2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Strategy and validation of a nonclinical generic plug-and-play antidrug antibody method for human monoclonal antibody biotherapeutics.

The measurement of antidrug antibodies (ADA) in nonclinical studies provides limited value because the formation and incidence of nonclinical ADA does not translate to clinical experience. The formation and presence of ADA in nonclinical species can, however, correlate to reduced drug exposure and safety observations including vasculitis and immune complex disease. Generic ADA methods for humanized monoclonal antibody biotherapeutics mitigate the need to develop bespoke ADA methods during nonclinical drug development. A drug-tolerant, sensitive, generic ADA immunoassay has been developed and validated for measuring ADA in cynomolgus monkey serum samples, allowing for immediate qualification of future monoclonal antibody biotherapeutics. This approach allows us to differentiate complexed and free ADA in a rapidly deployable manner when needed.

The testing of antidrug antibodies (ADA) in animal studies offers low value because the presence of animal ADA does not translate to human studies. However, the impact of ADA can be seen with reduced drug levels and/or safety findings in animal studies. Generic ADA methods offer a way to measure ADA leading to time and cost savings. This article details the testing of a generic plug-and-play method to measure ADA in monkey serum and how to qualify future drugs. To date, 16 drugs have been qualified using this method, which has also been applied to mouse, rat and rabbit serum.

Bovine mortality: the utility of two data sources for the provision of population-level surveillance intelligence.

Introduction: The use of existing data to provide surveillance intelligence is widely advocated but often presents considerable challenges. Two data sources could be used as proxies for the mortality experienced by the Scottish cattle population: deaths recorded in the mandatory register [Cattle Tracing System (CTS)] and fallen stock collections by the National Fallen Stock Company (NSFCo) with a nationwide voluntary membership. Methods: Data for the period 2011-2016 were described and compared to establish their strengths and limitations. Similarities and differences in their temporal, seasonal and spatial patterns were examined overall, at postcode area level and for different age groups. Temporal aberration detection algorithms (TADA) were fitted. Results: Broadly, similar patterns were observed in the two datasets; however, there were some notable differences. The observed seasonal, annual and spatial patterns match expectations, given knowledge of Scottish cattle production systems. The registry data provide more comprehensive coverage of all areas of Scotland, while collections data provide a more comprehensive measure of the mortality experienced in 0-1-month-old calves. Discussion: Consequently, estimates of early calf mortality and their impact on the livestock sector made using CTS, or successor registers, will be under-estimates. This may apply to other registry-based systems. Fitted TADA detected points of deviations from expected norms some of which coincided in the two datasets; one with a known external event that caused increased mortality. We have demonstrated that both data sources do have the potential to be utilized to provide measures of mortality in the Scottish cattle population that could inform surveillance activities. While neither is perfect, they are complementary. Each has strengths and weaknesses, so ideally, a system where they are analyzed and interpreted in parallel would optimize the information obtained for surveillance purposes for epidemiologists, risk managers, animal health policy-makers and the wider livestock industry sector. This study provides a foundation on which to build an operational system. Further development will require improvements in the timeliness of data availability and further investment of resources.

Social media network analysis of Smallholder livestock farming communities in the United Kingdom.

The creation of targeted policies and actions to help small-scale livestock keepers and reduce the risks associated with disease outbreaks in this sector is hampered by the scarcity of information about smallholder farmers. Smallholders play a crucial part in disease outbreaks containment, hence there is a need for better monitoring methods that take this population into account while gathering data. According to the literature, these communities frequently use social media as a channel for communication and information exchange. In this study we conducted social network analysis of an influential smallholder within the UK and visualised the user follower network. Additionally, we performed influential user analysis, Twitter user categorisation, and community detection to uncover more insights into the livestock farming networks. Our findings reveal distinct communities within the smallholder farming sector and identify influential users with the potential to impact information dissemination and animal health practices. The study also highlights the role of community structure in surveillance and control of animal diseases and emphasises the need for further research to refine our understanding of these communities and their unique characteristics. This work contributes to the growing body of literature on small-scale livestock farming in the UK and underscores the importance of incorporating smallholder communities into disease surveillance and control efforts.

Spatio-temporal evaluation of social media as a tool for livestock disease surveillance.

