Thursday, November 21, 2024
11/21/2024
Title: Immune-based therapy against STEC intoxication and HUS Shiga toxin (Stx) producing E. coli (STEC) are the bacteria responsible for outbreaks and sporadic acute cases of diarrhea in humans, primarily children, that can lead to hemolytic uremic syndrome (HUS) resulting in acute kidney failure and CNS abnormalities. No therapy currently exists, and antibiotics are contraindicated. We, and others, have shown that human monoclonal antibodies (HuMab) against Shiga toxins (Stx1 and Stx2) were protective in animal models and safe demonstrating that these toxins indeed are the primary cause of STEC associated HUS, and that specific HuMab is an effective immune-based therapy. However, such treatment is costly and cumbersome with limited shelf-life and requires intravenous administration to patients at risk of developing HUS. Here we propose a much-simplified strategy using camelid-derived (VHH) antibodies delivered by mRNA nanoparticles to prevent or arrest development of HUS. It is expected to be a low-cost and safe therapy, given in a single intramuscular (IM) injection to patients exposed to or presenting with STEC infection or bloody diarrhea. The antitoxin product is based on a highly innovative VHH-based neutralizing agents (VNA) platform developed at our laboratory. Linking VHHs together into VNAs leads to enhanced toxin neutralization potency and permits the targeting of multiple toxins with a single agent. Such VNAs have now been successfully generated and tested for efficacy in animal models against botulinum neurotoxin, Clostridium difficile TcdA and TcdB, ricin, anthrax, and Stx1 and Stx2 toxins. A single VNA protein targeting both Stx1 and Stx2 (VNA-Stx), injected IP or IM, potently neutralized both Stx1 and Stx2. We previously reported data showing that: a) multiple IP or IM administrations of VNA-Stx protein protected STEC-infected piglets with diarrhea from developing systemic intoxication, and; b) a single IM injection of an adenovirus (Ad) vector with a secretory VNA-Stx1/Stx2 transgene was equally protective when given to infected piglets [2]. More recently, we developed a heterotetrameric VNA that more broadly neutralizes diverse Stx2 natural variants that cause human disease. Here we propose to complete development of a VNA capable of potent neutralization of all known STEC human pathogens, and explore and evaluate more practical, simplified VNA delivery systems using various mRNA delivery technologies (Aim 1). Our hypothesis is that the application of mRNA technology to express a single, multi-specific VNA will be more effective than HuMab, simpler to manufacture at a fraction of the cost of HuMab and permit the much more practical intramuscular administration. This hypothesis will be first tested in mice against toxicosis (Aim 2) and against oral infection of STEC in the gnotobiotic (GB) piglet model. This model leads to symptoms of acute diarrhea and serious colonic mucosal damage followed ~48 hours later with systemic vascular-mediated intoxication (fatal neurologic symptoms) that closely mimic some of the sequence of events observed in humans infected with STEC strains who go on to develop HUS (Aim 3).
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