Biology subjects

Laustsen, A. H.

Publications and source records attributed to Laustsen, A. H..

15 recordsLinked to original sources

bioRxiv2026

Accelerating multi-objective VHH discovery via integrated high-throughput selection and AlphaFold3-guided structure prediction

Discovering therapeutic antibodies that bind multiple related targets with high affinity and favourable biophysical properties remains challenging and resource intensive. For snakebite antivenoms, this challenge is critical as treatments must neutralise toxins across multiple snake species. We developed a pipeline combining high-throughput yeast screening, deep sequencing, and AlphaFold3 structure prediction to rapidly identify poly-specific variable domains of heavy-chain-only antibodies (VHHs) against long-chain -neurotoxins. Multiplexed yeast display screening generated a dataset of diverse candidates with varying binding specificities. AlphaFold3-generated VHH-toxin complex predictions enabled structure-based prioritisation that accurately predicted poly-specific binders targeting conserved epitopes across multiple toxins. These structural insights enabled computational optimisation of both affinity and solubility without disrupting target recognition. Experimental validation confirmed improved variants maintained broad specificity across toxins. This integrated approach accelerates multi-objective antibody discovery by predicting which candidates will bind multiple targets before extensive laboratory testing, providing a generalisable strategy applicable beyond antivenoms to any therapeutic requiring broad target coverage.

bioengineering↗

bioRxiv2025

Generalizable direct protein sequencing with InstaNexus

Protein-based therapeutics, such as antibodies and nanobodies, are not encoded in reference genomes, challenging their accurate characterization via standard proteomics. Current methods rely on indirect inference, fragmented outputs, and labor-intensive workflows, which hinder functional insights and routine application. Here, we present a generalizable, end-to-end workflow for direct protein sequencing, combining streamlined sample preparation, AI-driven de novo peptide sequencing, and tailored assembly to reconstruct contiguous protein sequences. A novel composite scoring framework prioritises longer assemblies and coverage, enhancing accuracy and reducing ambiguity. Validation across diverse protein modalities demonstrates its utility and ability to robustly sequence functionally critical regions of selected proteins. This workflow represents an advance in precision proteomics with promising applications in therapeutic discovery, immune profiling, and protein science.

bioinformatics↗

bioRxiv2024

Assessing AF2's ability to predict structural ensembles of proteins

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined, setting new benchmarks for accuracy and efficiency in the field. However, the fundamental mechanisms of biological processes at a molecular level are often connected to conformational changes of proteins. Molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein structures by manipulating the input multiple sequence alignment (MSA). These approaches operate under the assumption that the MSA also contains information about the heterogeneity of protein structures. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction focusing on the subsampling of the MSA as implemented in ColabFold and compare the obtained structures with ensembles obtained from MD simulations and NMR. As test cases, we chose four proteins namely the bovine pancreatic inhibitor protein (BPTI), thrombin and two antigen binding fragments (antibody Fv and nanobody), for which reliable experimentally validated structural information (X-ray and/or NMR) was available. Thus, we provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. In three out of the four test cases, we find structural variations fall within the predicted ensembles. Nevertheless, significant minima of the free energy surfaces remain undetected. This study highlights the possibilities and pitfalls when generating ensembles with AF2 and thus may guide the development of future tools while informing upon the results of currently available applications.

biophysics↗

bioRxiv2024

mRNA-delivered consensus allergens induce a neutralizing IgG response against food and pollen allergens

Pollen-food allergy syndrome (PFAS) affects a significant proportion of the global population with a major health and socioeconomic impact. Patients are generally treated against the major sensitized allergen which does not warrant protection against cross-reactive allergens, leading to long and ineffective treatment regimens. For food allergies, patient guidelines rely on source avoidance, leading to dietary restrictions and reduced quality of life - in particular for those suffering from PFAS. To overcome these limitations, we introduce a novel allergy immunotherapy (AIT) approach utilizing consensus allergens and mRNA technology to achieve broader, safer, and faster desensitization in PFAS patients. We first designed a consensus allergen of orthologs of non-specific Lipid Transfer Proteins (cnsLTP-1) representing a broad spectrum of nsLTP allergens prevalent in food and pollen sources. CnsLTP-1 was delivered to naive BALB/c mice using mRNA-lipid nanoparticles (mRNA-LNP) as vehicle, or by a traditional protein formulation, to assess if it elicits broad protection against allergens from different sources. Immunization with both mRNA-LNP and protein formulations demonstrated that cnsLTP-1-specific IgGs could be induced, whilst the mRNA-LNP formulation notably avoided the induction of allergen-specific IgEs. The induced antibodies were capable of recognizing and binding to a variety of nsLTPs, and effectively blocked the binding of allergens by allergic patient serum IgEs. This study thus demonstrates that the presented AIT strategy, based on mRNA-LNP technology and consensus allergens, could find clinical utility by addressing the limitations of current AIT. Further development of this technology platform could pave the way for more effective and patient-friendly treatments for PFAS and other cross-reactive allergies.

