides a host cell containing the said isovirus genome fragment, cDNA of the said isovirus genome fragment, or a vector containing cDNA of the said isovirus genome fragment.

[0013] This article also provides a sand-like virus particle containing the sand-like virus genome fragment and a second sand-like virus genome fragment, thereby the sand-like virus particle containing an S fragment and an L fragment.

[0014] In some embodiments, the sand-like virus particles are infectious and replicating. In some embodiments, the sand-like virus particles are attenuated. In other embodiments, the sand-like virus particles are infectious but cannot produce further infectious progeny in non-supplementary cells.

[0015] In some implementations, at least one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or functionally inactivated.

[0016] In some embodiments, at least one of the four ORFs encoding the GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than isopyrvirus. In other embodiments, only one of the four ORFs encoding the GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than isopyrvirus. In a more specific embodiment, the ORF encoding the GP protein is removed and replaced with a heterologous ORF from an organism other than isopyrvirus. In other embodiments, the ORF encoding the NP protein is removed and replaced with a heterologous ORF from an organism other than isopyrvirus. In some embodiments, the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than isopyrvirus. In other embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than isopyrvirus.

[0017] In some embodiments, the heterologous ORF encodes a reporter protein. In some embodiments, the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the heterologous ORF encoding the antigen is selected from human immunodeficiency virus antigen, hepatitis C virus antigen, hepatitis B surface antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen.

[0018] In some embodiments, the growth or infectivity of the sand virus particles is not affected by heterologous ORFs from organisms other than sand viruses.

[0019] This document also provides a method for producing a fragment of the arenavirus genome. In some embodiments, the method includes transcribing cDNA of the arenavirus genome fragment.

[0020] This document also provides a method for generating sand-like virus particles. In some embodiments, the method for generating sand-like virus particles includes: (i) Transfecting host cells with cDNA fragments of the arenavirus genome; (ii) Transfecting the host cell with a plasmid containing a fragment of the second arenavirus genome; (iii) Maintaining the host cell under conditions suitable for virus formation; and (iv) Harvest the sand-like virus particles.

[0021] In some implementations, transcription of the L and S fragments is performed using a bidirectional promoter.

[0022] In some embodiments, the method further includes transfecting a host cell with one or more nucleic acids encoding a arenavirus polymerase. In a more specific embodiment, the polymerase is an L protein. In other embodiments, the method further includes transfecting the host cell with one or more nucleic acids encoding an NP.

[0023] In some implementations, transcription of the L and S fragments is each controlled by a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0024] In another embodiment, the present invention provides a vaccine comprising arenavirus particles, wherein at least one of the four ORFs encoding GP, NP, Z, and L proteins is removed or functionally inactivated; or wherein at least one ORF encoding GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than arenavirus; or wherein only one of the four ORFs encoding GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than arenavirus. In a more specific embodiment, the vaccine further comprises a pharmaceutically acceptable vector.

[0025] In another embodiment, the present invention provides a pharmaceutical composition comprising arenavirus particles, wherein at least one of the four ORFs encoding GP, NP, Z, and L proteins is removed or functionally inactivated; or wherein at least one ORF encoding GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than arenavirus; or wherein only one of the four ORFs encoding GP, NP, Z, and L proteins is removed and replaced with a heterologous ORF from an organism other than arenavirus. In a more specific embodiment, the pharmaceutically acceptable carrier further comprises a pharmaceutically acceptable carrier.

[0026] In some embodiments, the arenavirus genome fragment or the arenavirus particle is derived from LCMV. In some embodiments, the arenavirus genome fragment or the arenavirus particle is derived from LCMV MP strain, Armstrong strain, or Armstrong Clone 13 strain. In other embodiments, the arenavirus genome fragment or the arenavirus particle is derived from Junin virus vaccine Candid #1 or Junin virus vaccine XJ Clone 3 strain.

[0027] 3.2 Tri-fragmented sand virus In one aspect, this article provides a three-segment arenavirus particle comprising one L segment and two S segments. The proliferation of the three-segment arenavirus particle occurs in the absence of type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1), and has been performed using 10... 4 Mice infected with the three-segment arenavirus particles of PFU did not produce replicating two-segment virus particles after 70 days of continuous infection. In some embodiments, intra-segment recombination of the two S segments of the two arenavirus ORFs, conjugated on only one rather than two separate segments, deactivates viral promoter activity.

[0028] In another aspect, this document provides a three-fragment isovirus particle comprising two L fragments and one S fragment. In some embodiments, the proliferation of said three-fragment isovirus particle occurs in the absence of type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1), and has been treated with 10... 4 Mice infected with the three-segment arenavirus particles of PFU did not produce replicating two-segment virus particles after 70 days of continuous infection. In some embodiments, intra-segment recombination of the two L segments of the two arenavirus ORFs, conjugated on only one rather than two separate segments, deactivates viral promoter activity.

[0029] In some implementations, one of the two S segments is selected from: (i) S fragment, in which the ORF encoding NP is under the control of the sand virus 5' UTR; (ii) S fragment, in which the ORF encoding the Z protein is under the control of the sand virus 5' UTR; (iii) S fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the sand virus; (iv) S fragment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; (v) S fragment, in which the ORF encoding the L protein is under the control of the arenavirus 3' UTR; and (vi) S fragment, in which the ORF encoding the Z protein is under the control of the 3' UTR of the sand virus.

[0030] In some implementations, one of the two L segments is selected from: (i)L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (ii) L segment, in which the ORF encoding NP is under the control of the sand virus 5' UTR; (iii) L fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR; (v) L segment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and (vi) L fragment, in which the ORF encoding the Z protein is under the control of the sand virus 3' UTR.

[0031] In some embodiments, the 3' UTR of the three-fragmented isovirus particle is the 3' UTR of either the S fragment or the L fragment of isovirus. In other embodiments, the 5' UTR of the three-fragmented isovirus particle is the 5' UTR of either the S fragment or the L fragment of isovirus.

[0032] In some implementations, the two S-fragments comprise (i) one or two heterologous ORFs from an organism other than a sand virus; or (ii) one or two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than a sand virus.

[0033] In some implementations, the two L fragments comprise (i) one or two heterologous ORFs from an organism other than a sand virus; or (ii) one or two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than a sand virus.

[0034] In some embodiments, the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the heterologous ORF encoding the antigen is selected from human immunodeficiency virus antigen, hepatitis C virus antigen, hepatitis B surface antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen.

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

[0036] In some embodiments, the three-fragmented isovirus particle contains all four isovirus ORFs. In some embodiments, the three-fragmented isovirus particle is infectious and replicating.

[0037] In some embodiments, the three-fragmented isovirus particles lack one or more of the four isovirus ORFs. In other embodiments, the three-fragmented isovirus particles are infectious but cannot produce further infectious progeny in non-supplementary cells.

[0038] In some embodiments, the trisegmental sand virus particles lack one of the four sand virus ORFs, wherein the trisegmental sand virus particles are infectious but cannot produce further infectious progeny in non-supplementary cells.

[0039] In some embodiments, the three-fragmented sand-like virus particles lack GF ORF.

[0040] In a further aspect, this document provides a three-segment isovirus particle comprising one L segment and two S segments. In some embodiments, the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 3' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 5' UTR. In some embodiments, the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 3' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 5' UTR.

[0041] In another aspect, this document provides a three-segment isovirus particle comprising an L segment and two S segments. In some embodiments, the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 5' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 3' UTR. In some embodiments, the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 5' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 3' UTR.

[0042] In some embodiments, the target gene encodes an antigen derived from an infectious organism, tumor, or allergen. In other embodiments, the target gene encodes an antigen selected from human immunodeficiency virus antigen, hepatitis C virus antigen, hepatitis B surface antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen. In yet another embodiment, at least one target gene encodes a fluorescent protein. In a specific embodiment, the fluorescent protein is GFP or RFP.

[0043] This document also provides isolated cDNA of the genome of the three-segment isovirus particle. This document also provides DNA expression vectors containing the cDNA of the genome of the three-segment isovirus particle. This document also provides one or more DNA expression vectors that individually or collectively contain the cDNA of the three-segment isovirus.

[0044] This article also provides a host cell containing the three-segment isovirus particles, cDNA of the genome of the three-segment isovirus particles, or a vector containing cDNA of the genome of the three-segment isovirus particles.

[0045] In some embodiments, the three-fragmented sand-like virus particles are attenuated.

[0046] This document also provides a method for generating the aforementioned three-fragment sand-like virus particles. In some embodiments, the method for generating sand-like virus particles includes: (i) Transfecting a host cell with one or more cDNAs, consisting of one L fragment and two S fragments; (ii) Maintaining the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

[0047] This document also provides a method for generating the aforementioned three-fragment sand-like virus particles. In some embodiments, the method for generating the three-fragment sand-like virus particles includes: (i) Transfecting a host cell with one or more cDNAs, consisting of two L fragments and one S fragment; (ii) Maintaining the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

[0048] In some embodiments, transcription of the one L fragment and the two S fragments is performed using a bidirectional promoter.

[0049] In some embodiments, the method further includes transfecting one or more nucleic acids encoding a arenavirus polymerase into the host cell. In a more specific embodiment, the polymerase is an L protein. In other embodiments, the method further includes transfecting one or more nucleic acids encoding an NP protein into the host cell.

[0050] In some implementations, the transcription of the one L fragment and the two S fragments is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0051] In some implementations, the transcription of the two L fragments and one S fragment is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0052] In some embodiments, the three-fragmented sand-like virus particles have the same tropism as the two-fragmented sand-like virus particles. In other embodiments, the three-fragmented sand-like virus particles are replication-defective.

[0053] In another embodiment, this document provides a vaccine comprising three-fragmented sand-like virus particles and a pharmaceutically acceptable vector.

[0054] In another embodiment, this document provides a pharmaceutical composition comprising three-fragmented sand virus particles and a pharmaceutically acceptable carrier.

[0055] In some embodiments, the three-fragmented isovirus particles are derived from LCMV. In some embodiments, the three-fragmented isovirus particles are derived from LCMV MP strain, Armstrong strain, or Armstrong Clone 13 strain. In other embodiments, the three-fragmented isovirus particles are derived from Junin virus vaccine Candid #1 or Junin virus vaccine XJ Clone 3 strain.

[0056] 3.3 Common Names and Abbreviations

[0057] 4. Brief description of the attached drawings Appendix Figure 1 The recombinant three-segment virus shows impaired growth compared to wild-type LCMV, independent of the location of the GP ORF in the genome. (AC) Schematic diagram of the genomic architecture of two-segment and three-segment LCMVs. The two-segment genome of wild-type LCMV consists of an S segment encoding GP and NP and an L segment encoding Z and L proteins (A). Both segments are flanked by their respective 5' and 3' UTRs. The genome of the recombinant three-segment LCMV (r3LCMV) consists of one L and two S segments, with a location for inserting the target gene (GFP in this case) into each S segment. (B) r3LCMV-GFP natural (nat) possesses all viral genes in their natural locations, while r3LCMV / GFP artificial (art) The GP ORF was artificially placed in the 3' UTR and expressed under the control of the 3' UTR. (D) Growth kinetics of the virus labeled in BHK-21 cells infected with a multiplicity of infection (moi) of 0.01 (wild-type LCMV: gray triangle; r3LCMV-GFP)nat Black circular shape; r3LCMV-GFP art (White square). Supernatant was collected at the indicated post-infection time points, and viral titers were determined by lesion formation analysis. The symbols and bars represent the mean ± SEM of three copies for each group. Error bars are obscured in the sign size.

[0058] Appendix Figure 2 The three-fragment viral product contains mostly two-fragment replication-defective particles (r2LCMV). (A) r2LCMV (white bar), r3LCMV-GFP / RFP art (Black bars, GFP-GP, RFP-NP) and r3LCMV-GFP / RFP nat (Gray bars, GP-GFP, RFP-NP) Infectivity of supernatants was determined on wild-type BHK-21 cells grown on wild-type non-supplementary BHK-21 cells (BHK21), GP-expressing BHK-21 cells (BHK-GP), or NP-expressing BHK-21 cells (BHK-NP). Titers on BHK-21 and BHK-GP cells were determined by staining NP-positive viral foci. Titers on NP-supplementary BHK-21 cells were determined by counting GP-positive foci. Titers were normalized to the mean titers obtained when assessed on BHK-21 cells and are therefore expressed as folds. Bars represent the mean ± SEM of 6 copies per group. ns.: not statistically significant (p ≥ 0.05); According to the Dunnett post-test using one-way ANOVA and then with r2LCMV as a reference, p < 0.01. (B) r2LCMV (left figure) and r3LCMV-GFP / RFP art (Middle and right panels) were grown on wild-type BHK-21 cells (BHK21; left and middle panels) or NP-expressing BHK-21 cells (BHK-NP, right panel), with fluorescence assessed by flow cytometry at 12 hours post-infection. r2LCMV-infected cells were used as a gate selection control. A representative panel is shown for each condition. (C) Using r3LCMV-GFP / RFP art Quantification of GFP+, RFP+, or GFP+RFP+ double-positive cells 12 hours after infection with BHK-21 or BHK-NP cells. Bars represent the mean ± SEM of three copies per group. ns.: not statistically significant (p≥0.05); p < 0.001, according to the unpaired two-tailed Student's T-cell test.

[0059] Appendix Figure 3 Design and growth kinetics of recombinant three-fragment viruses with partially codon-optimized GP ORFs or genetic tags in the IGR of the S fragment. (A) Schematic diagram of the genetically engineered S fragment, where the 255 C-terminal base pairs of the GP are codon-optimized and the NP is replaced with GFP (the GP ORF is referred to as "WE / WET"). r3LCMV-WEWET / GFP nat Growth kinetics were observed in BHK-21 cells, as shown in the attached diagram. Figure 1 As detailed in B, it consists of two S and one L fragments, modified with the GP-containing S fragment shown in (A). Supernatant was collected at time points indicated by infection at moi=0.01, and viral titers were determined by lesion formation analysis (B). Symbols and bars represent the mean ± SEM of three copies per group. Error bars are masked in the symbol size. (C) Schematic diagram of the S fragment encoding NP, where one base pair of IGR is deleted to genetically “mark” this non-coding RNA element. The deleted G residue (indicated by arrow) is located outside the key stem-loop structure of the IGR. Growth kinetics of the three-fragment virus were compared on BHK-21 cells at moi 0.01, with or without the genetic marker (r3LCMV-GFP) in the S fragment encoding NP. nat Black circular shape; r3LCMV-GFP nat IGR (White circle). Supernatant was collected at the indicated post-infection time point, and viral titer was determined by lesion formation analysis. Symbols and bars represent the mean ± SEM of three copies per group. Representative data from one of the two independent experiments are shown.

[0060] Appendix Figure 4 r3LCMV-GFP in immunodeficient mice nat Instead of r3LCMV-GFP art Persistent infection reached levels comparable to that of the two-fragment wild-type virus and caused loss of GFP expression. (A) AGRAG mice were infected with 1×10 4 r3LCMV-GFP of PFU nat (Black circle), r3LCMV-GFP art (White square) or control, two-fragment r2LCMV (gray triangle) intravenous inoculation was performed, and viremia was monitored over time. Symbols represent mean ± SEM of 3–7 mice per group. (B) shows the results using 1 × 10⁻⁶ cells. 4 r3LCMV-GFP of PFU nat or r3LCMV-GFP artLCMV viremia on day 127 after intravenous infection of AGRAG mice. Immunofocal analysis was performed to detect nucleoprotein NP (gray circles) or GFP (white circles). Symbols represent individual mice. ns.: not statistically significant (p≥0.05); The result was obtained by unpaired two-tailed Student's T cell test, p < 0.001. (CE) from r3LCMV-GFP nat r3LCMV-GFP art Blood samples from AGRAG mice infected with r2LCMV were analyzed by flow cytometry to detect the presence of GFP+ cells 120 days post-infection. Monocytes and macrophages were identified using the gating strategy described in (C). A representative FACS plot for each group, along with a representative histogram overlay of GFP expression, is shown in (D). (E) Quantification of the GFP+ population in CD11b+GR1- monocyte / macrophage populations. Symbols represent individual mice.

[0061] Appendix Figure 5 : mouse r3LCMV-GFP nat Persistent infection results in S-fragment recombination and loss of functional full-length transgenes. From 1×10 4 PFU r3LCMV-GFP nat or r3LCMV-GFP art Viral RNA was isolated from serum of AGRAG mice 127 days after intravenous infection. Retroviral RNA and cDNA containing NP and GP sequences were amplified by PCR using appropriate gene-specific primers. (A) DNA electrophoresis of PCR products obtained after prior reverse transcription of the RNA template (+RT, lanes 1-8) or without reverse transcription (-RT, negative control, lanes 9-12). Serum from native animals was used as an independent negative control (n, lane 8), and plasmid DNA encoding the wild-type LCMV S fragment was used as a positive control (p, lane 17). Amplicons from lanes 1-3 were subjected to Sanger sequencing. (B) DNA from animal #3 (r3LCMV-GFP) nat (#3) The representative cDNA sequence obtained shows the recombinant S fragment, which combines NP and GP sequences, two IGRs (bold), and a C-terminal GFP portion (highlighted in gray) (SEQ ID NO: 17). (C) Schematic diagram of three recombinant viral S fragment sequences isolated 127 days post-infection, each of which dominates a viral population in a representative AGRAG mouse. IGRs from the S fragment carrying NP are marked with an asterisk ( The sequenced extensions are indicated by double arrows (<-->). The base pair (bp) length index describes the above-mentioned GFP remnants and truncated (shortened) IGR elements.

[0062] Appendix Figure 6 The growth kinetics of recombinant viruses with two IGRs on the S fragment are similar to those of dual-fragment viruses. BHK-21 cells were treated with dual-fragment LCMV (grey triangle) with the wild-type S fragment at 0.01 moi, and with triple-fragment r3LCMV-GFP. nat (Black circular), or infected with r2LCMV_2IGRs (white diamond-shaped), which carry an S fragment corresponding to the recombinant product recovered from infected AGRAG mice (compare with appendix). Figure 5 Supernatant was collected at the indicated time points, and viral titer was determined by lesion formation analysis. Symbols and bars represent the mean ± SEM of three copies per group. Error bars are masked in sign size. ns.: not statistically significant (p ≥ 0.05); ***: p < 0.001 (one-way ANOVA, and subsequent Bonferroni's post-test for multiple comparisons).

[0063] Appendix Figure 7 : Can explain r3LCMV-GP nat Recombination event model of transgenic loss and r3LCMV-GP art The hypothetical mechanism of genetic stability. This model itself is based on sequence data of LCMV transcription termination (Meyer and Southern, 1993, J Virol, 67(5):2621-2627), and on the reverse genetic evidence of IGR as a transcription termination signal (Pinschewer). et al.,(2005, J Virol, 79(7):4519-4526) combined. Together, these findings suggest structure-dependent polymerase pausing during the completion of the hairpin structure of the IGR. The discovery that the GFP remnant between the two IGRs in the recombinant S fragment originates from one or both S fragments supports a model of polymerase template switching (also known as copy selection) occurring during polymerase pausing during genome or antigenome synthesis (cases A and B below, respectively). (A) During antigenome synthesis, RNA-dependent RNA polymerase (RdRp) is initiated at the 3' UTR of the genome S fragment template and then reads through the NP ORF and IGR. At the end of the IGR, the polymerase pauses due to secondary structure ("structure-dependent polymerase pausing"). The pausing of the polymerase facilitates copy selection, with RNA replication continuing on the selectable template (in this case: the S fragment genome encoding the GP). Template switching must occur upstream of the GP stop codon, which is obviously most likely to target a sequence near the IGR hairpin or at the bottom of the IGR hairpin. The polymerase continues to read the C-terminus of GFP through the second template, and then synthesizes the second IGR, GP ORF, and 5' UTR. (B) During genome synthesis, RdRp initiates RNA synthesis at the 3' end of the antigenomic S fragment template containing GP, synthesizing the 5' UTR, GP, and most or all of the IGR, followed by a structure-dependent polymerase pause. Copy selection occurs, switching to the C-terminal portion of the GFP ORF near the IGR containing the NP. Thus, the fragment with added GFP is replicated, followed by the full-length IGR, NP, and 3' UTR. (C – D) Template switching similar to cases (A) and (B) can also occur in r3LCMV-GFP. art This process occurs during the synthesis of the genome or antigenome. It can also combine NPs and GP ORFs onto a single RNA fragment. However, the latter consists of two 3' UTRs, not a 3' UTR and a 5' UTR, which together form a functional viral promoter. Therefore, such a molecule cannot be amplified by RdRp and thus does not form a recombinant replicating virus.

[0064] Appendix Figure 8 It produced a GFP with characteristics similar to r3LCMV-GFP. art The genome architecture (see Appendix) Figure 1 C) r3LCMV-OVA art The vaccine vector contains two ovalbumin (OVA) genes, replacing the corresponding GFP genes in the previous virus. Using 10... 4 PFU's r3LCMV-OVA art , or use 10 8C57BL / 6 mice were immunized intramuscularly (im) with a vector expressing OVA based on a replication-defective E1 deletion-based adenovirus 5. Animals were euthanized 8 days later, and T-cell responses induced by immunization were analyzed. A: The frequency of OVA-specific CD8+ T cells in the spleen was determined using class I MHC tetramers loaded with SIINFEKL peptide. Epitope-specific cell frequencies were determined in B220-negative CD8+ lymphocytes. B: OVA-specific CD8+ T cell function was analyzed by intracellular cytokine analysis using SIINFEKL peptide for restimulation. Bars represent mean + / - SEM values ​​from five mice per group. p<0.05; Since p>0.01, we can use the unpaired two-tailed Student's t-test.

[0065] Appendix Figure 9 Three-segment LCMV induces multifunctional memory CD8+ T cells. C57BL / 6 mice were treated with 1×10-1 5 PFUr3LCMV OVA or 1×10 8 PFU rAd-OVA iv infection. Spleens were harvested 25 days post-infection, and OVA-specific CD8+ T cell function was analyzed by intracellular cytokine staining. Cytokine distribution (IFN-γ, TNF-α, and IL-2) of OVA-specific T cells induced by r3LCMV-OVA (black bars) or rAd-OVA (white bars) is shown as the percentage of CD8+ T cells (A) or the absolute number per spleen (B). Symbols and bars represent the mean ± SEM of five mice per group. Unpaired two-tailed Student's t-tests were 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 study from two similar experiments is shown.

[0066] Appendix Figure 10 The LCMV encoding the antigen induced specific T cell responses against exogenous and self-antigens. C57BL / 6 mice were treated with 1×10... 5 PFU encoding rat, human, or mouse Her2 peptides (A, B, and C, respectively) was administered via r3LCMV iv infection. Spleens were harvested nine days post-infection, and the induction of functional antigen-specific CD8+ T cells was analyzed by intracellular cytokine staining and flow cytometry. Cytokine distribution (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 mean ± SEM of three mice per group.

[0067] Appendix Figure 11Interferon-α was induced in r3LCMV infection, but not in infection with recombinant adenovirus or vaccinia virus. C57BL / 6 mice were treated with 1×10 5 PFU's r3LCMV OVA, 1×10 8 rAd-OVA of PFU or 1×10 6 PFU was administered via rVacc-OVA iv. Blood samples were collected at indicated post-infection time points, and serum interferon-α levels were measured by ELISA. Symbols and bars represent the mean ± SEM of the following formula for each group of four instruments. p < 0.001 (two-way ANOVA, followed by Bonferroni's post-test for multiple comparisons). Representative data from one of the two independent experiments are shown.

[0068] Appendix Figure 12 : with r3JUNV-GFP nat r3JUNV-GFP compared to r2JUNV-wt art Cell cultures grow. r3JUNV-GFP art and r3JUNV-GFP nat Similar to appendix Figure 1 The corresponding r3LCMV vectors were constructed as illustrated in the diagram. To avoid their cell culture growth properties, 293T cells were infected with r2LCMV-wt and r3JUNV-GFP at a multiplicity of infection (MOI) of 0.01. art and r3JUNV-GFP nat Infection was achieved, and supernatant was harvested at the indicated time points. Infectious units (FFU) in the supernatant were determined by immunofocal analysis. Symbols and bars represent the mean ± SEM of three copies per group, masked within the symbol size.

[0069] Appendix Figure 13 The three-segment JUNV was significantly attenuated in vivo, causing only detectable viremia upon GFP loss. (A) AGRAG mice were treated with 7 × 10 4 r3JUNV-GFP of PFU nat (Gray square), r3JUNV-GFP art (White triangle) or control double-fragment r2JUNV strain Candid#1 (black circle) IV infection, monitoring for viremia over time. Symbols represent individual mice (n=3–7 per group). (B) Inoculated with 7 × 10 4 r3JUNV-GFP of PFU nat or r3JUNV-GFP artJUNV viremia was measured 120 days after intravenous infection of AGRAG mice. Immunofocal analysis was performed to detect nucleoprotein NP (gray circles) or GFP (white circles). A staining control was used to inoculate the mouse virus stockpile for analysis. Symbols represent individual mice and inoculum, respectively.

[0070] Appendix Figure 14 Homologous and heterologous priming-boost combinations of vaccine vectors based on three fragments of LCMV and JUNV induced strong P1A autoantigen-specific CD8+ T cell responses. (A) On days 0 and 35 of the experiment, BALB / c mice were treated with 8.5 × 10⁻⁶ LCMV and JUNV. 4 PFU r3JUNV-P1A art (r3JUNV-P1A) and r3LCMV-P1A art (r3LCMV-P1A) was administered intravenously via homologous or heterologous combinations as indicated in the chart. Epitope-specific CD8+ T cells were stained with MHC class I tetramers loaded with the P1A epitope in combination with anti-CD8a antibody. The frequency of P1A-tetramer-bound cells in the peripheral blood CD8+ T cell fraction (A) and the absolute number of P1A-tetramer-bound CD8+ T cells per μL of peripheral blood were calculated (B). Symbols represent mean + / - SEM values ​​for 3–5 mice per group and time points.

[0071] Detailed description of the invention 4.1 Sand-borne viruses with open reading frames in non-natural locations This document provides an isovirus with ORF rearrangement. In some embodiments, such an isovirus is replicating and infectious. This document provides the genome sequence of such an isovirus. In one aspect, this document provides an isovirus genome fragment, wherein the isovirus genome fragment is engineered to carry an isovirus ORF at a location (i.e., a non-natural location) different from the location where the corresponding gene of the ORF in wild-isolated viruses, such as LCMV-MP (see SEQ ID NO: 4 and 5), is present (referred to herein as the "wild-type location"). In one embodiment, the isovirus particle is an LCMV.

[0072] The wild-type arenavirus genome fragments and ORFs are known in the art. Specifically, the arenavirus genome consists of an S fragment and an L fragment. The S fragment carries an ORF encoding GP and NP. The L fragment encodes the L and Z proteins. Both fragments are flanked by their respective 5' and 3' UTRs (see Appendix). Figure 1 A). Illustrative wild-type arenavirus genome fragments are provided in SEQ ID NO: 1-10.

