Use of Staby^®technology for development and production of DNA vaccines free of antibiotic resistance gene

Abstract

The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1).

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

“…This ineffectiveness of ampicillin, in addition to the deprecation by regulatory authorities of its use in biotherapeutics and biomaterials production due to safety concerns232425, prompted us to investigate other plasmid stabilisation systems. Indeed, use of β-lactam antibiotics such as ampicillin poses safety risks associated with their potential for causing serious hypersensitivity reactions in patients and for leading to the spread of antibiotic resistance traits to the environments2526. Furthermore, their use, and the necessity for additional purification and testing to ensure their absence in the final product leads to higher process costs.…”

“…Furthermore, their use, and the necessity for additional purification and testing to ensure their absence in the final product leads to higher process costs. The antibiotic kanamycin is more readily accepted25 but a recently developed antibiotic free system, the separate-component-stabilisation system (SCS)2627, offers a means of avoiding the use of costly antibiotics and potentially improving both plasmid segregational stability and product yield and safety. This post-segregational stabilisation system employs a toxin producing host which kills host cells that have lost the plasmid and an anti-toxin producing expression vector which inactivates the toxin, thereby ensuring persistence of plasmid-containing cells only.…”

“…This post-segregational stabilisation system employs a toxin producing host which kills host cells that have lost the plasmid and an anti-toxin producing expression vector which inactivates the toxin, thereby ensuring persistence of plasmid-containing cells only. In addition, as compared to antibiotic resistance markers, SCS has an improved safety profile as it is a naturally occurring system innocuous to eukaryotic cells26. Furthermore, it is expected to impose a reduced metabolic burden on host cells due to the small sizes of the toxin (100 amino acids) and anti-toxin (90 amino acids) as well as the regulated expression of these (toxin production only in the absence of anti-toxin)252627.…”

“…In addition, as compared to antibiotic resistance markers, SCS has an improved safety profile as it is a naturally occurring system innocuous to eukaryotic cells26. Furthermore, it is expected to impose a reduced metabolic burden on host cells due to the small sizes of the toxin (100 amino acids) and anti-toxin (90 amino acids) as well as the regulated expression of these (toxin production only in the absence of anti-toxin)252627. In contrast, the relatively large antibiotic-resistance proteins (β-lactamase: 263 amino acids + 23 amino acid signal sequence; aminoglycoside 3′-phosphotransferase: 271 amino acids) are constitutively expressed from the pET expression vectors and indeed have already been shown to have a deleterious effect on host cells242829.…”

Antibiotic-Free Selection in Biotherapeutics: Now and Forever

“…The Merial’s Porcine Rotavirus vaccine based on expression of NSP4, VP4 and VP6 rotavirus antigens that prevents pigs from gastroenteritis (US 2013/0209507A1). This antibiotic free selection system uses the stabilization system StabyExpress based on the use of toxin-antitoxin genes [ 28 , 93 ] and anticipates future regulatory requirements.…”