Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model.

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Cost of Manufacturing for Recombinant Snakebite Antivenoms.

Snakebite envenoming is a neglected tropical disease that affects millions of people across the globe. It has been suggested that recombinant antivenoms based on mixtures of human monoclonal antibodies, which target key toxins of medically important snake venom, could present a promising avenue toward the reduction of morbidity and mortality of envenomated patients. However, since snakebite envenoming is a disease of poverty, it is pivotal that next-generation therapies are affordable to those most in need; this warrants analysis of the cost dynamics of recombinant antivenom manufacture. Therefore, we present, for the first time, a bottom-up analysis of the cost dynamics surrounding the production of future recombinant antivenoms based on available industry data. We unravel the potential impact that venom volume, abundance of medically relevant toxins in a venom, and the molecular weight of these toxins may have on the final product cost. Furthermore, we assess the roles that antibody molar mass, manufacturing and purification strategies, formulation, antibody efficacy, and potential cross-reactivity play in the complex cost dynamics of recombinant antivenom manufacture. Notably, according to our calculations, it appears that such next-generation antivenoms based on cocktails of monoclonal immunoglobulin Gs (IgGs) could be manufacturable at a comparable or lower cost to current plasma-derived antivenoms, which are priced at USD 13-1120 per treatment. We found that monovalent recombinant antivenoms based on IgGs could be manufactured for USD 20-225 per treatment, while more complex polyvalent recombinant antivenoms based on IgGs could be manufactured for USD 48-1354 per treatment. Finally, we investigated the prospective cost of manufacturing for recombinant antivenoms based on alternative protein scaffolds, such as DARPins and nanobodies, and highlight the potential utility of such scaffolds in the context of low-cost manufacturing. In conclusion, the development of recombinant antivenoms not only holds a promise for improving therapeutic parameters, such as safety and efficacy, but could possibly also lead to a more competetive cost of manufacture of antivenom products for patients worldwide.

Toxin Neutralization Using Alternative Binding Proteins.

Animal toxins present a major threat to human health worldwide, predominantly through snakebite envenomings, which are responsible for over 100,000 deaths each year. To date, the only available treatment against snakebite envenoming is plasma-derived antivenom. However, despite being key to limiting morbidity and mortality among snakebite victims, current antivenoms suffer from several drawbacks, such as immunogenicity and high cost of production. Consequently, avenues for improving envenoming therapy, such as the discovery of toxin-sequestering monoclonal antibodies against medically important target toxins through phage display selection, are being explored. However, alternative binding protein scaffolds that exhibit certain advantages compared to the well-known immunoglobulin G scaffold, including high stability under harsh conditions and low cost of production, may pose as possible low-cost alternatives to antibody-based therapeutics. There is now a plethora of alternative binding protein scaffolds, ranging from antibody derivatives (e.g., nanobodies), through rationally designed derivatives of other human proteins (e.g., DARPins), to derivatives of non-human proteins (e.g., affibodies), all exhibiting different biochemical and pharmacokinetic profiles. Undeniably, the high level of engineerability and potentially low cost of production, associated with many alternative protein scaffolds, present an exciting possibility for the future of snakebite therapeutics and merit thorough investigation. In this review, a comprehensive overview of the different types of binding protein scaffolds is provided together with a discussion on their relevance as potential modalities for use as next-generation antivenoms.