[0073] In some embodiments, the arenavirus genome fragment can be engineered to carry two or more arenavirus ORFs at a location other than the wild-type location. In other embodiments, the arenavirus genome fragment can be engineered to carry two, three, or four arenavirus ORFs at a location other than the wild-type location.

[0074] In some implementations, the arenavirus genome fragments provided herein may be: (i) Sand virus S segment, in which the ORF encoding NP is under the control of sand virus 5' UTR; The isovirus S fragment, in which the ORF encoding the Z protein is under the control of the isovirus 5' UTR; The isovirus S fragment, in which the ORF encoding the L protein is under the control of the isovirus 5' UTR; The sand virus S segment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; The isovirus S fragment, in which the ORF encoding the L protein is under the control of the isovirus 3' UTR; and The isovirus S fragment, in which the ORF encoding the Z protein is under the control of the isovirus 3'UTR; The L segment of the sand virus, in which the ORF encoding GP is under the control of the sand virus 5' UTR; The L segment of the sand virus, in which the ORF encoding NP is under the control of the sand virus 5' UTR; The L fragment of the isoplasmosis virus, in which the ORF encoding the L protein is under the control of the isoplasmosis virus 5' UTR; The L segment of the sand virus, in which the ORF encoding GP is under the control of the sand virus 3'UTR; The L segment of the sand virus, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and The L fragment of the isovirus, in which the ORF encoding the Z protein is under the control of the isovirus 3' UTR.

[0075] In some embodiments, the ORF at a non-natural location of the arenavirus genome fragment described herein may be under the control of the arenavirus 3' UTR or the arenavirus 5' UTR. In a more specific embodiment, the arenavirus 3' UTR is the 3' UTR of the arenavirus S fragment. In another specific embodiment, the arenavirus 3' UTR is the 3' UTR of the arenavirus L fragment. In a more specific embodiment, the arenavirus 5' UTR is the 5' UTR of the arenavirus S fragment. In other specific embodiments, the 5' UTR is the 5' UTR of the L fragment.

[0076] In other embodiments, the ORF in the non-natural location of the arenavirus genome fragment described herein may be under the control of conserved terminal sequence elements of the arenavirus (5'- and 3' end 19-20-nt regions) (see, for example, Perez & de la Torre, 2003, J Virol. 77(2): 1184–1194).

[0077] In some implementations, the ORF at a non-natural location in the arenavirus genome fragment can be under the control of the promoter element of the 5' UTR (see, for example, Albariño). et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORF at a non-natural location of the arenavirus genome fragment can be under the control of the promoter element of the 3' UTR (see, for example, Albariño). et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, the promoter element of the 5' UTR is a promoter element of the S-fraction or L-fraction 5' URT. In another specific embodiment, the promoter element of the 3' UTR is a promoter element of the S-fraction or L-fraction 3' URT.

[0078] In some implementations, the ORF at a non-natural location of the arenavirus genome fragment may be under the control of a truncated arenavirus 3' UTR or a truncated arenavirus 5' UTR (see, for example, Perez & de la Torre, 2003, J Virol. 77(2): 1184–1194; Albariño). et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, the truncated 3' UTR is the 3' UTR of the isovirus S fragment or L fragment. In a more specific embodiment, the truncated 5' UTR is the 5' UTR of the isovirus S fragment or L fragment.

[0079] This document also provides a sand-like virus particle comprising a first genome fragment and a second sand-like virus genome fragment, wherein the first genome fragment is engineered to carry an ORF at a location other than the wild-type location of the ORF, thereby the sand-like virus particle comprising an S fragment and an L fragment. In a specific embodiment, the ORF at the location other than the wild-type location of the ORF is one of the sand-like virus ORFs.

[0080] In some specific embodiments, the sand-virus particle may contain the entirety of all four sand-virus ORFs. In another specific embodiment, the second sand-virus genome fragment is engineered to carry the ORF at a location other than the wild-type location of the ORF. In yet another specific embodiment, the second sand-virus genome fragment may be a wild-type genome fragment (i.e., containing the ORF at the wild-type location).

[0081] In some embodiments, the first isovirus genome fragment is an L fragment and the second isovirus genome fragment is an S fragment. In other embodiments, the first isovirus genome fragment is an S fragment and the second isovirus genome fragment is an L fragment.

[0082] Non-restricted examples of sand-like viral particles, including genomic fragments of the ORF located outside the wild-type position of the ORF and second genomic fragments, are listed in Table 1.

[0083] Table 1 Sand-like virus particles Position 1 is under the control of the sand virus S fragment 5' UTR; Position 2 is under the control of the sand virus S fragment 3' URT; Position 3 is under the control of the sand virus L fragment 5' UTR; Position 4 is under the control of the sand virus L fragment 3' UTR.

[0084] This document also provides cDNAs engineered to carry arenavirus genome fragments of the ORF at locations other than the wild-type location of the ORF. In a more specific embodiment, this document provides cDNAs or collections of arenavirus genomes listed in Table 1.

[0085] In some embodiments, cDNA engineered to carry an arenavirus genome fragment of the ORF at a location other than the wild-type location of the ORF is part of or incorporated into a DNA expression vector. In a specific embodiment, cDNA engineered to carry an arenavirus genome fragment of the ORF at a location other than the wild-type location of the ORF is part of or incorporated into a DNA expression vector that facilitates the production of the arenavirus genome fragment described herein. In another embodiment, the cDNA described herein may 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. Techniques for producing cDNA are conventional and routine techniques in molecular biology and DNA manipulation and production. Any cloning technique known to a skilled technician may be used. Such techniques are well known and available to a skilled technician from laboratory manuals, e.g., Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory NY (2001).

[0086] In some embodiments, cDNA of an arenavirus genome fragment engineered to carry the ORF at a location other than the wild-type location of the ORF is introduced (e.g., transfected) into a host cell. Thus, in some embodiments, a host cell is provided herein containing cDNA of an arenavirus genome fragment engineered to carry the ORF at a location other than the wild-type location of the ORF (i.e., cDNA of the genome fragment). In other embodiments, the cDNA described herein is a portion of or incorporated into a DNA expression vector and is introduced into a host cell. Thus, in some embodiments, a host cell is provided herein containing the cDNA described herein incorporated into a vector. In other embodiments, the arenavirus genome fragment described herein is introduced into a host cell.

[0087] In some embodiments, this document describes a method for generating a isoplasmic virus genome fragment, wherein the method includes transcribing cDNA of the isoplasmic virus genome fragment. In some embodiments, a viral polymerase protein may be present during transcription of the isoplasmic virus genome fragment, either in vitro or in vivo.

[0088] In some embodiments, transcription of the arenavirus genome fragment is performed using a bidirectional promoter. In other embodiments, transcription of the arenavirus genome fragment is performed using a bidirectional expression cassette (see, for example, Ortiz-Riaño). et al.,2013, J Gen Virol., 94(Pt 6): 1175–1188). In a more specific embodiment, the bidirectional expression cassette includes polymerase I and polymerase II promoters, which read from the contralateral ends of the inserted arenavirus genome fragment, respectively. In an even more specific embodiment, the bidirectional expression cassette having pol-I and pol-II promoters reads from the contralateral L and S fragments.

[0089] In other embodiments, transcription of the cDNA of the arenavirus genome fragment described herein includes a promoter. Specific examples of promoters include RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter, SP6 promoter, or T3 promoter.

[0090] In some embodiments, the method for producing a arenavirus genome fragment may further include introducing cDNA of the arenavirus genome fragment into a host cell. In some embodiments, the method for producing a arenavirus genome fragment may further include introducing cDNA of the arenavirus genome fragment into a host cell, wherein the host cell expresses all other elements for producing the arenavirus genome fragment; and purifying the arenavirus genome fragment from the supernatant of the host cell. Such methods are well known to those skilled in the art.

[0091] This document provides cell lines, cultures, and methods for culturing cells infected with the nucleic acids, vectors, and compositions provided herein. More detailed descriptions of the nucleic acid, vector systems, and cell lines described herein are provided in Section 4.5.

[0092] In some embodiments, the arenavirus particles described herein produce infectious and replicating arenavirus particles. In specific embodiments, the arenavirus particles described herein are attenuated. In particular embodiments, the arenavirus particles are attenuated such that the virus retains, at least partially retains, its transmissibility and can replicate in vivo, but only produces a low viral load, resulting in a non-pathogenic, subclinical level of infection. Such attenuated virus can be used as an immunogenic composition. Immunogenic compositions are provided herein that comprise arenaviruses having ORFs at non-natural locations as described in Section 4.7.

[0093] 4.1.1 Replication-defective sand-like virus particles with open reading frames at non-natural locations In some embodiments, this document provides arenavirus particles in which (i) the ORF is located in a position other than the wild-type position of the ORF; and (ii) the ORF encoding the GP, NP, Z, and L proteins is removed or functionally inactivated, so that the resulting virus cannot further produce infectious progeny virus particles. Arenavirus particles containing a genetically modified genome with one or more deleted or functionally inactivated ORFs can be generated in supplementary cells (i.e., cells expressing deleted or functionally inactivated arenavirus ORFs). The genetic material of the generated arenavirus particles can be transferred to host cells upon infection, where the genetic material can be expressed and amplified. Furthermore, the genome of the genetically modified arenavirus particles described herein can encode heterologous ORFs from organisms other than arenavirus particles.

[0094] In some embodiments, at least one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or replaced with a heterologous ORF from an organism other than isopyvirus. In another embodiment, at least one, at least two, at least three, or at least four ORFs encoding the GP, NP, Z, and L proteins are removed or replaced with a heterologous ORF from an organism other than isopyvirus. In a specific embodiment, only one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or replaced with a heterologous ORF from an organism other than the isopyvirus particle. In a more specific embodiment, the ORF encoding the GP protein from the isopyvirus genome fragment is removed. In another specific embodiment, the ORF encoding the NP protein from the isopyvirus genome fragment is removed. In a more specific embodiment, the ORF encoding the Z protein from the isopyvirus genome fragment is removed. In yet another specific embodiment, the ORF encoding the L protein is removed.

[0095] Therefore, in some embodiments, the sand virus particles provided herein contain a genome fragment that (i) is engineered to carry an ORF in a non-natural location; (ii) the ORF encoding a GP, NP, Z, or L protein is removed; and (iii) the removed ORF is replaced by a heterologous ORF from an organism other than a sand virus.

[0096] In some embodiments, the length of the heterologous ORF is 8 to 100 nucleotides, 15 to 100 nucleotides, 25 to 100 nucleotides, 50 to 200 nucleotides, 50 to 400 nucleotides, 200 to 500 nucleotides, or 400 to 600 nucleotides, or 500 to 800 nucleotides. In other embodiments, the length of the heterologous ORF is 750 to 900 nucleotides, 800 to 100 nucleotides, 850 to 1000 nucleotides, 900 to 1200 nucleotides, 1000 to 1200 nucleotides, 1000 to 1500 nucleotides, or 10 to 1500 nucleotides, 1500 to 2000 nucleotides, 1700 to 2000 nucleotides, 2000 to 2300 nucleotides, 2200 to 2500 nucleotides, 2500 to 3000 nucleotides, 3000 to 3200 nucleotides, 3000 to 3500 nucleotides, 3200 to 3600 nucleotides, 3300 to 3800 nucleotides, or 4000 nucleotides. 4400 to 4200 to 4700 nucleotides, 4800 to 5000 nucleotides, 5000 to 5200 nucleotides, 5200 to 5500 nucleotides, 5500 to 5800 nucleotides, 5800 to 6000 nucleotides, 6000 to 6400 nucleotides, 6200 to 6800 nucleotides, 6600 to 7000 nucleotides, 7000 to 7200 nucleotides, 7200 to 7500 nucleotides, or 7500 nucleotides. In some embodiments, the heterologous ORF encodes a peptide or polypeptide with a length of 5 to 10 amino acids, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 100 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 750 amino acids, 750 to 1000 amino acids, 1000 to 1250 amino acids, 1250 to 1500 amino acids, 1500 to 1750 amino acids, 1750 to 2000 amino acids, 2000 to 2500 amino acids, or more than 2500 or more amino acids. In some embodiments, the heterologous ORF encodes a polypeptide with a length not exceeding 2500 amino acids. In a specific embodiment, the heterologous ORF does not contain a stop codon. In some embodiments, the heterologous ORF is codon-optimized. In some embodiments, the nucleotide composition, or both, may be optimized. Such optimization techniques are known in the art and can be used to optimize heterologous ORFs.

[0097] Any heterologous ORF from an organism other than isoniavirus can be included in the isoniavirus genome fragment. In one embodiment, the heterologous ORF encodes a reporter protein. A more detailed description of the reporter protein is given in Section 4.3. In another embodiment, the heterologous ORF encodes an antigen of an infectious pathogen capable of evoking an immune response or an antigen associated with any disease. In a specific embodiment, the antigen is derived from an infectious organism, a tumor (i.e., cancer), or an allergen. A more detailed description of the heterologous ORF is given in Section 4.3.

[0098] In some embodiments, the growth and infectivity of the sand virus particles are not affected by heterologous ORFs from organisms other than sand viruses.

[0099] Techniques known to those skilled in the art can be used to generate arenavirus particles containing arenavirus genome fragments engineered to carry arenavirus ORFs at locations other than the wild-type location. For example, reverse genetics techniques can be used to generate such arenavirus particles. In other embodiments, replication-defective arenavirus particles (i.e., arenavirus genome fragments engineered to carry arenavirus ORFs at locations other than the wild-type location, wherein the ORFs encoding GP, NP, Z, and L proteins are deleted) can be generated in supplementary cells.

[0100] In some implementations, the sand virus genome fragments or sand virus particles used, depending on the current application, may be Old World viruses, such as LCMV.

[0101] In some implementations, the current application relates to the sand-like virus particles described herein that are suitable for use as vaccines, and to methods of using such sand-like virus particles in vaccination and, for example, the treatment or prevention of infection or cancer. More detailed descriptions of the methods of using the sand-like virus particles described herein are provided in section 4.6.

[0102] In some embodiments, this document provides a kit containing one or more cDNAs described herein in one or more containers. In specific embodiments, the kit contains a sand-like virus genome fragment or sand-like virus particle described herein in one or two or more containers. The kit may further contain one or more of the following: host cells suitable for rescuing sand-like virus genome fragments or sand-like virus particles, reagents suitable for transfecting plasmid cDNA into host cells, helper viruses, plasmids encoding viral proteins, and / or one or more primers specific to the modified sand-like virus genome fragment or sand-like virus particle or its cDNA.

[0103] In some embodiments, the current application relates to the sand-like virus particles described herein, suitable for use as pharmaceutical compositions, and to methods of using such sand-like virus particles in vaccination and, for example, the treatment or prevention of infections and cancers. More detailed descriptions of the methods of using the sand-like virus particles described herein are provided in section 4.7.

[0104] 4.2 Three-fragment sand-like virus particles This document provides a three-segment isovirus particle with ORF rearrangement. In one aspect, this document provides a three-segment isovirus particle comprising one L segment and two S segments, or two L segments and one S segment. In some embodiments, the three-segment isovirus particle does not recombine into a replicating two-segment isovirus particle. More specifically, in some embodiments, the two segments of the genome (e.g., two S segments or two L segments, respectively) cannot recombine in any way to produce a single viral fragment that can replace the two parental segments. In a specific embodiment, the three-segment isovirus particle contains an ORF located outside the wild-type position of the ORF. In yet another specific embodiment, the three-segment isovirus particle contains all four isovirus ORFs. Thus, in some embodiments, the three-segment isovirus particle is replicating and infectious. In other embodiments, the three-segment isovirus particle lacks one of the four isovirus ORFs. Thus, in some embodiments, the three-segment isovirus particle is infectious but cannot produce further infectious progeny in non-compensatory cells.

[0105] In some embodiments, the ORF encoding the GP, NP, Z, or L protein of the three-fragment isovirus particle described herein may be under the control of the isovirus 3' UTR or the isovirus 5' UTR. In a more specific embodiment, the three-fragment isovirus 3' UTR is the 3' UTR of the isovirus S fragment. In another specific embodiment, the three-fragment isovirus 3' UTR is the 3' UTR of the three-fragment isovirus L fragment. In a more specific embodiment, the three-fragment isovirus 5' UTR is the 5' UTR of the isovirus S fragment. In other specific embodiments, the 5' UTR is the 5' UTR of the L fragment.

[0106] In other embodiments, the ORF encoding the GP, NP, Z, or L protein of the three-segment arenavirus particle described herein may be under the control of conserved terminal sequence elements of the arenavirus (5'- and 3'-terminal 19-20-nt regions) (see, for example, Perez & de la Torre, 2003, J Virol. 77(2): 1184–1194).

[0107] In some embodiments, the ORF encoding the GP, NP, Z, or L protein of the three-segment sand-borne virus particle can be under the control of the promoter element of the 5' UTR (see, for example, Albariño). et al., 2011, J Virol., 85(8): 4020-4). In another embodiment, the ORF encoding the GP, NP, Z, and L proteins of the three-segment sand-like virus particle can be under the control of the promoter element of the 3' UTR (see, for example, Albariño). et al., 2011, JVirol., 85(8):4020-4). In a more specific embodiment, the promoter element of the 5' UTR is an S-fraction or L-fraction 5' UTR promoter element. In another specific embodiment, the promoter element of the 3' UTR is an S-fraction or L-fraction 3' UTR promoter element.

[0108] In some embodiments, the ORF encoding the GP, NP, Z, or L proteins of the three-segment arenavirus particle can be under the control of a truncated arenavirus 3' UTR or a truncated arenavirus 5' UTR (see, for example, Perez & dela Torre, 2003, J Virol. 77(2): 1184–1194; Albariño). et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, the truncated 3' URT is the 3' UTR of the isovirus S fragment or L fragment. In a more specific embodiment, the truncated 5' UTR is the 5' UTR of the isovirus S fragment or L fragment.

[0109] This document also provides cDNA of the three-fragment sand-like virus particles. In a more specific embodiment, this document provides DNA nucleotide sequences or sets of DNA nucleotide sequences encoding the three-fragment sand-like virus particles, as listed in Table 2 or Table 3.

[0110] In some embodiments, the nucleic acid encoding the three-segment arecavirus genome is a portion of or incorporated into one or more DNA expression vectors. In a specific embodiment, the nucleic acid encoding the genome of a three-segment arecavirus particle is a portion of or incorporated into one or more DNA expression vectors that facilitate the production of the three-segment arecavirus particles described herein. In another embodiment, the cDNA described herein may be incorporated into a plasmid. More detailed descriptions of the cDNA and expression system are provided in section 4.5.1. The techniques for producing cDNA are conventional and routine techniques in molecular biology and DNA manipulation and production. Any cloning technique known to a skilled technician may be used. Such techniques are well-known and available to a skilled technician from laboratory manuals, e.g., Sambrook and Russell, Molecular Cloning: A laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory NY (2001).

[0111] In some embodiments, the cDNA of the three-fragment isavirus is introduced (e.g., transfected) into a host cell. Thus, in some embodiments, a host cell is provided herein containing the cDNA of the three-fragment isavirus particle (i.e., the cDNA of the genomic fragments of the three-fragment isavirus particle). In other embodiments, the cDNA described herein is a portion of a DNA expression vector or may be incorporated into a DNA expression vector and introduced into a host cell. Thus, in some embodiments, a host cell is provided herein containing the cDNA described herein incorporated into a vector. In other embodiments, the three-fragment isavirus genomic fragments described herein (i.e., the L fragment and / or the S fragment or fragment) are introduced into a host cell.

[0112] In some embodiments, this document describes a method for producing three-fragment arenavirus particles, wherein the method includes transcribing the cDNA of the three-fragment arenavirus particles. In some embodiments, a viral polymerase protein may be present during transcription of the three-fragment arenavirus particles in vitro or in vivo. In some embodiments, transcription of the arenavirus genome fragments is performed using a bidirectional promoter.

[0113] In other embodiments, transcription of the arenavirus genome fragment is performed using a bidirectional expression cassette (see, for example, Ortiz-Riaño). et al.,2013, J Gen Virol., 94(Pt 6): 1175–1188). In a more specific embodiment, the bidirectional expression cassette contains polymerase I and polymerase II promoters, which read from the opposite side to the two ends of the inserted arenavirus genome fragment.

[0114] In other embodiments, transcription of the cDNA of the arenavirus genome fragment described herein includes a promoter. Specific examples of promoters include RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter, SP6 promoter, or T3 promoter.

[0115] In some embodiments, the method for producing three-fragment isopyrvirus particles may further include introducing cDNA of the three-fragment isopyrvirus particles into a host cell. In some embodiments, the method for producing three-fragment isopyrvirus particles may further include introducing cDNA of the three-fragment isopyrvirus particles into a host cell, wherein the host cell expresses all other elements for producing the three-fragment isopyrvirus particles; and purifying the three-fragment isopyrvirus particles from the supernatant of the host cell. Such methods are well known to those skilled in the art.

[0116] This document provides cell lines, cultures, and methods for culturing cells infected with the nucleic acids, vectors, and compositions provided herein. More detailed descriptions of the nucleic acid, vector systems, and cell lines described herein are provided in Section 4.5.

[0117] In some embodiments, the three-fragmented isovirus particles described herein produce infectious and replicating isovirus particles. In specific embodiments, the isovirus particles described herein are attenuated. In particular embodiments, the three-fragmented isovirus particles are attenuated such that the virus retains, at least partially retains, its replicative capacity and can replicate in vivo, but only produces a low viral load, resulting in a non-pathogenic, subclinical level of infection. Such attenuated virus can be used as an immunogenic composition.

[0118] In some embodiments, the three-fragment sand-like virus particles have the same tropism as the two-fragment sand-like virus particles.

[0119] This document also provides a kit containing one or more cDNAs described herein in one or more containers. In a specific embodiment, the kit contains three-fragment isopyrvirus particles described herein in one, two, or more containers. The kit may further contain one or more of the following: host cells suitable for rescuing the three-fragment isopyrvirus particles, reagents suitable for transfecting plasmid cDNA into the host cells, helper viruses, plasmids encoding viral proteins, and / or one or more oligonucleotide primers specific to the modified isopyrvirus genome fragments or isopyrvirus particles or nucleic acids encoding them.

[0120] This article also provides immunogenic compositions comprising the three-segment sand virus particles described in sections 4.6 and 4.7.

[0121] 4.2.1 Three-fragment sand-like virus particles containing one L fragment and two S fragments In one aspect, this document provides a three-fragment isovirus particle comprising one L fragment and two S fragments. In some embodiments, the replication of the three-fragment isovirus particle comprising one L fragment and two S fragments does not produce replicating two-fragment virus particles. In a specific embodiment, in the absence of type I interferon receptor, type II interferon receptor, and recombinant activation gene (RAG1), using 10 4 In mice infected with the three-segment isovirus particles of PFU, replication of the three-segment isovirus particles containing one L fragment and two S fragments does not produce replicating two-segment virus particles after at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days of sustained infection (see section 4.8.13). In other embodiments, replication of the three-segment isovirus particles containing one L fragment and two S fragments does not produce replicating two-segment virus particles after at least 10, 20, 30, 40, or 50 passages.

[0122] Three-segment sand-like virus particles, in which all viral genes are located at their corresponding wild-type positions, are known in the art (e.g., Emonet). et al., 2011 J. Virol., 85(4):1473; Popkin et al.,2011, J. Virol, 85(15):7928). Specifically, the three-segment arenavirus genome consists of one L segment and two S segments, wherein a heterologous ORF (e.g., GFP) is inserted into a position on each S segment. More specifically, one S segment encodes GP and GFP respectively. The other S segments encode GFP and NP respectively. The L segment encodes the L protein and the Z protein. All segments are flanked by their respective 5' and 3' UTRs.

[0123] In some embodiments, intra-fragment recombination of the two S fragments of the three-fragmented isovirus particle provided herein unifies the two isovirus ORFs into one fragment instead of two separate fragments, producing a nonfunctional promoter (i.e., a genomic fragment with the following structure: 5' UTR-----------5' UTR or 3' UTR------------3' UTR), wherein each UTR forming one end of the genome is an inverted repeat sequence at the other end of the same genome.

[0124] In some embodiments, the three-fragment isovirus particle containing one L fragment and two S fragments is engineered to carry isovirus ORFs at a location other than the wild-type location of the ORF. In other embodiments, the three-fragment isovirus particle containing one L fragment and two S fragments is engineered to carry two, three, four, five, or six isovirus ORFs at a location other than the wild-type location. In a specific embodiment, the three-fragment isovirus particle containing one L fragment and two S fragments comprises all four types of isovirus ORFs. Thus, in some embodiments, the three-fragment isovirus particle is an infectious and replicating three-fragment isovirus particle. In a specific embodiment, the two S fragments of the three-fragment isovirus particle are engineered to carry one of their ORFs at a location other than the wild-type location. In a more specific embodiment, the two S fragments comprise all S fragment ORFs. In some specific implementations, the L fragment is engineered to carry the ORF at a location other than the wild-type location, or the L fragment may be a wild-type genome fragment.

[0125] In some implementations, one of the two S segments may be: (i) The S fragment of the isoplasmosis virus, wherein the ORF encoding the Z protein is under the control of the isoplasmosis virus 5' UTR; (ii) The S fragment of the isoplasmosis virus, wherein the ORF encoding the L protein is under the control of the isoplasmosis virus 5' UTR; (iii) Sand virus S fragment, wherein the ORF encoding NP is under the control of the sand virus 5'UTR; (iv) Sand virus S fragment, wherein the ORF encoding GP is under the control of sand virus 3' UTR; (v) Sand virus S fragment, wherein the ORF encoding L is under the control of the sand virus 3' UTR; and (vi) The S fragment of the isoplasmosis virus, in which the ORF encoding the Z protein is under the control of the isoplasmosis virus 3' UTR.

[0126] In some embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments may contain duplicate ORFs (i.e., two wild-type S fragment ORFs, such as GP or NP). In specific embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments may contain one duplicate ORF (e.g., (GP, GP)) or two duplicate ORFs (e.g., (GP, GP) and (NP, NP)).

[0127] Table 2 below is an example of the genomic architecture of a trisegmental arenavirus particle containing one L fragment and two S fragments, wherein intersegmental recombination of the two S fragments in the trisegmental arenavirus genome does not produce a replicative two-segmental virus particle and cancels arenavirus promoter activity (i.e., the resulting recombinant S fragment consists of two 3'UTRs instead of 3'UTR and 5'UTR).

[0128] Table 2A Triple-fragment sand virus particle containing one L fragment and two S fragments Location 1 is under the control of the 5' UTR of the S segment of the sand virus; Location 2 is under the control of the 3' UTR of the S segment of the sand virus; Location 3 is under the control of the 5' UTR of the S segment of the sand virus; Location 4 is under the control of the 3' UTR of the S segment of the sand virus; Location 5 is under the control of the 5' UTR of the L segment of the sand virus; Location 6 is under the control of the 3' UTR of the L segment of the sand virus.

[0129] ORF stands for inserted heterogeneous ORF.

