Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle

Abstract

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves’ age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids

“…Colostral nStx1Ab and nStx2Ab were effectively transferred to calves with nStx1Ab titers clearly exceeding nStx2Ab titers in the VAC+ as well as the VAC− group similar to what was observed after natural Stx exposure and after rStx MUT vaccination [ 12 , 24 , 40 ]. Active rStx MUT immunization in week 5 and 8 did not result in a detectable increase of nStx1Ab in calves’ sera, different from calves vaccinated after vanishing of maternal antibodies [ 24 ]. High maternal titers may have impaired the success of the rStx1 MUT vaccination but a significant humoral immune response was achieved by rStx2 MUT immunization of calves with no or low serum nStx2Ab titers at the time of active vaccination.…”

“…Recombinant Stx (rStx1 WT and rStx2 WT ) and genetically inactivated recombinant Stx toxoids (rStx1 MUT and rStx2 MUT ) were previously generated and tested by Kerner et al [ 24 ]. rStx1 MUT and rStx2 MUT preparation were adjusted separately with NaCl solution (0.89%) to 0.75 Mio verocytotoxic doses 50% (CD 50 ) equivalents [ 24 ] each in 1.4 mL and frozen at −20 °C.…”

“…0.75 µg/mL, or rStx1 MUT or rStx2 MUT f.c. 200 CD 50 equivalent [ 24 ] were added in 1 mL/well. Plates were incubated for 5 days at 5% CO 2 and 37 °C.…”

“…Inactivation of Stx by genetic modification located within the enzymatically active cleft of Stx resulted in toxoids (rStx1 MUT and rStx2 MUT ) with retained antigenicity and immunogenicity but lost immunomodulatory properties in cattle [ 24 ]. Immunisation of sows with Stx2e toxoid [ 25 , 26 ] was shown to trigger maternal immunity which protects offspring against edema disease [ 27 ] and fully protected the animals when challenged with native Stx2e [ 28 ].…”

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7

“…As mentioned above, Stx might act as an immunomodulating agent during STEC infections in cattle and is a virulence factor harboured by all STEC strains, which makes them interesting vaccine candidates [36]. Considering that Stx2 is the most pathogenic Stx toxin[37], we chose a Stx2B-based immunogen to raise antibodies against Stx2.…”