Recent outbreaks of Avian Influenza across Europe have highlighted the potential for syndromic surveillance systems that consider other modes of data, namely social media. This study investigates the feasibility of using social media, primarily Twitter, to monitor illness outbreaks such as avian flu. Using temporal, geographical, and correlation analyses, we investigated the association between avian influenza tweets and officially verified cases in the United Kingdom in 2021 and 2022. Pearson correlation coefficient, bivariate Moran's I analysis and time series analysis, were among the methodologies used. The findings show a weak, statistically insignificant relationship between the number of tweets and confirmed cases in a temporal context, implying that relying simply on social media data for surveillance may be insufficient. The spatial analysis provided insights into the overlaps between confirmed cases and tweet locations, shedding light on regionally targeted interventions during outbreaks. Although social media can be useful for understanding public sentiment and concerns during outbreaks, it must be combined with traditional surveillance methods and official data sources for a more accurate and comprehensive approach. Improved data mining techniques and real-time analysis can improve outbreak detection and response even further. This study underscores the need of having a strong surveillance system in place to properly monitor and manage disease outbreaks and protect public health.

Phylogenetic relationship and virulence composition of Escherichia coli O26:H11 cattle and human strain collections in Scotland; 2002-2020.

O26 is the commonest non-O157 Shiga toxin (stx)-producing Escherichia coli serogroup reported in human infections worldwide. Ruminants, particularly cattle, are the primary reservoir source for human infection. In this study, we compared the whole genomes and virulence profiles of O26:H11 strains (n = 99) isolated from Scottish cattle with strains from human infections (n = 96) held by the Scottish Escherichia coli O157/STEC Reference Laboratory, isolated between 2002 and 2020. Bovine strains were from two national cross-sectional cattle surveys conducted between 2002-2004 and 2014-2015. A maximum likelihood phylogeny was constructed from a core-genome alignment with the O26:H11 strain 11368 reference genome. Genomes were screened against a panel of 2,710 virulence genes using the Virulence Finder Database. All stx-positive bovine O26:H11 strains belonged to the ST21 lineage and were grouped into three main clades. Bovine and human source strains were interspersed, and the stx subtype was relatively clade-specific. Highly pathogenic stx2a-only ST21 strains were identified in two herds sampled in the second cattle survey and in human clinical infections from 2010 onwards. The closest pairwise distance was 9 single-nucleotide polymorphisms (SNPs) between Scottish bovine and human strains and 69 SNPs between the two cattle surveys. Bovine O26:H11 was compared to public EnteroBase ST29 complex genomes and found to have the greatest commonality with O26:H11 strains from the rest of the UK, followed by France, Italy, and Belgium. Virulence profiles of stx-positive bovine and human strains were similar but more conserved for the stx2a subtype. O26:H11 stx-negative ST29 (n = 17) and ST396 strains (n = 5) were isolated from 19 cattle herds; all were eae-positive, and 10 of these herds yielded strains positive for ehxA, espK, and Z2098, gene markers suggestive of enterohaemorrhagic potential. There was a significant association (p < 0.001) between nucleotide sequence percent identity and stx status for the bacteriophage insertion site genes yecE for stx2 and yehV for stx1. Acquired antimicrobial resistance genes were identified in silico in 12.1% of bovine and 17.7% of human O26:H11 strains, with sul2, tet, aph(3″), and aph(6″) being most common. This study describes the diversity among Scottish bovine O26:H11 strains and investigates their relationship to human STEC infections.

Analysis of Escherichia coli O157 strains in cattle and humans between Scotland and England & Wales: implications for human health.

