Recombineering Vectors: pKD46-RecA and pKD46-RecA_paChromosome Engineering in E. coliX|XRIG-I Activating DNA Vaccine VectorsX|XMammalian Expression VectorsX|XAntibiotic-Free RNA-OUT Marker for Mammalian Expression and DNA Vaccine VectorsX|XpDNAVACCUltra™ DNA Vaccine VectorsX|XAutolytic E. Coli Cell Lines

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Rig-1 Activating DNA Vaccine Vectors

• Enhanced antigen expression in mammalian cells
• Compliance with FDA guidance
•  Improved antibody response
• Improved antibody avidity
• Kanamycin or RNA-OUT (antibiotic free) selectable markers
•Improved DNA backbone for maximum plasmid production (>2.5g/L)

Description:

Co-stimulation, in addition to antigen presentation, is an essential property of vaccines that was often ignored in early stage DNA vaccine backbones.  Antigen expression in the absence of co-stimulation can result in anergy and tolerance. NTC’s RNA elements (eRNA^tm) are designed to mimic Pathogen Associated Molecular Patterns (PAMPS), such as double stranded RNA and RNA hairpins, which are agonists of the innate response genes, retinoic acid inducible gene 1 (RIG-1) and melanoma differentiation antigen 5 (mda5).  Synergistic activation of these pathways by RNAe^™ (Fig. 1) resulted in dramatic enhancement of Type I interferon production, as well as in improved antibody responses and avidity.

RNAe^™ vaccine vectors are available either as traditional Kanamycin resistance vectors, or as antibiotic-free versions of our popular NTC7482 and NTC7485 vaccine/expression vectors.  The RNAe^tm, 41H, is a 760bp region inserted into the vector backbone (Fig. 2). The 41H vector series was rationally designed as a safe, minimalized selection set, for the expression of antigens in vivo for genetic immunization (DNA vaccines).


Features:

• Enhanced co-stimulatory RNAe^tm activates RIG-1 pathway, Type I interferon production

• Optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)
• Superior E. coli plasmid production yields (>2.5g/L)

• Optional TPA secretion tag

Versions:


Kanamycin resistance selectable markert (KanR gene):

NTC7485-eRNA41H-EGFP – native, Kanamycin selection, EGFP control gene (can be used to clone a gene of

NTC7482-eRNA1H – secreted (TPA tag), Kanamycin selection, can be used to clone a gene of interest


 
Antibiotic-Free (SacB gene, sucrose resistance):

NTC8485-eRNA41H-EGFP – equivalent to NTC7485-eRNA41H-EGFP, above, except for antibiotic-free selection on sucrose

NTC8482-eRNA41H – secreted (TPA tag), equivalent to NTC7482-eRNA41H, above, except for antibiotic-free selection on sucrose

Fig. 1. Left: RIG-1 activation of IFNβ promoter-luciferase reporter (ratio of luciferase to EGFP internal transfection control).  Synergistic RIG-1 activation by plasmid-borne eRNA VA RNA1 and eRNA 11a combination (eRNA41H). Right: Antibody avidity after prime (T=21 days) and boost (T=49 days) IM immunizations).