Tri-segmented arenaviruses as vaccine vectors

Abstract

The present application relates to arenaviruses with rearrangements of their open reading frames (“ORF”) in their genomes. In particular, described herein is a modified arenavirus genomic segment, wherein the arenavirus genomic segment is engineered to carry a viral ORF in a position other than the wild-type position of the ORF. Also described herein are trisegmented arenavirus particles comprising one L segment and two S segments or two L segments and one S segment. The arenavirus, described herein may be suitable for vaccines and/or treatment of diseases and/or for the use in immunotherapies.

1 - 21 . (canceled) 
     
     
         22 . A method of generating an arenavirus particle, wherein the method comprises:
 transfecting into a host cell a complementary deoxyribonucleic acid (cDNA) of a first arenavirus genomic segment;   (ii) transfecting into the host cell a cDNA of a second arenavirus genomic segment;   (iii) maintaining the host cell under conditions suitable for virus formation; and   (iv) harvesting the arenavirus particle,   
       wherein the first arenavirus genomic segment is engineered to carry a viral open reading frame (“ORF”) in a position other than the wild-type position of the ORF and is selected from the group consisting of:
 (i) an S segment, wherein an ORF encoding a nucleoprotein (“NP”) is under control of an arenavirus 5′ untranslated region (“UTR”); 
 (ii) an S segment, wherein an ORF encoding a matrix protein Z (“Z protein”) is under control of an arenavirus 5′ UTR; 
 (iii) an S segment, wherein an ORF encoding an RNA dependent RNA polymerase L (“L protein”) is under control of an arenavirus 5′ UTR; 
 (iv) an S segment, wherein an ORF encoding a viral glycoprotein (“GP”) is under control of an arenavirus 3′ UTR; 
 (v) an S segment, wherein an ORF encoding the L protein is under control of an arenavirus 3′ UTR; and 
 (vi) an S segment, wherein an ORF encoding the Z protein is under control of an arenavirus 3′ UTR. 
 
     
     
         23 . The method of  claim 22 , wherein the method further comprises transcribing the cDNA of the first arenavirus genomic segment and the second arenavirus genomic segment using a promoter. 
     
     
         24 . The method of  claim 22 , wherein the method further comprises transcribing the cDNA of the first arenavirus genomic segment and the second arenavirus genomic segment using a bi-directional expression cassette. 
     
     
         25 . The method of  claim 22 , wherein the second arenavirus genomic segment is an L segment. 
     
     
         26 . The method of  claim 22 , wherein the method further comprises transfecting into the host cell a cDNA of a third arenavirus genomic segment. 
     
     
         27 . The method of  claim 23 , wherein the promoter is selected from the group consisting of:
 (i) an RNA polymerase I promoter;   (ii) an RNA polymerase II promoter; and   (iii) a T7 promoter.   
     
     
         28 - 59 . (canceled) 
     
     
         60 . A method of generating a tri-segmented arenavirus particle comprising one L segment and two S segments, wherein the method comprises:
 (i) transfecting into a host cell one or more cDNAs of the one L segment and two S segments;   (ii) maintaining the host cell under conditions suitable for virus formation; and   (iii) harvesting the tri-segmented arenavirus particle comprising one L segment and two S segments,   
       wherein one of the two S segments is selected from the group consisting of:
 (i) an S segment, wherein an ORF encoding an NP is under control of an arenavirus 5′ UTR, 
 (ii) an S segment, wherein an ORF encoding a Z protein is under control of an arenavirus 5′ UTR, 
 (iii) an S segment, wherein an ORF encoding a L protein is under control of an arenavirus 5′ UTR, 
 (iv) an S segment, wherein an ORF encoding a GP is under control of an arenavirus 3′ UTR, 
 (v) an S segment, wherein an ORF encoding the L protein is under control of an arenavirus 3′ UTR, and 
 (vi) an S segment, wherein an ORF encoding the Z protein is under control of an arenavirus 3′ UTR. 
 
     
     
         61 . (canceled) 
     
     
         62 . The method of  claim 60 , wherein the method further comprises transcribing the one or more cDNAs of the one L segment and two S segments using a promoter. 
     
     
         63 . The method of  claim 60 , wherein the method further comprises transcribing the one or more cDNAs of the one L segment and two S segments using a bi-directional expression cassette. 
     
     
         64 . The method of  claim 60 , wherein the method further comprises transfecting into the host cell one or more nucleic acids encoding an arenavirus polymerase. 
     
     
         65 . The method of  claim 64 , wherein the arenavirus polymerase is the L protein. 
     
     
         66 . The method of  claim 60 , wherein the method further comprises transfecting into the host cell one or more nucleic acids encoding the arenavirus NP. 
     
     
         67 . The method of  claim 62 , wherein the promoter is selected from the group consisting of:
 (i) an RNA polymerase I promoter;   (ii) an RNA polymerase II promoter; and   (iii) a T7 promoter.   
     
     
         68 . The method of  claim 60 , wherein the tri-segmented arenavirus particle comprising one L segment and two S segments comprises all four arenavirus ORFs, and wherein the tri-segmented arenavirus particle comprising one L segment and two S segments is infectious and replication competent. 
     
     
         69 - 75 . (canceled) 
     
     
         76 . The method of  claim 60 , wherein the two S segments of the tri-segmented arenavirus particle comprising one L segment and two S segments comprise (i) one or two heterologous ORFs from an organism other than an arenavirus; or (ii) one or two duplicated arenavirus ORFs; or (iii) one heterologous ORF from an organism other than an arenavirus and one duplicated arenavirus ORF. 
     
     
         77 . The method of  claim 26 , wherein the third arenavirus genomic segment is an S segment. 
     
     
         78 . The method of  claim 22 , wherein the arenavirus 3′ UTR is the 3′ UTR of the S segment, and wherein the arenavirus 5′ UTR is the 5′ UTR of the S segment. 
     
     
         79 . The method of  claim 22 , wherein the arenavirus particle generated by the method is: (i) infectious and replication competent; (ii) attenuated; or (iii) infectious but unable to produce further infectious progeny in non-complementing cells. 
     
     
         80 . The method of  claim 79 , wherein: (i) at least one of the ORFs encoding GP, NP, Z protein, or L protein is removed or functionally inactivated; (ii) at least one of the ORFs encoding GP, NP, Z protein, or L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus; (iii) only one of the ORFs encoding GP, NP, Z protein or L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus; (iv) the ORF encoding GP is removed and replaced with a heterologous ORF from an organism other than an arenavirus; (v) the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than an arenavirus; (vi) the ORF encoding the Z protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus; or (vii) the ORF encoding the L protein is removed and replaced with a heterologous ORF from an organism other than an arenavirus 
     
     
         81 . The method of  claim 80 , wherein the heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen. 
     
     
         82 . The method of  claim 24 , wherein the bi-directional expression cassette comprises both a polymerase I and a polymerase II promoter reading from opposite sides into the two termini of the first arenavirus genomic segment and the second arenavirus genomic segment. 
     
     
         83 . The method of  claim 63 , wherein the bi-directional expression cassette comprises both a polymerase I and a polymerase II promoter reading from opposite sides into the two termini of the first arenavirus genomic segment and the second arenavirus genomic segment. 
     
     
         84 . The method of  claim 60 , wherein the tri-segmented arenavirus particle comprising one L segment and two S segments is derived from a lymphocytic choriomeningitis virus (LCMV). 
     
     
         85 . The method of  claim 76 , wherein the one or two heterologous ORFs or the one heterologous ORF encodes an antigen derived from an infectious organism, tumor, or allergen.