Our findings do indicate that augmenting immunity to Stx with immunity to PNAG might have the potential to reduce diarrhea or shedding of STEC in either animals or humans

Our findings do indicate that augmenting immunity to Stx with immunity to PNAG might have the potential to reduce diarrhea or shedding of STEC in either animals or humans. Immunity to pathogens confined to the GI tract requires that immune effectors such as antibodies, complement, and phagocytes gain access to the infecting organisms. to challenge with ~8 108 to 9 108 CFU of strain EDL933/mouse. strain J. Strain J, a UTI isolate, was injected i.p. 24?h after Bifenazate i.p. injection of 200?l of the indicated serum. After 48 h, the mice were euthanized, their livers were removed and homogenized, and bacterial levels were enumerated. Each symbol represents the value (CFU/gram) for an individual mouse. The black horizontal lines represent the median values for Bifenazate the groups of mice. The and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 Bifenazate strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups. IMPORTANCE The presence of Bifenazate poly-(STEC) bacteria are serious causes of infection whose virulence is dependent on elaboration of Stx, we prepared a vaccine containing a synthetic nonamer of PNAG (9GlcNH2) conjugated to Shiga toxin 1b subunit (9GlcNH2-Stx1b) to evaluate bacterial killing, toxin neutralization, and protective efficacy in infant mice. All nine (100%) clinical strains of STEC from different serogroups expressed PNAG. Vaccine-induced antibody mediated killing of STEC and neutralization of both Stx1 and Stx2. Passive administration of antibody to the conjugate showed protection requiring immunity to both PNAG and Stx for O157 strains, although for an O104 strain, antibody to PNAG alone was protective. Immunity to PNAG may contribute to protection against STEC infections. INTRODUCTION Outbreaks and sporadic cases of intestinal infections caused by Shiga toxin (Stx)-producing O157:H7 remains the most common STEC serogroup (1), additional STEC serogroups are being reported more frequently as causes of infection (2). In 2011, a large outbreak of diarrhea and hemolytic-uremic syndrome (HUS) caused by a novel Stx-producing strain of serogroup O104:H4 occurred in Germany (3), and a recent study of virulence factors of this strain in an infant rabbit model of intestinal colonization and diarrhea indicated that Stx and chromosomally encoded autotransporters, but not the aggregative adherence plasmid pAA, were required for disease induction (4). STEC bacteria are estimated to cause over 265,000 cases of infection annually in the United States, with more than 3,600 hospitalizations and 30 deaths (5). The gastrointestinal illnesses caused by STEC range from nonbloody diarrhea to hemorrhagic colitis, and approximately 5% to 10% of patients with STEC infections develop HUS, a life-threatening complication, with a case fatality rate of 3% to 5% (6). In the 2011 epidemic in Germany, HUS developed in >20% of individuals infected with the O104:H4 strain (3, 7). Shiga toxins are the principal cause of the diarrhea and HUS associated with STEC infections (8). They are categorized into two antigenically distinct groups, Stx1 and Stx2, which are potent cytotoxins composed of a single toxic A subunit and five B subunits (8). Stx2 is more frequently associated with severe disease and is more potent in toxicity and lethality models in mice (9). Treatment of STEC infection is supportive, and antibiotic use is controversial (10), as it has been reported to promote production and release of the phage-encoded Stxs (11, 12) and may increase the risk of HUS (13). However, not all studies have detected an impact DLL4 of antibiotic treatment on the development of HUS (14). In light of this controversy, vaccines and/or immunotherapeutics Bifenazate that target Shiga toxins are considered a valuable approach for prevention and treatment of these common infections. To date, several Shiga toxin-based vaccine strategies using either nontoxic subunits, recombinant proteins, or inactivated holotoxins have been undertaken (15,C17), and several protein-based vaccines have been shown to reduce fecal shedding and carriage in cows (14). In addition to Stx, other vaccine antigens that are under development include the lipopolysaccharide (LPS).