A Conjugate Enterobactin Vaccine Decreases Mortality from Escherichia coli K1 in a Mouse Model of Neonatal Early Onset Sepsis

Background: E. coli containing the K1 antigen (E. coli K1) does not cause disease in healthy adults but is the most common cause of early-onset sepsis-related death in neonates. Similarly, intraperitoneal (IP) injection of E. coli K1 in mice has minimal to no effect in adults but causes sepsis in neonates. This difference is due to higher levels of iron within the neonatal peritoneal cavity. Enterobactin (ENT) is an important iron acquisition compound secreted by E. coli K1.

Objectives: We hypothesized that immunization against ENT will decrease inflammation and mortality in an in vivo E. coli K1 murine neonatal early-onset sepsis model.

Design/Methods: ENT was conjugated to bovine serum albumin (BSA) to create an ENT-BSA conjugate vaccine. 6-8 week old female C57Bl/6J mice were intranasally immunized with ENT-BSA or ENT, BSA, or PBS as controls. They were subsequently mated and their neonatal offspring were intraperitoneally injected with 1000 CFU E. coli K1 between P4-6. Mice were followed for mortality. Additionally, 12 hours after inoculation, select cytokine and chemokine levels were quantified and CFU E. coli K1 in blood and peritoneal wash fluid (PWF) was quantified.

Results: Neonatal offspring born to mice immunized with ENT-BSA had decreased mortality compared to those born to mothers receiving PBS, ENT, or BSA [% mortality at 120 hours: ENT-BSA 68 (n=53), PBS 89 (n=56), ENT 89 (n=49), BSA 97 (n=30); p< 0.01]. 12 hours after inoculation, bacterial burden in PWF was decreased by the ENT-BSA conjugate vaccine [log CFU: ENT-BSA 3.347 (n=20), PBS 4.634 (n=17), ENT 4.731 (n=12), BSA 4.505 (n=19); p< 0.05] but there was no difference in blood [log CFU: ENT-BSA 1.551 (n=19), PBS 2.229 (n=18), ENT 2.496 (n=12), BSA 1.931 (n=18)]. However, there was a decrease in bacteremia [% bacteremia: ENT-BSA 74, PBS 100, ENT 100, BSA 88; p< 0.05]. Of measured cytokines and chemokines, MCP-1 was increased in the PWF of ENT-BSA offspring (ENT-BSA 36301 vs PBS 16743 pg/mL; p< 0.05) and IL-6, MCP-1, IL-10, MIP-1α, and TNFα were decreased in the blood.

Conclusions: Immunization of adult female mice with an ENT-BSA conjugate vaccine protected their offspring from mortality due to E. coli K1 early-onset sepsis and was associated with decreased peritoneal bacterial burden, decreased rates of bacteremia, increased peritoneal MCP-1, and decreased systemic inflammation. These studies suggest that, by targeting ENT with a conjugate vaccine, we may dramatically reduce neonatal morbidity and mortality due to infection.