Protection from hemolytic uremic syndrome by eyedrop vaccination with modified Enterohemorrhagic E. coli\00A0outer membrane vesicles

Abstract

Enterohemorrhagic E. coli (EHEC) can cause severe diarrhea, hemorrhagic colitis, which is often accompanied by hemolytic anemia, thrombocytopenia, and acute renal failure. But up until now, specific treatments except public health and sanitation are not available. Accordingly, the development of effective vaccines preventing EHEC O157:H7 infection is of prime research interest. Outer membrane vesicles (OMVs) are spherical membrane blebs shed by Gram-negative bacteria and vaccination with OMVs has been reported to induce an immune response and protect vaccinated organisms. However, an effective and safe OMV vaccine for protection from EHEC O157:H7 infection has not been reported, presumably because OMVs generated from EHEC O157:H7 are intrinsically toxic due to presence of Shiga toxin (STx) and lipopolysaccharide (LPS) endotoxin. Modified OMVs (mOMVs) from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria have less toxic penta-acylated lipid A moiety. Moreover, they do not contain toxic STxA subunit proteins. WaaJ-mOMVs with additional waaJ mutation do not have the polymeric O-antigen of O157 LPS.This study is to explore whether eyedrop vaccination using mOMVs and waaJ-mOMVs is effective for protecting against hemolytic uremic syndrome (HUS) caused by EHEC O157:H7 infection.Modified OMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB) were administered to BALB/c mice by eyedrop at the onset of experiments. Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. In order to increase intestinal mucosal immunity, cholera toxins (CT) or polyinosinic:polycytidylic acid (polyI:C) were administered with mOMVs. As parameters for evaluation of the mOMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. Lastly, to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice.The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses. mOMVs plus CT increase systemic IgG antibody response and intestinal IgA antibody response. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not.In conclusion, the current study showed that eyedrop vaccination using mOMVs of EHEC O157 bacteria sufficiently induced immunogenicity in mice and intact O157 LPS antigen could be a critical component for enhancing the immunogenicity of the mOMVs. Moreover, eyedrop vaccination with mOMVs was shown to be effective for preventing HUS pathogenesis in mice against challenge with HUS-causative wtOMVs and thus hold promise for the prevent of EHEC-related pathogenicity.