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Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria by bacteriophage ɸX174 gene  E  mediated lysis. They represent a novel inactivated vaccine platform; however, the practical application of BGs for human vaccines seems to be limited due to the safety concerns on the presence of viable cells in BGs. Therefore, to improve the lysis efficiency of the gene  E , we exploited the peptidoglycan hydrolyzing ability of the  λ  phage holin-endolysins to expedite the process of current BG production system. In this report, we constructed a novel ghost plasmid encoding protein E and holin-endolysins in tandem. We observed that sequential expressions of the gene  E  and the holin-endolysins elicited rapid and highly efficient  Salmonella  lysis compared to the lysis mediated by gene  E  only. These lysed BGs displayed improved immunogenicity in mice compared to the gene  E  mediated BGs. Consequently, seventy percent of the mice immunized with these novel ghosts survived against a lethal challenge while all the mice vaccinated with gene  E  mediated ghosts died by day 9 post-infection. We conclude that this novel strategy has the potential to generate highly efficient inactivated candidate vaccines that could replace the currently available bacterial vaccines.

Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by co-expression of λ phage holin-endolysin and ɸX174 gene E