Vectors and methods for genetic immunization
Improved DNA vaccine plasmids are disclosed that contain novel immunostimulatory RNA compositions. The improved plasmids eliminate all extraneous sequences, incorporate a novel antibiotic free short RNA based selectable marker, increase eukaryotic expression using a novel chimeric promoter, improve yield and stability during bacterial production, and improve immunostimulation. These vectors are utilized in immunization to elicit improved immune responses or therapy to induce type 1 interferon production.
1. A composition of matter for a antibiotic marker-free covalently closed, super-coiled DNA replicon comprising:
a. an RNA-OUT antisense RNA gene sequence comprising an internal insertion sequence 10 derived portion comprising a RNA-OUT promoter sequence and a RNA-OUT sequence portion that is complementary to an RNA-IN gene sequence and a terminator sequence wherein said RNA-OUT antisense RNA gene sequence has at least 90% sequence identity to the sequence set forth as SEQ ID NO: 15, operably linked to,
b. an antibiotic marker-free covalently closed, super-coiled DNA replicon comprising a gene of interest.
2. The composition of claim 1 wherein said antibiotic marker-free covalently closed, super-coiled DNA replicon comprises a promoter comprising SEQ ID NO:6 operably linked to said gene of interest.
3. The composition of claim 2 wherein the antibiotic marker-free covalently closed, super-coiled DNA replicon comprises a VARNA.
4. The composition of claim 3 wherein the antibiotic marker-free covalently closed, super-coiled DNA replicon further comprises at least one immunostimulatory RNA element.
5. The composition of claim 1 , wherein said RNA-OUT antisense RNA gene sequence comprises SEQ ID NO: 15.
6. The composition of claim 1 , wherein said RNA-OUT antisense RNA gene comprises an RNA-OUT promoter sequence modified to increase expression.