Chimeric alphavirus replicon particles
Chimeric alphaviruses and alphavirus replicon particles are provided including methods of making and using same. Specifically, alphavirus particles are provided having nucleic acid molecules derived from one or more alphaviruses and structural proteins (capsid and/or envelope) from at least two or more alphaviruses. Methods of making, using, and therapeutic preparations containing the chimeric alphavirus particle, are disclosed.
1. A chimeric alphavirus replicon particle comprising:
a) an alphavirus replicon RNA, comprising:
(1) a nucleotide sequence encoding one or more nonstructural proteins from a first alphavirus,
(2) a packaging signal derived from a second alphavirus , and
(3) a heterologous nucleic acid sequence encoding an antigen, wherein said packaging signal is inserted into a site selected from the group consisting of (i) the junction of nsP3 with nsP4, (ii) following the nsP4 open reading frame and (iii) a deletion in a nonstructural protein gene;
b) a capsid protein comprising:
(1) an RNA binding domain derived from the second alphavirus ; and
(2) an envelope glycoprotein interaction domain;
c) an alphavirus E2 glycoprotein comprising an E2 cytoplasmic tail, wherein the E2 cytoplasmic tail and the envelope glycoprotein interaction domain are derived from the same alphavirus ; and
d) an alphavirus E1 glycoprotein,
wherein at least one of the E2 cytoplasmic tail or the E1 glycoprotein is derived from said second alphavirus virus, and
wherein said first alphavirus is a Venezuelan equine encephalitis (VEE) virus and said second alphavirus is not a VEE virus.
2. The chimeric alphavirus replicon particle of claim 1 , wherein the envelope glycoprotein interaction domain is derived from the second alphavirus .
3. The chimeric alphavirus replicon particle of claim 1 , wherein the envelope glycoprotein interaction domain is derived from VEE.
4. The chimeric alphavirus replicon particle of claim 1 , wherein the envelope glycoprotein interaction domain and the alphavirus envelope glycoproteins E2 and E1 are derived from a Sindbis virus.
5. An immunogenic composition comprising the chimeric alphavirus replicon particle of claim 1 and a pharmaceutically acceptable excipient.
6. An immunogenic composition comprising the chimeric alphavirus replicon particle of claim 2 and a pharmaceutically acceptable excipient.
7. An immunogenic composition comprising the chimeric alphavirus replicon particle of claim 3 and a pharmaceutically acceptable excipient.
8. An immunogenic composition comprising the chimeric alphavirus replicon particle of claim 4 and a pharmaceutically acceptable excipient.
9. A method of generating an immune response in a subject comprising administering to the subject the immunogenic composition of claim 5 .
10. A method of generating an immune response in a subject comprising administering to the subject the immunogenic composition of claim 6 .
11. A method of generating an immune response in a subject comprising administering to the subject the immunogenic composition of claim 7 .
12. A method of generating an immune response in a subject comprising administering to the subject the immunogenic composition of claim 8 .
13. A method for producing an alphavirus replicon particle, comprising introducing into a host cell:
a) a nucleic acid sequence or vector particle encoding an alphavirus replicon RNA, said alphavirus replicon RNA encoding one or more nonstructural proteins from a first alphavirus , a packaging signal derived from a second alphavirus and one or more heterologous nucleic acid sequences, wherein said packaging signal is inserted into a site selected from the group consisting of the junction of nsP3 with nsP4, following the nsP4 open reading frame and a deletion in a nonstructural protein gene; and
b) at least one helper nucleic acid sequence encoding a defective helper RNA, said defective helper RNA encoding at least three structural proteins derived from alphavirus , wherein said structural proteins comprise a capsid protein, an E1 envelope glycoprotein, and an E2 envelope glycoprotein,
wherein the capsid protein comprises
(1) an RNA binding domain derived from said second alphavirus, and
(2) an envelope glycoprotein interaction domain; and
wherein glycoprotein E2 comprises a cytoplasmic tail, wherein the E2 cytoplasmic tail and envelope glycoprotein interaction domain are derived from the same alphavirus and wherein at least one of the E2 cytoplasmic tail or the E1 glycoprotein is derived from the second alphavirus .
14. The method of claim 13 , wherein two helper nucleic acid sequences are introduced into the host cell, each said helper nucleic acid sequence encoding a defective helper RNA encoding at least one structural protein derived from alphavirus , wherein at least one defective helper RNA encodes a capsid protein and a second defective helper RNA encodes an E2 envelope glycoprotein and an E1 envelope glycoprotein,
wherein the capsid protein comprises
(1) an RNA binding domain derived from the second alphavirus , and
(2) an envelope glycoprotein interaction domain; and
wherein the E2 glycoprotein comprises a cytoplasmic tail, wherein the E2 cytoplasmic tail and envelope glycoprotein interaction domain are derived from the same alphavirus and wherein at least one of the E2 cytoplasmic tail or E1 glycoprotein is derived from said second alphavirus .
15. The method of claim 13 , wherein said first alphavirus is a VEE virus and said second alphavirus is not a VEE virus.
16. The method of claim 14 , wherein said first alphavirus is a VEE virus and said second alphavirus is not a VEE virus.
17. The method of claim 13 , wherein said first alphavirus is a VEE virus and said structural proteins are not derived from a VEE virus.
18. The method of claim 14 , wherein said first alphavirus is a VEE virus and said structural proteins are not derived from a VEE virus.
19. The method of claim 13 , wherein said capsid protein is a hybrid capsid protein.
20. The method of claim 14 , wherein said capsid protein is a hybrid capsid protein.
21. A chimeric alphavirus replicon particle comprising:
a) a chimeric alphavirus RNA genome, wherein a first portion of said genome is derived from a first alphavirus and a second portion of said genome is derived from a second alphavirus , wherein said first alphavirusis a VEE virus and said second alphavirus is not a VEE virus, and further wherein said first portion encodes VEE virus nonstructural proteins and said second portion encodes alphavirus envelope glycoproteins E2 and E1,
b) a capsid protein comprising
(1) an RNA binding domain derived from the second alphavirus , and
(2) an envelope glycoprotein interaction domain; and
c) , an alphavirus E2 glycoprotein comprising an E2 cytoplasmic tail, wherein the E2 cytoplasmic tail and envelope glycoprotein interaction domain are derived from the same alphavirus , and
d) an alphavirus E1 glycoprotein,
wherein at least one of the E2 cytoplasmic tail or E1 glycoprotein is derived from said second alphavirus virus.
22. The chimeric alphavirus replicon particle of claim 21 , wherein said second portion encodes envelope glycoproteins E2 and E1 from a second alphavirus .
23. The chimeric alphavirus replicon particle of claim 21 , wherein the envelope glycoprotein interaction domain and said alphavirus envelope glycoproteins E2 and E1 are derived from said second alphavirus .