Two-tiered biological containment strategy for Lactococcus lactis-based vaccine or immunotherapy vectors

Abstract

The concept of biological containment was developed as a strategy to prevent environmental dissemination of engineered live vaccine or drug delivery vehicles. A mutation in the gene encoding thymidylate synthase (thyA), a key enzyme in the pyrimidine biosynthetic pathway, has previously been shown to limit growth of L. lactis vectors under restrictive conditions. We hypothesized that further mutations in the pyrimidine biosynthetic pathway might enhance the stability and safety of live L. lactis vectors. We show that a double mutation in the genes encoding ThyA and CTP synthase (PyrG) in L. lactis confers double auxotrophy for both thymidine and cytidine. However, the combination of two mutations failed to enhance the biological containment phenotype of the engineered strain. In the absence of thymine/thymidine, the thyA mutant exhibited a strong bactericidal phenotype. However, creation of the double mutant caused the loss of this phenotype, though survival in the mouse GI tract was enhanced. The implications for biological containment of live L. lactis based delivery vectors are discussed.

Keywords: :

• biological containment
• vaccine
• Lactococcus lactis
• thyA
• pyrG

10.4161/hv.26954

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors’ work was supported by grants from Science Foundation Ireland in the form of a center grant (Alimentary Pharmabiotic Centre; Grant Numbers SFI/12/RC/2273 and 12/RC/2273).