[0130] In some embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment or L-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In specific embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In some embodiments, other combinations are also possible. For example, a three-fragment isoprev. particle containing one L-fragment and two S-fragments, wherein intersegmental recombination of the two S-fragments in the three-fragment isoprev. genome does not produce a replicative two-fragment virus particle, and the isoprev. promoter activity is cancelled (i.e., the resulting recombinant S-fragment consists of two 5'UTRs instead of 3'UTR and 5'UTR).

[0131] In some embodiments, recombination between the S and L fragments in the tri-fragmented isovirus particle containing one L fragment and two S fragments restores a functional fragment having two viral genes on only one fragment, rather than two separate fragments. In other embodiments, recombination between the S and L fragments in the tri-fragmented isovirus particle containing one L fragment and two S fragments does not produce a replicating two-fragmented virus particle.

[0132] Table 2B below is an example of the genomic architecture of a trisegmental isovirus particle containing one L fragment and two S fragments, wherein intersegmental recombination of the S and L fragments in the trisegmental isovirus genome does not produce a replicative two-segmental virus particle and cancels isovirus promoter activity (i.e., the resulting recombinant S fragment consists of two 3'UTRs instead of 3'UTR and 5'UTR).

[0133] Table 2B Triple-fragment sand virus particle containing one L fragment and two S fragments Location 1 is under the control of the 5' UTR of the S segment of the sand virus; Location 2 is under the control of the 3' UTR of the S segment of the sand virus; Location 3 is under the control of the 5' UTR of the S segment of the sand virus; Location 4 is under the control of the 3' UTR of the S segment of the sand virus; Location 5 is under the control of the 5' UTR of the L segment of the sand virus; Location 6 is under the control of the 3' UTR of the L segment of the sand virus.

[0134] ORF stands for inserted heterogeneous ORF.

[0135] In some embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment or L-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In specific embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In some embodiments, other combinations are also possible. For example, a three-fragment isoprev. particle containing one L-fragment and two S-fragments, wherein intersegmental recombination of the two S-fragments in the three-fragment isoprev. genome does not produce a replicative two-fragment virus particle, and the isoprev. promoter activity is cancelled (i.e., the resulting recombinant S-fragment consists of two 5'UTRs instead of 3'UTR and 5'UTR).

[0136] In some implementations, those skilled in the art can construct a sand-like virus genome having the architecture described in Table 2A or 2B and as described herein, and then use the analysis described in Section 4.8 to determine whether the three-segment sand-like virus particles are genetically stable, i.e., do not produce the replicating two-segment virus particles discussed herein.

[0137] 4.2.2 Tri-fragment sand virus particles containing two L fragments and one S fragment In one aspect, this document provides a trisegmental isovirus particle comprising two L fragments and one S fragment. In some embodiments, the replication of the trisegmental isovirus particle comprising two L fragments and one S fragment does not produce replicating bisegmental virus particles. In a specific embodiment, in the absence of type I interferon receptor, type II interferon receptor, and recombinant activation gene (RAG1), using 10 4 In mice infected with the three-segment isovirus particles of PFU, replication of the three-segment isovirus particles containing two L fragments and one S fragment does not produce replicating two-segment virus particles after at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days of sustained infection (see section 4.8.13). In other embodiments, replication of the three-segment isovirus particles containing two L fragments and one S fragment does not produce replicating two-segment virus particles after at least 10, 20, 30, 40, or 50 passages.

[0138] In some embodiments, intra-fragment recombination of the two L fragments of the three-fragmented isovirus particle provided herein unifies the two isovirus ORFs into one fragment instead of two separate fragments, producing a nonfunctional promoter (i.e., a genomic fragment with the following structure: 5' UTR-----------5' UTR or 3' UTR------------3' UTR), wherein each UTR forming one end of the genome is an inverted repeat sequence at the other end of the same genome.

[0139] In some embodiments, the three-fragment isovirus particle containing two L fragments and one S fragment is engineered to carry isovirus ORFs at a location other than the wild-type location of the ORF. In other embodiments, the three-fragment isovirus particle containing two L fragments and one S fragment is engineered to carry one, three, four, five, or six isovirus ORFs at a location other than the wild-type location. In a specific embodiment, the three-fragment isovirus particle containing two L fragments and one S fragment contains all four types of isovirus ORFs. Thus, in some embodiments, the three-fragment isovirus particle is an infectious and replicating three-fragment isovirus particle. In a specific embodiment, the two L fragments of the three-fragment isovirus particle are engineered to carry one of their ORFs at a location other than the wild-type location. In a more specific embodiment, the two L fragments contain all L fragment ORFs. In some specific implementations, the S fragment is engineered to carry one of its ORFs at a location other than the wild-type location, or the S fragment may be a wild-type genome fragment.

[0140] In some implementations, one of the two L segments can be: (i)L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (ii) L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR; (iii) L fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR; (v) L segment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and (vi) L fragment, in which the ORF encoding the Z protein is under the control of the sand virus 3' UTR.

[0141] In some embodiments, the tri-fragmented arenavirus particle containing one L fragment and two L fragments may contain duplicate ORFs (i.e., two wild-type L fragment ORFs, such as Z protein or L protein). In specific embodiments, the tri-fragmented arenavirus particle containing two L fragments and one S fragment may contain one duplicate ORF (e.g., (Z protein, Z protein)) or two duplicate ORFs (e.g., (Z protein, Z protein) and (L protein, L protein)).

[0142] Table 3 below provides an example of the genomic architecture of a trisegmental arenavirus particle containing two L segments and one S segment. In this trisegmental arenavirus genome, intersegmental recombination of the two L segments does not produce replicative two-segment viral particles and cancels arenavirus promoter activity (i.e., it is presumed that the resulting recombinant L segments will consist of two 3' UTRs or two 5' UTRs, rather than both 3' UTRs and 5' UTRs). Based on Table 3, similar combinations can be predicted for producing arenavirus particles consisting of two 5' UTRs instead of both 3' UTRs and 5' UTRs.

[0143] Table 3 Triple-fragment sand virus particle containing two L fragments and one S fragment Location 1 is under the control of the 5' UTR of the L segment of the sand virus; Location 2 is under the control of the 3' UTR of the L segment of the sand virus; Location 3 is under the control of the 5' UTR of the L segment of the sand virus; Location 4 is under the control of the 3' UTR of the L segment of the sand virus; Location 5 is under the control of the 5' UTR of the S segment of the sand virus; Location 6 is under the control of the 3' UTR of the S segment of the sand virus.

[0144] ORF stands for inserted heterogeneous ORF.

[0145] In some embodiments, the IGR between positions one and two can be an S-fragment or L-fragment IGR of a sand-like virus; the IGR between positions two and three can be an S-fragment or L-fragment IGR of a sand-like virus; and the IGR between positions five and six can be an S-fragment or L-fragment IGR of a sand-like virus. In a specific embodiment, the IGR between positions one and two can be an L-fragment IGR of a sand-like virus; the IGR between positions two and three can be an L-fragment IGR of a sand-like virus; and the IGR between positions five and six can be an S-fragment IGR of a sand-like virus. In some embodiments, other combinations are also possible.

[0146] In some embodiments, recombination between the L and S fragments of a trisegmental arenavirus particle containing two L fragments and one S fragment restores a functional fragment, having two viral genes located on only one fragment rather than two separate fragments. In other embodiments, recombination between the L fragments in a trisegmental arenavirus particle containing two L fragments and one S fragment does not produce a replicating two-fragment virus particle.

[0147] Table 3B below is an example of the genomic architecture of a trisegmental isovirus particle containing two L segments and one S segment, wherein intersegmental recombination of the L and S segments in the trisegmental isovirus genome does not produce a replicative two-segmental virus particle and cancels isovirus promoter activity (i.e., the resulting recombinant S segment will consist of two 3'UTRs instead of 3'UTR and 5'UTR).

[0148] Table 3B Triple-fragment sand virus particle containing two L fragments and one S fragment Location 1 is under the control of the 5' UTR of the L segment of the sand virus; Location 2 is under the control of the 3' UTR of the L segment of the sand virus; Location 3 is under the control of the 5' UTR of the L segment of the sand virus; Location 4 is under the control of the 3' UTR of the L segment of the sand virus; Location 5 is under the control of the 5' UTR of the S segment of the sand virus; Location 6 is under the control of the 3' UTR of the S segment of the sand virus.

[0149] ORF stands for inserted heterogeneous ORF.

[0150] In some embodiments, the IGR between positions one and two can be an S-fragment or L-fragment IGR of a sand-like virus; the IGR between positions two and three can be an S-fragment or L-fragment IGR of a sand-like virus; and the IGR between positions five and six can be an S-fragment or L-fragment IGR of a sand-like virus. In a specific embodiment, the IGR between positions one and two can be an L-fragment IGR of a sand-like virus; the IGR between positions two and three can be an L-fragment IGR of a sand-like virus; and the IGR between positions five and six can be an S-fragment IGR of a sand-like virus. In some embodiments, other combinations are also possible.

[0151] In some implementations, those skilled in the art can construct a sand-like virus genome having the architecture described in Table 3A or 3B and as described herein, and then use the analysis described in Section 4.8 to determine whether the three-segment sand-like virus particles are genetically stable, i.e., do not produce the replicating two-segment virus particles discussed herein.

[0152] 4.2.3 Three-fragment sand-like virus particles with replication defects In some embodiments, this document provides a three-segment arenavirus particle, wherein (i) the ORF is located outside the wild-type position of the ORF; and (ii) the ORF encoding the GP, NP, Z, or L protein is removed or functionally inactivated, resulting in a virus that cannot produce further infectious progeny virus particles (i.e., is replication-defective). In some embodiments, the third arenavirus fragment may be an S fragment. In other embodiments, the third arenavirus fragment may be an L fragment. In a more specific embodiment, the third arenavirus fragment may be engineered to carry the ORF outside the wild-type position of the ORF, or the third arenavirus fragment may be a wild-type arenavirus genome fragment. In yet another more specific embodiment, the third arenavirus fragment lacks an arenavirus ORF encoding the GP, NP, Z, or L protein gene.

[0153] In some embodiments, the three-fragment genome can be an S or L fragment hybrid (i.e., the genome fragment can be a combination of S and L fragments). In other embodiments, the hybrid fragment is an S fragment containing an L fragment IGR. In another embodiment, the hybrid fragment is an L fragment containing an S fragment IGR. In another embodiment, the hybrid fragment is an S fragment UTR with an L fragment IGR. In yet another embodiment, the hybrid fragment is an L fragment UTR with an S fragment IGR. In a specific embodiment, the hybrid fragment is an S fragment 5' UTR with an L fragment IGR or an S fragment 3' UTR with an L fragment IGR. In other specific embodiments, the hybrid fragment is an L fragment 5' UTR with an S fragment IGR or an L fragment 3' UTR with an S fragment IGR.

[0154] A three-segment isovirus particle containing a genetically modified genome with one or more deleted or inactivated ORFs can be generated in supplementary cells (i.e., cells expressing deleted or inactivated isovirus ORFs). The genetic material of the generated isovirus particle can be transferred to a host cell upon infection, where the genetic material can be expressed and amplified. Furthermore, the genome of the genetically modified isovirus particle described herein can encode a heterologous ORF from an organism other than the isovirus particle.

[0155] In some embodiments, at least one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or replaced with a heterologous ORF from an organism other than isopyvirus. In another embodiment, at least one, at least two, at least three, or at least four ORFs encoding the GP, NP, Z, and L proteins are removed or replaced with a heterologous ORF from an organism other than isopyvirus. In a specific embodiment, only one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or replaced with a heterologous ORF from an organism other than the isopyvirus particle. In a more specific embodiment, the ORF encoding the GP protein from the isopyvirus genome fragment is removed. In another specific embodiment, the ORF encoding the NP protein from the isopyvirus genome fragment is removed. In a more specific embodiment, the ORF encoding the Z protein from the isopyvirus genome fragment is removed. In yet another specific embodiment, the ORF encoding the L protein is removed.

[0156] In some embodiments, this document provides a three-fragment isovirus particle comprising one L fragment and two S fragments, wherein (i) the ORF is located outside the wild-type position of the ORF; and (ii) the ORF encoding GP or NP is removed or functionally inactivated, resulting in a replication-defective and non-infectious virus. In a specific embodiment, one ORF is removed or replaced with a heterologous ORF from an organism other than isovirus. In another specific embodiment, two ORFs are removed or replaced with a heterologous ORF from an organism other than isovirus. In yet another specific embodiment, three ORFs are removed or replaced with a heterologous ORF from an organism other than isovirus. In a specific embodiment, the ORF encoding GP is removed or replaced with a heterologous ORF from an organism other than isovirus. In yet another specific embodiment, the ORF encoding NP is removed or replaced with a heterologous ORF from an organism other than isovirus. In yet another more specific embodiment, the ORF encoding NP and the ORF encoding GP are removed and replaced with one or both heterologous ORFs from an organism other than isovirus particle. Therefore, in some embodiments, the three-fragmented sand virus particle comprises (i) an L fragment and two S fragments; (ii) an ORF at a location other than the wild-type location of the ORF; and (iii) one or more heterologous ORFs from an organism other than the sand virus.

[0157] In some embodiments, this document provides a three-fragment isovirus particle comprising two L fragments and one S fragment, wherein (i) the ORF is located outside the wild-type position; and (ii) the ORF encoding the Z protein and / or L protein is removed or functionally inactivated, resulting in a replication-defective and non-infectious virus. In a specific embodiment, one ORF is removed or replaced with a heterologous ORF from an organism other than isovirus. In another specific embodiment, both ORFs are removed or replaced with heterologous ORFs from an organism other than isovirus. In a specific embodiment, the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than isovirus. In other specific embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than isovirus. In yet more specific embodiments, both the ORF encoding the Z protein and the ORF encoding the L protein are removed or replaced with heterologous ORFs from an organism other than isovirus. Therefore, in some embodiments, the three-fragmented sand virus particle comprises (i) two L fragments and one S fragment; (ii) an ORF at a location other than the wild-type location of the ORF; and (iii) a heterologous ORF from an organism other than the sand virus.

[0158] Therefore, in some embodiments, the three-fragmented sand virus particle provided herein comprises a three-fragmented sand virus particle (i.e., one L fragment and two S fragments, or two L fragments and one S fragment), which (i) is engineered to carry an ORF in a non-natural location, (ii) has an ORF encoding a GP, NP, Z, or L protein removed, and (iii) has the removed ORF replaced with one or more heterologous ORFs from an organism other than sand virus.

[0159] In some embodiments, the length of the heterologous ORF is 8 to 100 nucleotides, 15 to 100 nucleotides, 25 to 100 nucleotides, 50 to 200 nucleotides, 50 to 400 nucleotides, 200 to 500 nucleotides, or 400 to 600 nucleotides, or 500 to 800 nucleotides. In other embodiments, the length of the heterologous ORF is 750 to 900 nucleotides, 800 to 100 nucleotides, 850 to 1000 nucleotides, 900 to 1200 nucleotides, 1000 to 1200 nucleotides, 1000 to 1500 nucleotides, or 10 to 1500 nucleotides, 1500 to 2000 nucleotides, 1700 to 2000 nucleotides, 2000 to 2300 nucleotides, 2200 to 2500 nucleotides, 2500 to 3000 nucleotides, or 3000 to 3200 nucleotides. 00 nucleotides, 3000 to 3500 nucleotides, 3200 to 3600 nucleotides, 3300 to 3800 nucleotides, 4000 to 4400 nucleotides, 4200 to 4700 nucleotides, 4800 to 5000 nucleotides, 5000 to 5200 nucleotides, 6000 to 6400 nucleotides, 6200 to 6800 nucleotides, 6600 to 7000 nucleotides, 7000 to 7200 nucleotides, 7200 to 7500 nucleotides, or 7500 nucleotides. In some embodiments, the heterologous ORF peptide or polypeptide has a length of 5 to 10 amino acids, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 100 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 750 amino acids, 750 to 1000 amino acids, 1000 to 1250 amino acids, 1250 to 1500 amino acids, 1500 to 1750 amino acids, 1750 to 2000 amino acids, 2000 to 2500 amino acids, or more than 2500 or more amino acids. In some embodiments, the heterologous ORF encodes a polypeptide with a length not exceeding 2500 amino acids. In a specific embodiment, the heterologous ORF does not contain a stop codon. In some embodiments, the heterologous ORF is codon-optimized. In some embodiments, the nucleotide composition, or both, may be optimized. Such optimization techniques are known in the art and can be used to optimize heterologous ORFs.

[0160] Any heterologous ORF from an organism other than isoninvirus can be included in the three-segment isoninvirus particle. In one embodiment, the heterologous ORF encodes a reporter protein. A more detailed description of the reporter protein is described in section 4.3. In another embodiment, the heterologous ORF encodes an antigen of an infectious pathogen capable of evoking an immune response or an antigen associated with any disease. In a specific embodiment, the antigen is derived from an infectious organism, a tumor (i.e., cancer), or an allergen. A more detailed description of the heterologous ORF is given in section 4.3.

[0161] In some embodiments, the growth and infectivity of the sand virus particles are not affected by heterologous ORFs from organisms other than sand viruses.

[0162] Techniques known to those skilled in the art can be used to generate arenavirus particles containing arenavirus genome fragments engineered to carry arenavirus ORFs at locations other than the wild-type location. For example, reverse genetics techniques can be used to generate such arenavirus particles. In other embodiments, replication-defective arenavirus particles (i.e., arenavirus genome fragments engineered to carry arenavirus ORFs at locations other than the wild-type location, wherein the ORFs encoding GP, NP, Z, and L proteins are deleted) can be generated in supplementary cells.

[0163] In some implementations, the three-fragmented sand-like virus particles used, depending on the current application, can be Old World viruses, such as LCMV.

[0164] In some implementations, the current application relates to the sand-like virus particles described herein that are suitable for use as vaccines, and to methods of using such sand-like virus particles in vaccination and, for example, the treatment or prevention of infections and cancers. More detailed descriptions of the methods of using the sand-like virus particles described herein are provided in section 4.6.

[0165] In some embodiments, the present application relates to the sand-like virus particles described herein, suitable for use as pharmaceutical compositions, and methods of using such sand-like virus particles in vaccination or, for example, the treatment or prevention of infection or cancer. More detailed descriptions of the methods of using the sand-like virus particles described herein are provided in section 4.6.

[0166] 4.3 Arenavirus particles expressing heterologous ORF or tri-fragment arenavirus particles In some embodiments, the arenavirus genome fragment and the corresponding arenavirus particle or three-fragment arenavirus particle may contain a heterologous ORF. In other embodiments, the arenavirus genome fragment and the corresponding arenavirus particle or three-fragment arenavirus particle may contain a target gene. In a more specific embodiment, the heterologous ORF or the target gene encodes an antigen. In a more specific embodiment, the heterologous ORF or the target gene encodes a reporter protein or a fluorescent protein.

[0167] In some embodiments, the isovirus genome fragment, the isovirus particle, or the three-fragment isovirus particle may contain one or more heterologous ORFs or one or more target genes. In other embodiments, the isovirus genome fragment, the isovirus particle, or the three-fragment isovirus particle may contain at least one heterologous ORF, at least two heterologous ORFs, at least three heterologous ORFs, or more heterologous ORFs. In other embodiments, the isovirus particle or the three-fragment isovirus particle contains at least one target gene, at least two target genes, at least three target genes, or more target genes.

[0168] A wide variety of antigens can be expressed from the arenavirus genome fragments, arenavirus particles, or three-fragment arenavirus particles of this application. In one embodiment, the heterologous ORF encodes antigens of infectious pathogens capable of evoking an immune response or antigens associated with any disease. In some embodiments, the heterologous ORF may encode antigens derived from viruses, bacteria, fungi, parasites, or may be expressed in tumors or tumor-related diseases (i.e., cancer), autoimmune diseases, degenerative diseases, genetic diseases, substance dependence, obesity, or allergic diseases.

[0169] In some embodiments, the heterologous ORF encodes a viral antigen. Non-limiting examples of viral antigens include antigens from: Adenoviridae (e.g., mammalian adenoviruses and avian adenoviruses), Herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, herpes simplex virus 6, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), Pneumoviridae (e.g., polioviruses, enterobacterial MS2 bacteriophage, alloleviruses), Poxviridae (e.g., chordopoxyirinae, parapoxviruses, fowlpoxviruses, etc.). Capripoxvirus, leporiipoxvirus, suipoxvirus, molluscum cipoxvirus, and insectopoxviruses; Papillomaviruses (e.g., polyomaviruses and papillomas); Paramyxoviridae (e.g., paramyxoviruses, parainfluenza virus 1, mobilliviruses); and rubulaviruses (e.g., mumps virus). Viruses, including the Pneumoviridae family (e.g., pneumovirus, human respiratory syncytial virus), human respiratory syncytial virus and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus), Picornaviridae family (e.g., enterovirus, rhinovirus, hepatvirus (e.g., human hepatitis A virus), cardiovirus and apthovirus), Reoviridae family (e.g., ororeovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus and oryzavirus), Retroviridae family (e.g., mammalian B retrovirus, mammalian C retrovirus, avian C retrovirus, D retrovirus group, BLV-HTLV retrovirus), Lentivirals (e.g., human immunodeficiency virus (HIV) 1 and HIV-2, e.g., HIV... gp160), spumaviruses, flaviviruses (e.g., hepatitis C virus, dengue virus, West Nile virus), hepatDNAviruses (e.g., hepatitis B virus), togaviridae (e.g., alpha viruses (e.g., Sindbis virus), and rubiviruses (e.g.,Rubella virus, rhabdoviridae (e.g., vesiculovirus, rabies virus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and Lassa virus), and coronavirusidae (e.g., coronavirus and circovirus). In a specific embodiment, the viral antigen is HIV gp120, gp41, HIV Nef, RSV F glycoprotein, RSV G glycoprotein, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE), or hepatitis B surface antigen, hepatitis C virus E protein, or coronavirus spike protein. In one embodiment, the viral antigen is not an HIV antigen.

[0170] In other embodiments, the heterologous ORF encodes bacterial antigens (e.g., bacterial coat proteins). In other embodiments, the heterologous ORF encodes parasitic antigens (e.g., protozoan antigens). In yet another embodiment, the heterologous nucleotide sequence encodes fungal antigens.

[0171] Non-limiting examples of bacterial antigens include antigens from the following bacteria: Aquaspirillum family, Azospirillum family, Azotobacteraceae family, Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylobacter species (…). Campylobacter species), Chlamydia species ( Chlamydia species) (e.g., Chlamydia pneumoniae) Chlamydia pneumonia Clostridium ( )), Clostridium spp. ( Clostridium ), Enterobacteriaceae family ( Enterobacteriaceae family (e.g., species of the genus Citric acid bacteria) Citrobacter species), Edwardsiella genus ( Edwardsiella Enterobacter aerogenes ( Enterobacter aerogenes Erwinia species ( Envy species), Escherichia coli ( Escherichia coli ), Hafnia species ( Hafnia species), species of the genus Klebsiella ( Klebsiella species), Morganella species ( Morganella species), Proteus vulgaris ( Common Proteus Providencia spp. Providence Salmonella species ( Salmonella species), Serratia marcescens ( Serratia marcescens ) and Shigella freundii ( Shigella flexible ), the Pterygomycetes family ( Gardenia family), Haemophilus influenzae ( Haemophilus influenzae, the family Halobacteriaceae ( Halobacteriaceae family), Helicobacter genus ( Helicobacter family), Regionallaceae family ( Legionella family), Listeria species ( Listeria species), the Methylococcaceae family, and the genus *mycobacteria* (e.g., *Mycobacterium tuberculosis*). Mycobacterium tuberculosis ), Neisseriaceae family, Oceanospirillum family, Pasteurellaceae family, Pneumococcus species ( Pneumococcus species, species of the genus *Pseudomonas*, family Rhizobiaceae, family *Spirillum*, family *Spirosomaceae*, genus *Staphylococcus* ( Staphylococcus (For example, methicillin-resistant Staphylococcus aureus) Staphylococcus aureus ) and Staphylococcal pyrogens ( Staphylococcus pyrogenes), Streptococcus ( Streptococcus (For example, enterococci (Streptococcus enterica) Streptococcus enteritidis ), Streptococcus fasciae and Streptococcus pneumoniae ( Streptococcus pneumoniae ), the Helicobacter family of Vibrio batrachnifolia ( Vampirevibr Helicobacter Family), Yersinia genus ( Yersinia family), Bacillus anthracis ( Bacillus anthrax ) and the Vibrio genus ( Vampirovibrio family).

[0172] Non-limiting examples of parasitic antigens include antigens from parasites such as amoebas, Plasmodium, Plasmodium species, and Trypanosoma cruzi. Non-limiting examples of fungal antigens include antigens from fungi such as *Plasmodium* species. Absidia Species (For example, *Pterocarya stenoptera*) Absidia corymbifera ) and branched plowshare mold ( Absidia ramosa Aspergillus species ()), Aspergillus Species (For example, Aspergillus flavus) Aspergillus flavus Aspergillus fumigatus ( ) Aspergillus fumigatus Aspergillus nidus ( ) Aspergillus nidulans Aspergillus niger ( ) Aspergillus black ) and Aspergillus terreus ( Aspergillus terreus ), frog droppings mold ( Basidiobolus of frogs ), dermatitis blastomyces ( Blastomyces dermatitidis ), species of the genus Candida ( Candida Species (For example, Candida albicans) Candida albicans ), Candida glabrata ( Candida glabrata ), Candida albicans ( Candida kerr ), Candida krusei ( Candida krusei ), Candida parapsilosis ( Candida parapsilosis ), Candida pseudotropica ( Candida pscudotropicalis ), and Candida albicans ( Candida quillermondii ), Candida folds ( Candida rugosa ), Candida albicans ( Candida stellatoidea ) and Tropical Candida ( Candida tropicalis ), coccidioides ( Coccidioides immitis ), species of the genus *Otomycetes* ( Conidiobolus species), Cryptococcus neoformans ( Cryptococcus neoforms ), small silver hygrophytes species ( Cunninghamella species), dermatophytes, histoplasmosis ( Histoplasma capsulatum ), Microsporum gypseum ( Microsporum gypseum ), Micromold ( Mucor pusillus ), Paracoccidioides brasiliensis ( Paracoccidioides brasiliensis ), Pseudomystoma bortneri ( Pseudallescheria boydii ), Rhinospora sibiricum ( Rhinosporidium seeberi Pneumocystis carinii ( Pneumocystis carinii ), species of the genus Rhizopus ( Rhizopus species) (e.g., Rhizopus septemlobus) Rhizopus arrhizus ), Rhizopus oryzae ( Rhizopus oryzae ) and Microsporum ( Rhizopus microsporus )), species of the genus Yeast ( Saccharomyces species), Schenck's Sporothrix ( Sporthrix schenckii The classification includes zygomycetes, ascomycetes, basidiomycetes, deuteromycetes, and oomycetes.