For the last two decades, the human infection frequency of Escherichia coli O157 (O157) in Scotland has been 2.5-fold higher than in England and Wales. Results from national cattle surveys conducted in Scotland and England and Wales in 2014/2015 were combined with data on reported human clinical cases from the same time frame to determine if strain differences in national populations of O157 in cattle could be associated with higher human infection rates in Scotland. Shiga toxin subtype (Stx) and phage type (PT) were examined within and between host (cattle vs human) and nation (Scotland vs England and Wales). For a subset of the strains, whole genome sequencing (WGS) provided further insights into geographical and host association. All three major O157 lineages (I, II, I/II) and most sub-lineages (Ia, Ib, Ic, IIa, IIb, IIc) were represented in cattle and humans in both nations. While the relative contribution of different reservoir hosts to human infection is unknown, WGS analysis indicated that the majority of O157 diversity in human cases was captured by isolates from cattle. Despite comparable cattle O157 prevalence between nations, strain types were localized. PT21/28 (sub-lineage Ic, Stx2a+) was significantly more prevalent in Scottish cattle [odds ratio (OR) 8.7 (2.3-33.7; P<0.001] and humans [OR 2.2 (1.5-3.2); P<0.001]. In England and Wales, cattle had a significantly higher association with sub-lineage IIa strains [PT54, Stx2c; OR 5.6 (1.27-33.3); P=0.011] while humans were significantly more closely associated with sub-lineage IIb [PT8, Stx1 and Stx2c; OR 29 (4.9-1161); P<0.001]. Therefore, cattle farms in Scotland were more likely to harbour Stx2a+O157 strains compared to farms in E and W (P<0.001). There was evidence of limited cattle strain migration between nations and clinical isolates from one nation were more similar to cattle isolates from the same nation, with sub-lineage Ic (mainly PT21/28) exhibiting clear national association and evidence of local transmission in Scotland. While we propose the higher rate of O157 clinical cases in Scotland, compared to England and Wales, is a consequence of the nationally higher level of Stx2a+O157 strains in Scottish cattle, we discuss the multiple additional factors that may also contribute to the different infection rates between these nations.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

The GSK Bioanalytical Hub model in bioanalysis: maximizing the synergies of co-locating various platforms in support of regulated and nonregulated study end points.

The term "bioanalytical" encompasses a much greater breadth of analytical deliverables than ever before. Circulating drug concentration data are complemented by experimental evidence of drug in biophase, immunogenicity, target engagement and subsequent pathway modulation. Many bioanalytical assays bridge the traditional divide across discovery and development. Our approach is the Bioanalytical Hub model bringing together a wide breadth of bioanalytical support (GxP and non-GxP), multiple end points (pharmacokinetics, anti-drug antibodies and biomarkers) and analytical platforms (LC/MS, immunoassay, flow cytometry, genomics, immunohistochemistry) onto a common lab footprint. This maximizes instrument utilization, facilitates workforce agility and enhances data interpretation capability while reducing the number of hand-offs as assays evolve from their origins as exploratory end points to fully characterized to support primary and secondary end points.

Linking human tick bite risk with tick abundance in the environment: A novel approach to quantify tick bite risk using orienteers in Scotland.

The rate that people are bitten by ticks is critical in determining the risk of tick-borne infections but is rarely quantified accurately. Often tick abundance in the environment is used as a proxy for tick bite risk, but the relationship with risk is poorly understood. We used a novel citizen science approach to measure tick bite rate in orienteers, to assess the relationship between tick abundance and tick bite risk and to identify risk factors for tick bites. Eleven orienteering events were attended in Scotland between August 2018 and September 2019. The number of tick bites in orienteers, and the time and distance of activity were collected using an online questionnaire. Tick abundance in the same areas used for the orienteering events was estimated by surveying ticks on ground vegetation using blanket drags. Among orienteers, mean incidence was 409 tick bites per 1,000 person-hours. Tick abundance and tick bite rate were strongly correlated, indicating that data from questing tick surveys is a useful proxy for the risk of human tick bites. Tick bite rate was better explained by the activity duration than distance covered and was higher in orienteers that ran earlier in the day, exposed to higher temperatures and in woodland habitats. This study highlights the value of the citizen science approach used, which crucially included submission of activity reports both with and without ticks, to generate robust data on tick bite rate. Accurately measuring tick bite rate and understanding environmental factors that influence it are essential in mitigating the risk of tick-borne diseases.

Determination of label efficiency and label degree of critical reagents by LC-MS and native MS.

