Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

Abstract

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.The enterohemorrhagic group of Escherichia coli (EHEC) causes hemorrhagic colitis and, in anywhere from 5 to 15% of infected individuals, primarily very young and elderly subjects, a serious clinical complication called hemolytic-uremic syndrome (HUS) (8,23,45). HUS is characterized by a triad of clinical features, including hemolytic anemia, thrombocytopenia, and ultimately, acute renal failure. As well, in the most severe cases, various degrees of central nervous system involvement can become apparent. EHEC is also referred to as Shiga toxigenic E. coli because this organism expresses exotoxins that are biochemically related to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (43). Once EHEC has colonized the intestines, it is possible for Shiga toxins to be translocated into the submucosal compartment of the gut (3,19). From there, the toxins can be transported, possibly on the surface of polymorphonuclear leukocytes (23,46,47), to extraintestinal organs and tissues, primarily the kidneys, where Shiga toxinmediated damage to endothelial cells in the glomerular capillaries induces a cascade of microangiopathic events leading ultimately to HUS (45).The Shiga toxins produced by EHEC are classified into two families, Stx1 and Stx2, also commonly referred to as verotoxin or verocytotoxin 1 and 2, according to their genetic and antigenic relatedness to the prototypic Stx produced by S. dysenteriae. In this classification scheme, Stx1 is virtually identical to the prototypic S. dysenteriae Stx (3...

Live Attenuated Shigella dysenteriae Type 1 Vaccine Strains Overexpressing Shiga Toxin B Subunit

“…Shiga toxin is composed of a single A subunit that is enzymatically active and five B subunits that form a pentamer which is responsible for target cell binding. While the A subunit must be deleted in a live vaccine strain to completely abolish toxin enzymatic activity, the B subunit has been demonstrated to elicit antibodies that can neutralize toxin activity (1,6,8,27). Here, a series of vaccine candidates were evaluated in the guinea pig model and found to protect against challenge with wild-type S. dysenteriae 1 and induce Shiga toxin neutralizing antibody responses.…”

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

“…The anti-Stx2B sera employed to monitor the antigen in bacterial extracts detected a minimum of 120 ng of Stx2B toxin, while other sera, such as those previously used to quantify LTB, had a higher sensitivity (16,17). Additionally, the low immunogenicity of Stx2B, under different vaccine formulations, has been previously reported (1,2,3,11,12). Thus, it is possible that either the low amount of Stx2B produced by the B.subtilis vaccine strains and/or the reduced immunogenicity of the protein contributed to the low antibody levels detected in serum and fecal samples collected from vaccinated mice.…”

“…There is neither a vaccine nor an effective treatment to avoid EHEC infection or HUS control (9) but, given the importance of Stx in the EHECassociated diseases, several attempts to use Stx toxoids or the Stx B subunit (Stx2B) have been reported (1,3,11,12,20,22).…”

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part II: Vaccines forShigella,Salmonella,enterotoxigenicE. coli(ETEC) enterohemorragicE. coli(EHEC) andCampylobacter jejuni

“…These include the use of Stx toxoids, 139,140 hybrid Stx toxoids, 141,142 proteins secreted by TTSS, [143][144][145][146][147][148] chimeric proteins, [148][149][150][151][152][153] DNA vaccines, 154,155 live attenuated vaccines [156][157][158][159] and phantom strains of O157: H7. [160][161][162] These vaccine candidates have had variable success, and their results are discussed extensively in 2 recent reviews.…”