Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

FIG. 1.

Schematic drawing of plasmid pML1 and construction of plasmid pSNUCIQ3. Plasmid pML1, encoding the lysis protein E, and plasmid pSNUCIQ3, encoding SNUC, were used for the coexpression of gene E and SNUC in E. coli O157:H7. The expression of the killing genes was triggered either by the addition of a chemical inducer (pSNUCIQ3) or by a thermal shift from 28°C to 42°C (pML1). E, lysis gene E; Amp, ampicillin resistance gene; Kan, kanamycin resistance gene; Tet, tetracycline resistance gene; ColE1 and p15A, origins of replication; A1-O4/O3, synthetic, chemically inducible promoter; P_RMand P_R, rightward “maintenance” and rightward promoters of bacteriophage lambda, respectively; cI857, gene encoding the thermosensitive repressor for the lambda P_R promoter; lacI^q, gene encoding the repressor for the synthetic A1-O4/O3 promoter; XhoI, PstI, and SalI, restriction sites used for the construction of pSNUCIQ3.

FIG. 2.

Growth and lysis kinetics of E. coli O157:H7 strain CIP 105282(pML1, pSNUCIQ3). Growth and lysis were monitored by the measurement of the OD₆₀₀ (•) and the determination of the number of CFU (○). The dashed line indicates the reduction of cell viability to zero after the freeze-drying process. The time points of supplementation with IPTG (−45 min), the temperature shift to 42°C (0 min), and the addition of CaCl₂ and MgCl₂ (90 min) are indicated by arrows at the top of the figure.

FIG. 3.

Antibody levels in mice following oral immunization with EHEC O157:H7 ghosts and challenge with E. coli O157:H7. Each value represents the mean for three mice. (A) Serum IgA antibody titers against EHEC O157:H7 ghosts. (B) Colon IgA antibody titers against EHEC O157:H7 ghosts. (C) Serum IgG antibody titers against EHEC O157:H7 ghosts. Mice were immunized intragastrically with 1 mg EHEC O157:H7 ghosts once (group A) or twice (group B) (⇑). All mice were challenged with 1 × 10⁸ CFU EHEC O157:H7 (CIP 103571) on day 55 (↑).