Since 1961, there has been an ongoing cholera pandemic, with a rise in cases in 2022. Cholera, caused by the bacterium Vibrio cholerae, is known for its hallmark symptom: severe watery diarrhea, driven by cholera toxin (CTX). It affects all age groups, but especially children under the age of five.
Marcus Petersson recently concluded his doctoral research with this study and works at Bactolife alongside Thrane.
Bactolife A/S
Although oral cholera vaccines exist, the recent spike in cases and limited production capacity have outpaced the global vaccine supply, prompting researchers to explore alternative protection strategies against CTX. One promising approach is the use of single-domain antibodies (VHHs, or nanobodies) seen in camels, llamas, and alpacas. Unlike conventional antibody proteins, which have heavy and light chains, VHHs only consist of heavy chains. These are small, highly specific, and stable in the harsh environment of the gastrointestinal tract.
In a collaboration between the Technical University of Denmark, Harvard Medical School, and Bactolife—a biotech company focused on gut health through binding proteins—biotechnologists Marcus Petersson, Sandra Wingaard Thrane, and their colleagues developed an orally delivered VHH protein that binds to CTX. This engineered protein blocks the interaction between the toxin and a gut receptor, effectively preventing the domino effect that causes diarrhea. Published in Nature Communications, the study suggests this VHH-based approach could serve as a dietary supplement to help reduce cholera symptoms.1
CTX consists of five B-subunits (CTXB), which bind to the GM1 ganglioside receptor on human intestinal cells, and one A-subunit, which is then released into the cells and triggers the cellular response that leads to diarrhea. This CTX–GM1 receptor interaction is central to the development of symptoms.
Because of this, the researchers hypothesized that blocking this toxin with a binding protein would have protective effects. To develop their ideal nanobody, the team conducted a functional screen from two alpacas immunized with CTXB and created a library of VHHs. “We identified several good binding proteins that block this interaction between the toxin and receptor, but there was one of them that really stood out,” said Petersson. That standout was the monovalent BL3.1, which has one binding site. To boost its binding power, they linked two BL3.1 units together to engineer BL3.2.
They found that BL3.2 was stable in a simulated gastric fluid environment. Then, in a human intestinal cell assay, they incubated cells with a mixture of BL3.2 and CTX. The team found that BL3.2 also latched onto the GM1-binding pocket of CTXB and blocked toxin activity.
Encouraged by these results, the team moved to an infant mouse cholera model—ideal for mimicking human infection due to the mice’s immature immune system.2 Mice given oral delivery of BL3.2 before and after CTX exposure showed no signs of diarrhea and had significantly less weight loss and fluid accumulation than mice not given BL3.2.
Sandra Wingaard Thrane leverages her background in molecular medical microbiology to develop binding proteins to maintain healthy gut microbiomes.
Bactolife A/S
They then tested BL3.2 against a virulent V. cholerae strain from a 2022 clinical isolate and saw similar protection in the BL3.2-treated group. The harmful effects of the CTX abrogated, but the researchers also observed a 10-fold reduction of V. cholerae colonization levels in the small intestine of mice. “That was really cool to see,” said Petersson.
Michiel Harmsen, an independent consultant who has studied VHHs from a veterinary perspective for therapeutic and diagnostic applications, noted, “It was nice that they showed that the bivalent version was more potent than the monovalent version.”
Harmsen, who has worked with other toxins, explained that botulinum, for instance, can be neutralized not by blocking receptor binding but through toxin aggregation. This made him curious if BL3.2 or another VHH might perform similarly without blocking GM1 binding. He added that studying whether a bivalent VHH could cause toxin aggregation may offer deeper insights into the mode of action.
Meanwhile, the team plans to evaluate different activities, such as dosing and translatability to other gastrointestinal pathogens, of these binding proteins, in hopes of scaling their application to humans. “This is kind of like a new, exciting potential tool in the toolbox that can further both our understanding bacterial pathogenesis, but also of how we can fortify our gut in these types of situations where diarrheal outbreaks occur,” remarked Thrane.
Disclosure of Conflicts of Interest: Marcus Petersson and Sandra Wingaard Thrane are both affiliated with Bactolife A/S.
- Petersson M, et al. Orally delivered toxin-binding protein protects against diarrhoea in a murine cholera model. Nat Commun. 2025;16(1):2722.
- Sit B, et al. Animal models for dissecting Vibrio cholerae intestinal pathogenesis and immunity. Curr Opin Microbiol. 2022;65:1-7.
