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Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper, in a mouse model of envenoming that mimics a snakebite. While clinical ADET related to snake venom has not yet been reported in humans, this report of ADET of a toxin from the animal kingdom highlights the necessity of assessing even well-known antibody formats in representative preclinical models to evaluate their therapeutic utility against toxins or venoms. This is essential to avoid potential deleterious effects as exemplified in the present study., (© 2024. The Author(s).)

Antibodies with cross-reactive binding and broad toxin-neutralizing capabilities are advantageous for treating indications such as infectious diseases and animal envenomings. Such antibodies have been successfully selected against closely related antigens using phage display technology. However, the mechanisms driving antibody cross-reactivity typically remain to be elucidated. Therefore, we sought to explore how a previously reported phage display-based cross-panning strategy drives the selection of cross-reactive antibodies using seven different snake toxins belonging to three protein (sub-)families: phospholipases A 2 , long-chain α-neurotoxins, and short-chain α-neurotoxins. We showcase how cross-panning can increase the chances of discovering cross-reactive single-chain variable fragments (scFvs) from phage display campaigns. Further, we find that the feasibility of discovering cross-reactive antibodies using cross-panning cannot easily be predicted by analyzing the sequence, structural, or surface similarity of the antigens alone. However, when antigens share the (exact) same functions, this seems to increase the chances of selecting cross-reactive antibodies, which may possibly be due to the existence of structurally similar motifs on the antigens., (© 2023. The Author(s).)

Viruses, such as influenza A, typically bind to the plasma membrane of their host by engaging multiple membrane receptors in parallel, thereby forming so-called multivalent interactions that are created by the collective action of multiple weak ligand-receptor bonds. The overall interaction strength can be modulated by changing the number of engaged receptors. This feature is used by viruses to achieve a sufficiently firm attachment to the host's plasma membrane but also allows progeny viruses to leave the plasma membrane after completing the virus replication cycle. Design of strategies to prevent infection, for example, by disturbing these attachment and detachment processes upon application of antivirals, requires quantification of the underlying multivalent interaction in absence and presence of antivirals. This is still an unresolved problem, as there is currently no approach available that allows for determining the valency (i.e., of the number of receptors bound to a particular virus) on the level of single viruses under equilibrium conditions. Herein, we track the motion of single influenza A/X31 viruses (IAVs; interacting with the ganglioside GD1a incorporated in a supported lipid bilayer) using total internal reflection fluorescence microscopy and show that IAV residence time distributions can be deconvoluted from valency effects by taking the IAV mobility into account. The so-derived off-rate distributions, expressed in dependence of an average, apparent valency, show the expected decrease in off-rate with increasing valency but also show an unexpected peak structure, which can be linked to a competition in the opposing functionalities of the two influenza A virus spike proteins, hemagglutinin (HA), and neuraminidase (NA). By application of the antiviral zanamivir that inhibits the activity of NA, we provide direct evidence, how the HA/NA balance modulates this virus-receptor interaction, allowing us to assess the inhibition concentration of zanamivir based on its effect on the multivalent interaction.