An anti-Shiga toxin VHH nanobody multimer protects mice against fatal toxicosis when administered intramuscularly as repRNA

Abstract

Hemolytic uremic syndrome (HUS) is a systemic sequelae from gastrointestinal infection with Shiga toxin (Stx) producing Escherichia coli (STEC) that can result in acute kidney injury, lasting renal disease, and death. Despite a window for intervention between hemorrhagic diarrhea and onset of HUS, no specific therapies exist to prevent or treat HUS following STEC infection. Furthermore, there is no way to predict which patients with STEC will develop HUS or any rapid way to determine which Stx variant is present. To address this, we have broadened the therpay to neutralize additional toxin variants. It contains a multimer of nanobodies derived from camelid heavy chain antibody fragments (VHHs). An improved VHH-based neutralizing agent (VNA2) is delivered intramuscularly as RNA combined with LION nanoparticles rather than mRNA, that replicates on administration (repRNA), resulting in a rapidly circulating VNA that can bind systemic toxin. The RNA/VNA2-Stx administered intramuscularly prevents toxicity and death in a mouse model of acute Stx toxicity.

Keywords: E. coli O157; Shiga toxins; Stx mouse toxicity model; Stx treatment.

Fig 1

Stx2d-binding ELISA comparing various Stx2a-neutralizing VHHs. Stx2d-binding was assessed using a mAb-capture ELISA as described in Materials and Methods. VHHs or VHH dimers were tested in a dilution series to assess their apparent affinities for Stx2d.

Fig 2

Stx2a toxicity as demonstrated by the probability of survival for mice given a dose ranging from 15 to 90 ng (n = 5 mice each). Stx2a was delivered intraperitoneally.

Fig 3

RNA/VNA2-Stx delivered intramuscularly at different timepoints relative to Stx2a (15 ng) administration. Comparison of survival (left) and body weight (right) for the three studies: (top) iteration 1, (middle) iteration 2, and (bottom) iteration 3.

Fig 4

Comparison of survival for RNA/VNA2-Stx administration across the study iterations. Protection from lethal toxicity was dependent on timing of RNA/VNA2-Stx administration in relation to Stx2a challenge. Mice given RNA/VNA2-Stx 3, 2, or 1 day before Stx2a had protection while those given on the same day or 1 day after Stx2a did not survive beyond 3 days post-challenge.

Fig 5

RNA/VNA2-Stx treatment was protective in both male (left) and female (right) mice. Data are from iteration 3 where RNA/VNA2-Stx was administered 1 day before Stx2a challenge.

Fig 6

VHH levels in serum quantified by ELISA 4 days after RNA/VNA2-Stx administration. Range 190–919 pM. [VHH] did not differ by sex (P = 0.1827).