Efficacy of EHEC gold nanoparticle vaccines evaluated with the Shiga toxin-producing Citrobacter rodentium mouse model

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a group of pathogenic bacteria responsible for several foodborne-associated outbreaks of human diarrheal disease. Although EHEC is a major cause of morbidity as well as the condition known as hemolytic uremic syndrome, linked to the production of Shiga toxins (Stx), there are no licensed vaccines approved for human use. Fully assessing vaccine efficacy against EHEC infections is challenging because conventional adult mice are inherently resistant to EHEC infection and do not develop hallmark symptoms of the disease. Therefore, in this study, we utilized a strain of the murine pathogen Citrobacter rodentium that has been lysogenized to produce Stx2d, which consistently causes lethal infection and other measurable disease outcomes following oral challenge, to further evaluate the protective efficacy of our previously formulated EHEC gold nanoparticle (AuNP) vaccines. Using this model, along with a non-Stx2d-producing C. rodentium strain, we assessed the protection conferred by AuNPs conjugated to three EHEC antigens: EscC, LomW, and intimin (Eae). We demonstrated that intranasally immunizing mice with AuNP-Eae provides partial protection against mortality caused by Stx2d-producing C. rodentium and that AuNP-EscC and AuNP-Eae moderately reduce intestinal burden of the non-Stx2d-producing C. rodentium strain compared with adjuvant-only-treated mice. Additionally, the AuNP-protein vaccines induced antigen- and pathogen-specific serum IgG and fecal IgA against both EHEC and C. rodentium while in vitro functional assays indicate that the elicited antibodies are bactericidal and prevent adherence of C. rodentium to intestinal epithelial cells. We propose that C. rodentium murine data could translate into correlates of protection. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) remains an important cause of diarrheal disease and complications worldwide, especially in children, yet there are no available vaccines for human use. Inadequate pre-clinical evaluation due to inconsistent animal models remains a major barrier to novel vaccine development. We demonstrate the usefulness of Stx2d-producing Citrobacter rodentium in assessing vaccine effectiveness because it more closely recapitulates human disease caused by EHEC.

Further Evaluation of Enterohemorrhagic Escherichia coli Gold Nanoparticle Vaccines Utilizing Citrobacter rodentium as the Model Organism

“…Further in vitro functional studies demonstrated that the generated sera antibodies were bactericidal and reduced attachment of C. rodentium to IECs [24]. Additionally, we found that immunization with AuNP-Eae prevented death and limited intestinal damage in over 30% of DBS770-infected mice [24]. Also, both AuNP-EscC and AuNP-Eae moderately reduced the intestinal bacterial burden at 14 days post-infection (dpi) with DBS771 compared to control mice [24].…”

“…Before assessing the EHEC AuNP vaccines, we first confirmed that we could recapitulate the established disease kinetics of both DBS770 and a non-Stx2d-producing C. rodentium strain (ATCC DBS771) [24]. We found that infection of female 10-12-week-old C57BL/6 mice with either strain resulted in robust colonization over 14 days and a significant increase in intestinal inflammation markers (like LCN-2), and that infection with DBS770 led to 100% mortality, significant weight loss, and inflammatory intestinal damage [24]. Next, we intranasally immunized mice using AuNPs linked to the EHEC antigens EscC, LomW, or Eae, and all three antigens stimulated both antigen-and pathogen-specific sera IgG and fecal IgA.…”

“…Next, we intranasally immunized mice using AuNPs linked to the EHEC antigens EscC, LomW, or Eae, and all three antigens stimulated both antigen-and pathogen-specific sera IgG and fecal IgA. Further in vitro functional studies demonstrated that the generated sera antibodies were bactericidal and reduced attachment of C. rodentium to IECs [24]. Additionally, we found that immunization with AuNP-Eae prevented death and limited intestinal damage in over 30% of DBS770-infected mice [24].…”

“…Bacterial antigen cloning was performed as previously described [24]. Briefly, DNA from EHEC strain EDL933 was extracted with a DNeasy Blood and Tissue kit (Qiagen, Germantown, MD, USA), according to the manufacturer's instructions.…”

“…Additionally, we found that immunization with AuNP-Eae prevented death and limited intestinal damage in over 30% of DBS770-infected mice [24]. Also, both AuNP-EscC and AuNP-Eae moderately reduced the intestinal bacterial burden at 14 days post-infection (dpi) with DBS771 compared to control mice [24]. Therefore, the purpose of the current study was to use the valuable information collected from our first vaccination trial to further evaluate our EHEC AuNP vaccines.…”