bioengineering↗

bioRxiv2023

Simplified post-assembly plasmid library amplification for increased transformation yields in E. coli and S. cerevisiae

Many biological disciplines rely upon the transformation of host cells with heterologous DNA, the limited efficiency of which can significantly hinder a researchers work. Directed evolution in particular typically requires the screening of large (thousand-billion member) libraries to identify sequences of interest, and the creation of these libraries at large enough scales to overcome transformation inefficiencies is a cost-and time-intensive process. We simplify this process by using Rolling Circle Amplification (RCA) to amplify in vitro plasmid DNA assembly reactions, followed by facile resolution of the concatomeric products to monomers through treatment with specific endonucleases and subsequent efficient transformation of the linear DNA products into host cells for in vivo circularisation. We demonstrate that use of a nicking endonuclease to generate homologous single-stranded ends increases the efficiency of E. coli chemical transformation versus both linear DNA with double-stranded homologous ends, and circular golden-gate assembly products, whilst use of a restriction endonuclease to generate linear DNA with double-stranded homologous ends increases the efficiency of chemical and electrotransformation of S. cerevisiae. Importantly, we also optimise the process such that both RCA and endonuclease treatment occur efficiently in the same buffer, streamlining the workflow and reducing product loss through purification steps. We expect our approach to have utility beyond directed evolution in E. coli and S. cerevisiae, to areas such as genome engineering and the manipulation of alternative organisms with even poorer transformation efficiencies.

synthetic biology↗

bioRxiv2023

Machine-learning guided Venom Induced Dermonecrosis Analysis tooL: VIDAL

Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snakes venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tool (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease.

pharmacology and toxicology↗

bioRxiv2023

Phage display assisted discovery of a pH-dependent anti-alpha-cobratoxin antibody from a natural variable domain library

Recycling antibodies can bind to their target antigen at neutral pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the antibodies to be recycled into circulation via FcRn-mediated pathway, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve the same therapeutic effect at lower doses than their non-recyclable counterparts. The development of such antibodies is typically achieved by histidine doping of the variable regions of specific antibodies or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. While often successful, these strategies may introduce sequence liabilities, as they often involve mutations that may render the resultant antibodies to be non-natural. Here, we present a methodology that employs a naive antibody phage display library, consisting of natural variable domains, to discover antibodies that bind -cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. Upon screening of the discovered antibodies with immunoassays and bio-layer interferometry, a pH-dependent antibody was discovered that exhibits an 8-fold higher dissociation rate at pH 5.5 than 7.4. Interestingly, the variable domains of the pH-dependent antibody were found to be entirely devoid of histidines, demonstrating that pH-dependency may not always be driven by this amino acid. Further, given the high diversity available in a naive antibody library, the methodology presented here can likely be applied to discover pH-dependent antibodies against different targets ab initio without the need of histidine doping. For broader audienceHere, we present the discovery of an -cobratoxin targeting pH-dependent antibody, with a variable region devoid of histidines, from a naive antibody library with natural variable domains. Our findings suggest that the commonly taken approach of histidine doping to find pH-dependent antibodies may not always be required, and thus offer an alternative strategy for the discovery of pH-dependent antibodies.

bioengineering↗

bioRxiv2023

A Comparative Study of Protein Structure Prediction Tools for Challenging Targets: Snake Venom Toxins

Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins, and comparisons using proteins with poor reference templates are lacking. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures with no reference templates. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins that have poor reference templates, are large, and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes. StatementRecent advances in machine learning have led to the development of new protein structure prediction tools. However, these tools have mainly been tested on well-known proteins and their performance on proteins without known templates is unclear. This study evaluated the performance of three tools on over 1000 snake venom toxins. We found that while caution is required when studying poorly characterised proteins, these tools offer valuable opportunities to understand protein function and applications.

bioinformatics↗

bioRxiv2023

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by -neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting -neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the {gamma}-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying -neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

pharmacology and toxicology↗

bioRxiv2023

Discovery of broadly-neutralizing antibodies against brown recluse spider and Gadim scorpion sphingomyelinases using consensus toxins as antigens

Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and developing broadly-neutralizing antibodies can be cumbersome using traditional low-throughput iterative protein engineering methods. Here, we present a new high-throughput approach for the standardized discovery of broadly-neutralizing monoclonal antibodies relying on phage display technology and consensus antigens representing an average sequence of related proteins. We showcase the utility of this approach by applying it to toxic sphingomyelinases from the venoms of very distant orders of the animal kingdom, the recluse spider and Gadim scorpion. First, we designed a consensus sphingomyelinase and performed three rounds of phage display selection, followed by DELFIA-based screening and ranking, and benchmarked this to a similar campaign involving cross-panning against recombinant versions of the native toxins. Second, we identified two scFvs that not only bind the consensus toxins, but which can also neutralize sphingomyelinase activity in vitro. Finally, we conclude that the phage display campaign involving the use of the consensus toxin was more successful in yielding cross-neutralizing scFvs than the phage display campaign involving cross-panning.

bioengineering↗

bioRxiv2022

Prototyping of a lateral flow assay based on monoclonal antibodies for detection of Bothrops venoms

BackgroundBrazil is home to a multitude of venomous snakes, perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice in antivenom that is administered depends not only on its availability and how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of a photo of the snake or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. ResultsIn this paper, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp. specific antibody sandwich pairs. The sandwich pairs were used to develop a prototype LFA that was able to detect venom from several different Bothrops spp. The limit of detection (LoD) of the prototype was evaluated using Brazilian B. atrox whole venom and was determined to be 8.0 ng/mL in spiked serum samples and 9.5 ng/mL in spiked urine samples, when using a portable reader, and < 25 ng/mL in spiked buffer when reading by eye. SignificanceThe work presented here serves as a proof of concept of a genus-specific venom detection kit, which could support physicians in diagnosing Bothrops envenomings. Although further optimization and testing is needed before the LFA can find clinical use, such a device could aid in decentralizing antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.

immunology↗

bioRxiv2022

ALOHA: AI-guided tool for the quantification of venom-induced haemorrhage in mice

Venom-induced haemorrhage constitutes a severe pathology in snakebite envenomings, especially those inflicted by viperid species. In order to both explore venom compositions accurately, and evaluate the efficacy of viperid antivenoms for the neutralisation of haemorrhagic activity it is essential to have available a precise, quantitative tool for empirically determining venom-induced haemorrhage. Thus, we have built on our prior approach and developed a new AI-guided tool (ALOHA) for the quantification of venom-induced haemorrhage in mice. Using a smartphone, it takes less than a minute to take a photo, upload the image, and receive accurate information on the magnitude of a venom-induced haemorrhagic lesion in mice. This substantially decreases analysis time, reduces human error, and does not require expert haemorrhage analysis skills. Furthermore, its open access web-based graphical user interface makes it easy to use and implement in laboratories across the globe. Together, this will reduce the resources required to preclinically assess and control the quality of antivenoms, whilst also expediting the profiling of hemorrhagic activity in venoms for the wider toxinology community.

bioinformatics↗

bioRxiv2022

Discovery of a broadly-neutralizing human antibody that can rescue mice challenged with neurotoxin-rich snake venoms

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and improved neutralization capacity. However, as new antivenom products may need to be polyvalent, i.e., target multiple different snake species, without dramatically increasing dose or cost of manufacture, such monoclonal antibodies need to be broadly-neutralizing. Here, we report the establishment of a pipeline for the discovery of high affinity broadly-neutralizing human monoclonal antibodies. We further demonstrate its utility by discovering an antibody that can prevent lethality induced by N. kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin, and which also prolongs survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

bioengineering↗

bioRxiv2021

Black-necked spitting cobra (Naja nigricollis) phospholipases A2 cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to a glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components including proteases and phospholipases. Here, we investigated the effect of Naja nigricollis on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VGS hydrolysis however N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) a cell cycle arrest prior to cytokinesis. A single protein family, the phospholipases A2s present in N. nigricollis venom, was necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.

microbiology↗

bioRxiv2021

In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom

The monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain -neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against -cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified -cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic. One Sentence SummaryA recombinant human monoclonal immunoglobulin G antibody, discovered and optimized using in vitro methods, was demonstrated to neutralize the lethal effect of whole venom from the monocled cobra in mice via abrogation of -neurotoxin-mediated neurotoxicity.

molecular biology↗