A recombinant O-polysaccharide-protein conjugate approach to develop highly specific monoclonal antibodies to Shiga toxin-producing Escherichia coli O157 and O145 serogroups.

Daniela S Castillo,Luciano J Melli,Andrés E Ciocchini,Diego A Rey Serantes,Diego J Comerci,Alejandro Cassola,Juan E Ugalde,Chandra Shekhar Bakshi

doi:10.1371/journal.pone.0182452

Abstract

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.

Highlights

Shiga toxin-producing Escherichia coli (STEC) pathovar is associated with sporadic cases and outbreaks of diarrhea, bloody diarrhea (BD) and hemolytic-uremic syndrome (HUS), a systemic illness defined by the clinical triad of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure [1, 2]
We observed a 33% titer increase at a 1:6400 sera dilution and a 184% titer increase at a 1:25600 sera dilution after the immunization with O157-AcrA and O145-AcrA, respectively (S1 Table). This suggests that the final boosts with bacterial engineered O-PS-protein conjugates led to a selective proliferation of B-cell clones specific to O157 or O145 antigens, probably enhancing the affinity of the secreted monoclonal antibodies (mAbs) against the respective O-PS
Urbana or Y. enterocolitica O9 (Fig 2D), given that E. coli O145 does not share common structural epitopes in the O-PS repeating unit of its LPS with these enterobacteria [42]. These results suggest that the O157 and O145 mAbs are highly specific to the corresponding O-PS and do not present cross-reactions with the O antigen of other STEC priority serogroups or other enterobacteria that are associated with similar clinical manifestations

Summary

Introduction

Shiga toxin-producing Escherichia coli (STEC) pathovar is associated with sporadic cases and outbreaks of diarrhea, bloody diarrhea (BD) and hemolytic-uremic syndrome (HUS), a systemic illness defined by the clinical triad of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure [1, 2]. STEC strains are characterized by the production of the cytotoxins Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2), which have a pivotal role in BD and HUS pathogenesis [3, 4]. Ruminants such as cattle are major reservoirs for pathogenic STEC and exposure to their fecal matter is considered the most frequent source of human illness. The mortality rate in acute phase is 2 to 5%, but since 20 to 30% of HUS-affected children develop long-term renal sequelae, HUS constitutes the second cause of chronic renal failure and accounts for 20% of kidney transplants in children and adolescents [11, 15,16,17,18]

Methods

Results

Conclusion