[0173] In some embodiments, the heterologous ORF encodes a tumor antigen or tumor-associated antigen. In some embodiments, the tumor antigen or tumor-associated antigen includes antigens from tumor-related diseases, including acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancers, Kaposi's sarcoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic (see cholangiocarcinoma), bladder cancer, osteosarcoma / malignant fibrous histiocytoma, etc. Brainstem glioma, brain cancer, brain tumor, cerebellar astrocytoma, astrocytoma / malignant glioma, ependymoma, medulloblastoma, optic nerve primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, carcinoid gastrointestinal tumor, unknown primary cancer, central nervous system lymphoma, primary, cerebellar astrocytoma, astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic bronchitis, chronic lymphocytic leukemia. Chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, fibroblastic small round cell tumor, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma (a member of the Ewing tumor family), extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct carcinoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, germ cell tumors: extracranial, extragonadal, or ovarian trophoblastomas during pregnancy, brainstem glioma, glioma, childhood astrocytoma, pediatric visual pathway and hypothalamus, gastric carcinoid, piloblastic leukemia, head and neck cancer, cardiac cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas. Intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, renal cell carcinoma, laryngeal cancer, lymphoblastic lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lip and oral cancer, liposarcoma, primary liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, HIV-associated lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma. Lymphoma, primary central nervous system cancer, macroglobulinemia, Waldenström, male breast cancer, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, intraocular (eye), Merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, occult primary and metastatic squamous neck cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma / plasma cell vegetation, mycosis fungoides, myelodysplastic syndrome.Myelodysplastic disorders / myelogenic diseases, myeloid leukemia, chronic myeloid leukemia, adult acute myeloid leukemia, childhood acute myeloma, multiple myeloma (myeloma), myelogenic disorders, chronic nasal cavity and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal carcinoma, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumor, low-grade potential ovarian tumor, pancreatic cancer, islet cells, paranasal sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor. Pineal cell carcinoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasmacytoma formation / multiple myeloma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (renal carcinoma), renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, childhood cancer, salivary gland cancer, sarcoma, Ewing tumor family, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, seminal fluid syndrome, skin cancer ( Non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma - see skin cancer (non-melanoma), occult primary metastatic squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin - see mycosis fungoides and Sézary syndrome, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, childhood transitional cells of the renal pelvis and ureter, Gestational trophoblastic tumor, cancer of unknown primary site, cancer of unknown primary site in adults, transitional cell carcinoma of the ureter and renal pelvis in children, pharyngeal cancer, uterine cancer, endometrial uterine sarcoma, bronchial tumors, embryonal tumors of the central nervous system; chordoma in children, colorectal cancer, craniopharyngioma, ependymoblastoma, Langerhans cell histiocytosis, acute lymphoblastic leukemia, acute myeloid leukemia (adult / child), small cell lung cancer, myeloid epithelioma, oral cancer, papilloma, intermediately differentiated pineal parenchymal tumors, respiratory tract cancers involving the NUT gene on chromosome 15, spinal cord tumors, thymoma, thyroid cancer, vaginal cancer; vulvar cancer and Wilms' tumor.

[0174] Non-restricted examples of tumors or tumor-associated antigens include lipophilic acid, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CPSF, cyclin D1, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, G250 / MN / CAIX, HER-2 / neu, IDO1, IGF2B3, IL13Ralpha2, intestinal carboxyl esterase, alpha-fetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, seternin 1. SOX10, STEAP1, survivin, telomerase, VEGF, or WT1, EGF-R, CEA, CD52, gp100 protein, MELANA / MART1, NY-ESO-1, p53 MAGE1, MAGE3 and CDK4, alpha-actin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2, elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase AS fusion protein, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triose phosphate isomerase, Lengsin, M-CSF, MCSP, or mdm-2.

[0175] In some embodiments, the heterologous ORF encodes a respiratory pathogen antigen. In specific embodiments, the respiratory pathogen is a virus, such as RSV, coronavirus, human metapneumovirus, parainfluenza virus, Hendra virus, Nipah virus, adenovirus, rhinovirus, or PRRSV. Non-limiting examples of respiratory viral antigens include respiratory syncytial virus F, G, and M2 proteins; coronavirus (SARS, HuCoV) spike protein (S); human metapneumovirus fusion protein; parainfluenza virus fusion protein and hemagglutinin protein (F, HN); Hendra virus (HeV) and Nipah virus (NiV) attachment glycoproteins (G and F); adenovirus capsid protein; rhinovirus protein; and wild-type or modified GP5 and M proteins of PRRSV.

[0176] In a specific implementation, the respiratory pathogen is a bacterium, such as Bacillus anthracis (Bacillus anthracis). Bacillus anthracis ), Mycobacterium tuberculosis ( mycobacterium tuberculosis Bordetella pertussis ( ) Bordetella pertussis Streptococcus pneumoniae () streptococcus pneumoniae) Yersinia pestis (Yersinia pestis) yersinia pestis Staphylococcus aureus ( staphylococcus aureus Tula Francisella ( Francisella tularensis Legionella pneumophila ( legionella pneumophila Chlamydia pneumoniae ( Chlamydiapneumoniae ), Pseudomonas aeruginosa ( pseudomonasaeruginosa ), Neisseria meningitidis ( neisseriameningitides ) and Haemophilus influenzae ( haemophilusinfluenzae Non-limiting examples of respiratory bacterial antigens include the anthrax protective antigen PA, Mycobacterium tuberculosis antigen 85A and heat shock protein (Hsp65), Bordetella pertussis toxoid (PT) and filamentous hemagglutinin (FHA), Streptococcus pneumoniae isolate A and surface adhesin A (PsaA), Yersinia pestis F1 and V subunits, and proteins from Staphylococcus aureus, Francisella tularensis, Legionella pneumophila, Chlamydia pneumoniae, Pseudomonas aeruginosa, Neisseria meningitidis, and Haemophilus influenzae.

[0177] In some embodiments, the heterologous ORF encodes a T-cell epitope. In other embodiments, the heterologous ORF encodes a cytokine or growth factor.

[0178] In other embodiments, the heterologous ORF encodes an antigen expressed in an autoimmune disease. In a more specific embodiment, the autoimmune disease may be type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, and psoriasis. Non-limiting examples of autoimmune disease antigens include Ro60, dsDNA, or RNP.

[0179] In other embodiments, the ORF encodes an antigen expressed in an allergic disease. In a more specific embodiment, the allergic disease includes, but is not limited to, seasonal and perennial rhinoconjunctivitis, asthma, and eczema. Non-limiting examples of allergic antigens include Bet v1 and Fel d1.

[0180] In other embodiments, the arenavirus genome fragment, the arenavirus particle, or the three-fragment arenavirus particle further comprises a reporter protein. The reporter protein is capable of co-expression with the antigen described herein. Ideally, the expression is visible under normal light or other wavelengths of light. In some embodiments, the intensity of the effect produced by the reporter protein is used to directly measure and monitor the arenavirus particle or the three-fragment arenavirus particle.

[0181] The reporter gene will be readily identifiable to those skilled in the art. In some embodiments, the sand virus particle is a fluorescent protein. In other embodiments, the reporter gene is GFP. GFP emits a bright green light when exposed to UV or blue light.

[0182] Non-limiting examples of reporter proteins include various enzymes, such as, but not limited to, β-galactosidase, chloramphenicol acetyltransferase, neomycin phosphotransferase, luciferase, or RFP.

[0183] In some embodiments, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle has the desired properties for use as a vector for vaccination (see, for example, section 4.6). In another embodiment, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle is capable of inducing an immune response in a host (e.g., mouse, rabbit, goat, donkey, human). In other embodiments, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle described herein induces an innate immune response. In other embodiments, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle induces an adaptive immune response. In a more specific embodiment, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle simultaneously induces both innate and adaptive immune responses.

[0184] In another embodiment, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle induces a T cell response. In yet another more specific embodiment, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle induces a CD8+ T cell response. In other embodiments, arenavirus particles carrying a target exogenous gene induce a high-frequency and functionally potent CD8+ T cell response. The arenavirus genome fragment, the arenavirus particle, or the three-segment arenavirus particle expressing an arenavirus derived from an infectious organism, cancer, or allergen induces a CD8+ T cell response specific to one or more epitopes of the corresponding target exogenous gene.

[0185] In some embodiments, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle can induce T helper cell 1 differentiation, CD4+ T cell memory formation, and / or trigger a durable antibody response. These antibodies can be neutralizing, modulating, toxic to tumor cells, or possess other advantageous biological characteristics. In other embodiments, the arenavirus genome fragment expressing a heterologous ORF, the arenavirus particle, or the three-segment arenavirus particle has a strong tropism for dendritic cells and activates them upon infection. This enhances antigen presentation by antigen-presenting cells.

[0186] In some embodiments, the arenavirus genome fragment, the arenavirus particle, or the three-segment arenavirus particle expressing an antigen derived from an infectious organism, cancer, or allergen induces low or undetectable neutralizing antibody titers against LCMV, and high protective neutralizing antibody responses against the corresponding exogenous transgene. The particle-forming arenavirus backbone or three-segment arenavirus particle expressing antigens derived from infectious organisms, cancer, or allergens has a very low capacity to induce immunity against the arenavirus backbone component.

[0187] 4.4 Production of Arenavirus Particles and Tri-fragment Arenavirus Particles Generally, sand virus particles can be recombinantly generated using standard reverse genetic techniques described for LCMV (see Flatz). et al., 2006, Proc Natl Acad Sci USA 103:4663-4668; Sanchez et al., 2006, Virology 350:370; Ortiz-Riano et al.,2013, J Gen Virol. 94:1175-88, which is incorporated herein by reference. To generate the sand-like virus particles presented herein, these techniques can be applied as described below. The viral genome can be modified as described in Sections 4.1 and 4.2, respectively.

[0188] 4.4.1 Open reading frame in non-natural locations The generation of sand-like virus particles, which are engineered to carry genomic fragments of the viral ORF at locations other than the wild-type location of the ORF, can be recombinantly generated by any reverse genetics technique known to those skilled in the art.

[0189] (i) Infectious and replicating sand virus particles In some embodiments, the method for generating arenavirus particles includes (i) transfecting a first arenavirus genome fragment cDNA into a host cell; (ii) transfecting a second arenavirus genome fragment cDNA into the host cell; (iii) transfecting a plasmid expressing the minimal trans-acting factors NP and L of arenavirus into the host cell; (iv) maintaining the host cell under conditions suitable for virus formation; and (v) harvesting the arenavirus particles. In some more specific embodiments, the cDNA is contained within a plasmid.

[0190] Once generated from cDNA, arenavirus particles (i.e., infectious and replicating) can proliferate. In some embodiments, the arenavirus particles can proliferate in any host cell that allows the virus to grow to a titer that permits the use of the virus described herein. In one embodiment, the host cell allows the arenavirus particles to grow to a titer comparable to that determined for the corresponding wild type.

[0191] In some embodiments, the arenavirus particles can be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO, or others. In specific embodiments, the arenavirus particles can be propagated in cell lines.

[0192] In some embodiments, the host cell is held in a culture and transfected with one or more plasmids. Under the control of one or more expression cassettes suitable for expression in mammalian cells, the plasmid expresses the desired isovirus genome fragment, for example, the expression cassette consisting of a polymerase I promoter and a terminator.

[0193] Plasmids that can be used to generate arenavirus particles may include: i) plasmids encoding the S genome segment, such as pol-I S, and ii) plasmids encoding the L genome segment, such as pol-I L. In some embodiments, plasmids encoding arenavirus polymerases that direct the intracellular synthesis of viral L and S segments may be incorporated into the transfection mixture. For example, plasmids encoding the L protein and / or plasmids encoding NP (pC-L and pC-NP, respectively) may be present. The L protein and NP are minimal trans-acting factors essential for viral RNA transcription and replication. Alternatively, along with the NP and L protein, the intracellular synthesis of viral L and S segments may be performed using expression cassettes with pol-I and pol-II promoters having L and S segment cDNAs that read from the contralateral side to two separate plasmids, respectively.

[0194] In some embodiments, the arenavirus genome fragment is under the control of a promoter. Generally, an RNA polymerase I-driven expression cassette, an RNA polymerase II-driven expression cassette, or a T7 phage RNA polymerase-driven expression cassette can be used. In some embodiments, the plasmid encoding the arenavirus genome fragment can be the same; that is, the genome sequence and trans-acting factor can be transcribed from a single plasmid via a promoter. Specific examples of promoters include the RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter, SP6 promoter, or T3 promoter.

[0195] In addition, the plasmid may contain mammalian selection markers, such as puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells, such as the polymerase II expression cassette described above, or the viral gene transcript may be followed by an internal ribosome entry site, such as that of encephalocardiitis virus, and then a mammalian resistance marker. For production in E. coli, the plasmid may also contain bacterial selection markers, such as an ampicillin resistance cassette.

[0196] Plasmid transfection of host cells can be performed using any commonly used strategy, such as calcium phosphate-based, liposome-based protocols, or electroporation. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and high-expressing clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry.

[0197] To recover the sand-like virus particles described herein, the following procedure is envisioned. Day 1: Cells, typically 80% confluent on M6-well plates, are transfected with a mixture of plasmids as described above. For this, any commonly used strategy can be employed, such as calcium phosphate-based, liposome-based, or electroporation protocols.

[0198] 3-5 days later: Harvest the culture supernatant (arenavirus vector product), aliquot it, and store it at 4°C, -20°C, or -80°C, depending on the storage time required before use. Assess the infectious titer of the arenavirus vector product using immunofocal analysis. Alternatively, transfected cells and supernatant can be passaged into larger containers (e.g., T75 tissue culture flasks) 3-5 days after transfection, and the culture supernatant can be harvested five days after passage.

[0199] This application further relates to the expression of heterologous ORFs, wherein plasmids encoding genomic fragments are modified to incorporate heterologous ORFs. Heterologous ORFs can be incorporated into plasmids using restriction enzymes.

[0200] (ii) Infectious, replication-defective sand-like virus particles Infectious, replication-defective arenavirus particles can be rescued as described above. However, once generated from cDNA, the infectious, replication-defective arenaviruses presented herein can proliferate in supplementary cells. Supplementary cells are cells that provide function that has been eliminated from replication-defective arenaviruses through modifications to their genome (e.g., supplementary cells provide the GP protein if the ORF encoding the GP protein is deleted or its function is inactivated).

[0201] Due to the removal or functional inactivation of one or more ORFs in the isovirus vector (here, the deletion of glycoprotein GP is an example), the isovirus vector can be generated and amplified in cells that trans-provide the deleted viral gene, for example, GP in the current embodiment. Such supplementary cell lines, hereinafter referred to as C-cells, are generated by transfecting cell lines such as BHK-21, HEK 293, VERO, or others with one or more plasmids expressing the target viral gene (supplementary plasmids, referred to as C-plasmids). Under the control of one or more expression cassettes suitable for expression in mammalian cells, such as mammalian polymerase II promoters, such as the EF1alpha promoter with a polyadenylation signal, the C-plasmid expresses the deleted viral gene from the isovirus vector to be produced. In addition, the supplementary plasmid may have mammalian selection markers, such as puromycin resistance, under the control of expression cassettes suitable for gene expression in mammalian cells, such as the aforementioned polymerase II expression cassette, or the viral gene transcript may be followed by an internal ribosome entry site, such as one of the encephalocardiitis viruses, followed by a mammalian resistance marker. For production in E. coli, the plasmid also contains bacterial selection markers, such as ampicillin resistance kits.

[0202] Usable cells, such as BHK-21, HEK 293, MC57G, or others that can be maintained in culture, can be transfected using any commonly used strategy, such as calcium phosphate-based, liposome-based, or electroporation with supplemental plasmids. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and highly expressed C-cell clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry. As an alternative to using stably transfected C-cells, transient transfection of normal cells can supplement missing viral genes in each step below using C-cells. Additionally, helper viruses can be used to trans-provide the missing function.

[0203] Plasmids can be of two types: i) two plasmids, called TF-platinum, for intracellular expression of the minimal trans-acting factor of arenavirus in C-cells, derived in the current embodiment from, for example, the NP and L proteins of LCMV; and ii) a plasmid, called GS-platinum, for intracellular expression of a arenavirus vector genomic fragment in C-cells, for example, a fragment with designed modifications. The TF-platinum expresses the NP and L proteins of the corresponding arenavirus vector under the control of an expression cassette suitable for protein expression in mammalian cells, generally a mammalian polymerase II promoter, such as the CMV or EF1alpha promoter, either preferably combined with a polyadenylation signal. The GS-platinum expresses the small (S) and large (L) genomic fragments of the vector. Generally, a polymerase I-driven expression cassette or a T7 phage RNA polymerase (T7-)-driven expression cassette can be used, the latter preferably having a 3'-terminal ribozyme for processing the master transcript to produce the correct ends. When using a T7-based system, it is necessary to provide additional T7 expression in a stable manner by constructing other expression plasmids that provide T7, such as those similar to TF-plasmids, during the recovery process, or by constructing C-cells to provide T7 expression in C-cells. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via T7, polI, and polII promoters.

[0204] To recover the arenavirus vector, the following procedure can be used. Day 1: C cells, typically 80% confluent on M6-well plates, are transfected with a mixture of two TF-plasmids and two GS-plasmids. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via the T7, polI, and polII promoters. For this purpose, any commonly used strategy can be utilized, such as calcium phosphate-based, liposome-based protocols, or electroporation.

[0205] 3-5 days later: Harvest the culture supernatant (arenavirus vector product), aliquot it, and store it at 4°C, -20°C, or -80°C, depending on how long the arenavirus vector needs to be stored before use. Then assess the infectious titer of the arenavirus vector product using immunofocal analysis on C-cells. Alternatively, transfected cells and supernatant can be passaged into larger containers (e.g., T75 tissue culture flasks) 3-5 days after transfection, and the culture supernatant can be harvested five days after passage.

[0206] The present invention further relates to the expression of antigens in cell cultures, wherein the cell cultures are infected with an infectious, replication-defective isovirus expressing the antigen. When used to express antigens in cultured cells, the following two processes can be used: i) Infect the target cell type with one or more, such as two, three or four multiples of infection (MOI), using the sand-like virus vector product described herein, causing the production of the antigen described in all cells shortly after infection.

[0207] ii) Alternatively, a lower MOI can be used, allowing individual cell clones to be selected based on the virus-driven antigen expression level. Subsequently, due to the non-cytolytic nature of the arenavirus vector, individual clones can be expanded indefinitely. Regardless of the pathway, antigens can then be collected (and purified) from the culture supernatant or from the cells themselves, depending on the nature of the resulting antigens. However, the invention is not limited to these two strategies, and other methods using infectious, replication-defective arenaviruses as vectors to drive antigen expression can also be considered.

[0208] 4.4.2 Production of Tri-Fragment Sand Virus Particles Three-segment sand-like virus particles can be recombinantly generated using reverse genetics techniques known in the art, such as Emonet. et al., 2008, PNAS, 106(9):3473-3478; Popkin et al., The method described in 2011, J. Virol., 85(15):7928–7932, is incorporated herein by reference. The generation of the three-segment sand-like virus particles presented herein can be modified as described in Section 4.2.

[0209] (i) Infectious and replicating three-fragmented sand virus particles In some embodiments, the method for generating three-fragmented sand virus particles includes (i) transfecting a host cell with cDNA of one L fragment and two S fragments, or two L fragments and one S fragment; (ii) transfecting a host cell with a plasmid expressing the minimum trans-acting factors NP and L of the sand virus; (iii) maintaining the host cell under conditions suitable for virus formation; and (iv) harvesting the sand virus particles.

[0210] Once generated from cDNA, the three-segment arenavirus particles (i.e., infectious and replicative) can proliferate. In some embodiments, the three-segment arenavirus particles can proliferate in any host cell that allows the virus to grow to a titer that permits the use of the virus described herein. In one embodiment, the host cell allows the three-segment arenavirus particles to grow to a titer comparable to that determined for the corresponding wild type.

[0211] In some embodiments, the three-fragmented isovirus particles can be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO, or others. In specific embodiments, the three-fragmented isovirus particles can be propagated in cell lines.

[0212] In some embodiments, the host cell is held in a culture and transfected with one or more plasmids. Under the control of one or more expression cassettes suitable for expression in mammalian cells, the plasmid expresses the desired isovirus genome fragment, for example, the expression cassette consisting of a polymerase I promoter and a terminator.

[0213] In a specific implementation, the host cell is held in a culture and transfected with one or more plasmids. Under the control of one or more expression cassettes suitable for expression in mammalian cells, the plasmid expresses the viral gene to be produced; for example, the expression cassette consists of a polymerase I promoter and a terminator.

[0214] Plasmids that can be used to produce a three-fragment isovirus containing one L fragment and two S fragments can include: 1) two plasmids, each encoding an S genomic fragment, such as pol-I S; and ii) a plasmid encoding an L genomic fragment, such as pol-I L. The plasmids required for a three-fragment isovirus containing two L fragments and one S fragment are: i) two plasmids, each encoding an L genomic fragment, such as pol-L; and ii) a plasmid encoding an S genomic fragment, such as pol-I S.

[0215] In some embodiments, plasmids encoding arenavirus polymerases that guide the intracellular synthesis of the L and S fragments of the virus can be incorporated into the transfection mixture. For example, plasmids encoding the L protein and / or plasmids encoding NP (pC-L and pC-NP, respectively). The L protein and NP are minimal trans-acting factors essential for viral RNA transcription and replication. Alternatively, along with the NP and L protein, the intracellular synthesis of the viral L and S fragments can be performed using an expression cassette with pol-I and pol-II promoters having cDNA for the L and S fragments of two separate plasmids, respectively, read from the contralateral side.

[0216] In addition, the plasmid may contain mammalian selection markers, such as puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells, such as the polymerase II expression cassette described above, or the viral gene transcript may be followed by an internal ribosome entry site, such as that of encephalocardiitis virus, and then a mammalian resistance marker. For production in E. coli, the plasmid may also contain bacterial selection markers, such as an ampicillin resistance cassette.

[0217] Plasmid transfection of BHK-21 cells can be performed using any commonly used strategy, such as calcium phosphate-based, liposome-based, or electroporation protocols. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and high-expressing clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry.

[0218] Generally, polymerase I-driven expression cassettes, RNA polymerase II-driven cassettes, or T7 phage RNA polymerase-driven cassettes can be used, the latter preferably having a 3' phage RNA polymerase terminal ribozyme for processing the main transcript to produce the correct ends. In some embodiments, the plasmids encoding arenavirus genome fragments can be the same, i.e., the genome sequence and trans-acting factors can be transcribed from a single plasmid via T7, polI, and polII promoters.

[0219] To recover the three-fragment isovirus vector, the following procedure is envisioned. Day 1: Cells, typically 80% confluent on M6-well plates, are transfected with a mixture of plasmids as described above. For this, any commonly used strategy can be employed, such as calcium phosphate-based, liposome-based, or electroporation protocols.

[0220] 3-5 days later: Harvest the culture supernatant (arenavirus vector product), aliquot it, and store it at 4°C, -20°C, or -80°C, depending on how long the arenavirus vector needs to be stored before use. Assess the infectious titer of the arenavirus vector product using immunofocal analysis. Alternatively, transfected cells and supernatant can be passaged into larger containers (e.g., T75 tissue culture flasks) 3-5 days after transfection, and the culture supernatant can be harvested five days after passage.

[0221] This application also relates to the expression of heterologous ORFs and / or target genes, wherein plasmids encoding genomic fragments are modified to incorporate heterologous ORFs and / or target genes. Heterologous ORFs and / or target genes can be incorporated into plasmids using restriction enzymes.

[0222] (ii) Infectious, replication-defective three-segment sand virus particles Infectious, replication-defective three-segment arenavirus particles can be rescued as described above. However, once generated from cDNA, the infectious, replication-defective arenaviruses presented herein can proliferate in supplementary cells. Supplementary cells are cells that provide function that has been eliminated from replication-defective arenaviruses through modifications to their genome (e.g., supplementary cells provide the GP protein if the ORF encoding the GP protein is deleted or its function is inactivated).

[0223] Due to the removal or functional inactivation of one or more ORFs in the arenavirus vector (here, the deletion of glycoprotein GP is an example), the arenavirus vector can be generated and amplified in cells that trans-provide the deleted viral gene, for example, GP in the current embodiment. Such supplementary mammalian cell lines, hereinafter referred to as C-cells, are generated by transfecting mammalian cell lines such as BHK-21, HEK 293, VERO, or others (here, BHK-21 is an example) with one or more plasmids expressing the target viral gene (supplementary plasmids, referred to as C-plasmids). Under the control of one or more expression cassettes suitable for expression in mammalian cells, such as mammalian polymerase II promoters, such as CMV or EF1alpha promoters with polyadenylation signals, the C-plasmid expresses the deleted viral gene from the arenavirus vector to be produced. In addition, supplemental plasmids may contain mammalian selection markers, such as puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells, such as the aforementioned polymerase II expression cassette, or the viral gene transcript may be followed by an internal ribosome entry site, such as one of the encephalocardiitis viruses, followed by a mammalian resistance marker. For production in E. coli, the plasmid additionally contains bacterial selection markers, such as an ampicillin resistance cassette.

[0224] Usable cells, such as BHK-21, HEK 293, MC57G, or others that can be maintained in culture, can be transfected using any commonly used strategy, such as calcium phosphate-based, liposome-based, or electroporation with supplemental plasmids. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and highly expressed C-cell clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry. As an alternative to using stably transfected C-cells, transient transfection of normal cells can supplement missing viral genes in each step below using C-cells. Additionally, helper viruses can be used to trans-provide the missing function.

[0225] Two types of plasmids can be used: i) two plasmids, called TF-platinum, for intracellular expression of minimal trans-acting factors of arenavirus in C-cells, derived in the current embodiment from, for example, the NP and L proteins of LCMV; and ii) a plasmid, called GS-platinum, for intracellular expression of arenavirus vector genomic fragments in C-cells, for example, fragments with designed modifications. The TF-platinum expresses the NP and L proteins of the corresponding arenavirus vector under the control of an expression cassette suitable for protein expression in mammalian cells, generally a mammalian polymerase II promoter, such as the CMV or EF1alpha promoter, either preferably combined with a polyadenylation signal. The GS-platinum expresses the small (S) and large (L) genomic fragments of the vector. Generally, a polymerase I-driven expression cassette or a T7 phage RNA polymerase (T7-)-driven expression cassette can be used, the latter preferably having a 3'-terminal ribozyme for processing the master transcript to produce the correct ends. When using a T7-based system, it is necessary to provide additional T7 expression in a stable manner by constructing other expression plasmids that provide T7, such as those similar to TF-plasmids, during the recovery process, or by constructing C-cells to provide T7 expression in C-cells. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via T7, polI, and polII promoters.

[0226] To recover the arenavirus vector, the following procedure can be used. Day 1: C cells, typically 80% confluent on M6-well plates, are transfected with a mixture of two TF-plasmids and two GS-plasmids. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via the T7, polI, and polII promoters. For this purpose, any commonly used strategy can be utilized, such as calcium phosphate-based, liposome-based protocols, or electroporation.

