Flunixin (FLU) is a nonsteroidal drug that is widely used in animals, causing severe drug residues in animal-derived foods and environment. The development of antibody-based rapid immunoassay methods is of great significance for the monitoring of FLU and its metabolite 5-hydroxyflunixin (5-FLU). We prepared monoclonal antibodies (mAbs) with different recognition spectra through FLU-keyhole limpet hemocyanin conjugates as immunogen coupled with antibody screening strategies. mAb5E6 and mAb6D7 recognized FLU with high affinity, and mAb2H5 and mAb4A4 recognized FLU and 5-FLU with broad specificity. Through evaluating the recognition of these mAbs against more than 11 structural analogues and employing computational chemistry, molecular docking, and molecular dynamics methodologies, we preliminarily determined the recognition epitope and recognition mechanism of these mAbs. Finally, an indirect competitive enzyme-linked immunosorbent assay for FLU based on mAb6D7 was developed, which exhibited limits of detection as low as 0.016-0.042 μg kg -1 (L-1) in milk and muscle samples.

The COVID-19 pandemic, which began at the end of 2019 in Wuhan, has affected 220 countries and territories to date. In the present study, we studied humoral immunity in samples of the blood sera of COVID-19 convalescents of varying severity and patients who died due to this infection, using native SARS-CoV-2 and its individual recombinant proteins. The cross-reactivity with SARS-CoV (2002) was also assessed. We used infectious and inactivated SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 strain, inactivated SARS-CoV strain (strain Frankfurt 1, 2002), recombinant proteins, and blood sera of patients diagnosed with COVID-19. The blood sera from patients were analyzed by the Virus Neutralization test, Immunoblotting, and ELISA. The median values and mean ± SD of titers of specific and cross-reactive antibodies in blood sera tested in ELISA were mainly distributed in the following descending order: N > trimer S > RBD. ELISA and immunoblotting revealed a high cross-activity of antibodies specific to SARS-CoV-2 with the SARS-CoV antigen (2002), mainly with the N protein. The presence of antibodies specific to RBD corresponds with the data on the neutralizing activity of blood sera. According to the neutralization test in a number of cases, higher levels of antibodies that neutralize SARS-CoV-2 were detected in blood serum taken from patients several days before their death than in convalescents with a ranging disease severity. This high level of neutralizing antibodies specific to SARS-CoV-2 in the blood sera of patients who subsequently died in hospital from COVID-19 requires a thorough study of the role of humoral immunity as well as comorbidity and other factors affecting the humoral response in this disease.

BACKGROUND: Shigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis.
METHODS: In this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed.
RESULTS: Sixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002).
CONCLUSIONS: The Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FUNDING: Funding for this study was through a grant from the Wellcome Trust.

BACKGROUND: Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality.
METHODS: Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here.
RESULTS: Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection.
CONCLUSIONS: Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field.
BACKGROUND: Funding for this study was provided through a Wellcome Trust grant.

Breastfeeding provides defense against infectious disease during early life. The mechanisms underlying this protection are complex but likely include the vast array of immune cells and components, such as immunoglobulins, in milk. Simply characterizing the concentrations of these bioactives, however, provides only limited information regarding their potential relationships with disease risk in the recipient infant. Rather, understanding pathogen and antigen specificity profiles of milk-borne immunoglobulins might lead to a more complete understanding of how maternal immunity impacts infant health and wellbeing. Milk produced by women living in 11 geographically dispersed populations was applied to a protein microarray containing antigens from 16 pathogens, including diarrheagenic E. coli, Shigella spp., Salmonella enterica serovar Typhi, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis and other pathogens of global health concern, and specific IgA and IgG binding was measured. Our analysis identified novel disease-specific antigen responses and suggests that some IgA and IgG responses vary substantially within and among populations. Patterns of antibody reactivity analyzed by principal component analysis and differential reactivity analysis were associated with either lower-to-middle-income countries (LMICs) or high-income countries (HICs). Antibody levels were generally higher in LMICs than HICs, particularly for Shigella and diarrheagenic E. coli antigens, although sets of S. aureus, S. pneumoniae, and some M. tuberculosis antigens were more reactive in HICs. Differential responses were typically specific to canonical immunodominant antigens, but a set of nondifferential but highly reactive antibodies were specific to antigens possibly universally recognized by antibodies in human milk. This approach provides a promising means to understand how breastfeeding and human milk protect (or do not protect) infants from environmentally relevant pathogens. Furthermore, this approach might lead to interventions to boost population-specific immunity in at-risk breastfeeding mothers and their infants.