Degree of labeling and label efficiency are key factors for optimal characterization of critical reagents that are used in ligand binding assays. Here, three case studies are shown demonstrating how liquid chromatography-mass spectrometry (LC-MS) was utilized to characterize critical reagents using three unique methodologies. Critical reagent batches were prepared for LC-MS analysis by use of: 20 mM dithiothreitol (DTT) (Case 1), rapid PNGaseF (Case 2), and a mobile phase diluent (Case 3). LC-MS was run at three different MS method conditions in each troubleshooting case specific for reduced IgG, intact IgG, and native LC-MS, respectively. Specified LC-MS methods based on sample type and configuration elucidated clear MS profiles, allowing for degree of labeling and label efficiencies to be calculated. Ultimately the LC-MS analyses were fine-tuned for critical reagent characterization, and practices for analyzing similar reagents in the future can be established.

Corrigendum: Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

[This corrects the article DOI: 10.3389/fvets.2021.688078.].

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Field evaluation of diagnostic sensitivity (DSe) and specificity (DSp) of common tests for amoebic gill disease (AGD) and complex gill disease (CGD) in cultured Atlantic salmon (Salmo salar) in Scotland using Bayesian latent class models.

Amoebic gill disease (AGD) and complex gill disease (CGD) are the most significant marine gill diseases in salmon aquaculture in Scotland. Little is published about diagnostic performance of tests to detect these diseases, making it difficult to interpret test results. We estimated diagnostic sensitivity (DSe) and specificity (DSp) of common tests for AGD (gross AGD score, qPCR for Neoparamoeba perurans, histopathology) and CGD (gross proliferative gill disease (PGD) score, gross total gill score, histopathology). Because specifications in our sampling protocol implemented to encourage consistency across the farms might affect diagnostic performance of histopathology (historically the reference standard for gill diseases), we used Bayesian latent class models without reference standard. Cases and non-cases were based on less, medium, and severe stringent case definitions, representing different cut-off levels for the different tests. Gross gill scores for both diseases were excellent in designating non-diseased fish, DSps were generally around 1. To detect CGD, DSe of gross total gill score and gross PGD score were between respectively 0.81 (0.73 - 0.91 lower to upper 95% credible interval) and 0.53 (0.46 - 0.64) for medium stringent case definitions, and to detect AGD the DSe for the gross AGD score was between 0.53 (0.48-0.57) and 0.14 (0.07 - 0.22) for respectively the less and severe stringent case definition. Thus, gross gill scores were medium to good in designating truly diseased fish, implying some false negatives are expected. For CGD the DSe for gross total gill scores were the highest, for AGD it was the qPCR test at a DSe of 0.92 (0.86 - 0.99). For both diseases, DSe was lowest for histopathology, e.g. 0.23 (0.16 - 0.30) for AGD and 0.1 (0.07 - 0.14) for CGD under medium stringent case definitions, perhaps due to collecting the second gill arch on the right rather than the worst affected arch, whilst PCR sampling and gross gill scoring included multiple (PCR) or all (gross scoring) gill arches. The diagnostic goals of these tests differ; gross gill scoring provides a low-cost presumptive diagnosis, PCR a non-lethal confirmation of the presence of a specific pathogen and histopathology provides information on the underlying aetiology of gill damage as well as the extent, severity, and chronology of gill disease. An effective gill health surveillance strategy is likely to incorporate multiple diagnostic tools used in a complementary manner.

Phylogeny and potential virulence of cryptic clade Escherichia coli species complex isolates derived from an arable field trial.

Analysis of Escherichia coli taxonomy has expanded into a species-complex with the identification of divergent cryptic clades. A key question is the evolutionary trajectory of these clades and their relationship to isolates of clinical or veterinary importance. Since they have some environmental association, we screened a collection of E. coli isolated from a long-term spring barley field trial for their presence. While most isolates clustered into the enteric-clade, four of them clustered into Clade-V, and one in Clade-IV. The Clade -V isolates shared >96% intra-clade average nucleotide sequence identity but <91% with other clades. Although pan-genomics analysis confirmed their taxonomy as Clade -V (E. marmotae), retrospective phylogroup PCR did not discriminate them correctly. Differences in metabolic and adherence gene alleles occurred in the Clade -V isolates compared to E. coli sensu scricto. They also encoded the bacteriophage phage-associated cyto-lethal distending toxin (CDT) and antimicrobial resistance (AMR) genes, including an ESBL, blaOXA-453. Thus, the isolate collection encompassed a genetic diversity, and included cryptic clade isolates that encode potential virulence factors. The analysis has determined the phylogenetic relationship of cryptic clade isolates with E. coli sensu scricto and indicates a potential for horizontal transfer of virulence factors.