[0227] 3-5 days later: Harvest the culture supernatant (arenavirus vector product), aliquot it, and store it at 4°C, -20°C, or -80°C, depending on how long the arenavirus vector needs to be stored before use. Then, assess the infectious titer of the arenavirus vector product using immunofocal analysis on C-cells. Alternatively, transfected cells and supernatant can be passaged into larger containers (e.g., T75 tissue culture flasks) 3-5 days after transfection, and the culture supernatant can be harvested five days after passage.

[0228] This invention further relates to the expression of antigens in cell cultures, wherein the cell cultures are infected with an infectious, replication-defective, three-fragment isovirus expressing the antigen. When used to express CMV antigens in cultured cells, the following two processes can be used: i) Infect the target cell type with one or more, such as two, three or four multiples of infection (MOI), using the sand-like virus vector product described herein, causing the production of the antigen described in all cells shortly after infection.

[0229] ii) Alternatively, a lower MOI can be used, and individual cell clones can be selected based on the virus-driven antigen expression level. Subsequently, due to the non-cytolytic nature of the arenavirus vector, individual clones can be expanded indefinitely. Regardless of the pathway, the antigen can then be collected (and purified) from the culture supernatant or from the cells themselves, depending on the nature of the antigen produced. However, the invention is not limited to these two strategies, and other methods of using infectious, replication-defective arenaviruses as vectors to drive CMV antigen expression can also be considered.

[0230] 4.5 Nucleic Acids, Vector Systems, and Cell Lines In some embodiments, this document provides cDNA comprising or composed of sand virus genome fragments or three sand virus particles as described in Sections 4.1 and 4.2, respectively.

[0231] 4.5.1 Open reading frame in non-natural locations In one embodiment, this document provides a nucleic acid encoding a segment of the arenavirus genome described in Section 4.1. In a more specific embodiment, this document provides a DNA nucleotide sequence or a collection of DNA nucleotide sequences as listed in Table 1. Host cells containing such nucleic acids are also provided in Section 4.1.

[0232] In a specific implementation, this document provides cDNA of a sand-like virus genome fragment engineered to carry an ORF at a location other than the wild-type location of the ORF, wherein the sand-like virus genome fragment encodes a heterologous ORF as described in Section 4.1.

[0233] In one embodiment, this document provides a DNA expression vector system encoding a segment of the arenavirus genome engineered to carry the ORF at a location other than the wild-type location of the ORF. Specifically, this document provides a DNA expression vector system in which one or more vectors encode two segments of the arenavirus genome, namely, the L segment and the S segment, of the arenavirus particle described herein. Such a vector system can encode (one or more independent DNA molecules).

[0234] In another embodiment, the present invention provides cDNA of the isoplasmic virus S fragment, engineered to carry an ORF at a location other than the wild-type location, as part of or incorporated into a DNA expression system. In other embodiments, cDNA of the isoplasmic virus L fragment, engineered to carry an ORF at a location other than the wild-type location, as part of or incorporated into a DNA expression system. In some embodiments, cDNA of an isoplasmic virus genome fragment is engineered to carry (i) an ORF at a location other than the wild-type location of the ORF; and (ii) an ORF encoding a GP, NP, Z, or L protein that has been removed or replaced with a heterologous ORF from an organism other than isoplasmic virus.

[0235] In some embodiments, the cDNA provided herein may be derived from a specific LCMV strain. 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, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and their derivatives. In a specific embodiment, the cDNA is derived from LCMV clone 13. In other specific embodiments, the cDNA is derived from the LCMV MP strain.

[0236] In some embodiments, the vector generated to encode the arenavirus particle or three-fragment arenavirus particle described herein may be based on a specific LCMV strain. 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, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and derivatives thereof. In some embodiments, the arenavirus particle or three-fragment arenavirus particle described herein may be based on LCMV clone 13. In other embodiments, the vector generated to encode the arenavirus particle or three-fragment arenavirus particle described herein is the LCMV MP strain. The sequence of the S fragment of LCMV clone 13 is as listed in SEQ ID NO: 2. In some embodiments, the sequence of the S fragment of LCMV clone 13 is the sequence listed in SEQ ID NO: 1. The sequence of the L fragment of LCMV clone 13 is as listed in LEQ ID NO: 5. The sequence of the S fragment of LCMV strain MP is as listed in SEQ ID NO: 53. The sequence of the L fragment of LCMV strain MP is as listed in LEQ ID NO: 4.

[0237] In another embodiment, this document provides cells comprising the cDNA or vector systems described above in this section. Cell lines derived from such cells, cultures comprising such cells, and methods for culturing infected such cells are also provided. In some embodiments, this document provides cells comprising cDNA engineered to carry a fragment of the arenavirus genome of the ORF at a location other than the wild-type location of the ORF. In some embodiments, the cells comprise S and / or L fragments.

[0238] 4.5.2 Three-fragment sand-like virus particles In one embodiment, this document provides a nucleic acid encoding a three-segment sand-borne virus particle as described in Section 4.2. In a more specific embodiment, this document provides a DNA nucleotide sequence or a collection of DNA nucleotide sequences, for example, as listed in Table 2 or Table 3. Host cells containing such nucleic acids are also provided in Section 4.2.

[0239] In a specific embodiment, this document provides a cDNA composed of three fragments of isovirus particles, the three fragments of which are engineered to carry an ORF at a location other than the wild-type location of the ORF. In other embodiments, it is a cDNA of three fragments of isovirus particles, the three fragments of which are engineered to (i) carry an isovirus ORF at a location other than the wild-type location of the ORF; and (ii) wherein the three fragments of areovirus particles encode a heterologous ORF as described in section 4.2.

[0240] In one embodiment, this document provides a DNA expression vector system that collectively encodes the three-segment isopyrvirus particle described herein. Specifically, this document provides a DNA expression vector system in which one or more vectors encode three isopyrvirus genomic segments, namely, one L segment and two S segments, or two L segments and one S segment, of the three-segment isopyrvirus particle described herein. Such a vector system can encode (one or more independent DNA molecules).

[0241] In another embodiment, the present invention provides cDNA of a isopyrvirus S fragment engineered to carry an ORF at a location other than the wild-type location, which is part of or incorporated into a DNA expression system. In other embodiments, cDNA of an isopyrvirus L fragment engineered to carry an ORF at a location other than the wild-type location, which is part of or incorporated into a DNA expression system. In some embodiments, cDNA of a three-fragment isopyrvirus particle engineered to carry (i) an ORF at a location other than the wild-type location of the ORF; and (ii) an ORF encoding a GP, NP, Z, or L protein that has been removed or replaced with a heterologous ORF from an organism other than isopyrvirus.

[0242] In some embodiments, the cDNA provided herein may be derived from a specific LCMV strain. 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, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and their derivatives. In a specific embodiment, the cDNA is derived from LCMV clone 13. In other specific embodiments, the cDNA is derived from the LCMV MP strain.

[0243] In some embodiments, the vector generated to encode the arenavirus particle or three-fragment arenavirus particle described herein may be based on a specific LCMV strain. 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, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and derivatives thereof. In some embodiments, the arenavirus particle or three-fragment arenavirus particle described herein may be based on LCMV clone 13. In other embodiments, the vector generated to encode the arenavirus particle or three-fragment arenavirus particle described herein is the LCMV MP strain. The sequence of the S fragment of LCMV clone 13 is as listed in SEQ ID NO: 2. In some embodiments, the sequence of the S fragment of LCMV clone 13 is the sequence listed in SEQ ID NO: 1. The sequence of the L fragment of LCMV clone 13 is as listed in LEQ ID NO: 5. The sequence of the S fragment of LCMV strain MP is as listed in SEQ ID NO: 53. The sequence of the L fragment of LCMV strain MP is as listed in LEQ ID NO: 4.

[0244] In another embodiment, this document provides cells containing the cDNA or vector systems described above in this section. Cell lines derived from such cells, cultures containing such cells, and methods for culturing infected such cells are also provided. In some embodiments, this document provides cells containing cDNA of three fragments of a isonavirus particle. In some embodiments, the cells contain an S fragment and / or an L fragment.

[0245] 4.6 Usage Method Vaccines have been successfully used to prevent and / or treat infectious diseases, such as those for poliovirus and measles. However, therapeutic immunization is rarely successful in the case of established chronic diseases, including chronic infections and cancer. The ability to produce isovirus particles and / or trisegmented isovirus particles represents a novel vaccine strategy.

[0246] In one embodiment, this document provides a method for treating an infection and / or cancer in a subject, comprising administering to the subject one or more types of arenavirus particles or trisegmented arenavirus particles, or combinations thereof, as described herein. In a specific embodiment, the method for treating an infection and / or cancer described herein comprises administering to a subject in need an effective amount of one or more types of arenavirus particles or trisegmented arenavirus particles, or combinations thereof, as described herein. The subject may be a mammal, such as, but not limited to, a human, mouse, rat, guinea pig, or a domesticated animal, such as, but not limited to, a cow, horse, sheep, pig, goat, cat, dog, hamster, or donkey. In a specific embodiment, the subject is a human. Human subjects may be males, females, adults, children, elderly (65 years of age or older), and those suffering from multiple diseases (i.e., multi-disease subjects). In some embodiments, the subject is a subject whose disease has progressed after treatment with chemotherapy, radiation therapy, surgery, and / or biological agents.

[0247] In another embodiment, this document provides a method for inducing an immune response in a subject against an antigen derived from an infectious organism, a tumor, or an allergen, comprising administering to the subject sand-like virus particles or trisegmented sand-like virus particles expressing antigens derived from an infectious organism, a tumor, or an allergen, or a combination thereof.

[0248] Subjects receiving the arenavirus particles or trisegmented arenavirus particles described herein are infected with, susceptible to, or at risk of infection, cancer, or allergy, or exhibit precancerous lesions, wherein the arenavirus particles or trisegmented arenavirus particles express antigens derived from infectious organisms, tumors, or allergens. In another specific embodiment, the subjects receiving the arenavirus particles or trisegmented arenavirus particles are infected with, susceptible to, at risk of, or diagnosed with an infection, cancer, precancerous lesions, or allergy, wherein the arenavirus particles or trisegmented arenavirus particles express antigens derived from infectious organisms, tumors, or allergens, or combinations thereof, as described herein.

[0249] In another embodiment, the subject receiving isovirus particles or trisegmented isovirus particles has, is susceptible to, or is at risk of infection, cancer, precancerous lesions, or allergies in the pulmonary, central nervous, lymphatic, gastrointestinal, or circulatory systems, wherein the isovirus particles or trisegmented isovirus particles express antigens or combinations thereof derived from infectious organisms, tumors, or allergens as described herein. In a specific embodiment, the subject receiving isovirus particles or trisegmented isovirus particles has, is susceptible to, or is at risk of infection, cancer, precancerous lesions, or allergies in one or more organs of the body, including but not limited to the brain, liver, lungs, eyes, ears, intestines, esophagus, uterus, nasopharynx, or salivary glands, wherein the isovirus particles or trisegmented isovirus particles express antigens or combinations thereof derived from infectious organisms, tumors, or allergens as described herein.

[0250] In another embodiment, the symptoms experienced by subjects to whom arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens described herein are administered include, but are not limited to, fever, night sweats, fatigue, malaise, restlessness, pharyngitis, glandular swelling, joint pain, muscle pain, loss of appetite, diarrhea, gastrointestinal ulcers, gastrointestinal bleeding, shortness of breath, pneumonia, oral ulcers, vision problems, hepatitis, jaundice, encephalitis, epilepsy, coma, pruritus, erythema, hyperpigmentation, lymph node changes, or hearing loss.

[0251] In another embodiment, arenavirus or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to subjects of any age group who have, are susceptible to, or are at risk of infection, cancer, or allergy. In a specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to subjects with compromised immune systems, pregnant subjects, subjects who have undergone organ or bone marrow transplants, subjects taking immunosuppressive drugs, subjects undergoing hemodialysis, subjects with cancer, or subjects who have, are susceptible to, or are at risk of infection, cancer, or allergy. In a more specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to children aged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years who have, are susceptible to, or are at risk of infection, cancer, or allergy. In yet another specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to subjects who are infants suffering from, susceptible to, or at risk of infection, cancer, or allergies. In yet another specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to subjects who are infants aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months of age suffering from, susceptible to, or at risk of infection, cancer, or allergies. In yet another specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to elderly subjects who suffer from, are susceptible to, or at risk of infection, cancer, or allergies. In a more specific implementation, isovirus particles or trisegmented isovirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to elderly subjects aged 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 years of age.

[0252] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to subjects at increased risk of transmitting the infection, cancer, or allergy. In a specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to neonatal subjects with a nascent and therefore immature immune system.

[0253] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to a subject suffering from a latent infection, cancer, or allergy. In a specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein are administered to a subject suffering from a latent infection, a latent dormant infection, or a latent allergy that may be reactivated in a compromised immune system. Thus, this document provides a method for preventing the reactivation of infections, cancers, or allergies.

[0254] In another embodiment, isovirus particles or trisegmented isovirus particles expressing antigens derived from infectious organisms, cancers, or allergens, or combinations thereof, as described herein, are administered to a subject suffering from recurrent infections, cancer, or allergies.

[0255] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergens, or combinations thereof, as described herein, are administered to a subject with a genetic predisposition to infection, cancer, or allergy. In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergens, or combinations thereof, as described herein, are administered to a subject. In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergens are administered to a subject with risk factors. Exemplary risk factors include aging, tobacco use, sunlight exposure, radiation exposure, chemical exposure, family history, alcohol consumption, poor diet, lack of physical activity, or being overweight.

[0256] In another embodiment, administration of arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergens reduces symptoms of infection, cancer, or allergies. In another embodiment, administration of arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergens reduces asymptomatic infection, cancer, or allergies.

[0257] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from or in combination with antigens of the infectious organisms described herein are administered to a subject or animal infected with one or more strains of influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus, infectious laryngotracheitis virus, avian anemia virus, Marek's disease virus, avian leukosis virus, avian adenovirus or avian pneumovirus, SARS-causing virus, human respiratory syncytial virus, human immunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, poliovirus, rabies virus, Hendra virus, Nipah virus, human parainfluenza 3 virus, measles virus, mumps virus, Ebola virus, Marburg virus, West Nile virus, Japanese encephalitis virus, dengue virus, Hantavirus, Rift Valley fever virus, Lassa fever virus, herpes simplex virus, and yellow fever virus.

[0258] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing cancer-derived antigens or combinations thereof described herein are administered to a subject suffering from one or more types of cancer. In other embodiments, any type of cancer that is therapeutically sensitive to the vaccine described herein may be targeted. In a more specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing cancer-derived antigens or combinations thereof described herein are administered to a subject suffering from, for example, melanoma, prostate cancer, breast cancer, lung cancer, neuroblastoma, hepatocellular carcinoma, cervical cancer, and gastric cancer, Burkitt lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma; nasopharyngeal carcinoma (cancer of the upper pharynx on the posterior side of the nose), leukocyte cancer, and mucosa-associated lymphoid tissue lymphoma.

[0259] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from allergens or compositions thereof as described herein are administered to a subject suffering from one or more allergies. In a more specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from allergens or compositions thereof as described herein are administered to a subject suffering from, for example, seasonal allergies, perennial allergies, rhinoceros conjunctivitis, asthma, eczema, or food allergies.

[0260] In another embodiment, isabinovirus particles or trisegmented isabinovirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens, as described herein, are administered to a subject to confer cell-mediated immunity (CMI) against the infection, cancer, or allergen. Not limited to this theory, in another embodiment, isabinovirus particles or trisegmented isabinovirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens, as described herein, are used to infect and express the target antigen in antigen-presenting cells (APCs) of a host (e.g., macrophages, dendritic cells, or B cells) to guide antigen presentation on major histocompatibility complexes (MHCs) of classes I and II. In another embodiment, isabinovirus particles or trisegmented isabinovirus particles expressing compressed antigens or combinations thereof derived from infectious organisms, cancers, or allergens, as described herein, are administered to a subject to induce pluripotent cell lysis and the production of IFN-γ and TNF-α in conjunction with high-level CMV-specific CD4+ and CD8+ T cell responses to treat or prevent infection, cancer, or allergy.

[0261] In another implementation, compared with the risk of infection, cancer, or allergy without treatment, administration of isovirus particles or trisegmented isovirus particles expressing antigens derived from infectious organisms, cancers, or allergens or combinations thereof reduces an individual's risk of infection, cancer, or allergy by at least about 10%, at least about 20%, at least about 25%, at least about 30%, 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 higher.

[0262] In another implementation, compared with the symptom presentation of infection, cancer, or allergy without treatment, administration of isovirus particles or trisegmented isovirus particles expressing antigens derived from infectious organisms, cancers, or allergens, or combinations thereof, reduces the symptom presentation of infection, cancer, or allergy by at least about 10%, at least about 20%, at least about 25%, at least about 30%, 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 higher.

[0263] In some embodiments, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergens are preferably administered at multiple sites (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 14 sites) via multiple injections (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 45, or 50 injections). In some embodiments, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergens are administered in two or more independent injections over a period of 6 months, 12 months, 24 months, or 48 months. In some embodiments, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergens are administered with a first dose on a selected date, a second dose at least 2 months after the first dose, and a third dose at least 6 months after the first dose.

[0264] In one instance, multiple skin injections are administered at various body sites to reduce the severity of local skin reactions. On a given day of vaccination, the patient receives a specified total cellular dose administered from a single syringe in 3 to 5 separate intradermal injections of a dose (e.g., at least 0.4 ml, 0.2 ml, or 0.1 ml), with the needle inserted at least approximately 5 cm (e.g., at least 4.5, 5, 6, 7, 8, 9, or 1 cm) from the nearest injection site. On subsequent days of vaccination, the injection sites are rotated clockwise or counterclockwise to different limbs.

[0265] In another embodiment, administration of an infectious replication-defective isoplasmic virus expressing a CMV antigen or a combination thereof to a subject with a newly formed and thus immune system induces a cell-mediated immune (CMI) response against an infection, cancer, or allergy exceeding 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 about 80%, at least about 90%, or more of the CMI response to the infection, cancer, or allergy in the absence of such treatment.

[0266] In some embodiments, administration of isabinovirus particles or trisegmented isabinovirus particles expressing antigens derived from infectious organisms, cancers, or allergens, as described herein, to a subject induces a detectable antibody titer that lasts for at least four weeks. In another embodiment, administration of isabinovirus particles or trisegmented isabinovirus particles expressing antigens derived from infectious organisms, cancers, or allergens, as described herein, increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0267] In some embodiments, the initial antigen exposure elicits functional, (neutralizing), and minimum antibody titers that are at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the mean control serum from infected-immunized human subjects. In a more specific embodiment, the initial neutralizing geometric mean antibody titer increases to a peak of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 at least 4 weeks post-immunization. In another embodiment, immunization with isanoxic virus particles or trisegmented isanoxic virus particles expressing antigens derived from infectious organisms, cancer, or allergies, as described herein, produces high antibody titers at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post-single administration of the vaccine, or after two or more consecutive immunizations.

[0268] In yet another embodiment, the second antigen exposure increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In another embodiment, the second antigen exposure induces a functional, (neutralizing), and minimum antibody titer that is at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the mean control serum from infected-immunized human subjects. In a more specific embodiment, the second neutralizing geometric mean antibody titer increases to a peak of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 at least 4 weeks post-immunization. In another embodiment, a second immunization with sand virus particles or trisegmented sand virus particles expressing antigens derived from infectious organisms, cancer, or allergies, as described herein, produces high titers of antibodies for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after sustained immunization.

[0269] In yet another embodiment, the third booster immunization increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In another embodiment, the booster immunization induces functional, (neutralizing), and minimum antibody titers of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the average control serum from infected-immunized human subjects. In a more specific embodiment, the third booster immunization induces functional, (neutralizing), and minimum antibody titers of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the average control serum from infected-immunized human subjects. In yet another embodiment, the third booster immunization prolongs the antibody titer to at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post-immunization.

[0270] In some embodiments, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergies elicit T cell-independent or T cell-dependent responses. In other embodiments, arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergies elicit T cell responses. In other embodiments, the arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergies, as described herein, elicit T helper cell responses. In yet another embodiment, the arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergies, as described herein, elicit Th1-directed or Th2-directed responses.

[0271] In a more specific embodiment, the Th1-oriented response is demonstrated by the dominance of IgG1 antibody against IgG2. In other embodiments, the IgG1:IgG2 ratio is greater than 1:1, greater than 2:1, greater than 3:1, or greater than 4:1. In yet another embodiment, infectious arenavirus particles or trisegmental arenavirus particles expressing antigens derived from infectious organisms, cancers, or allergies as described herein are demonstrated by the dominance of antibodies against IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, or IgE.

[0272] In some embodiments, the infectious, replication-defective arenavirus expressing CMV antigens or fragments thereof elicits a CD8+ T cell response. In another embodiment, arenavirus particles or tri-fragmented arenavirus particles expressing antigens derived from infectious organisms, cancer, or allergies simultaneously elicit CD4+ and CD8+ T cell responses, with or without antibodies.

[0273] In some embodiments, the expression of arenavirus particles or trisegmented arenavirus particles derived from antigens of infectious organisms, cancer, or allergies, as described herein, elicits high titers of neutralizing antibodies. In another embodiment, the expression of arenavirus particles or trisegmented arenavirus particles derived from antigens of infectious organisms, cancer, or allergies, as described herein, elicits even higher titers of neutralizing antibodies compared to the expression of the protein complex component alone.

[0274] In another implementation, isovirus particles expressing one, two, three, four, five or more antigens derived from infectious organisms, cancer or allergens elicit higher titers of neutralizing antibodies compared to isovirus particles or trisegmented isovirus particles expressing one antigen derived from an infectious organism, cancer or allergen.

[0275] In some embodiments, the method further includes co-administering the arenavirus particles or trisegmented arenavirus particles with at least one other therapy. In some embodiments, the co-administration is simultaneous. In another embodiment, the arenavirus particles or trisegmented arenavirus particles are administered before the administration of the other therapy. In other embodiments, the arenavirus particles or trisegmented arenavirus particles are administered after the administration of the other therapy. In some embodiments, the administration of the arenavirus particles or trisegmented arenavirus particles and the other therapy is approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, or approximately 12 hours. In some embodiments, the interval between the administration of the arenavirus particles or trisegmented arenavirus particles and the other therapy is approximately 1 day, 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, or approximately 12 weeks. In some embodiments, the interval between the administration of the sand virus particles or trisegmented sand virus particles and the other treatments is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

[0276] In some embodiments, administration of arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens reduces the number of antibodies detected in a patient's blood or serum samples. In some embodiments, administration of arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens reduces the number of infectious organisms, cancers, or allergens detected in urine, saliva, blood, tears, semen, exfoliated cell samples, or breast milk.

[0277] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein may further include a reporter protein. In a more specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein, along with a reporter protein, are administered to a subject for the treatment and / or prevention of infection, cancer, or allergy. In yet another specific embodiment, the reporter protein may be used for in vivo, in situ, and real-time monitoring of gene expression, protein localization, and vaccine delivery.

[0278] In another embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein may further include a fluorescent protein. In a more specific embodiment, arenavirus particles or trisegmented arenavirus particles expressing antigens or combinations thereof derived from infectious organisms, cancers, or allergens as described herein, and reporter proteins, are administered to a subject for the treatment and / or prevention of infection, cancer, or allergy. In yet another specific embodiment, the fluorescent protein may be a reporter protein, which can be used for in vivo, in situ, and real-time monitoring of gene expression, protein localization, and vaccine delivery.

[0279] The alteration in CMI response function induced in subjects by administration of arenavirus particles or trisegmented arenavirus particles expressing antigens derived from infectious organisms, cancers, allergens, or combinations thereof, can be achieved by any analysis known to those skilled in the art, including but not limited to flow cytometry (see, for example, Perfetto SP). et al., 2004, Nat Rev Immun., 4(8):648-55), lymphocyte proliferation analysis (see, for example, Bonilla FA et al., 2008, Ann Allergy Asthma Immunol, 101:101-4 and Hicks MJ et al., (1983, Am J Clin Pathol., 80:159-63), an analysis measuring lymphocyte activation, including determining changes in surface marker expression after measuring T lymphocyte cytokines (see, e.g., CarusoA. et al., Cytometry. 1997;27:71-6), ELISPOT analysis (see, for example, Czerkinsky CC) et al.,1983, J Immunol Methods, 65:109-121 and Hutchings PR et al., 1989, J Immunol Methods, 120:1-8), or natural killer cell cytotoxicity assay (see, for example, Bonilla FA et al., 2006, Ann Allergy Asthma Immunol., 94 (5 Suppl 1): S1-63) to measure.

[0280] Successful treatment of cancer patients can be assessed as prolonging expected survival, inducing anti-tumor immune responses, and improving specific cancer characteristics. Examples of cancer characteristics that can be improved include tumor size (e.g., T0, T, or T1-4), metastatic status (e.g., M0, M1), number of visible tumors, lymph node involvement (e.g., N0, N1-4, Nx), grade (i.e., grade 1, 2, 3, or 4), stage (i.e., stage 0, I, II, III, or IV), and the presence or concentration of certain markers in cells or body fluids (e.g., AFP, B2M, beta-HCG, BTA, CA 15-3, CA 27.29, CA 125, CA 72.4, CA 19-9, calcitonin, CEA, chromogranin A). A) EGFR, hormone receptors, HER2, HCG, immunoglobulins, NSE, NMP22, PSA, PAP, PSMA, S-100, TA-90, and thyroglobulin) and / or related pathologies (e.g., ascites or edema) or symptoms (e.g., cachexia, fever, anorexia, or pain). If measurable as a percentage, the improvement may be at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., survival, or tumor volume or linear dimension).

[0281] In another embodiment, this document describes a method for using sand-borne virus particles (e.g., LCMV) expressing antigens derived from infectious organisms, cancers, or allergens, wherein at least one ORF encoding GP, NP, Z, and L proteins is replaced by a nucleotide sequence encoding an antigen derived from an infectious organism, cancer, allergen, or an antigenic fragment thereof.

[0282] 4.7 Composition, Application and Dosage This application also relates to vaccines, immunogenic compositions (e.g., vaccine formulations), and pharmaceutical compositions comprising arenavirus particles or trisegmented arenavirus particles as described herein. Such vaccines, immunogenic compositions, and pharmaceutical compositions can be formulated according to standard procedures in the art.

[0283] It will be apparent to those skilled in the art that suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any of its embodiments.

