pBOOST2-sahIRF3 Adjuvants

• 20 µg of lyophilized pBOOST2-sahIRF3 plasmid expressing a human super-activated IRF3 gene
• 4 pouches of E. coli Fast-Media Zeo (2 TB and 2 Agar)

• Products are shipped at room temperature.
• Lyophilized DNA should be resuspended upon receipt and stored at -20°C.
• Lyophilized DNA is stable 12 months at -20°C. Resuspended DNA is stable more than one year at -20°C. Avoid repeated freeze-thaw cycles.
• Store E. coli Fast-Media Zeo at room temperature. Fast-Media pouches are stable 18 months when stored properly.

General Product Use

Plasmid Features

• sahIRF3 (super-activated human interferon regulatory factor 3) IRF-3 primarily increases Th1 T-cell responses. A constitutively active form of IRF-3 was generated by creating a single point mutation of Ser396 to Asp. This super-activated IRF-3 presents a >10-fold enhanced transactivating potential over the wild-type IRF-3 for the IFN-α and IFN-ß promoters.
• hEF1 / HTLV prom is a composite promoter comprising the Elongation Factor-1α (EF-1α) core promoter4 and the R segment and part of the U5 sequence (R-U5') of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5' has been coupled to the EF-1α core promoter to enhance stability of RNA.
• SV40 pAn : The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.
• Ori is a minimal E. coli origin of replication with the same activity as the longer Ori.
• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.
• Sh-∆CpG (Synthetic Zeocin gene) : The Sh ble gene from Streptoalloteichus hindustanus encodes a small protein that confers resistance to Zeocin by binding to the antibiotic. To reduce the amount of CpG motifs that may skew the raised antigen-specific immune response, pBOOST2 contains a CpG-free allele of the Zeo gene. All CpGs from the wild-type gene (50) were removed by synthesizing a new allele that contains no CpGs but encodes the exact same protein sequence.

E. coli Fast-Media Zeo is a fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. Fast-Media Zeo is a TB (liquid) or LB (solid) based medium with zeocin.

Method

1. Pour the contents of a pouch of Fast-Media into a clean borosilicate glass bottle or flask.
2. Add 200 ml of distilled water to the flask
3. Heat in a microwave on MEDIUM power setting (about 400Watts), until bubbles start appearing (approximately 3 minutes). Do not heat a closed container. Do not autoclave Fast-Media.
4. Swirl gently to mix the preparation. Be careful, the bottle and media are hot, use heatproof pads or gloves and care when handling.
5. Reheat the media for 30 seconds and gently swirl again. Repeat as necessary to completely dissolve the powder into solution. But be careful to avoid overboiling and volume loss.
6. Let agar medium cool to 45°C before pouring plates. Let liquid media cool to 37°C before seeding bacteria.

Note: Do not reheat solidified Fast-Media as the antibiotic will be permanently destroyed by the procedure.

Plasmid DNA solution

• - Prepare the vaccine plasmid solution by resuspending 10 µg of the vaccine plasmid DNA in 50 µl saline solution.
• - Prepare the pBOOST2 solution by mixing 10 µg of pBOOST2-sahIRF3 and 90 µg of the mock plasmid pBOOST2-null in 50 µl saline solution for low dose, or 100 µg of pBOOST2-sahIRF3 in 50 µl saline solution for high dose.
• - Combine both solutions to obtain a total of 110 µg DNA in 100 µl saline solution.

Note: The quantities are per mouse.

Intramuscular injections

• - Inoculate 6 to 8-week old female BALB/c mice with 100 µl plasmid DNA solution (described above) into the quadriceps at 0 and 4 weeks.
• - Collect sera and analyze for antibodies at 8 weeks.