Type I Secretion System for Heterologous Antigen Expression in a Tularemia Vaccine

Francisella tularensis is a highly infectious bacteria that causes the disease tularemia; It has a very low infectious does and is classified as a type 1 select agent for its potential for use as a bioweapon. Developing a vaccine to target is of importance to investigators as a defense against potential weaponization of the bacteria. The Klose laboratory has developed a potential live attenuated vaccine for F. tularensis and now we are engineering it to be bivalent for multiple bacterial diseases. This strain will express heterologous antigens so that the vaccine will provide cross-protection against additional bacterial pathogens. The goal of this project is to develop an expression system to allow expression and secretion of Yersinia pestis protective antigens and potentially design a safe live attenuated bivalent vaccine against Francisella Tularensis and Yersinia Pestis. We are utilizing the Type I secretion system (T1SS) from E. coli that secretes the hemolysin A (HlyA) protein using the transmembrane proteins HlyB and HlyD. The C-terminus of the HlyA protein has been implicated to be a signal to initiate the secretion through the E. coli T1SS. Our belief is that a fusion protein of the Y. Pestis protective antigens and the C-terminus of HlyA will allow for secretion of the full-length fusion protein, allowing for good protection and vaccination to challenge against Y. Pestis. The HlyA, HlyB and HlyD genes will be inserted into the chromosome of the current vaccine strain (KKF768) in place of the FTN0715 gene. FTN0715 is a likely putative, repeated protein with no known function and serves as a suitable target to be replaced with the T1SS genes HlyB and HlyD. The Y. Pestis F1-LcrV fusion protein will be expressed with a HlyA secretion signal at the C-terminal end. We have been able to clone the F1-LcrV antigen to the cHlyA secretion signal as well as deleted FTN0715 from the chromosome of the current vaccine strain. Our belief is that we will be able to make a bivalent live attenuated vaccine for tularemia and plague with this strain.