[0284] In another embodiment, this document provides a composition comprising isovirus particles or trisegmented isovirus particles as described herein. Such compositions can be used in methods of treating and preventing disease. In a specific embodiment, the compositions described herein are used to treat infected or susceptible subjects. In other embodiments, the compositions described herein are used to treat subjects susceptible to cancer or tumorigenesis, exhibiting symptoms characteristic of cancer or tumorigenesis, or diagnosed with cancer. In another specific embodiment, the immunogenic compositions provided herein can be used to induce an immune response in a host to which the composition has been administered. The immunogenic compositions described herein can be used as vaccines and thus can be formulated as pharmaceutical compositions. In a specific embodiment, the immunogenic compositions described herein are used to prevent infection or cancer in subjects (e.g., human subjects). In other embodiments, the vaccines, immunogenic compositions, or pharmaceutical compositions are suitable for veterinary and / or human administration.

[0285] In some embodiments, this document provides an immunogenic composition comprising the arenavirus vector described herein. In some embodiments, such an immunogenic composition further comprises a pharmaceutically acceptable excipient. In some embodiments, such an immunogenic composition further comprises an adjuvant. The adjuvant for co-administration with the composition described herein may be administered before, concurrently with, or after administration of the composition. In some embodiments, the term "adjuvant" refers to a compound that, when administered with or as part of the composition described herein, increases, enhances, and / or strengthens the immune response to arenavirus particles or trisegmental arenavirus particles, and most importantly, the immune response to the gene product carried thereon, but when the compound is administered alone, does not produce an immune response to arenavirus particles or trisegmental arenavirus particles or the gene product carried thereon. In some embodiments, the adjuvant produces an immune response to the arenavirus particles or trisegmental arenavirus particles or the gene product carried thereon without causing allergic reactions or other adverse reactions. Adjuvants can enhance immune responses through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages or dendritic cells. When the vaccine or immunogenic composition of the present invention contains an adjuvant, or is administered in combination with one or more adjuvants, the adjuvants that may be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticle adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Application WO2007 / 109813), and saponins, such as QS21 (see eds. Powell & Newman, Plenum Press, NY); U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (e.g., squalene or peanut oil), optionally combined with an immunostimulant, such as monophosphoryl lipid A (see Stoute). et al.,1997, N. Engl. J. Med. 336, 86-91).

[0286] The composition comprises individual isovirus particles or trisegmented isovirus particles as described herein, or together with a pharmaceutically acceptable carrier. Suspensions or dispersions of isovirus particles or trisegmented isovirus particles, particularly isotonic aqueous suspensions, can be used. The pharmaceutical composition may be sterilized and / or may contain excipients, such as preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, osmotic pressure-adjusting salts and / or buffers, prepared in a manner known in the art, for example, by conventional dispersion and suspension processing. In some embodiments, such dispersions or suspensions may contain viscosity modifiers. The suspensions or dispersions are maintained at about 2°C to 8°C, and preferably for longer storage, may be frozen and then thawed before use, or alternatively may be lyophilized for preservation. For injectable formulations, the vaccine or immunogenic product may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulated pharmaceutical agents such as suspending agents, stabilizers, and / or dispersants.

[0287] In some embodiments, the compositions described herein additionally contain a preservative, such as the mercury derivative thimerosal. In a specific embodiment, the pharmaceutical compositions described herein contain 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not contain a preservative.

[0288] The pharmaceutical composition contains about 10 3 To about 10 11 Sand virus particles that form a single lesion unit or three fragments of sand virus particles.

[0289] In one embodiment, the pharmaceutical composition is administered parenterally. Parenterally administration can be intravenous or subcutaneous. Therefore, the unit dose form for parenterally administration is, for example, an ampoule or vial containing approximately 10... 3 Up to 10 10 One lesion forming unit or 10 5 Up to 10 15 Sand virus particles that are physically separate particles or three-segment sand virus particles.

[0290] In another embodiment, the vaccine or immunogenic composition provided herein is administered to a subject via routes including but not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, local, subcutaneous, transdermal, intranasal, and inhalation, as well as through skin rupture (scraping away the top layer of skin, e.g., using a bifurcated needle). Specifically, subcutaneous or intravenous routes may be used.

[0291] For intranasal or inhalation administration, a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide, or other suitable gas, is used to conveniently deliver the article for use according to the invention in the form of an aerosol spray from a pressurized package or nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the measured amount. For example, gelatin capsules and cartridges used in inhalers or blowpipes can be formulated as a powder mixture containing a compound and a suitable powder base, such as lactose or starch.

[0292] The dosage of the active ingredient depends on the type of vaccine administered and the subject, their age, weight, individual condition, individual pharmacokinetic data, and route of administration. In some implementations, in vitro analysis is used to help identify the optimal dosage range. An effective dosage can be deduced from dose-response curves derived from in vitro or animal model testing systems.

[0293] In some embodiments, the vaccine, immunogenic composition, or pharmaceutical composition comprising arenavirus particles or trisegmented arenavirus particles can be used as a live vaccine. An exemplary dose of live arenavirus particles can be 10 to 100 or higher PFU of live virus per dose. In some embodiments, a suitable dose of arenavirus particles or trisegmented arenavirus particles is 10 PFU. 2 5×10 2 10 3 5×10 3 10 4 5×10 4 10 5 5×10 5 10 6 5×10 6 10 7 5×10 7 10 8 5×10 8 1×10 9 5×10 9 1×10 10 5×10 10 1×10 11 5×10 11 Or 10 12PFU can be administered to the subject once, twice, three times, or more at the usual required intervals. In another embodiment, live isovirus is prepared such that a 0.2 mL dose contains 10% live isovirus particles. 6.5 -10 7.5 One fluorescent hemolytic focus unit. In another embodiment, an inactivated vaccine is formulated such that it contains about 15 μg to about 100 μg, about 15 μg to about 75 μg, about 15 μg to about 50 μg, or about 15 μg to about 30 μg of isovirus.

[0294] In some embodiments, for administration to children, two doses of the arenavirus particles or trisegmental arenavirus particles or combinations thereof described herein are administered at intervals of at least one month. In specific embodiments for administration to adults, a single dose of the arenavirus particles or trisegmental arenavirus particles or combinations thereof described herein is administered. In another embodiment, two doses of the arenavirus particles or trisegmental arenavirus particles or combinations thereof described herein are administered to adults at intervals of at least one month. In another embodiment, young children (six months to nine years old) may receive the arenavirus particles or trisegmental arenavirus particles or combinations thereof described herein for the first administration in two doses at monthly intervals. In certain embodiments, children who receive only one dose in the first year of vaccination should receive two doses in the second year. In some embodiments, two doses administered at 4-week intervals are preferred for children aged 2–8 years who are receiving the immunogenic composition described herein for the first time. In some embodiments, half the dose (0.25 ml) may be preferred for children aged 6–35 months, compared to 0.5 ml for subjects older than 3 years.

[0295] In some embodiments, the composition may be administered to a subject in a single dose comprising a therapeutically effective amount of isovirus particles or trisegmented isovirus particles. In some embodiments, the isovirus particles or trisegmented isovirus particles may be administered to a patient in a single dose comprising a therapeutically effective amount of isovirus particles or trisegmented isovirus particles and one or more pharmaceutical compositions, each at a therapeutically effective amount.

[0296] In some embodiments, the composition is administered to the patient as a single dose, followed by a second dose three to six weeks later. According to these embodiments, booster vaccination may be administered to the subject at an interval of six to twelve months following the second vaccination. In some embodiments, the booster vaccination may use different arenaviruses or combinations thereof. In some embodiments, administration of the same composition described herein may be repeated, spaced at intervals of at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0297] This document also provides a process and use for manufacturing vaccines in the form of pharmaceutical articles containing the aforementioned isovirus particles or trisegmented isovirus particles as active ingredients. The pharmaceutical compositions of this application are prepared in a manner known per se, for example, by conventional mixing and / or dispersion processes.

[0298] 4.8 Analysis 4.8.1 Detection and Analysis of Arena Virus Skilled technicians can use techniques known in the art to detect the arenavirus genomic fragments or trisegment arenavirus particles described herein. For example, RT-PCR, using primers specific to the arenavirus to be detected, can be used to detect and quantify arenavirus genomic fragments or trisegment arenavirus particles engineered to carry the ORF at a location other than the wild-type location of the ORF. Western blot, ELISA, radioimmunoassay, immunoprecipitation, immunohistochemistry, or immunocytochemistry, in conjunction with FACS, can be used to quantify the gene products of arenavirus genomic fragments or trisegment arenavirus particles.

[0299] 4.8.2 Analysis of infectivity measurement Any analysis known to a skilled technician can be used to measure the infectivity of isovirus vector preparations. For example, virus / vector titers can be determined by “focal formation unit analysis” (FFU analysis). In short, supplementary cells, such as MC57 cells, are seeded with virus / vector samples at different dilutions. After the incubation period, cells are allowed to form a monolayer and the virus is allowed to attach to the cells; the monolayer is covered with methylcellulose. When the plates are further incubated, the originally infected cells release viral progeny. Due to the methylcellulose covering, the spread of new virus is confined to neighboring cells. Thus, each infectious particle produces a circular area of ​​infected cells called a foci. Such foci can become visible and countable using antibodies against LCMV-NP or other proteins expressed by the isovirus particle or trisegmental isovirus particles, along with an HRP-based display reaction. The virus / vector titer can be calculated in focal formation units per milliliter (FFU / mL).

[0300] 4.8.3 Growth of Sand Virus Particles The growth of the arenavirus particles described herein can be assessed by any method known in the art or described herein (e.g., cell culture). Viral growth can be determined by inoculating serial dilutions of the arenavirus particles described herein into cell cultures (e.g., Vero cells or BHK-21 cells). After incubation for a specified time, the virus is isolated using standard methods.

[0301] 4.8.4 Serum ELISA In animal vaccination (e.g., mice, guinea pigs), the humoral immune response can be measured using an antigen-specific serum ELISA (enzyme-linked immunosorbent assay). In short, plates are coated with an antigen (e.g., recombinant protein), blocked to prevent non-specific antibody binding, and incubated with serial dilutions of serum. After incubation, bound serum-antibodies can be detected, for example, using enzyme-linked anti-species (e.g., mouse, guinea pig) specific antibodies (detecting total IgG or IgG subclasses) and a subsequent color reaction. Antibody titers can be determined, for example, as an endpoint geometric mean titer.

[0302] 4.8.5 Analysis of the neutralizing activity of induced antibodies The determination of neutralizing antibodies in serum was performed using the following cellular assay with ARPE-19 cells from ATCC and GFP-tagged virus. Supplemented guinea pig serum was used as a source of exogenous complement. Analysis began one day or two days prior to neutralization in 6.5 × 10⁶ cells seeded in 384-well plates. 3 Cells / well (50 μl / well). Neutralization was performed for 1 hour at 37°C on 96-well sterile tissue culture plates for Key Cells. After the neutralization incubation step, the mixture was added to the cells and incubated for an additional 4 days for GFP detection using a plate reader. Positive neutralized human serum was used as an analytical positive control on each plate to check the reliability of all results. Titers (EC50) were determined using 4-parameter logistic curve fitting. As an additional test, the reaction wells were examined using a fluorescence microscope.

[0303] 4.8.6 Pockmark Subtraction Analysis In short, plaque reduction (neutralization) analysis of LCMV can be performed using replicated or defective LCMV labeled with green fluorescent protein, with 5% rabbit serum used as a source of exogenous complement, and plaques counted by fluorescence microscopy. The neutralization titer can be defined as the highest serum dilution that causes a 50%, 75%, 90%, or 95% reduction in plaques compared to a control (pre-immunization) serum sample.

[0304] Following the manufacturer's instructions, the qPCR LCMV RNA genome was isolated using the QIAamp Viral RNA mini kit (QIAGEN). The LCMV RNA genome equivalent was detected by quantitative PCR using the SuperScript® III Platinum® One-Step qRT-PCR kit (Invitrogen) and primers and probes (FAM reporter and NFQ-MGB quencher) specific to a portion of the LCMV NP coding region, or another genome extension of a isovirus particle or a three-segment isovirus particle. The reaction temperature distribution was: 60°C for 30 min, 95°C for 2 min, followed by 45 cycles of 95°C for 15 s and 56°C for 30 s. RNA can be quantified by comparing sample results with a standard curve prepared from a log10 dilution series of in vitro transcribed RNA fragments quantified by spectrophotometry, the RNA fragments corresponding to LCMV NP coding sequences containing primer and probe binding sites, or fragments of another genome extension of a sand virus particle or a three-fragment sand virus particle.

[0305] 4.8.7 Western Imprint Infected cells grown in tissue culture flasks or suspensions were lysed at the time points indicated post-infection using RIPA buffer (ThermoScientific), or used directly without cell lysis. Samples were heated to 99°C for 10 minutes with reducing agent and NuPage LDS sample buffer (NOVEX), cooled to room temperature, and then loaded onto 4–12% SDS-PAGE gels for electrophoresis. Protein spots were stained onto the membrane using an Invitrogens iBlot Gel transfer device and visualized by Ponceau staining. Finally, the samples were probed with a primary antibody against the target protein and a secondary antibody that binds to alkaline phosphatase, followed by staining with 1-Step NBT / BCIP solution (INVITROGEN).

[0306] 4.8.8 MHC-peptide multimer staining analysis for detecting antigen-specific CD8+ T cell proliferation. Any analysis known to a skilled technician can be used to test for antigen-specific CD8+ T cell responses. For example, MHC-peptide tetramer staining analysis can be used (see, for example, Altman JD). et al., Science. 1996;274:94-96; and Murali-Krishna K. et al.,Immunity. 1998; 8:177-187). Briefly, the analysis comprises the following steps: tetramer analysis is used to detect the presence of antigen-specific T cells. To detect T cell-specific peptides, it is necessary to identify the peptide and a tetramer of an MHC molecule tailored for a defined antigen specificity, as well as the MHC monomer of the T cell (typically fluorescently labeled). The tetramer is then detected by flow cytometry using fluorescent labeling.

[0307] 4.8.9 ELISPOT analysis for detecting antigen-specific CD4+ T cell proliferation Any analysis known to a skilled technician can be used to test for antigen-specific CD4+ T cell responses. For example, the ELISPOT assay (see, for example, Czerkinsky CC) can be used. et al., J Immunol Methods.1983;65:109-121 and Hutchings PR et al., J Immunol Methods. 1989; 120:1-8). Briefly, the analysis comprises the following steps: an immunospot plate is coated with an anti-cytokine antibody. Cells are incubated on the immunospot plate. Cells secrete cytokines, which are then washed away. The plate is then coated with a second biotinylated anti-cytokine antibody and visualized using the avidin-HRP system.

[0308] 4.8.10 Intracellular cytokine analysis to detect the function of CD8+ and CD4+ T cell responses Any analysis known to a skilled technician can be used to test the function of CD8+ and CD4+ T cell responses. For example, intracellular cytokine analysis in combination with flow cytometry (see, for example, Suni MA) can be used. et al., JImmunol Methods. 1998;212:89-98; Nomura LE et al., Cytometry. 2000;40:60-68 and Ghanekar SA et al., Clinical and Diagnostic Laboratory Immunology. 2001; 8:628-63. Briefly, the analysis comprises the following steps: cell activation via specific peptides or proteins, and retention of cytokines within the cells by adding an inhibitor of protein transport (e.g., brefidobacterium A). After a defined incubation period, typically 5 hours, a washing step is performed, during which antibodies against other cellular markers can be added to the cells. The cells are then fixed and permeabilized. Anti-cytokine antibodies bound to fluorophores are added, and the cells can be analyzed by flow cytometry.

[0309] 4.8.11 Analysis confirming the replication defects of the viral vector Any analysis known to a skilled technician for determining the concentration of infectious and replicating viral particles can also be used to measure replication-defective viral particles in a sample. For example, FFU analysis using non-supplementary cells can be used for this purpose.

[0310] Furthermore, plaque-based analysis is a standard method for determining the concentration of virus in a viral sample according to plaque-forming units (PFU). Specifically, a confluent monolayer of non-supplementary host cells is infected with varying dilutions of virus and covered with a semi-solid medium, such as agar, to prevent uncontrolled spread of viral infection. Viral plaques are formed when the virus successfully infects and replicates itself within the cells of the fixed cell monolayer and spreads to surrounding cells (see, e.g., Kaufmann, SH; Kabelitz, D. (2002). Methods in Microbiology Vol.32: Immunology of Infection. Academic Press. ISBN 0-12-521532-0). Plaque formation can take 2–14 days, depending on the virus being analyzed. Plaques are generally counted manually, and this result, along with the dilution factor used to prepare the plate, is used to calculate the number of plaque-forming units per unit volume of sample (PFU / mL). The PFU / mL result represents the number of infectious replicating particles within the sample. When using C-cells, the same analysis can be used to titrate replication-defective arenavirus particles or trisegmented arenavirus particles.

[0311] 4.8.12 Analysis of viral antigen expression Any analysis known to a skilled technician can be used to measure viral antigen expression. For example, FFU analysis can be performed. For detection, a monoclonal or monoclonal antibody product (gene-specific FFU) against the corresponding viral antigen is used.

[0312] 4.8.13 Animal Models To study the recombination and infectivity of the arenavirus particles described herein, in vivo animal models can be used. In some embodiments, animal models that can be used to study the recombination and infectivity of the three-segment arenavirus particles include mice, guinea pigs, rabbits, and monkeys. In a preferred embodiment, animal models that can be used to study the recombination and infectivity of arenaviruses include mice. In a more specific embodiment, mice that can be used to study the recombination and infectivity of arenavirus particles are triple-deficient in type I interferon receptor, type II interferon receptor, and recombinant activation gene 1 (RAG1).

[0313] In some embodiments, the animal model can be used to determine arenavirus infectivity and transgenic stability. In some embodiments, viral RNA can be isolated from the serum of the animal model. These techniques are readily understood by those skilled in the art. Viral RNA can be reverse transcribed, and cDNA carrying the arenavirus ORF can be amplified by PCR using gene-specific primers. Flow cytometry can also be used to study arenavirus infectivity and transgenic stability.

[0314] 5. Examples These embodiments demonstrate that LCMV-based vector technology can be used to successfully develop (1) sand-like virus genome fragments with viral ORFs located outside the wild-type position of the ORF, and (2) three-fragment sand-like virus particles that do not produce replicative two-fragment viral particles.

[0315] 5.1 Materials and Methods 5.1.1 Cells BHK-21 cells were cultured in high-glucose Dulbecco's Eagle medium (DMEM; Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FCS; Biochrom), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), and 1× tryptone phosphate broth. MC57 cells were maintained in minimally essential medium (MEM; Sigma) supplemented with 5% heat-inactivated FCS, 2 mM L-glutamine (Gibco), and penicillin-streptomycin (100,000 U / ml penicillin and 50 mg / L chloramphenicol; Gibco). Both cell lines were cultured at 37°C in a humidified 5% CO2 incubator.

[0316] According to the manufacturer's protocol, NP-expressing BHK-21 cells were generated by transfecting BHK-21 cells with a plasmid expressing NP under the control of the eukaryotic EF1-α promoter and encoding a puromycin resistance gene. Forty-eight hours post-transfection, 4 μg / ml puromycin was added to the culture medium. Forty-eight hours later, the cells were passaged into T150 flasks. Once isolated clones began to appear, the cells were harvested and serially diluted into 96-well plates to obtain individual clones. Growth from single clones to cell populations in the reaction wells was optically examined, and once a confluent monolayer formed, the corresponding cells were passaged into 6-well plates. NP-expressing BHK-21 cells were cultured in BHK-21 medium containing 4 μg / ml puromycin.

[0317] Previously, BHK-21 cells expressing GP were described. Briefly, BHK-21 cells were stably transfected with a plasmid expressing codon-optimized LCMV-GP cDNA and a puromycin resistance cassette. Clones expressing GP were selected by adding 4 μg / ml puromycin to the culture medium, and individual clones were obtained by serial dilutions as described for BHK-21 cells expressing NP.

[0318] 5.1.2 Plasmids The pol-I L, pC-NP, and pC-L plasmids have been described previously. For the generation of the pol-I S plasmid encoding GFP or RFP as a reporter gene and NP or GP, we used the pol-I Bbs / Bsm cloning plasmid as a base (pol-I 5'-BsmBI_IGR_BbsI_3'). This plasmid encodes the 5' untranslated region (5' UTR) of the viral S fragment, followed by two BsmBI restriction sites, an intergenic region (IGR), flanked by BbsI restriction sites, and NP rest and CAT open reading frames (ORF) of the 3' UTR of the S fragment. The pol-I S plasmid encodes the GP at its native 5' position and the GFP at the antisense position at 3' (pol-I5'-GP_IGR_GfP-3'), which is cloned by inserting the GP through BsmBI site-specific restriction and ligating it to the pol-I Bbs / Bsm plasmid. In a second step, GFP is inserted via BbsI digestion and ligation. To obtain the pol-I S plasmid (pol-I 5'-GFP_IGR_GP-3') encoding GP at the artificial 3' orientation, GP is inserted into the pol-I Bbs / Bsm plasmid at the 3' position via BBSI digestion, and GFP is inserted at the 5' position via BsmBI restriction / ligation. For pol-I S plasmids encoding GFP or RFP with NP (pol-I 5'-GFP_IGR_NP-3' or pol-I 5'RFP_IGR_NP-3'), NP is inserted via BBSI digestion and ligated into the pol-I Bbs / Bsm cloning plasmid, and GFP or RFP is cloned via BsmBI cloning. For pol-I plasmids containing GP from LCMV strain WE and NP from LCMV strain clone 13 (Cl13), the corresponding genes are inserted into the pol-I Bbs / Bsm cloning plasmid at the appropriate sites via Bbs and Bsm site-specific restriction / ligation.

[0319] The S fragment encoding the WE / WET fusion GP was obtained by replacing the last 255 base pairs of the WE ORF with a codon-optimized sequence called "WET". This was achieved by PCR amplification of the WE GP fragment in a first step using a WE-specific primer (SEQ ID NO: 11) and a WE-specific fusion primer (SEQ ID NO: 12) with a protrusion complementary to the WET sequence. In parallel, the WET sequence was amplified by PCR using a WET-specific primer (SEQ ID NO: 13) and a WET-specific fusion primer (SEQ ID NO: 14) complementary to the WE sequence. In a third PCR reaction, the two PCR products were fused by PCR fusion using the two mentioned fusion primers. The resulting WE / WET fusion fragment was digested with BsmBI and ligated into the pol-I BsmBI_IGR_GFP-3' plasmid, which had already been digested with the same restriction enzyme.

[0320] By using site-specific restriction / ligation of SacI and XmaI, a synthesized DNA fragment (synthesized by the GenScript gene) was inserted into a plasmid encoding the wild-type S fragment under the control of the pol-I promoter (pol-I GP_IGR_NP), generating pol-I GP_IGR_GFPrest_IGR_NP, to clone the in vivo recombinant viral r3LCMV-GFP. nat The pol-I plasmid of the recombinant S fragment of #3.

[0321] 5.1.3 DNA transfection of cells and rescue of recombinant viruses With 4×10 5 BHK-21 cells were seeded into 6-well plates at a density of 1 cell / well. After 24 hours, cells were transfected with different amounts of DNA using lipofectamine (3 μl / μg DNA; Invitrogen) or jetPRIME (2 μl / μg DNA; Polyplus) according to the manufacturer's instructions. For complete rescue of recombinant two-fragment virus from plasmid DNA, the two minimal viral trans-acting factors NP and L were released from the pol-II driven plasmid (0.8 μg pC-NP, 1 μg pC-L) and co-transfected with 1.4 μg pol-I L and 0.8 μg pol-I S. For rescue of three-fragment r3LCMV consisting of one L and two L fragments, 0.8 μg of each pol-I driven S fragment were included in the transfection mixture. 72 hours post-transfection, the supernatant was harvested and passaged in BHK-21 cells for further viral amplification. Viral titers in the supernatant were determined by lesion formation analysis.

[0322] 5.1.4 Viruses and Viral Growth Dynamics Wild-type Cl13 LCMV, originally derived from Armstrong's wild-type LCMV, has been previously described. Stocks of wild-type and recombinant viruses were produced by infecting BHK-21 cells at a multiplicity of infection (MOI) of 0.01, with the supernatant harvested 48 hours post-infection. Viral growth curves were plotted in vitro in a 6-well format. BHK-21 cells were cultured at 6 × 10⁻⁶ wells. 5 Cells were seeded at a density of 100 cells / well. Twenty-four hours after infection, cells were incubated for 90 minutes at 37°C and 5% CO2 with 500 μl of viral inoculum at 0.01 mol. Fresh culture medium was added, and cells were incubated at 37°C / 5% CO2 for 72 to 96 hours. Supernatants were collected at given time points (typically 18, 24, 48, and 72 hours) to analyze viral titers using lesion formation analysis.

[0323] 5.1.5 Lesion Formation Analysis Next, the titer of LCMV was determined by lesion formation analysis. LCMV is a non-cytolytic virus that does not lyse its host cells and therefore does not produce plaques. Nevertheless, the units used in this work will be expressed using the more commonly used term Plaque Forming Unit (PFU) rather than the correct term Lesion Forming Unit (FFU). Unless otherwise stated, MC57 cells were used for lesion formation analysis. Cells were grown at a density of 1.6 × 10⁻⁶. 5Cells were seeded at a density of 10 cells per well in 24-well plates and mixed with 200 μl of a 10-fold serially diluted virus prepared in MEM / 2% FCS. After incubation at 37°C for 2–4 hours, 200 μl of viscous medium (2×2% methylcellulose in supplemented DMEM) was added to each well to ensure that virus particles spread only to neighboring cells. After 48 hours at 37°C, the supernatant was removed, and cells were fixed by adding 200 μl of 4% paraformaldehyde (PFA) in PBS at room temperature for 30 minutes (all subsequent steps were performed at room temperature). Cells were infiltrated with 200 μl of BSS / 1% Triton X-100 (Merck Millipore) per well for 20 minutes, followed by blocking with PBS / 5% FCS for 60 minutes. For anti-NP staining, rat anti-LCMV-NP monoclonal antibody was used as the primary staining antibody at a 1:30 dilution in PBS / 2.5% FCS for 60 minutes. For anti-GFP staining, purified rat anti-GFP antibody (Biolegend 338002) was used at a dilution of 1:2000 in PBS / 2.5% FCS. Plates were washed three times with tap water, and HRP-goat anti-rat IgG was added at a dilution of 1:100 in PBS / 2.5% FCS and incubated for 1 hour. Plates were washed three more times with tap water. The color reaction was added (0.5 g / L DAB (Sigma D-5637), 0.5 g / L nickel ammonium sulfate in PBS / 0.015% H2O2), and the reaction was stopped with tap water after 10 minutes. The stained lesions were manually counted, and the final titer was calculated based on the dilution.

[0324] For anti-GP staining of cells, plates were fixed with 50% MeOH / 50% acetone for 5 minutes and washed with PBS. Blocking was performed as described. The first antibody, anti-GP GP83.4 (derived from hybridoma), was diluted 1:10 in PBS / 2.5% FCS and incubated for 60 minutes. After three washes with tap water, the second HRP-rabbit anti-mouse IgG antibody was added at a 1:50 dilution in PBS / 2.5% FCS and incubated for 60 minutes. After three more washes with tap water, the color reaction was performed as described above.