Quantifying changes in the British cattle movement network.

The modelling of disease spread is crucial to the farming industry and policy makers. In some of these industries, excellent data exist on animal movements, along with the networks that these movements create, and allow researchers to model spread of disease (both epidemic and endemic). The Cattle Tracing System is an online recording system for cattle births, deaths and between-herd movements in the United Kingdom and is an excellent resource for any researchers interested in networks or modelling infectious disease spread through the UK cattle system. Data exist that cover many years, and it can be useful to know how much change is occurring in a network, to help judge the merit of using historical data within a modelling context. This article uses the data to construct weighted directed monthly movement networks for two distinct periods of time, 2004-2006 and 2015-2017, to quantify by how much the underlying structure of the network has changed. Substantial changes in network structure may influence policy-makers directly or may influence models built upon the network data, and these in turn could impact policy-makers and their assessment of risk. We examined 13 network metrics, ranging from general descriptive metrics such as total number of nodes with movements and total movements, through to metrics to describe the network (e.g., Giant weakly and strongly connected components) and metrics calculated per node (betweenness, degree and strength). Mixed effect models show that there is a statistically significant effect of the period (2004-2006 vs 2015-2017) in the values of nine of the 13 network metrics. For example median total degree decreased by 19%. In addition to examining networks for two time periods, two updates of the data were examined to determine by how much the movement data stored for 2004-2006 had been cleansed between updates. Examination of these updates shows that there are small decreases in problem movements (such as animals leaving slaughterhouses) and therefore evidence of historical data being improved between updates. In combination with the significant effect of period on many of the network metrics, the modification of data between updates provides further evidence that the most recent available data should be used for network modelling. This will ensure that the most representative descriptions of the network are available to provide accurate modelling results to best inform policy makers.

Simple method to determine the concentration and incorporation ratio of ruthenium-labeled antibodies.

Aim: Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeled antibodies by UV/VIS spectroscopy. Materials & methods: Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292 nm. In contrast, antibodies demonstrate UV absorbance at 280 nm. After experimentally determining the extinction coefficients at 280 and 292 nm of free ruthenium and antibody, we generated a formula based on the Beer-Lambert law that calculates both concentration and DOL of these ruthenium-labeled antibodies. Conclusion: The concentration and DOL values determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.

Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

The COST action "Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control)," aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min-max: 1-13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.

Characterization of the robust humoral immune response to GSK2618960, a humanized anti-IL-7 receptor monoclonal antibody, observed in healthy subjects in a Phase 1 study.

Interleukin-7 (IL-7) signaling modulates T cell activity and is implicated in numerous autoimmune diseases. An anti-IL-7 receptor monoclonal antibody (GSK2618960) biotherapeutic was evaluated in healthy subjects for safety, pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity in a single-dose escalation phase I study. We found that antibodies against GSK2618960 (i.e., anti-drug antibodies or ADA) developed in 83% and 100% of GSK2618960-treated subjects in the 0.6 and 2.0 mg/kg dose cohorts, respectively. Of the ADA positive subjects, 64% (7 of 11) had detectable neutralizing activity. Further investigation revealed the presence of GSK2618960-specific memory B cells, indicating the development of immunological memory for the ADAs. Ex vivo stimulation of peripheral blood mononuclear cell (PBMC) samples demonstrated a relatively strong CD4+ T cell proliferation response to GSK2618960 as compared to the control anti-RSV antibody (which is known to have only low immunogenic potential), confirming the high immunogenic potential of GSK2618960. Furthermore, GSK2618960 was found to bind in vitro monocyte-derived dendritic cells (DCs). GSK2618960 treatment of PBMCs increased the proportion of DC cells showing an increase in expression of CD83, CD86 and CD209, which indicated enhanced DC differentiation and activation relative to the isotype control anti-ß amyloid antibody. Collectively, the evidence supports that the high incidence of observed clinical immunogenicity was likely related to the receptor-mediated activity by GSK2618960.