Compositions and methods for generating reversion free attenuated and/or replication incompetent vaccine vectors
Provided herein are methods for generating reversion free attenuated and/or replication incompetent vaccine vectors and their use in vaccine compositions and vaccination. In particular, the use of a codon shuffled helper gene is used to produce compositions comprising replication incompetent virus.
1. A method of preventing or reducing viral reversion of a virus during culture, the method comprising:
a) stably expressing a codon shuffled helper gene in a host cell, wherein the helper gene is codon modified to reduce expression of the helper gene in the host cell relative to the expression of an unmodified helper gene; and
b) culturing the host cell under conditions conducive to the propagation of a replication incompetent virus, wherein the helper gene is deleted from the virus thereby rendering the virus replication incompetent.
2. The method of claim 1 , wherein the virus is a DNA virus.
3. The method of claim 1 , wherein the virus is a gene therapy vector.
4. The method of claim 2 , wherein the virus is a gammaherpesvirus.
5. The method of claim 1 , wherein the replication incompetent virus comprises a deletion of replication and transcriptional activator (RTA) and the codon shuffled helper gene is ORF50.
6. The method of claim 1 , wherein the helper gene comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
7. The method of claim 1 , wherein viral reversion is prevented.
8. The method of claim 1 , wherein the cell line is a fibroblast cell line.
9. The method of claim 1 , wherein the codon shuffled helper gene is stably expressed via retroviral transduction.
10. A method of producing a viral stock composition, the method comprising:
a) stably expressing a codon shuffled helper gene in a host cell, wherein the helper gene is codon modified to reduce expression of the helper gene in the host cell relative to the expression of an unmodified helper gene;
b) culturing the host cell under conditions conducive to the propagation of a replication incompetent virus, wherein the helper gene is deleted from the virus thereby rendering the virus replication incompetent; and
c) collecting the replicated virus.
11. The method of claim 10 , wherein the virus is a DNA virus.
12. The method of claim 10 , wherein the virus is a gene therapy vector expressing one or more genes of interest.
13. The method of claim 10 , wherein the helper gene comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
14. The method of claim 10 , wherein viral reversion is prevented.
15. The method of claim 12 , further comprising step d) forming the composition comprising a gene therapy viral vector.
16. A composition comprising a gene therapy viral vector, wherein the viral vector was produced by the method of claim 15 .
17. A vaccine composition, wherein the vaccine composition comprises virus produced by a method comprising:
a) stably expressing a codon shuffled helper gene in a host cell, wherein the helper gene is codon modified to reduce expression of the helper gene in the host cell relative to the expression of an unmodified helper gene;
b) culturing the host cell under conditions conducive to the propagation of a replication incompetent virus, wherein the viral genome comprises the deleted helper gene thereby rendering the virus replication incompetent;
c) collecting the replicated virus; and
d) forming the vaccine composition.
18. The composition of claim 17 , wherein a pharmaceutically acceptable carrier or excipient is added to the vaccine composition.
19. The composition of claim 17 , wherein the virus is a DNA virus.
20. The composition of claim 17 , wherein the vaccine composition is formulated for oral administration, topical administration, parenteral administration or inhalation.