[0325] To determine viremia in mice, one drop of blood (relative to 50 μl volume) was collected into 950 μl of PBS-heparin (Na-heparin, Braun, final 1 IE / ml), mixed by inversion, and stored at -80°C until use.

[0326] 5.1.6 Mice AGRAG mice (IFNα / βR- / -, IFNγR- / -, RAG- / -) have been previously described, bred and housed under specific pathogen-free (SPF) conditions. They were bred at the Labortierkunde Institute at the University of Zurich, Switzerland. All animal experiments were conducted at the Universities of Geneva and Basel in accordance with Swiss animal protection laws and with the permission of the respective cantonal governments of Geneva and Basel. The experiment was conducted at a rate of 1 × 10⁶ mice per mouse. 4 Mice were infected with PFU intravenously.

[0327] 5.1.7 Preparation and sequencing of viral RNA Viral RNA was extracted from cell culture supernatant or from the serum of infected mice using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was performed using a ThermoScript RT-PCR system (Invitrogen) and primers specific to LCMV NPs (SEQ ID NO: 15) according to the manufacturer's protocol. PCR amplification was performed using 2 μl of cDNA from the RT step, with NP- and GP-specific primers (SEQ ID NO: 16). PCR was performed using Phusion High-Fidelity DNA polymerase (NEB). The amplified products were analyzed on a 2% agarose gel, excised from the gel, purified using the QIAquick Gel Extraction Kit (Qiagen), and sent for DNA Sanger sequencing (Microsynth) using NP- and GP-specific primers.

[0328] 5.1.8 Flow Cytometer Blood was stained with antibodies targeting CD11c (N418), CD11b (M1 / 70), CD19 (6D5), NK1.1 (PK136), CD90.2 (30-H12), and GR-1 (RB6-8C5). The expression of surface molecules stained with specific antibodies, as well as GFP and RFP expression, was analyzed using FlowJo software (Tree Star, Ashland, OR) on a BDLSR Fortessa flow cytometer.

[0329] 5.1.9 Statistical Analysis Statistical significance was determined using Graphpad Prism software (version 6.0d) via a two-tailed unpaired t-test or a one-way ANOVA followed by Dunnett's or Bonferroni's post-test for multiple comparisons (version 6.0d). A p-value > 0.5 was considered insignificant, while a p-value < 0.5 was considered significant. ), p<0.01 ( ) and p<0.001 ( () is highly significant.

[0330] 5.2 Results 5.2.1 Compared with wild-type LCMV, the recombinant three-fragment virus grew to a lower titer. The genome of wild-type LCMV consists of two single-stranded RNA segments of negative polarity (one L segment and one S segment) (see appendix). Figure 1 A). Recent years have shown that it is possible to introduce other exogenous genes into the normal two-segment genome present in LCMV particles. The NP and GP genes are separated on two S-segment analogs, and the target gene is inserted into each generated S-segment of the LCMV, producing a replicating viral particle with three RNA segments (two S + one L). The only currently disclosed strategy is to keep the NP and GP in their native positions within the S-segments, thus placing GFP or other transgenes at the corresponding free sites (r3LCMV-GFP). nat (Appendix) Figure 1 B). This is an intuitive way to minimize the potential risk to the viability of the genome obtained by removing the genetic shuffling of the S segment. However, this study speculates that it should also be possible to juxtapose the GP with the 3' UTR, causing it to be expressed from the promoter element that normally drives the NP (r3LCMV-GFP). art Appendix Figure 1 C). The corresponding expression plasmids were generated through recombinant cDNA cloning, and all three viral constructs were completely rescued from the plasmid DNA. Growth curves were compared using the three viruses (see attached). Figure 1 (D) All three viruses showed peak titers at 48 hours post-infection, with the peak titers of the three-fragment viruses being 10-100 times lower than those of the wild-type virus. The wild-type LCMV reached 3.4 × 10⁻⁶. 6 PFU / ml, r3LCMV-GFP nat The peak value is 2.7 × 10⁻⁶. 4 PFU / ml, r3LCMV-GFP art 2.2×10 5PFU / ml. Regardless of their similarly decreased peak titers, r3LCMV-GFP nat It showed better performance than r3LCMV-GFP at earlier time points. art Slightly higher cell-free infectivity.

[0331] 5.2.2 Packaging of three-fragment viral particles is less efficient than that of two-fragment viruses. These observations indicate that adding a second S fragment impairs and delays viral growth. The hypothesis is that this reduction in viral fitness may be due to the inefficient packaging of all three RNA fragments into the viral particle, resulting in excessive double-fragment particles that cannot replicate productively when infecting fresh cells. For these experiments, r3LCMVs with two different reporter genes were used: GFP along with GP on one S fragment, and NP near RFP on the second S fragment. This produced a variant called r3LCMV-GFP / RFP. nat and r3LCMV-GFP / RFP art The two viruses differ only in the arrangement of GFP and GP on their respective S fragments. BHK-21 uses r3LCMV-GFP-RFP. nat Alternatively, lesion formation analysis can be performed on normal BHK-21 cells by infection with either dual-fragment r2LCMV or, in parallel, by using stably transfected BHK-21 cells expressing GP (BHK-GP) or NP (BHK-NP) as the cellular basis to trans-supplement the lack of the corresponding viral genome. Wild-type and GP-supplemented cells are stained for viral lesions expressing nucleoproteins, while NP-supplemented cells are stained for GP-positive lesions. Thus, immunofoci formation on wild-type BHK-21 cells is detected only by three-fragment viral particles. Without being bound by theory, BHK-GP cells should replicate three-fragment viral particles as well as a dual-fragment virus containing an L fragment and an S fragment expressing NP (but not an S fragment expressing GP). Conversely, BHK-NP cells should replicate three-fragment LCMV, as well as an additional NP-deficient viral particle consisting of an L fragment and an S fragment expressing GP (but not an S fragment expressing NP). Compared to testing infectivity on wt BHK-21 cells, r3LCMV-GFP / RFP showed significantly higher infectivity when evaluated on BHK-GP or BHK-NP cells. nat and r3LCMV-GFP / RFP art The infectiousness titer is consistently higher. Conversely, regardless of the cellular substrate used to assess its infectivity, the titer of r2LCMV is similar.To correct for potential inherent differences in LCMV infectivity for each cell line, each viral titer on BHK-21 cells was normalized to 1, with BHK-GP and BHK-NP titers expressed as folds thereof. This reflects cell-clonal-related titer differences in viral infectivity that are associated with potential inherent clonal differences. On any supplementary cell line, for r3LCMV-GFP / RFP... nat and r3LCMV-GFP / RFP art A titer difference of approximately five to ten times was observed, significantly higher than that observed with r2LCMV. This indicates that most viral particles formed from the two tri-fragmented viruses contain only one of the two S fragments, encoding either a fragment expressing only NP (NP-only particles) or a fragment expressing only GP (GP-only particles). A five-fold or greater difference in titer suggests that NP-only particles and GP-only particles each outnumbered tri-fragmented particles by approximately five times, with tri-fragmented particles constituting less than 10% of the viral particles. This is consistent with the delayed growth and reduced peak viral titer when grown on non-supplementary cells. These findings were further validated by flow cytometry. Non-supplementary BHK-21 cells or BHK-NP cells were cultured with r3LCMV-GFP / RFP. art Alternatively, r2LCMV infection could be used as a gate control, and the fluorescence intensity of GFP and RFP could be assessed by flow cytometry (see attached). Figure 2 B). Since wild-type BHK-21 cells do not provide minimal trans-acting factors, only viral particles containing at least the L fragment and the S fragment expressing NP can initiate the infection cycle and produce a fluorescent signal (RFP) after cell entry. Therefore, the population of RFP+GFP- cells observed during BHK-21 infection reflects particles containing only NP. RFP+GFP+ double-positive cells are evidence of true three-fragment particles. The observation of gated RFP-GFP+ cells, which have a higher RFP MFI than RFP-GFP- cells, suggests they represent an early stage of three-fragment particle infection, an interpretation supported by the continuity between this population and the RFP+GFP+ double-positive population. However, when three-fragment r3LCMV-GFP / RFP is grown on BHK-NP cells... art In this case, instead of this minimal trans-acting factor, we observed more than 10-fold higher numbers of RFP-GFP+ cells compared to infection with unsupplemented BHK-21 cells. Conversely, RFP+GFP- (evidence of NP particles only) and GFP+RFP+ double-positive cells (triple-fragment particles) were detected at comparable abundance (see appendix). Figure 2 C). These results confirmed the findings obtained from the lesion formation analysis at the single-cell level, thus confirming that the three-fragment viral product contained mostly two-fragment replication-defective particles. These findings support the r3LCMV-GFP / RFP...nat and r3LCMV-GFP / RFP art The reduced growth provides a possible explanation and offers insight into the apparently rather inefficient random packaging of the three-fragment virus.

[0332] 5.2.3 Cloning and rescue of recombinant viruses to track in vivo recombination Because the three-fragment virus exhibited impaired growth kinetics, as shown in the appendix Figure 1 The assumption is that there should be high selective pressure on the recombination of NP and GP genetic information by viruses on only one S segment. It is speculated that intrasegmental recombination in arenaviruses caused the phylogenetic evolution of the North American clade, thus suggesting a possible mechanism by which three-segment viruses can reconstruct functional two-segment genomes. Without being limited by theory, let's examine the genomic architecture of two three-segment viruses, assuming r3LCMV-GFP... nat Selection pressure may favor recombination events in the IGR region, bringing GP and NP to the same fragment while removing GFP. In r3LCMV-GFP art Within the population, the selection pressure should be equally high; however, if not impossible, the reorganization of the GP and its proximity to the 3'UTR should make it extremely difficult for this virus to combine its two S segments into a single functional segment (see appendix below). Figure 7 Given the considerations for identifying RNA recombination and to reliably distinguish it from potential cDNA contamination, we cloned the S fragment carrying GFP and a recombinant GP ORF, in which the terminal 255 nucleotides of the recombinant GP ORF were codon-optimized. The resulting GP had a different nucleotide sequence than the wild-type WE strain GP but the same translation product (WE / WET-GP, appended). Figure 3 A). However, this recombinant WE / WET GP ORF does not exist as an infectious two-segment virus, and the laboratory does not possess a cDNA construct associated with the NP. Therefore, any possible two-segment virus containing WE / WET on the same fragment as the NP is considered clear evidence of intersegment recombination, distinguishing such a virus from potentially contaminating cDNA or RNA in the corresponding analysis. To test whether the chimeric GP affects viral fitness, a recombinant three-segment virus (r3LCMV-WEWET / GFP) carrying a WE / WET fusion GP was used. nat The cell culture growth curve of ) and the three-fragment virus (r3LCMV-WE / GFP) carrying wild-type WE GP. nat ) for comparison (with appendix) Figure 3 B). The growth kinetics and peak titers of the two viruses are comparable (r3LCMV-WE / GFP). nat 1.7×106 PFU / ml, r3LCMV-WEWET / GFP nat 2.3×10 6 (PFU / ml). Therefore, the chimeric WEWET glycoprotein does not detectably affect viral growth.

[0333] To test whether possible recombination events could occur between the NP and GP genes containing the S fragment of IGR, a single nucleotide deletion was introduced in the intergenic region of the S fragment encoding NP, acting as a genetic tag. This nucleotide deletion was chosen because it is located in an extension that differs from most of the S fragment IGR, which is not conserved in sequence or length between strains. In the case of recombination events, this "tag" (labeled throughout the figures and text) becomes the tag. The intergenic regions should allow for the identification of the genetic origin of the S fragment IGR sequence. A schematic diagram of the location of the deleted cytosine (indicated by arrows) and the resulting S fragment with NPs is attached. Figure 3 As shown in C. To test whether the deletion introduced in the IGR affects viral growth, recombinant r3LCMV-GFP with or without this single nucleotide deletion was rescued. nat Growth curve experiments were performed on BHK-21 cells (moi=0.01). The three-fragment virus (r3LCMV-GFP) with wild-type IGR... nat () and a comparison of IGR with mutation (r3LCMV-GFP) nat IGR They grow at similar rates and reach indistinguishable peak titers (see appendix). Figure 3 (D). Therefore, the IGR tag on the S fragment carrying NP has no detectable effect on viral fitness, which validates its use in subsequent in vivo experiments.

[0334] 5.2.4 r3LCMV-GFP in mice nat Instead of r3LCMV-GFP art Persistent infection reached levels comparable to that of the two-fragment wt virus and caused loss of GFP expression. In evidence recombination of r3LCMV-GFP nat The aim was to investigate whether the three-fragment virus could recombine in vivo. For this purpose, AGRAG mice were treated with r3LCMV-GFP. nat r3LCMV-GFP artAlternatively, two-fragment r2LCMV infection could be used as a control. AGRAG mice exhibit targeted deletions in the genes encoding interferon-α / β receptors, interferon-γ receptors, and RAG1, resulting in an immunodeficient phenotype and the establishment of chronic viremia after infection with three-fragment LCMV. Blood samples were collected over time, and viral titers were assessed using lesion formation analysis (see attached). Figure 4 A). Within 5 days of infection, carriers of the two-fragment LCMV showed a level of 5 × 10 5 High titers of viremia within the range of PFU / ml blood, followed by 10 4 -10 5 Stable viremia within the PFU / ml range persisted for at least 50 days post-infection. Mice infected with trisegmented LCMV showed approximately 5 × 10⁻⁶ PFU / ml. 3 The viral load of PFU / ml blood remained consistent with diminished growth in cell culture until day 20 (as opposed to the attached...). Figure 1 (D comparison). From day 30 onwards, r3LCMV-GFP nat Carriers of this virus showed elevated viral load, which was observed in those infected with r3LCMV-GFP. art No difference of more than 10-fold in viremia was observed in the animals by day 50. To determine whether the major viral population still carried the GFP reporter gene and thus GFP expression in infected cells, r3LCMV-GFP samples collected on day 127 post-infection were used. nat and r3LCMV-GFP art Blood samples from carriers were analyzed for viral lesion formation, and nucleoprotein or reporter gene GFP staining was performed (see attached image). Figure 4 B). From r3LCMV-GFP art Staining of blood isolated from carriers produced a comparable number of lesions to detections of anti-NP and anti-GFP antibodies (both assessments independently showed 10). 3 Viral titers in the PFU / ml range, with at least 100-fold higher total (NP+)r3LCMV-GFP levels compared to GFP-expressing lesions. nat The lesions were obvious. At least 10 were detected based on anti-NP testing. 4 Viral titers of PFU / ml were measured, while two of the three mice failed to show any detectable GFP-positive infection, and one mouse had residual GFP-positive lesions in the range of 100 PFU / ml, corresponding to the detection limit of our analysis. GFP expression in infected cells was also assessed by fluorescence microscopy (data not shown). Analysis of r3LCMV-GFP... nat In the blood of carriers, GFP fluorescent lesions are practically undetectable, while those from r3LCMV-GFP are... artThe titer results obtained from manual counting of GFP-positive lesions in carriers matched those obtained from anti-NP lesion formation analysis. Reporter gene expression was further validated by flow cytometry analysis of PBMCs from infected mice at 120 days post-infection. We found that in r3LCMV-GFP... art More than 10% of CD11b+GR1-monocytes / macrophages in infected animals were GFP-positive, while those derived from r3LCMV-GFP... nat The blood showed only background levels of GFP, which was comparable to that of animals infected with non-fluorescent r2LCMV (see appendix). Figure 4 (CE). This finding further supports the hypothesis that tri-segment viruses with GP in their natural location lose reporter gene expression over time, while translocation of GP in an artificial 3'UTR side-by-side position prevents transgene loss.

[0335] 5.2.5 Three-segment viruses with GP in their natural position can recombine their two S segments to produce a single S segment with partial or complete IGR repeats located on either side of the transgenic sequence remnant. Appendix Figure 4 This shows the effect of infection with r3LCMV-GFP. nat Increased viremia and loss of reporter gene expression were observed in mice. Therefore, it was hypothesized that a recombination event could explain this experimental result. Intersegment recombination should combine GP and NP onto the same S fragment, avoiding the need for a second S fragment during the viral replication cycle. Such an event could explain the viremia at wild-type virus levels, while simultaneously causing loss of reporter gene expression. To test this possibility, viral RNA was isolated from the serum of infected mice. A pair of primers binding NP and GP sequences were used to selectively amplify only the inferred recombinant RNA molecule by RT-PCR, which carried NP and GP ORFs in the antisense direction on an RNA fragment. The resulting PCR fragments were analyzed by gel electrophoresis (see attached image). Figure 5 A). All r3LCMV-GFP nat The carrier's serum yielded RT-dependent PCR products, while r3LCMV-GFP... art No specific bands were observed in carriers and natural controls. Control PCR reactions were performed on RNA samples treated with simulated RT to exclude cDNA contamination as a source of PCR products. Three separate r3LCMV-GFP samples were observed. nat The carrier's sequencing results are in the appendix Figure 5The diagram in C is schematic. Three mice contained viral RNA fragments with different sequences but similar patterns: C-terminal portions of GP and NP in the antisense orientation were found on one RNA fragment. Between them, two intergenic regions—the intergenic region expressing NP and the original intergenic region expressing GP—were at least partially preserved, separated by fragments of one or both of the GFP reporter genes from the parental S fragment of the three-fragment virus. The orientation and length of the GFP fragment varied among the three RNA species recovered from individual mice, evidence of independent recombination events. To further support this view, precisely identical recombinant RNA sequences were recovered from two consecutive samples collected from the same mouse with an interval of no more than three weeks between sampling. Based on the obtained recombinant S fragment sequences, a molecular mechanism is proposed for the recombinant RNA fragments from the parental S fragment of the three-fragment virus. Figure 7 The diagram illustrates, and is described in the legend of the accompanying figure, how r3LCMV-GFP utilizes this mechanism. nat Recombining its two S segments resulted in transgene loss and phenotypic reversion to the wild-type virus. (Appendix) Figure 7 The diagram also illustrates, according to the proposed S-fragment recombination mechanism, r3LCMV-GFP art Why can't it be recombined and its NP and GP ORF combined into a functional S fragment?

[0336] 5.2.6 Recombinant r2LCMV with two IGRs in the S fragment is viable and grows to a titer similar to that of bifragment LCMV with only one IGR in the S fragment.

[0337] The sequencing data above showed a consistent pattern of viral genetic elements in the recombinant S fragment, among which the (at least partial) doubling of IGRs was particularly noteworthy and characteristic. However, arenaviruses with repetition of intergenic regions on an S fragment are unknown. However, double stem-loops are naturally present in the Old World arenavirus Mopeia. Therefore, we will present r3LCMV-GFP with two IGRs and residual GFP. nat The rearranged S fragment of carrier #3 was cloned into a pol-I driven S fragment expression plasmid, rescuing the corresponding virus. The growth kinetics of this virus (r2LCMV_2IGRs) on BHK-21 cells were compared with those of the three-fragment r3LCMV-GFP. nat Compared with dual-fragment r2LCMV (with appendix) Figure 6 The infectious cell-free titer of r2LCMV_2IGRs exceeded that of r3LCMV-GFP at an early time point. nat Yes, it achieved the same peak titer as r2LCMV (1.7 × 10⁻⁶). 7 PFU / ml vs. 1.6×107 PFU / ml). Importantly, r2LCMV_2IGRs grew to a higher size than its parental three-fragment r3LCMV-GFP. nat The significantly higher peak titer demonstrates that despite the doubling of IGR during this process, inter-fragment recombination still retains a selective advantage.

[0338] 5.2.7 Recombinant r3LCMV expressing ovalbumin (OVA) induces rapid, strong, and multifunctional OVA-specific CD8+ T cell responses.

[0339] To test r3LCMV art To demonstrate the practicality of vector delivery technology for vaccination purposes, we developed r3LCMV-OVA. art The vaccine vector has the properties of r3LCMV-GFP. art Similar genome architecture (with appendix) Figure 1 C), but later viruses possessed two ovalbumin (OVA) genes instead of the corresponding GFP genes. We used 10 4 PFU's r3LCMV-OVA art C57Bl / 6 mice were immunized via intramuscular injection (im), and T cell responses in the spleen were analyzed eight days later. To compare with widely used vector platforms, we used 10... 8 A second group of C57BL / 6 mice were immunized with an adenovirus 5-based vector (rAd5-OVA) that also expressed OVA and had a replication-defective E1 deletion. The frequency of OVA-specific CD8+ T cells recognizing the immunodeterminant OVA-derived SIINFEKL epitope was observed in r3LCMV-OVA. art The percentage of CD8+ T cells in the vaccine group was within the top 10%, which was significantly higher than in the rAd5-OVA group (see attached image). Figure 8 A). r3LCMV-OVA was measured by intracellular cytokine analysis. art The induced CD8+ T cell response was not only of a higher order of magnitude but also of higher function, showing that most SIINFEKL-reactive r3LCMV-OVA cells... art Induced CD8+ T cells respond to peptide stimulation by producing IFN-γ, along with a significant proportion of co-produced TNF-α and / or IL-2. This demonstrates the efficacy of r3LCMV-OVA. art The practicality of carrier technology for vaccine delivery.

[0340] 5.2.8 Three-segment LCMV induces multifunctional memory CD8+ T cells.

[0341] To answer the question of whether the r3LCMV vector induces functional CD8+ T cell memory, we used 10e5 PFU of r3LCMV-OVA art C57BL / 6 mice were immunized intravenously, and OVA-specific (SIINFEKL-specific) CD8+ T cell responses in the spleen were analyzed on day 25. Control mice were immunized via the same route with a recombinant E1-deleted adenovirus vector (rAd) expressing OVA containing 10e8 viral particles (vp). OVA-specific CD8+ T cells producing IFN-γ, TNF-α, and / or IL-2 upon SIINFEKL peptide stimulation were assessed using standard intracellular cytokine assays. The frequencies of cytokine-producing cells as indicated in the table below were measured. Figure 9 A) and absolute quantity (attached) Figure 9 B). Compared with rAd-OVA-immunized mice, r3LCMV-OVA art Immunized mice exhibited significantly higher frequencies and quantities of multifunctional IFN-γ / TNF-α and IFN-γ / TNF-α / IL-2 that co-generate OVA-specific CD8+ T cells.

[0342] 5.2.9 The LCMV encoding the antigen induced specific T cell responses against exogenous and autoantigens.

[0343] To study r3LCMV art To investigate whether vectors can be used to induce CD8+ T cell responses against tumor-expressed autoantigens, we used r3LCMV vectors expressing Her2-derived CD8+ T cell epitopes from rats (TYVPANASL), humans (TYLPTNASL), or mice (TYLPANASL). art BALB / c mice immunized with vector (with) Figure 10 Nine days later, we measured specific CD8+ T cells that produced IFN-γ, TNF-α, and / or IL-2 upon stimulation with the corresponding peptides in an intracellular cytokine analysis. (Appendix) Figure 10 The frequency of epitope-specific CD8+ T cells stimulated by the relevant peptide was shown as the percentage of CD8+ T cells producing the labeled cytokine combination. The frequency of CD8+ T cells producing cytokines upon restimulation with culture medium was not significant. The results demonstrate that r3LCMV... art The carrier has the ability to induce a considerable frequency of tumor autoantigen-responsive CD8+ T cell responses.

[0344] 5.2.10 in r3LCMV art Interferon-α is induced upon infection, but not when infected with recombinant adenovirus or vaccinia virus vectors.

[0345] Type I interferon possesses a variety of immunostimulatory and antitumor effects. Therefore, type I interferon induction can represent a favorable characteristic of viral vector vaccines. We performed ELISA measurements to determine the efficacy of r3LCMV-OVA administered 24, 48, or 72 hours prior. art The concentration of interferon-α in the serum of mice immunized with rAd-OVA or recombinant vaccinia virus expressing OVA (rVacc) (see attached image). Figure 11 r3LCMV art Induces a detectable and sustained (at least 48 hours) systemic interferon-α response, which is not induced by rAd or rVacc. This demonstrates the efficacy of r3LCMV. art The ability of the vector to induce a strong innate immune response.

[0346] 5.2.11 with r3JUNV-GFP nat r3JUNV-GFP compared to the parental Junin strain Candid#1 art cell culture growth With carrying Figure 1 r3LCMV-GFP genome listed in B nat and r3LCMV-GFP art Similar to the vector, we engineered r3JUNV-GFP. nat and r3JUNV-GFP art It consists of three fragments of a vector based on the Junin vaccine strain Candid#1, with the GFP gene (r3JUNV-GFP) carried in each of their two corresponding S fragments. nat and r3JUNV-GFP art We tested their growth properties in 293T cells, infecting the cells with a multiplicity of infection of 0.01, and collecting the supernatant over time (see attached). Figure 12 We discovered r3JUNV-GFP. art It grows slower than its parental two-fragment Junin vaccine strain, Candid#1 (see appendix). Figure 12 However, it is better than r3JUNV-GFP. nat Faster growth (with appendix) Figure 12 The distinct growth behaviors of the three fragments of the Junin virus-based vector compared to r3LCMV-GFP nat and r3LCMV-GFP art The carriers are similar (see appendix) Figure 1 D).

[0347] 5.2.12 The three-segment JUNV is significantly attenuated in vivo, r3JUNV-GFP nat Instead of r3JUNV-GFPart GFP expression is lost during prolonged in vivo replication.

[0348] To investigate r3JUNV-GFP nat and r3JUNV-GFP art To assess genetic stability, we infected AGRAG mice with any of these GFP expression vectors at 7 × 10e4 PFU (IFNα / βR- / -, IFNγR- / -, RAG- / -). For comparison, a third group was infected with wild-type dual-fragment Candid#1 virus. The latter virus was readily detectable in the blood of all infected mice on day 20 post-infection (see appendix). Figure 13 A), while the three-fragmented virus remained undetectable for at least 40 days. This finding demonstrates the weakened in vivo growth as a result of genome remodeling, which will allow us to... Figure 4 The discovery in A using the r3LCMV-GFP vector was extended to Junin-based vectors. After 40 days, r3JUNV-GFP was effective in several animals in each group. nat and r3JUNV-GFP art It also becomes detectable (attached) Figure 13 A). Importantly, however, some r3JUNV-GFP nat Infected mice achieved viral loads in the range of wild-type Candid#1-infected mice, while viremia was observed in r3JUNV-GFP. art The infected mice maintained a lower viral load than the Candid#1-infected controls.

[0349] To determine whether the major viral population in these viremic animals still carries the GFP reporter gene, thereby inducing GFP expression in infected cells, we used r3JUNV-GFP samples collected 120 days post-infection. nat and r3JUNV-GFP art Blood samples from carriers were analyzed for viral lesion formation. We compared the infectious titers of viruses maintaining GFP expression (anti-GFP, anti ... Figure 13 B), and total Junin virus infectivity (anti-NP, appendix) Figure 13 B). r3JUNV-GFP art The titers were within a similar range as determined by anti-GFP or anti-NP immunofoci analysis, demonstrating that most of the viral population maintained GFP expression. Conversely, the four r3JUNV-GFP titers with the highest viremia showed significantly lower titers. nat In the blood of infected animals, the anti-GFP infectious titer (compared to wild-type Candid #1) was at least 10-fold lower than the total infectious titer determined by NP staining. This demonstrates the effectiveness of r3JUNV-GFP. artThe GFP transgene was stably maintained in vivo, while r3JUNV-GFP nat No.

