Development of dual reporter imaging system for Francisella tularensis to monitor the spatio-temporal pathogenesis and vaccine efficacy

PURPOSE: Study on the pathogen and the pathogen-related disease require the information at both cellular and organism level. However, lack of appropriate high-quality antibodies and the difference between the experimental animal models make it difficult to analyze in vivo mechanism of pathogen-related diseases. For more reliable research on the infection and immune-response of pathogen-related diseases, accurate analysis is essential to provide spatiotemporal information of pathogens and immune activity to avoid false-positive or mis-interpretations. In this regards, we have developed a method for tracking Francisella tularensis in the animal model without using the specific antibodies for the F. tularensis. MATERIALS AND METHODS: A dual reporter plasmid using GFP-Lux with putative bacterioferritin promoter (pBfr) was constructed and transformed to F. tularensis live vaccine strain to generate F. tularensis LVS (FtLVS)-GFP-Lux for both fluorescence and bioluminescence imaging. For vaccination to F. tularensis infection, FtLVS and lipopolysaccharide (LPS) from FtLVS were used. RESULTS: We visualized the bacterial replication of F. tularensis in the cells using fluorescence and bioluminescence imaging, and traced the spatio-temporal process of F. tularensis pathogenesis in mice. Vaccination with LPS purified from FtLVS greatly reduced the bacterial replication of FtLVS in animal model, and the effect of vaccination was also successfully monitored with in vivo imaging. CONCLUSION: We successfully established dual reporter labeled F. tularensis for cellular and whole body imaging. Our simple and integrated imaging analysis system would provide useful information for in vivo analysis of F. tularensis infection as well as in vitro experiments, which have not been fully explained yet with various technical problems.

Organoid as a culture system for viral vaccine strains

Organoid is an in vitro multicellular form mimicking in vivo organ. Its similarity to human organ including cellular organization, molecular expression patterns, as well as genetic signatures enables to study the characteristics of infectious agents and host-pathogen interaction. For the features of organoid, this system also can be potentially used to cultivate currently uncultivable viruses of vaccine candidates. This paper will briefly describe problems in the current culture system for virus production and the possibility of organoid as culture system for viral vaccine and their current limitations that should be solved to meet the goal.

Application of aptamers for assessment of vaccine efficacy

Assessing antigen concentration of vaccine is essential step in determining the quality of the vaccine prior to vaccination. After vaccination, vaccine-induced antibody titer should also be measured to verify the vaccine efficacy. Since conventional assay used for vaccine concentrations and induced Ab-titers is antibody-based enzyme-linked immunosorbent assay, the assay inevitably brings drawbacks of antibody such as high cost for production, limited stability, and inconsistent quality between lot-to-lots. Aptamer is single-stranded nucleic acid having three-dimensional structure and has features overcoming limitations of antibody. This review will briefly introduce the features of aptamer and potential of aptamer-based system for evaluation of vaccine efficacy.

Current status of vaccine development for tularemia preparedness

Tularemia is a high-risk infectious disease caused by Gram-negative bacterium Francisella tularensis. Due to its high fatality at very low colony-forming units (less than 10), F. tularensis is considered as a powerful potential bioterrorism agent. Vaccine could be the most efficient way to prevent the citizen from infection of F. tularensis when the bioterrorism happens, but officially approved vaccine with both efficacy and safety is not developed yet. Research for the development of tularemia vaccine has been focusing on the live attenuated vaccine strain (LVS) for long history, still there are no LVS confirmed for the safety which should be an essential factor for general vaccination program. Furthermore the LVS did not show protection efficacy against high-risk subspecies tularensis (type A) as high as the level against subspecies holarctica (type B) in human. Though the subunit or recombinant vaccine candidates have been considered for better safety, any results did not show better prevention efficacy than the LVS candidate against F. tularensis infection. Currently there are some more trials to develop vaccine using mutant strains or nonpathogenic F. novicida strain, but it did not reveal effective candidates overwhelming the LVS either. Difference in the protection efficacy of LVS against type A strain in human and the low level protection of many subunit or recombinant vaccine candidates lead the scientists to consider the live vaccine development using type A strain could be ultimate answer for the tularemia vaccine development.