Kevin Christopher Gough

The UK's 2025 Replacing Animals in Science strategy introduces a commitment to end polyclonal antibody reagent production by 2030. While this represents a significant milestone, focusing exclusively on polyclonal reagents raises scientific, ethical and implementation challenges, given that monoclonal and other recombinant antibody formats frequently remain dependent on animal immunisation. This study examines whether current UK licensing practice and reporting frameworks are positioned to deliver genuine replacement. A structured, Three Rs-aligned replacement framework is articulated, reflecting the principles set out in the UK Animals in Science Committee (ASC) 2022 review. This framework is applied to UK Non-Technical Summaries (NTS), to assess how antibody reagent production is justified at the project licence stage and the extent to which applications align with ASC expectations. In parallel, UK Home Office Annual Statistics of Scientific Procedures on Living Animals are analysed, to evaluate how antibody-related procedures are categorised, and to determine whether this national reporting enables progress toward the 2030 commitment to be monitored. The analysis identifies limited implementation of ASC replacement guidance in post-2022 licences, with few applications providing evidence-based interrogation of non-animal alternatives, documented use of validated in vitro discovery platforms, or robust justification for immunisation. The national statistics lack sufficient resolution to clearly capture immunisation-dependent antibody reagent production across commercial, basic and translational research contexts. In addition, inconsistent terminology complicates the interpretation of genuine replacement. Importantly, the findings indicate that, without complementary regulatory, reporting and infrastructural measures, the 2030 commitment risks being met through increased reliance on other immunisation-dependent antibody formats, geographical outsourcing, or reduced visibility of antibody production that is embedded within broader research categories, rather than through genuine replacement. Achieving the intended reduction in animal use will therefore require clearer terminology, improved reporting resolution, wider access to nonanimal discovery infrastructure and the integration of a Three Rs-aligned decision framework into licensing expectations.

The UK’s 2025 Replacing Animals in Science strategy introduces a commitment to end polyclonal antibody reagent production by 2030. While this represents a significant milestone, focusing exclusively on polyclonal reagents raises scientific, ethical and implementation challenges, given that monoclonal and other recombinant antibody formats frequently remain dependent on animal immunisation. This study examines whether current UK licensing practice and reporting frameworks are positioned to deliver genuine replacement . A structured, Three Rs-aligned replacement framework is articulated, reflecting the principles set out in the UK Animals in Science Committee (ASC) 2022 review. This framework is applied to UK Non-Technical Summaries (NTS), to assess how antibody reagent production is justified at the project licence stage and the extent to which applications align with ASC expectations. In parallel, UK Home Office Annual Statistics of Scientific Procedures on Living Animals are analysed, to evaluate how antibody-related procedures are categorised, and to determine whether this national reporting enables progress toward the 2030 commitment to be monitored. The analysis identifies limited implementation of ASC replacement guidance in post-2022 licences, with few applications providing evidence-based interrogation of non-animal alternatives, documented use of validated in vitro discovery platforms, or robust justification for immunisation. The national statistics lack sufficient resolution to clearly capture immunisation-dependent antibody reagent production across commercial, basic and translational research contexts. In addition, inconsistent terminology complicates the interpretation of genuine replacement . Importantly, the findings indicate that, without complementary regulatory, reporting and infrastructural measures, the 2030 commitment risks being met through increased reliance on other immunisation-dependent antibody formats, geographical outsourcing, or reduced visibility of antibody production that is embedded within broader research categories, rather than through genuine replacement . Achieving the intended reduction in animal use will therefore require clearer terminology, improved reporting resolution, wider access to non-animal discovery infrastructure and the integration of a Three Rs-aligned decision framework into licensing expectations.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen’s epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection. Key words Phage display Next generation sequencing Biopanning Bioinformatic analysis Epitope mapping Antibody Peptide ELISA

Prion diseases are progressive neurodegenerative disorders with no effective therapeutics. The central event leading to the pathology in the diseases is the conversion of PrP^C into PrP^Sc and its accumulation in the central nervous system. Previous studies demonstrated that recombinant PrP (rPrP) and PrP peptides can inhibit the formation of PrP^Sc. Here, the effectiveness of ovine rPrP mutants at codon 136 and peptides derived from this region were assessed for their ability to inhibit PrP^Sc replication, using protein misfolding cyclic amplification (PMCA). Based on a rPrP VRQ (rVRQ) genotype background (positions 136, 154 and 171) and mutations at position 136, the most effective inhibitors were V136R, V136K and V136P mutants, with IC50 values of 1 to 2 nM; activities much more potent than rVRQ (114 nM). rRRQ and rKRQ were also shown to effectively inhibit multiple ruminant prion amplification reactions that used distinct prion strain seeds and substrate PRNP genotypes. rRRQ, rKRQ and rPRQ were also shown to effectively protect Rov9 cells from scrapie infection when applied at 250 nM. The study demonstrates for the first time that the rPrP sequence can be mutated at sites known to be involved in prion disease susceptibility, to produce inhibitors with improved efficacy.

Driven by necessity, the COVID-19 pandemic caused by SARS-CoV-2 has accelerated the development and implementation of new vaccine platforms and other viral therapeutics. Among these is the therapeutic use of antibodies including single-domain antibodies, in particular the camelid variable heavy-chain fragment (VHH). Such therapies can provide a critical interim intervention when vaccines have not yet been developed for an emerging virus. It is evident that an increasing number of different viruses are emerging and causing epidemics and pandemics with increasing frequency. It is therefore imperative that we capitalize on the experience and knowledge gained from combatting COVID-19 to be better prepared for the next pandemic.

Prion diseases are progressive neurodegenerative disorders with no effective therapeutics. A central event leading to the pathology in the diseases is the conversion of PrP^C into PrP^Sc and its accumulation in the central nervous system. Previous studies demonstrated that PrP^C, recombinant PrP (rPrP) and PrP peptides can inhibit the formation of PrP^Sc. Here, the effectiveness of ovine rPrP mutants at codon 136 and peptides derived from this region were assessed for their ability to inhibit PrP^Sc replication, using protein misfolding cyclic amplification (PMCA). Based on a rPrP VRQ (rVRQ) genotype background (positions 136, 154 and 171) and mutations at position 136, the most effective inhibitors were V136R, V136K and V136P mutants, with IC50 values of 2–11 nM; activities up to 57 times more potent than rVRQ. rRRQ was also shown to inhibit a range of ruminant prion amplification reactions including with distinct prion strain seeds and substrate PRNP genotypes. The rRRQ was also shown to effectively protect Rov9 cells from scrapie infection when applied at 250 nM. The study demonstrates for the first time that the rPrP sequence can be mutated at sites known to be involved in prion disease susceptibility, to produce inhibitors with improved efficacy.

Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 10⁹ variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 10⁹ variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrP^Sc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrP^Sc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrP^Sc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.