John A. Hammond

Foot-and-mouth disease virus (FMDV) causes a devastating disease that threatens global food security. Vaccination is hindered by antigenic diversity across serotypes. To identify cross-serotype neutralising epitopes, we isolated 24 FMDV-specific antibodies from cattle sequentially vaccinated with antigens from four serotypes, of which three neutralised three vaccine strains. These three antibodies neutralised 21 and bound 59 additional topotypes across O, A, Asia 1, and C serotypes. Cryo-EM complexes of Fabs with FMD virus-like particles indicated all three recognise a common flexible epitope at the VP1 C-terminus, confirmed by binding competition. Crystallography and structural modelling revealed that a normally inaccessible surface of the hydrophobic VP1 C-terminal peptides inserts into a similar groove in all three antibodies. Comparison of neutralisation activity and integrin receptor blocking by whole antibodies, F(ab’) ₂ s, and Fabs suggests neutralisation is mediated by Fc steric hindrance of receptor binding. This cryptic, linear, and cross-serotype neutralising epitope may inform improved FMD vaccines.

The bighorn sheep ( Ovis canadensis ), despite its close relation to domestic sheep, suffer higher morbidity and mortality from respiratory disease complexes, likely due to genetic differences in immune responses. Unraveling highly repetitive regions such as immune loci and genetic differences was problematic until now. We generated a bighorn sheep telomere-to-telomere assembly, adding 14.28% of novel sequence compared to the previous reference. This enabled the first complete immune loci annotation revealing the IGL and TR loci are significantly short in bighorn sheep. Importantly, a critical immune gene GBP5 and ZNF501 , involved in Golgi-mediated immune response, are lacking in bighorn but present in domestic sheep. Re-analysis of a Mycoplasma ovipneumoniae carriage study, using this assembly, identified the immune gene CAPN2 as a key genetic marker for disease carriage, not observable in the original study. This work provides a critical resource for identifying phenotype-linked genetic variation and exploring evolutionary adaptations of bighorn sheep.

The IPD-MHC Database project (http://www.ebi.ac.uk/ipd/mhc/) serves as a comprehensive and expertly curated repository for major histocompatibility complex (MHC) sequences from non-human species, providing the necessary infrastructure and tools to study the function and evolution of this highly polymorphic genomic region. In its latest version, the IPD-MHC database has expanded both in content and in the tools for data visualization and comparison. The database now hosts over 18 000 MHC alleles from 125 species, organized into eleven taxonomic groups, all manually curated and named by the Comparative MHC Nomenclature Committee. A cetacean section has recently been included, offering researchers valuable data to study the immune system of whales, dolphins, and porpoises, as well establishing the official nomenclature platform for the Cetacea Leukocyte Antigens (CeLA). In response to user demand and reflecting broader trends in bioinformatics and immunogenetics, IPD-MHC now includes the predicted tertiary structure of over 8000 alleles and allows comparison and visualisation of allele variation within and between species at single residue resolution. These latest developments maintain the critically important link between official nomenclature of curated alleles and the ability to analyse this complex polymorphism using the most up to date methods within a single repository.

Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)’s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly and transcriptome atlas of a Babraham pig. The genome was de novo assembled using PacBio long reads and error-corrected using Illumina short reads. Assembled contigs were then mapped to the porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting TPI_Babraham_pig_v1 assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham MHC and characterize the allele content and tissue expression of several other immune-related gene complexes, including the antibody and T cell receptor loci, the natural killer complex, and the leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form β-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

Background: The advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map dynamic changes in polyclonal responses precise framework and complementarity determining region annotation of rearranging genes is pivotal. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. For this reason, data agnostic tools able to learn from presented data can be very useful with new species or with novel datasets. Results: We have developed IgMAT, which utilises a reduced amino acid alphabet, that incorporates multiple HMM alignments into a single consensus to automatically annotate immunoglobulin sequences from most organisms. Additionally, the software allows the incorporation of user defined databases to better represent the species and/or antibody class of interest. To demonstrate the accuracy and utility of IgMAT, we present analysis of sequences extracted from structural data and immunoglobulin sequence datasets from several different species. Conclusions: IgMAT is fully open-sourced and freely available on GitHub (https://github.com/TPI-Immunogenetics/igmat) for download under GPLv3 license. It can be used as a CLI application or as a python module to be integrated in custom scripts.

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly of a Babraham pig. The Babraham genome was de novo assembled using PacBio long-reads and error-corrected using Illumina short-reads. The assembled contigs were then mapped to the current porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting Babraham pig assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham pig MHC and characterize the allele content across several immune-related gene complexes, including the contiguous assemblies of the antibody heavy chain locus and leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical.