High-resolution map of the Fc functions mediated by COVID-19-neutralizing antibodies.

A growing body of evidence shows that fragment crystallizable (Fc)-dependent antibody effector functions play an important role in protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To unravel the mechanisms that drive these responses, we analyzed the phagocytosis and complement deposition mediated by a panel of 482 human monoclonal antibodies (nAbs) neutralizing the original Wuhan virus, expressed as recombinant IgG1. Our study confirmed that nAbs no longer neutralizing SARS-CoV-2 Omicron variants can retain their Fc functions. Surprisingly, we found that nAbs with the most potent Fc function recognize the N-terminal domain, followed by those targeting class 3 epitopes in the receptor binding domain. Interestingly, nAbs direct against the class 1/2 epitopes in the receptor binding motif, which are the most potent in neutralizing the virus, were the weakest in Fc functions. The divergent properties of the neutralizing and Fc function-mediating antibodies were confirmed by the use of different B cell germlines and by the observation that Fc functions of polyclonal sera differ from the profile observed with nAbs, suggesting that non-neutralizing antibodies also contribute to Fc functions. These data provide a high-resolution picture of the Fc-antibody response to SARS-CoV-2 and suggest that the Fc contribution should be considered for the design of improved vaccines, the selection of therapeutic antibodies, and the evaluation of correlates of protection.

IgMAT: immunoglobulin sequence multi-species annotation tool for any species including those with incomplete antibody annotation or unusual characteristics.

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.

mRNA vaccines and hybrid immunity use different B cell germlines against Omicron BA.4 and BA.5.

Severe acute respiratory syndrome 2 Omicron BA.4 and BA.5 are characterized by high transmissibility and ability to escape natural and vaccine induced immunity. Here we test the neutralizing activity of 482 human monoclonal antibodies isolated from people who received two or three mRNA vaccine doses or from people vaccinated after infection. The BA.4 and BA.5 variants are neutralized only by approximately 15% of antibodies. Remarkably, the antibodies isolated after three vaccine doses target mainly the receptor binding domain Class 1/2, while antibodies isolated after infection recognize mostly the receptor binding domain Class 3 epitope region and the N-terminal domain. Different B cell germlines are used by the analyzed cohorts. The observation that mRNA vaccination and hybrid immunity elicit a different immunity against the same antigen is intriguing and its understanding may help to design the next generation of therapeutics and vaccines against coronavirus disease 2019.

B cell analyses after SARS-CoV-2 mRNA third vaccination reveals a hybrid immunity like antibody response.

The continuous evolution of SARS-CoV-2 generated highly mutated variants able to escape natural and vaccine-induced primary immunity. The administration of a third mRNA vaccine dose induces a secondary response with increased protection. Here we investigate the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses at single-cell level. We sorted 4100 spike protein specific memory B cells identifying 350 neutralizing antibodies. The third dose increases the antibody neutralization potency and breadth against all SARS-CoV-2 variants as observed with hybrid immunity. However, the B cell repertoire generating this response is different. The increases of neutralizing antibody responses is largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization. Our data show that different immunization regimens induce specific molecular signatures which should be considered while designing new vaccines and immunization strategies.

The IPD-IMGT/HLA Database.

It is 24 years since the IPD-IMGT/HLA Database, http://www.ebi.ac.uk/ipd/imgt/hla/, was first released, providing the HLA community with a searchable repository of highly curated HLA sequences. The database now contains over 35 000 alleles of the human Major Histocompatibility Complex (MHC) named by the WHO Nomenclature Committee for Factors of the HLA System. This complex contains the most polymorphic genes in the human genome and is now considered hyperpolymorphic. The IPD-IMGT/HLA Database provides a stable and user-friendly repository for this information. Uptake of Next Generation Sequencing technology in recent years has driven an increase in the number of alleles and the length of sequences submitted. As the size of the database has grown the traditional methods of accessing and presenting this data have been challenged, in response, we have developed a suite of tools providing an enhanced user experience to our traditional web-based users while creating new programmatic access for our bioinformatics user base. This suite of tools is powered by the IPD-API, an Application Programming Interface (API), providing scalable and flexible access to the database. The IPD-API provides a stable platform for our future development allowing us to meet the future challenges of the HLA field and needs of the community.

Highly-contiguous bovine genomes underpin accurate functional analyses and updated nomenclature of MHC class I.

The major histocompatibility complex (MHC) class I region of cattle is both highly polymorphic and, unlike many species, highly variable in gene content between haplotypes. Cattle MHC class I alleles were historically grouped by sequence similarity in the more conserved 3' end of the coding sequence to form phylogenetic allele groups. This has formed the basis of current cattle MHC class I nomenclature. We presently describe and compare five fully assembled MHC class I haplotypes using the latest cattle and yak genome assemblies. Of the five previously described "pseudogenes" in the cattle MHC class I region, Pseudogene 3 is putatively functional in all haplotypes and Pseudogene 6 and Pseudogene 7 are putatively functional in some haplotypes. This was reinforced by evidence of transcription. Based on full gene sequences as well as 3' coding sequence, we identified distinct subgroups of BoLA-3 and BoLA-6 that represent distinct genetic loci. We further examined allele-specific expression using transcriptomic data revealing that certain alleles are consistently weakly expressed compared to others. These observations will help to inform further studies into how MHC class I region variability influences T cell and natural killer cell functions in cattle.

Common gene variants within 3'-untranslated regions as modulators of multiple myeloma risk and survival.

We evaluated the association between germline genetic variants located within the 3'-untranlsated region (polymorphic 3'UTR, ie, p3UTR) of candidate genes involved in multiple myeloma (MM). We performed a case-control study within the International Multiple Myeloma rESEarch (IMMEnSE) consortium, consisting of 3056 MM patients and 1960 controls recruited from eight countries. We selected p3UTR of six genes known to act in different pathways relevant in MM pathogenesis, namely KRAS (rs12587 and rs7973623), VEGFA (rs10434), SPP1 (rs1126772), IRF4 (rs12211228) and IL10 (rs3024496). We found that IL10-rs3024496 was associated with increased risk of developing MM and with a worse overall survival of MM patients. The variant allele was assayed in a vector expressing eGFP chimerized with the IL10 3'-UTR and it was found functionally active following transfection in human myeloma cells. In this experiment, the A-allele caused a lower expression of the reporter gene and this was also in agreement with the in vivo expression of mRNA measured in whole blood as reported in the GTEx portal. Overall, these data are suggestive of an effect of the IL10-rs3024496 SNP on the regulation of IL10 mRNA expression and it could have clinical implications for better characterization of MM patients in terms of prognosis.

The UK Veterinary Immunological Toolbox Website: promoting vaccine research by facilitating communication and removing reagent barriers.

Using the best animal models to study immune responses against specific pathogens or vaccines can dramatically accelerate our understanding. Veterinary species are well studied, particularly livestock, to reduce their disease burden. They have also proven to be powerful models, especially for zoonotic pathogens and novel vaccination strategies. A prerequisite for any model selection is having the right quality and range of species-specific immunological reagents. To help promote the widest possible use of veterinary species, an open access website (https://www.immunologicaltoolbox.co.uk) has been created as a central community annotated hub for veterinary immunological reagents. The website is also the portal into services offered by the UK Immunological Toolbox project that includes antibody generation, sequencing and recombinant expression. The funding for this effort is linked into sustainable sources, but ultimate success relies on community engagement to continually increase the quality and quantity of information. It is hoped that as more users and reagent owners engage, it will become an essential resource for researchers, veterinarians and clinicians alike by removing barriers that prevent the use of the most informative animal models.

Correction to: An Illumina approach to MHC typing of Atlantic salmon.

The original version of this article was published without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed.

The IPD Project: a centralised resource for the study of polymorphism in genes of the immune system.

The Immuno Polymorphism Database (IPD), https://www.ebi.ac.uk/ipd/, is a set of specialist databases that enable the study of polymorphic genes which function as part of the vertebrate immune system. The major focus is on the hyperpolymorphic major histocompatibility complex (MHC) genes and the killer-cell immunoglobulin-like receptor (KIR) genes, by providing the official repository and primary source of sequence data. Databases are centred around humans as well as animals important for food security, for companionship and as disease models. The IPD project works with specialist groups or nomenclature committees who provide and manually curate individual sections before they are submitted for online publication. To reflect the recent advance of allele sequencing technologies and the increasing demands of novel tools for the analysis of genomic variation, the IPD project is undergoing a progressive redesign and reorganisation. In this review, recent updates and future developments are discussed, with a focus on the core concepts to better future-proof the project.

Nomenclature report 2019: major histocompatibility complex genes and alleles of Great and Small Ape and Old and New World monkey species.

The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

An Illumina approach to MHC typing of Atlantic salmon.

The IPD-MHC Database represents the official repository for non-human major histocompatibility complex (MHC) sequences, overseen and supported by the Comparative MHC Nomenclature Committee, providing access to curated MHC data and associated analysis tools. IPD-MHC gathers allelic MHC class I and class II sequences from classical and non-classical MHC loci from various non-human animals including pets, farmed and experimental model animals. So far, Atlantic salmon and rainbow trout are the only teleost fish species with MHC class I and class II sequences present. For the remaining teleost or ray-finned species, data on alleles originating from given classical locus is scarce hampering their inclusion in the database. However, a fast expansion of sequenced genomes opens for identification of classical loci where high-throughput sequencing (HTS) will enable typing of allelic variants in a variety of new teleost or ray-finned species. HTS also opens for large-scale studies of salmonid MHC diversity challenging the current database nomenclature and analysis tools. Here we establish an Illumina approach to identify allelic MHC diversity in Atlantic salmon, using animals from an endangered wild population, and alter the salmonid MHC nomenclature to accommodate the expected sequence expansions.

Nomenclature report for killer-cell immunoglobulin-like receptors (KIR) in macaque species: new genes/alleles, renaming recombinant entities and IPD-NHKIR updates.

The Killer-cell Immunoglobulin-like Receptors (KIR) are encoded by a diverse group of genes, which are characterized by allelic polymorphism, gene duplications, and recombinations, which may generate recombinant entities. The number of reported macaque KIR sequences is steadily increasing, and these data illustrate a gene system that may match or exceed the complexity of the human KIR cluster. This report lists the names of quality controlled and annotated KIR genes/alleles with all the relevant references for two different macaque species: rhesus and cynomolgus macaques. Numerous recombinant KIR genes in these species necessitate a revision of some of the earlier-published nomenclature guidelines. In addition, this report summarizes the latest information on the Immuno Polymorphism Database (IPD)-NHKIR Database, which contains annotated KIR sequences from four non-human primate species.

Correction to: Nomenclature report 2019: major histocompatibility complex genes and alleles of great and small ape and old and new world monkey species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.

Comparative MHC nomenclature: report from the ISAG/IUIS-VIC committee 2018.

Significant progress has been made over the last decade in defining major histocompatibility complex (MHC) diversity at the nucleotide, allele, haplotype, diplotype, and population levels in many non-human species. Much of this progress has been driven by the increased availability and reduced costs associated with nucleotide sequencing technologies. This report provides an update on the activities of the comparative MHC nomenclature committee which is a standing committee of both the International Society for Animal Genetics (ISAG) and the International Union of Immunological Societies (IUIS) where it operates under the umbrella of the Veterinary Immunology Committee (VIC). A previous report from this committee in 2006 defined the role of the committee in providing guidance in the development of a standardized nomenclature for genes and alleles at MHC loci in non-human species. It described the establishment of the Immuno Polymorphism Database, IPD-MHC, which continues to provide public access to high quality MHC sequence data across a range of species. In this report, guidelines for the continued development of a universal MHC nomenclature framework are described, summarizing the continued development of each species section within the IPD-MHC project.

IPD-MHC: nomenclature requirements for the non-human major histocompatibility complex in the next-generation sequencing era.

The IPD-MHC Database is the official repository for non-human MHC sequences, overseen and supported by the Comparative MHC Nomenclature Committee, providing access to curated MHC data and associated analysis tools. To address the increasing amount and complexity of data being submitted, an entirely upgraded version of the IPD-MHC Database was recently released to maintain IPD-MHC as the central platform for the comparison of curated MHC data. As a consequence, a new level of nomenclature standardisation is required between the different species to enable data submission and to allow the unambiguous inter- and intra-species comparison of alleles. However, any changes must retain the flexibility demanded by the unique biology of different taxonomic groups. Here, we describe the rationale for a standardised nomenclature system and summarise the changes that have been driven by the requirements of implementing the IPD-MHC database. This modified nomenclature system is essential to maintain the current functionality of IPD-MHC and provide a scalable future-proof database organisation to fully exploit the bioinformatic tools used for analysis.

Nomenclature for the KIR of non-human species.

The increasing number of Killer Immunoglobulin-like Receptor (KIR) sequences available for non-human primate species and cattle has prompted development of a centralized database, guidelines for a standardized nomenclature, and minimum requirements for database submission. The guidelines and nomenclature are based on those used for human KIR and incorporate modifications made for inclusion of non-human species in the companion IPD-NHKIR database. Included in this first release are the rhesus macaque (Macaca mulatta), chimpanzee (Pan troglodytes), orangutan (Pongo abelii and Pongo pygmaeus), and cattle (Bos taurus).

The IPD Databases: Cataloguing and Understanding Allele Variants.

The IMGT/HLA Database has provided a repository for information regarding polymorphism in the genes of the immune system since 1998. In 2003, it was absorbed into the Immuno Polymorphism Database (IPD). The IPD project has enabled us to create and maintain a platform for curating and publishing locus-specific databases which are either involved directly with, or relate to, the function of the Major Histocompatibility Complex across a number of species. In collaboration with specialist groups and nomenclature committees individual sections have been curated prior to their submission to the IPD for online publication. The IPD consists of five core databases, with the primary database being the IMGT/HLA Database. With the work of various nomenclature committees, the HLA Informatics Group, and alongside the European Bioinformatics Institute, we provide access to this data through the website ( http://www.ebi.ac.uk/ipd/ ) to the public domain. The IPD project continually develops new tools in conjunction with on-going scientific developments-such as Next-Generation Sequencing-to maintain efficiency and usability in response to user feedback and requests. The website is updated on a regular basis to ensure that new and confirmatory sequences are distributed to the immunogenetics community, as well as the wider research and clinical communities.

Analogs of the Frog-skin Antimicrobial Peptide Temporin 1Tb Exhibit a Wider Spectrum of Activity and a Stronger Antibiofilm Potential as Compared to the Parental Peptide.

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 µM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 µM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analogs when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization.

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.