TC-83-derived alphavirus vectors, particles and methods
The present disclosure provides TC-83 VEE-derived replicons, alphaviral replicon particles and immunogenic compositions containing TC-83 alphaviral replicon particles which direct the expression of at least one antigen when introduced into a suitable host cell. The TC-83 VEE-derived ARPs described herein are improved in that they are subject to a lower vector-specific immune response than prior art ARPs.
1. A method for preparing propagation-defective TC-83 derived alphaviral replicon particles (ARPs), said method comprising:
(a) introducing an alphaviral replicon nucleic acid into a host cell, said replicon nucleic acid comprising at least a virus packaging signal and at least one heterologous coding or functional sequence expressible in said alphaviral replicon nucleic acid, said replicon nucleic acid lacking sequences encoding an alphavirus structural protein, wherein said host cell comprises at least one helper function encoding the TC-83 structural proteins comprising the TC-83 capsid protein and the TC-83 glycoproteins, to produce a modified host cell; and
(b) culturing said modified host cell under conditions allowing expression of the at least one helper function, allowing replication of said TC-83-derived alphaviral replicon nucleic acid and packaging of said alphaviral replicon nucleic acid to form ARPs.
2. The method of claim 1 , wherein the alphaviral replicon nucleic acid comprises (i) a 5′ sequence of Venezuelan equine encephalitis strain TC-83 which initiates transcription of alphavirus RNA; (ii) one or more nucleotide sequences which together encode those TC-83 alphavirus nonstructural proteins necessary for replication of the replicon RNA: (iii) a virus packaging signal; (iv) at least one heterologous coding or functional sequence expressible in said alphaviral replicon nucleic acid; and (v) a 3′ RNA polymerase recognition sequence of Venezuelan equine encephalitis strain TC-83.
3. The method of claim 1 , further comprising:
(c) contacting the modified host cells after step (b) with an aqueous solution having an ionic strength of at least 0.2 M to release the ARPs into the aqueous solution to produce an ARP-containing solution;
(d) collecting ARPs from the ARP-containing solution of step (c); and
(e) optionally applying an ion exchange chromatography step to the collected ARPs.
4. The method of claim 3 , further comprising a cell washing step, prior to step (c) wherein the cell washing solution contains no salt and further comprises DNAse.
5. The method of claim 1 , wherein the at least one helper function in the host cell of step (a) is encoded by a nucleic acid sequence stably integrated within the genome of said host cell.
6. The method of claim 1 , wherein the at least one helper function in the host cell is introduced on at least one helper nucleic acid which encodes a capsid protein capable of binding said alphaviral replicon nucleic acid, and at least one alphaviral glycoprotein, wherein said alphaviral glycoprotein associates with said alphaviral replicon nucleic acid and said capsid protein, wherein the at least one helper nucleic acid molecule is introduced into the host cell together with said alphaviral replicon nucleic acid.
7. The method of claim 1 , wherein the at least one helper function is encoded by at least two helper nucleic acid molecules wherein each of said two helper nucleic acid molecules encodes at least one viral helper function.
8. The method of claim 1 , wherein the ionic strength of the aqueous solution in step (c) is between 0.5 M and 5 M.
9. The method of claim 1 , wherein the at least one helper nucleic acid molecule is a DNA molecule.
10. The method of claim 1 , wherein the alphaviral replicon nucleic acid is introduced into said host cell by electroporation.
11. The method of claim 1 , wherein the alphavirus-permissible cell culture is a Vero cell culture.
12. The method of claim 1 , wherein the aqueous solution of step (c) comprises salt selected from the group consisting of NaCl, KCl, MgCl 2 , CaCl 2 , NH 4 Cl, (NH 4 ) 2 SO4, NH 4 HCO 3 and NH 4 Acetate.
13. A method of preparing propagation-defective TC-83 derived alphavirus replicon particles comprising introducing an alphavirus replicon vector and one or more helper nucleic acid molecules into alphavirus-permissible cells via electroporation, wherein said alphavirus replicon vector comprises a nucleic acid lacking sequences encoding an alphavirus structural protein, wherein said one or more helper nucleic acid molecules encode the TC-83 structural proteins comprising the TC-83 capsid protein and the TC-83 glycoproteins, and wherein the alphavirus-permissive cells in a culture medium during electroporation are at a concentration of least 10 8 cells/ml medium and wherein the alphavirus RNA replicon vector is added to the cells prior to electroporation at a concentration of approximately 35 μg per ml.
14. The method of claim 13 , wherein the one or more helper nucleic acid molecule is comprised in a single DNA molecule encoding all alphavirus structural proteins.