ENDOTOXICITY DOES NOT LIMIT THE USE OF BACTERIAL GHOSTS AS CANDIDATE VACCINES

Gram-negative bacterial ghosts produced by controlled expression of th e plasmid-encoded lysis gene E offers a promising approach in non-livi ng vaccine technology. Bacterial cell wall complex and hence the antig enic determinants of the living cells are not affected by denaturation due to cell killing. However, the endotoxin content of the Gram-negat ive cell wall has been discussed as a potential problem for this kind of whole cell or envelope vaccines. Here we show that bacterial ghosts prepared from Escherichia coli 026:B6 and Salmonella typhimurium C5 i nduce dose-dependent antibody responses against bacterial cells or the ir corresponding lipopolysaccharides (LPS) in doses 25 ng kg(-1) when administered intravenously to rabbits in a standard immunization proto col. No differences between the immune responses of the rabbits were o bserved when comparing equivalent doses of bacterial ghosts and antibi otic-treated whole cells. The results indicate that the bacterial ghos ts exhibit all the antigenic properties of the living cells. No signif icant fever responses in rabbits have been recorded in doses of <250 n g kg(-1) E. coli 026:B6 ghosts and up to doses of 250 ng kg-1 S. typhi murium C5 ghosts when applying test methods recommended by the US phar macopoeia. These findings correlate with cell culture experiments wher e doses 100 ng ml(-1) of bacterial ghosts were needed for the release of tumour necrosis factor alpha (TNF alpha) and prostaglandin E(2) (PG E(2)) from RAW mouse macrophage cultures. Free LPS of Salmonella abort us equi commonly used as a LPS-standard, however, stimulated TNF alpha and PGE(2) synthesis of RAW cells in doses of 1 ng ml(-1). The endoto xic activity of our bacterial preparations analysed by a standard limu lus amoebocyte lysate and 2-keto-3-deoxyoctonate assay correlated with the capacity to stimulate the release of PGE(2) and TNF alpha in RAW mouse macrophage cultures and the endotoxic responses in rabbits. It c an be concluded that these in vitro systems can be used as easy predic tive test systems for preparations of bacterial vaccines, particularly for bacterial ghosts. Copyright (C) 1997 Elsevier Science Ltd.