Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection

Abstract

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies. Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.

FIG. 1.

Western blot analysis of HuMAb binding to Stx2. Covalently cross-linked Stx2 was electrophoresed, transferred to a nylon membrane, and then divided into individual lanes. As indicated, each HuMAb was incubated in a separate lane. Polyclonal rabbit anti-Stx2 serum and the Stx2 B-subunit-specific murine MAb 3D1 were included as positive controls. The identities of significant major bands were determined from published molecular weights of the A and B subunits of Stx2 (17) and are indicated to the left of each panel. Anti-Stx2 contains antibodies which bind both the A and B subunits of Stx2; MAb 3D1 binds the B subunit and thus binds A-B complexes in addition to B-subunit monomers and multimers. The relative intensity of binding is determined not only by whether the particular Stx2 entity is recognized by a MAb but also by the percentage of each complex present within the preparation of cross-linked Stx2. With the exception of HuMAbs 3F6, 4G7, 5H8, and 6G3, all are specific for the A subunit based on the ability to bind A-B complexes but not B-subunit monomers or multimers. HuMAbs 3F6, 4G7, 5H8, and 6G3 are specific for the B subunit of Stx2 based on the ability to bind B-subunit monomers and multimers in addition to A-B-subunit complexes. Similar to the murine MAb 3D1, HuMAbs 3F6 and 4G7 bind the 2B and 3B multimers, whereas 5H8 binds the 2B multimer only and 6G3 binds the 1B monomer and 2B and 3B multimers, suggesting differential epitope specificity.