New DNA vaccine adjuvant of the pVAC plasmids expressing a super-activated IRF3 gene
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)
Storage and Stability
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
pBOOST2 plasmids were developed as genetic adjuvants for DNA vaccines to potentiate the immune response to a specific antigen. They feature different genes from the interferon regulatory factor family (IRF). IRFs are transcriptional activators for IFN-α, IFN-ß and IFN-stimulated genes. In particular IRF-1, IRF-3 and IRF-7 act as direct transducers of virus-mediated signaling pathways activating IFN-α and IFN-ß in infected cells. Recently, IRF-1, IRF-3 and IRF-7 were shown to be able to bias T cells towards type 1 or type 2 immune responses, leading to the activation of cytotoxic T cells and/or the production of antibodies. The method of plasmid DNA vaccine delivery is known to bias the immune response to a specific antigen towards a type 1 (T-cell) or type 2 (antibody) response. These biases can be further enhanced by the codelivery of IRFs to increase the efficacy of the vaccination.
• 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.
Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1µg/µl, resuspend the DNA in 200 µl of sterile H20. Store resuspended plasmid at -20°C.
Selection of Bacteria with E. coli Fast-Media Zeo
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.