Platelet transfusion refractoriness results in adverse outcomes and increased health care costs. Managing refractoriness resulting from HLA alloimmunization necessitates the use of HLA antigen-matched platelets but requires a large platelet donor pool and does not guarantee full matching. We report the first randomized, double-blind, noninferiority, crossover trial comparing HLA epitope-matched (HEM) platelets with HLA standard antigen-matched (HSM) platelet transfusions. Alloimmunized, platelet-refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia were eligible. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to 8 prophylactic HEM and HSM transfusions provided in random order. The primary outcome was 1-hour posttransfusion platelet count increment (PCI). Forty-nine patients were randomized at 14 UK hospitals. For intention to treat, numbers of evaluable transfusions were 107 and 112 for HEM and HSM methods, respectively. Unadjusted mean PCIs for HEM and HSM methods were 23.9 (standard deviation [SD], 15) and 23.5 (SD, 14.1), respectively (adjusted mean difference, -0.1; 95% confidence interval [CI], -2.9 to 2.8). Because the lower limit of the 95% CI was not greater than the predefined noninferiority limit, the HEM approach was declared noninferior to the HSM approach. There were no differences in secondary outcomes of platelet counts, transfusion requirements, and bleeding events. Adequate 1-hour PCI was more frequently observed, with a mean number of 3.2 epitope mismatches, compared with 5.5 epitope mismatches for inadequate 1-hour increments. For every additional epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope-matched platelets should be considered to support HLA alloimmunized patients. This trial was registered at www.isrctn.com as #ISRCTN23996532.

Chromatin immunoprecipitation (ChIP) is a method used to examine the genomic localization of a target of interest (e.g., proteins, protein posttranslational modifications, or DNA elements). As ChIP provides a snapshot of in vivo DNA-protein interactions, it lends insight to the mechanisms of gene expression and genome regulation. This chapter provides a detailed protocol focused on native-ChIP (N-ChIP), a robust approach to profile stable DNA-protein interactions. We also describe best practices for ChIP , including defined controls to ensure specific and efficient target enrichment and methods for data normalization.

The control of antibody specificity plays pivotal roles in key technological fields such as diagnostics and therapeutics. During the development of immunoassays (IAs) for the biosensing of pathogens in food matrices, we have found a way to rationalize and control the specificity of polyclonal antibodies (sera) for a complex analytical target (the Salmonella genus), in terms of number of analytes (Salmonella species) and potential cross-reactivity with similar analytes (other bacteria strains). Indeed, the biosensing of Salmonella required the development of sera and serum mixtures displaying homogeneous specificity for a large set of strains showing broad biochemical variety (54 Salmonella serovars tested in this study), which partially overlaps with the molecular features of other class of bacteria (like specific serogroups of E. coli). To achieve a trade-off between specificity harmonisation and maximization, we have developed a strategy based on the conversion of the specificity profiles of individual sera in to numerical descriptors, which allow predicting the capacity of serum mixtures to detect multiple bacteria strains. This approach does not imply laborious purification steps and results advantageous for process scaling-up, and may help in the customization of the specificity profiles of antibodies needed for diagnostic and therapeutic applications such as multi-analyte detection and recombinant antibody engineering, respectively.

BACKGROUND: Coronavirus disease in 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has emerged as a global pandemic. Published data suggests that patients with a history of or active malignancy are at increased risk of infection and developing COVID-19 related complications. To date, the published data has analyzed the seroprevalence of COVID-19 infection in the general population, but not in cancer patients. Here we present the results of prevalence of IgG and IgM antibodies against SARS-CoV-2 in cancer patients from the University Hospital of Torrejón (Torrejón de Ardoz, Madrid, Spain).
METHODS: SARS-CoV-2 IgG and IgM antibodies was assessed using a commercially available rapid test (Testsealabs® IgG/IgM Rapid Test Cassette) and collect the result from cancer outpatients who attended the medical oncology consult at University Hospital of Torrejón between June 1st and June 19th, 2020.
RESULTS: We analyzed the serological test results of 229 cancer patients. We estimated an overall seroprevalence (IgG or IgM positive) of 31.4%. The probability of SARS-CoV-2 seropositivity was similar between men and women, type of treatment and cancer stage. The probability of seropositivity was significantly higher in cancer patients with pneumonia compared with cancer patients without pneumonia (Odds Ratio (OR) 7.65 [95% confidence interval (CI) 1,85-31,58]).
CONCLUSIONS: Our results show a higher rate of SARS-CoV-2 antibodies in cancer patients than in the general population. The role of those antibodies in the immune response against the virus infection is unclear.

Mosunetuzumab, a T-cell dependent bispecific antibody that binds CD3 and CD20 to drive T-cell mediated B-cell killing, is currently being tested in non-Hodgkin lymphoma. However, potent immune stimulation with T-cell directed therapies poses the risk of cytokine release syndrome, potentially limiting dose and utility. To understand mechanisms behind safety and efficacy and explore safety mitigation strategies, we developed a novel mechanistic model of immune and antitumor responses to the T-cell bispecifics (mosunetuzumab and blinatumomab), including the dynamics of B- and T-lymphocytes in circulation, lymphoid tissues, and tumor. The model was developed and validated using mosunetuzumab nonclinical and blinatumomab clinical data. Simulations delineated mechanisms contributing to observed cell and cytokine (IL6) dynamics and predicted that initial step-fractionated dosing limits systemic T-cell activation and cytokine release without compromising tumor response. These results supported a change to a step-fractionated treatment schedule of mosunetuzumab in the ongoing Phase I clinical trial, enabling safer administration of higher doses.