Ty21a-vectored, Oral, Enteric Fever-Shigellosis Vaccine- Advance in Development

Our focus over the past decade has been to insert the known protective LPS biosynthetic genes (Dupont et al., 1972a,b) for key Shigella serotypes (Noriega et al, 1999) into separate Ty21a strains to construct a combined oral vaccine for protection against typhoid fever and predominant Shigella serotypes (Xu et al. 2002, 2007). Manipulation of plasmid-cloned LPS gene regions to remove a selectable antibiotic resistance marker, though successful, resulted in unacceptable genetic instability. To solve this problem, we modified existing recombination methods to stably insert the targeted Shigella serotype LPS genes (i.e. 10-20 kb inserts) into a silent region of the Ty21a chromosome. This has resulted in the development of genetically stable Ty21a derivative strains, each expressing the homologous S. Typhi LPS and one heterologous Shigella LPS. Further, mouse immunization studies demonstrate that these strains stimulate high serum IgG titers against both S. Typhi and heterologous Shigellla LPS’s, and provide 100% protection against respective virulent challenge. In addition, we have developed new manufacturing formulations/drying methods that result in increased shelf-life at 4C, survival at 25C for 12 months and with ~2 mo. survival at 37C, which should allow for vaccine administration without refrigeration. Recently, we have cloned the Shigella GAD acid-resistance genes into Ty21a, which make Ty21a resistant to low pH, that may allow for final product delivery as a more immunogenic, rapidly dissolvable wafer (i.e. child-friendly). We have also completed the genomic sequence of Ty21a and can speculate on the key mutations for attenuation. To date, we have completed construction of four Ty21a strains expressing the LPS genes from S. sonnei, S. dysenteriae 1, S. flexneri 2a, and S. flexneri 3a. We believe that a mixture of 3 strains will provide combined protection against both typhoid/some paratyphoid strains and the predominant Shigella serotypes, and which can be administered in the absence of cold-chain in as few as 2 doses, given over three days to children and adults, and potentially in the EPI schedule.