VHH AGAINST THE B SUBUNIT OF SHIGA TOXIN 2 (STX2B) AS NOVEL THERAPEUTIC TOOLS AGAINST HUS (PART II).

MEJIAS MP; FERNÁNDEZ BRANDO, ROMINA J; HIRIART, Y; BRUBALLA, A; RAMOS, M. VICTORIA; ABREY-RECALDE MARIA JIMENA; GOLDBAUM, FERNANDO; ZYLBERMAN, VANESA; PALERMO, MARINA S

Boston

Congreso; 9th International Symposium On Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections.; 2015

VTEC

Introduction: Infection with Stx2-producing Escherichia coli (STEC) can progress to Hemolytic UremicSyndrome (HUS), for which no effective therapy is presently available. Thevariable chains of antibodies produced by camelids (vhh/nanobodies) exhibitcharacteristics that make them attractive candidates as therapeutic agents. Theaim of this work was to evaluate anti-Stx2B nanobodies as protective anti-Stx2tools for therapeutic ends. Methods: Three alternative formats wereevaluated: a monomeric anti-Stx2B vhh (vhh 2vb27), a bivalent molecule (twocopies of vhh 2vb27) and a heterotrimeric molecule (two copies of vhh 2vb27 and1 copy of an anti-human serum albumin vhh). Invitro neutralizing activity was measured by the Vero cell  assay. For in vivo protection studies Balb/c mice were injected i.v. with 1LD100Stx2 or i.g. with 4x1011 CFU/kg of STEC and inoculated i.p. with thedifferent vhh formats. The half-life of different vhh formats was determined bypersistence of antibody in plasma. Resultsand discussion: The bivalent and trivalent nanobodies showed a higherin vitro neutralizing activity thanthe monovalent vhh (mean of pmoles that neutralize 1CD50 Stx2 ±SD) monovalent:0.5±0.13, bivalent: 0.04±0.007*, trivalent: 0.06±0.04* (*p<0.05). However onlythe trivalent nanobody (0.1 pmoles) was able to fully protect mice againstmortality and Stx2-associated renal damage after i.v Stx2 or i.g. STECchallenge. The in vivo half-life studiesshowed that most of the monovalent vhh was removed within 5 minutes from plasma,the bivalent nanobody within 5 hours and the trivalent nanobody within 10 days.Implications: The heterotrimeric format showed the highest in vivo protection in both experimentalmodels, probably due to the longer invivo half-life as a result of the binding to serum albumin. Thesenanobodies are promising therapeutic agents against HUS.