[0350] 5.2.13 Homologous and heterologous primary-enhancing combinations of vaccine vectors based on three fragments of LCMV and JUNV induced strong P1A autoantigen-specific CD8+ T cell responses.

[0351] Next, we investigated r3LCMV-based... art and r3JUNV art Can this vector be used in homologous and heterologous primary-enhancing combinations to induce tumor autoantigen-specific CD8+ T cell responses? We constructed an r3LCMV-based vector... art and r3JUNV art The vectors express the autoantigen P1A (SEQ ID NO: 24) derived from P815 mouse mast cell tumor (r3LCMV-P1A). art (SEQ ID NO: 18, 19, 20) and r3JUNV-P1A art (SEQ ID NO: 21, 22, 23)). These vaccine constructs are attached. Figure 14 The above lists the methods for iv immunizing BALB / c mice in both homologous and heterologous primiparous-boost combinations. When administered in homologous primiparous-boost vaccination, r3LCMV-P1A... art and r3JUNV-P1A art Both induced P1A epitope-specific CD8+ T cells, and were produced using H-2L cells loaded with LPYLGWLVF peptide. d -Tetramer was measured from blood. On day 63 of the experiment, the mean frequency of epitope-specific CD8+ T cells was 1.2% (r3JUNV-P1A). art ) and 3.9% (r3LCMV-P1A) art Additionally, r3JUNV-P1A is used in a heterogeneous manner. art Initial vaccination with r3LCMV-P1A art The enhanced animals produced even higher responses, with a mean epitope-specific CD8+ T cell frequency of 19.5% on day 63. r3LCMV-P1A art Initial vaccination and r3JUNV-P1A art The frequency of enhanced animals (3.1%) was higher than that of animals that experienced r3LCMV-P1A. art The animals that received the primary and booster immunizations were quite similar.

[0352] 6. Equivalent The viruses, nucleic acids, methods, host cells, and compositions disclosed herein are not limited in scope to the specific embodiments described herein. In fact, various modifications to the viruses, nucleic acids, methods, host cells, and compositions, in addition to those described, will be apparent to those skilled in the art based on the foregoing specification and figures. Such modifications will also fall within the scope of the appended claims.

[0353] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by way of full reference.

[0354] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by way of full reference.

[0355] 7. Sequence List

[0356] This application also involves the following items.

[0357] 1. A sand-like virus genome fragment, wherein the genome fragment is engineered to carry a viral ORF at a location other than the wild-type location of the open reading frame (“ORF”), wherein the sand-like virus genome fragment is selected from: (i)S fragment, in which the ORF encoding the nucleoprotein (“NP”) is under the control of the 5' untranslated region (“UTR”) of the arena virus; (ii) S fragment, wherein the ORF encoding matrix protein Z (“Z protein”) is under the control of the 5' UTR of the sand virus; (iii) S fragment, in which the ORF encoding RNA-dependent RNA polymerase L (“L protein”) is under the control of the 5'UTR of the sand virus; (iv) S fragment, in which the ORF encoding the viral glycoprotein (“GP”) is under the control of the arenavirus 3' UTR; (v)S fragment, in which the ORF encoding the L protein is under the control of the arena virus 3'UTR; (vi) S fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3'UTR; (vii) L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (viii) L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR; (ix) L fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (x)L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR; (xi) L fragment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and (xii) L fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR.

[0358] 2. The isopyrvirus genome fragment of Project 1, wherein the isopyrvirus 3' UTR is the 3' UTR of the isopyrvirus S fragment or the isopyrvirus L fragment, and wherein the isopyrvirus 5' UTR is the 5' UTR of the isopyrvirus S fragment or the isopyrvirus L fragment.

[0359] 3. cDNA of the arenavirus genome fragment from Project 1.

[0360] 4. DNA expression vectors containing cDNA from item 3.

[0361] 5. Host cells containing the arenavirus genome fragment of Project 1, the cDNA of Project 3, or the vector of Project 4.

[0362] 6. A sand-borne virus particle comprising a sand-borne virus genome fragment of Item 1 and a second sand-borne virus genome fragment, wherein the sand-borne virus particle comprises an S fragment and an L fragment.

[0363] 7. The sand-like virus particles of Item 6, wherein the sand-like virus particles are infectious and replicating.

[0364] 8. Sand-like virus particles from Project 6, wherein the sand-like virus particles are attenuated.

[0365] 9. The sand-like virus particles of Project 6, wherein the sand-like virus particles are infectious but cannot produce further infectious progeny in non-supplementary cells.

[0366] 10. Sand-like viral particles from Project 9, wherein at least one of the four ORFs encoding the GP, NP, Z, and L proteins is removed or functionally inactivated.

[0367] 11. The sand virus particles of Project 9, wherein at least one of the four ORFs encoding the GP, NP, Z and L proteins is removed or replaced with a heterologous ORF from an organism other than sand virus.

[0368] 12. In the sand virus particles of Project 9, only one of the four ORFs encoding the GP, NP, Z, and L proteins was removed or replaced with a heterologous ORF from an organism other than sand virus.

[0369] 13. Sand virus particles from Project 9, in which the ORF encoding GP was removed or replaced with a heterologous ORF from an organism other than sand virus.

[0370] 14. Sand virus particles from Project 9, wherein the ORF encoding NP was removed or replaced with a heterologous ORF from an organism other than sand virus.

[0371] 15. Sand virus particles from Project 9, wherein the ORF encoding the Z protein was removed or replaced with a heterologous ORF from an organism other than sand virus.

[0372] 16. Sand virus particles from Project 9, wherein the ORF encoding the L protein was removed or replaced with a heterologous ORF from an organism other than sand virus.

[0373] 17. A sand-like virus particle of any of items 11 to 16, wherein the heterologous ORF encodes a reporter protein.

[0374] 18. Sand-like virus particles of any of items 11 to 17, wherein the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen.

[0375] 19. The sand-like virus particles of Item 18, wherein the heterologous ORF encoding is selected from antigens of human immunodeficiency virus antigen, hepatitis C virus antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen.

[0376] 20. The sand-like virus particle of any one of items 11 to 18, wherein the growth or infectivity of the sand-like virus particle is not affected by a heterologous ORF from an organism other than the sand-like virus.

[0377] 21. A method for producing a sand virus genome fragment of Project 1, wherein the method comprises transcribing cDNA of Project 3.

[0378] 22. A method for generating sand-like virus particles of item 6, wherein the method comprises: (i) Transfecting the cDNA of item 3 into the host cell; (ii) Transfecting the host cell with a plasmid containing a fragment of the second sand virus genome; (iii) Maintaining the host cell under conditions suitable for virus formation; and (iv) Harvest the sand-like virus particles.

[0379] 23. The method of Project 22, in which transcription of the L and S fragments is performed using a bidirectional promoter.

[0380] 24. The method of Item 22, wherein the method further comprises transfecting a host cell with one or more nucleic acids encoding a sand virus polymerase.

[0381] 25. The method of Item 22, wherein the sand virus polymerase is the L protein.

[0382] 26. The method of item 22 or 24, wherein the method further comprises transfecting one or more nucleic acids encoding NP proteins into the host cell.

[0383] 27. The method of Item 22, wherein transcription of the L and S fragments is each controlled by a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0384] 28. A vaccine comprising sand-like virus particles of items 10 or 11 to 16 and a pharmaceutically acceptable vector.

[0385] 29. A pharmaceutical composition comprising sand virus particles of items 10 or 11 to 16 and a pharmaceutically acceptable carrier.

[0386] 30. The isovirus genome fragment of Project 1 or the isovirus particle of Project 6, wherein the isovirus genome fragment or isovirus particle is derived from lymphocytic choriomeningitis virus (“LCMV”).

[0387] 31. A sand virus genome fragment or sand virus particle of Item 30, wherein the LCMV is an MP strain, an Armstrong strain, or an Armstrong clone 13 strain.

[0388] 32. A sand virus genome fragment from Project 1 or a sand virus particle from Project 6, wherein the sand virus genome fragment or sand virus particle is derived from Junin virus vaccine Candid #1 or Junin virus vaccine XJ clone 3 strain.

[0389] 33. A three-segment sand-like virus particle comprising one L segment and two S segments, wherein in the presence of 10 4 In mice lacking type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1) and infected with the three-fragmented isavirus particles of PFU, the proliferation of the three-fragmented isavirus particles did not produce replicating two-fragmented virus particles after 70 days of continuous infection.

[0390] 34. The three-segment sand-like virus particle of Project 33, wherein intrafragmental recombination of the two S segments binds the two sand-like virus ORFs to only one segment instead of two separate segments, thereby deactivating the viral promoter.

[0391] 35. A three-segment sand-like virus particle comprising two L segments and one S segment, wherein in the presence of 10 4 In mice lacking type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1) and infected with the three-fragmented isavirus particles of PFU, the proliferation of the three-fragmented isavirus particles did not produce replicating two-fragmented virus particles after 70 days of continuous infection.

[0392] 36. The three-segment sand-like virus particles of Project 35, in which intra-segment recombination of the two L segments binds the two sand-like virus ORFs to only one segment instead of two separate segments, thus canceling the viral promoter activity.

[0393] 37. The three-segment sand-like virus particle of Item 33, wherein one of the two S segments is selected from: (i)S fragment, in which the ORF encoding NP is under the control of the sand grain virus 5'UTR; (ii) S fragment, in which the ORF encoding the Z protein is under the control of the 5' UTR of the sand virus; (iii) S fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) S fragment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; (v) S segment, in which the ORF encoding L is under the control of the sand virus 3' UTR; and (vi) S fragment, in which the ORF encoding the Z protein is under the control of the 3' UTR of the sand virus.

[0394] 38. The three-segment sand-like virus particle of Item 35, wherein one of the two L segments is selected from: (i)L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (ii) L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR; (iii) L fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR; (v) L segment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and (vi) L fragment, in which the ORF encoding the Z protein is under the control of the sand virus 3' UTR.

[0395] 39. The three-segment sand-like virus particle of item 37 or 38, wherein the sand-like virus 3' UTR is the 3' UTR of the sand-like virus S segment or the sand-like virus L segment, and wherein the sand-like virus 5' UTR is the 5' UTR of the sand-like virus S segment or the sand-like virus L segment.

[0396] 40. The three-segment sand virus particle of Item 33, wherein the two S segments comprise (i) one or two heterologous ORFs from an organism other than sand virus; or (ii) one or two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than sand virus.

[0397] 41. The three-segment sand virus particle of Item 35, wherein the two L segments comprise (i) one or two heterologous ORFs from an organism other than sand virus; or (ii) two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than sand virus.

[0398] 42. The three-segment sand-like virus particle of item 40 or 41, wherein the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen.

[0399] 43. The three-segment sand-like virus particle of Item 42, wherein the heterologous ORF encoding is selected from antigens of human immunodeficiency virus antigen, hepatitis C virus antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen.

[0400] 44. A three-segment sand-like virus particle from item 40 or 41, wherein at least one heterologous ORF encodes a fluorescent protein.

[0401] 45. The three-segment sand-like virus particle of Item 44, wherein the fluorescent protein is green fluorescent protein or red fluorescent protein.

[0402] 46. ​​A three-segment sand-like virus particle of any of items 33 to 43, wherein the three-segment sand-like virus particle contains all four sand-like virus ORFs, and wherein the three-segment sand-like virus particle is infectious and replicating.

[0403] 47. A three-segmented sand-like virus particle of any of items 33 to 45, wherein the three-segmented sand-like virus particle is missing one or more of the four sand-like virus ORFs, wherein the three-segmented sand-like virus particle is infectious but cannot produce further infectious progeny in non-supplementary cells.

[0404] 48. A three-segmented sand virus particle of any of items 33 to 45, wherein the three-segmented sand virus particle is missing one of the four sand virus ORFs, wherein the three-segmented sand virus particle is infectious but cannot produce further infectious progeny in non-supplementary cells.

[0405] 49. The three-segment sand-like virus particles of item 46 or 47, wherein the sand-like virus lacks GP ORF.

[0406] 50. A three-segment isovirus particle comprising an L segment and two S segments, wherein the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 3' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 5' UTR, and the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 3' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 5' UTR.

[0407] 51. A three-segment isovirus particle comprising an L segment and two S segments, wherein the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 5' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 3' UTR, and the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 5' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 3' UTR.

[0408] 52. A three-segment sand-like virus particle of item 50 or 51, wherein the target gene encodes an antigen derived from an infectious organism, tumor, or allergen.

[0409] 53. The three-segment sand-like virus particle of Project 52, wherein the target gene encodes an antigen selected from human immunodeficiency virus antigen, hepatitis C virus antigen, herpes zoster virus antigen, cytomegalovirus antigen, mycobacterium tuberculosis antigen, and tumor-associated antigen.

[0410] 54. Three-segment sand-like virus particles from item 50 or 51, wherein at least one target gene encodes a fluorescent protein.

[0411] 55. The three-segment sand-like virus particle of Item 54, wherein the fluorescent protein is green fluorescent protein or red fluorescent protein.

[0412] 56. cDNA of a three-segment sand virus particle genome from any of items 33, 35, 37, 38, 50, or 51.

[0413] 57. DNA expression vectors containing cDNA from item 56.

[0414] 58. A host cell comprising a three-segment sand-like virus particle of item 33 or 35, cDNA of item 56, or a vector of item 57.

[0415] 59. A three-segmented sand-like virus particle of any of items 33 to 51, wherein the three-segmented sand-like virus particle is attenuated.

[0416] 60. A method for generating the three-segment sand-like virus particles of item 33, wherein the method comprises: (i) Transfecting one or more cDNAs of the L fragment and two S fragments into a host cell; (ii) Maintaining the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

[0417] 61. A method for generating the three-fragment sand-like virus particles of item 35, wherein the method comprises: (i) Transfecting a host cell with one or more cDNAs, consisting of two L fragments and one S fragment; (ii) Maintaining the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

[0418] 62. The method of Project 60, in which transcription of one L fragment and two S fragments is performed using a bidirectional promoter.

[0419] 63. The method of Project 61, in which transcription of two L fragments and one S fragment is performed using a bidirectional promoter.

[0420] 64. The method of item 60 or 61, wherein the method further comprises transfecting the host cell with one or more nucleic acids encoding a sand virus polymerase.

[0421] 65. The method of item 62, wherein the sand virus polymerase is the L protein.

[0422] 66. The method of Items 60, 61, 62 or 63, wherein the method further comprises transfecting one or more nucleic acids encoding an NP protein into the host cell.

[0423] 67. The method of Project 60, wherein the transcription of the L fragment and the two S fragments is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0424] 68. The method of item 61, wherein the transcription of the two L fragments and the S fragment is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

[0425] 69. A three-segmented sand-like virus particle of any of items 33 to 51, wherein the three-segmented sand-like virus particle has the same tropism as a two-segmented sand-like virus particle.

[0426] 70. A three-segment sand-particle virus particle of any of items 33 to 51, wherein the three-segment sand-particle virus particle is replication-defective.

[0427] 71. A vaccine comprising a three-segmented sand virus particle of any one of items 33 to 51, 69 or 70 and a pharmaceutically acceptable vector.

[0428] 72. A pharmaceutical composition comprising a three-segment sand virus particle of any one of items 33 to 51, 69 or 70 and a pharmaceutically acceptable carrier.

[0429] 73. A three-segmented sand-like virus particle of any one of items 33 to 51, 69 or 70, wherein the three-segmented sand-like virus particle is derived from LCMV.

[0430] 74. The three-segment sand virus particle of Item 73, wherein the LCMV is the MP strain, the Armstrong strain, or the Armstrong clone 13 strain.

[0431] 75. A three-segmented sand-like virus particle of any one of items 33 to 51, 69 or 70, wherein the three-segmented sand-like virus particle is derived from the Junin virus vaccine Candid #1 or the Junin virus vaccine XJ clone 3 strain.

Claims

1. An infectious and replicating three-segment isopyrovirus particle comprising an L segment and two S segments, wherein one L segment and both S segments contain open reading frames ("ORFs") encoding isopyrovirus nucleoprotein ("NP"), isopyrovirus matrix protein Z ("Z protein"), isopyrovirus RNA polymerase L ("L protein"), and isopyrovirus glycoprotein ("GP"), and wherein each of the three segments contains at least one of the isopyrovirus ORFs encoding NP, Z protein, L protein, and GP, and wherein one of said two S segments is selected from: (i) S fragment, in which the ORF encoding NP is under the control of the sand virus 5' UTR; (ii) S fragment, in which the ORF encoding the Z protein is under the control of the 5' UTR of the sand virus; (iii) S fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) S fragment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; (v) S segment, in which the ORF encoding L is under the control of the sand virus 3' UTR; and (vi) S fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR; The intrafragmental recombination of the two S fragments binds the two sand-like virus ORFs to only one fragment, rather than two separate fragments, thereby deactivating the virus promoter.

2. An infectious and replicating three-segment isopyrovirus particle comprising two L segments and one S segment, wherein the two L segments and one S segment contain open reading frames ("ORFs") encoding isopyrovirus nucleoprotein ("NP"), isopyrovirus matrix protein Z ("Z protein"), isopyrovirus RNA polymerase L ("L protein"), and isopyrovirus glycoprotein ("GP"), and wherein each of the three segments contains at least one of the isopyrovirus ORFs encoding NP, Z protein, L protein, and GP, and wherein one of said two L segments is selected from: (i) L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (ii) L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR; (iii) L fragment, in which the ORF encoding the L protein is under the control of the 5' UTR of the isopyrvirus; (iv) L segment, in which the ORF encoding GP is under the control of the sand virus 3' UTR; (v) L segment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and (vi) L fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR; The intrafragmental recombination of the two L fragments binds the two sand virus ORFs to only one fragment, rather than two separate fragments, thereby deactivating the viral promoter.

3. The three-segment sand-like virus particle as described in any one of claims 1-2, wherein 10 4 In mice lacking type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1) and infected with the three-fragmented isavirus particles of PFU, the proliferation of the three-fragmented isavirus particles did not produce replicating two-fragmented virus particles after 70 days of continuous infection.

4. The three-segment sand virus particle as described in any one of claims 1-2, wherein the sand virus 3' UTR is the 3' UTR of the sand virus S segment or the sand virus L segment, and wherein the sand virus 5' UTR is the 5' UTR of the sand virus S segment or the sand virus L segment.

5. The three-segment sand virus particle of claim 1, wherein the two S segments comprise (i) one or two heterologous ORFs from an organism other than sand virus; or (ii) one or two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than sand virus.

6. The three-segment sand virus particle of claim 2, wherein the two L segments comprise (i) one or two heterologous ORFs from an organism other than sand virus; or (ii) one or two duplicated sand virus ORFs; or (iii) one heterologous ORF and one duplicated sand virus ORF from an organism other than sand virus.

7. The three-segment sand-like virus particle as described in claim 5 or 6, wherein the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen.

8. The three-segment sand-like virus particle as described in claim 7, wherein the heterologous ORF encoding is selected from antigens of human immunodeficiency virus, hepatitis C virus, herpes zoster virus, cytomegalovirus, Mycobacterium tuberculosis, tumor-associated antigens, and human papillomavirus.

9. The three-segment sand-like virus particle as described in claim 5 or 6, wherein at least one heterologous ORF encodes a fluorescent protein.

10. The three-segment sand-like virus particle of claim 9, wherein the fluorescent protein is a green fluorescent protein or a red fluorescent protein.

11. The three-fragmented sand-like virus particle as claimed in any one of claims 1, 2, 5 or 6, wherein the three-fragmented sand-like virus particle comprises all four sand-like virus ORFs.

12. The cDNA of the three-segment sand virus particle genome as described in any one of claims 1 to 11.

13. A DNA expression vector comprising the cDNA of claim 12.

14. A host cell comprising a three-segment sand virus particle of any one of claims 1-11, cDNA of claim 12, or a vector of claim 13.

15. The three-fragmented sand-like virus particle as claimed in any one of claims 1 to 2, wherein the three-fragmented sand-like virus particle is attenuated.

16. A method for generating the three-fragmented sand-like virus particle of claim 1, wherein the method comprises: (i) Transfected into the host cell nucleotide sequence, from which the one L fragment and the two S fragments can be transcribed; (ii) Maintain the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

17. A method for generating the three-fragmented sand-like virus particle of claim 2, wherein the method comprises: (i) A nucleotide sequence transfected into a host cell, from which the two L fragments and the one S fragment can be transcribed; (ii) Maintain the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

18. The method of claim 16, wherein the transcription of the one L fragment and the two S fragments is performed using a bidirectional promoter.

19. The method of claim 17, wherein the transcription of the two L fragments and the one S fragment is performed using a bidirectional promoter.

20. The method of claim 16 or 17, wherein the method further comprises transfecting the host cell with one or more nucleic acids encoding a sand virus polymerase.

21. The method of claim 20, wherein the sand virus polymerase is an L protein.

22. The method of claim 16, 17, 18 or 19, wherein the method further comprises transfecting one or more nucleic acids encoding NP proteins into the host cell.

23. The method of claim 16, wherein the transcription of the one L fragment and the two S fragments is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

24. The method of claim 17, wherein the transcription of the two L fragments and the one S fragment is each under the control of a promoter selected from the group consisting of: (i) RNA polymerase I promoter; (ii) RNA polymerase II promoter; and (iii) T7 promoter.

25. The three-segment sand-like virus particle as claimed in claim 1 or 2, wherein the three-segment sand-like virus particle has the same directional properties as the two-segment sand-like virus particle.

26. A vaccine comprising a three-segmented sand virus particle as described in any one of claims 1 to 11 and 25 and a pharmaceutically acceptable vector.

27. A pharmaceutical composition comprising a three-segmented sand virus particle as described in any one of claims 1 to 11 and 25 and a pharmaceutically acceptable carrier.

28. The three-fragmented sand-like virus particle as described in claim 1 or 2, wherein the three-fragmented sand-like virus particle is derived from LCMV.

29. The three-segment sand-like virus particle of claim 28, wherein the LCMV is an MP strain, an Armstrong strain, or an Armstrong clone 13 strain.

30. The three-fragmented sand-like virus particle as claimed in claim 1 or 2, wherein the three-fragmented sand-like virus particle is derived from the Junin virus vaccine Candid #1 or the Junin virus vaccine XJ clone 3 strain.

31. A sand-like virus genome fragment, wherein the genome fragment is engineered to carry a viral ORF at a location outside the wild-type position of the open reading frame ("ORF"), wherein the sand-like virus genome fragment is selected from: (i) S fragment, in which the ORF encoding the nucleoprotein ("NP") is under the control of the 5' untranslated region ("UTR") of the arena virus; (ii) S fragment, in which the ORF encoding matrix protein Z ("Z protein") is under the control of the 5' UTR of the sand virus; (iii) S fragment, in which the ORF encoding RNA-dependent RNA polymerase L ("L protein") is under the control of the sand virus 5' UTR; (iv)S fragment, in which the ORF encoding the viral glycoprotein ("GP") is under the control of the arenavirus 3' UTR; (v)S fragment, in which the ORF encoding the L protein is under the control of the arena virus 3'UTR; (vi)S fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3'UTR; (vii)L segment, in which the ORF encoding GP is under the control of the sand virus 5' UTR; (viii)L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR; (ix)L fragment, in which the ORF encoding the L protein is under the control of the arena virus 5' UTR; (x)L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR; The (xi)L fragment, in which the ORF encoding NP is under the control of the sand virus 3' UTR; and The (xii)L fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3' UTR.

32. The cDNA of the isovirus genome fragment of claim 31.

33. A DNA expression vector comprising the cDNA of claim 32.

34. A host cell comprising the isovirus genome fragment of claim 31, the cDNA of claim 32, or the vector of claim 33.

35. A sand virus particle comprising the sand virus genome fragment of claim 31 and a second sand virus genome fragment, wherein the sand virus particle comprises an S fragment and an L fragment.

36. A method for producing the arenavirus genome fragment of claim 31, wherein the method comprises transcribing the cDNA of claim 32.

37. A method for generating the sand-like virus particles of claim 35, wherein the method comprises: (i) Transfecting the cDNA of claim 32 into a host cell; (ii) Transfecting the host cell with a plasmid containing a fragment of the second sand virus genome; (iii) Maintaining the host cell under conditions suitable for virus formation; and (iv) Harvest the sand-like virus particles.

38. A vaccine comprising the sand virus particles of claim 35 and a pharmaceutically acceptable vector.

39. A pharmaceutical composition comprising the sand virus particles of claim 35 and a pharmaceutically acceptable carrier.

40. A three-segment sand-like virus particle comprising one L segment and two S segments, wherein in the presence of 10 4 In mice lacking type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1) and infected with the three-fragmented isavirus particles of PFU, the proliferation of the three-fragmented isavirus particles did not produce replicating two-fragmented virus particles after 70 days of continuous infection.

41. A three-segment sand-like virus particle comprising two L segments and one S segment, wherein in the presence of 10 4 In mice lacking type I interferon receptor, type II interferon receptor, and recombinant activated gene 1 (RAG1) and infected with the three-fragmented isavirus particles of PFU, the proliferation of the three-fragmented isavirus particles did not produce replicating two-fragmented virus particles after 70 days of continuous infection.

42. A three-segment isovirus particle comprising an L segment and two S segments, wherein the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 3' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 5' UTR, and the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 3' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 5' UTR.

43. A three-segment isovirus particle comprising an L segment and two S segments, wherein the first S segment is engineered to carry an ORF encoding a GP at a position controlled by the isovirus 5' UTR and an ORF encoding a first target gene at a position controlled by the isovirus 3' UTR, and the second S segment is engineered to carry an ORF encoding an NP at a position controlled by the isovirus 5' UTR and an ORF encoding a second target gene at a position controlled by the isovirus 3' UTR.

44. The cDNA of a three-segment sand virus particle genome of any one of claims 40, 41, 42 or 43.

45. A DNA expression vector comprising the cDNA of claim 44.

46. ​​A host cell comprising the three-segment sand virus particle of claim 40 or 41, the cDNA of claim 44, or the vector of claim 45.

47. A method for generating the three-fragmented sand-like virus particle of claim 40, wherein the method comprises: (i) Transfecting one or more cDNAs of the L fragment and two S fragments into a host cell; (ii) Maintain the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

48. A method for generating the three-fragmented sand-particle virus particle of claim 41, wherein the method comprises: (i) Transfecting a host cell with one or more cDNAs, consisting of two L fragments and one S fragment; (ii) Maintain the host cell under conditions suitable for virus formation; and (iii) Harvest the sand-like virus particles.

49. A vaccine comprising a three-segmented sand virus particle of any one of claims 40 to 43 and a pharmaceutically acceptable vector.

50. A pharmaceutical composition comprising a three-segment sand virus particle of any one of claims 40 to 43 and a pharmaceutically acceptable carrier.

Images