Pub Date : 2025-09-18DOI: 10.1016/j.jim.2025.113980
Ti Lu, Md Shafiullah Parvej, Sean K. Whittier, Suhrid Maiti, Zackary K. Dietz, Debaki R. Howlader, Mst Nusrat Zahan, Satabdi Biswas, William D. Picking, Wendy L. Picking
In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.
{"title":"A high-throughput multi-species platform using Biolayer Interferometry Immunosorbent Assay (BLI-ISA) as an alternative to indirect ELISA for vaccine development","authors":"Ti Lu, Md Shafiullah Parvej, Sean K. Whittier, Suhrid Maiti, Zackary K. Dietz, Debaki R. Howlader, Mst Nusrat Zahan, Satabdi Biswas, William D. Picking, Wendy L. Picking","doi":"10.1016/j.jim.2025.113980","DOIUrl":"10.1016/j.jim.2025.113980","url":null,"abstract":"<div><div>In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113980"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-08-05DOI: 10.1016/j.jim.2025.113918
Francesco Savino, Paola Montanari, Maddalena Dini, Anna Pau, Lorenzo Ferroglio, Sara Burdisso, Cristina Calvi, Anna Clemente, Stefano Gambarino, Ilaria Galliano, Massimiliano Bergallo
Bronchiolitis is an acute lower respiratory tract infection mainly affecting children under 24 months, primarily caused by Respiratory Syncytial Virus (RSV). Interferons (IFNs), cytokines central to innate and adaptive immunity, are secreted in response to viral infections. In RSV bronchiolitis, Type I and III IFN Signatures modulate antiviral responses and impact disease severity. This study aimed to evaluate the correlation between Type I and III IFNs transcriptional signatures in blood and nasal swabs with clinical presentation. We analyzed transcription levels of Type I and III IFN Signatures using a PCR real-time TaqMan assay in blood and nasal swabs from children hospitalized for RSV bronchiolitis and from healthy controls (HC). Interferon score I was significantly higher in bronchiolitis patients at admission than at discharge and compared to HC, in both blood and nasal swabs (p < 0.0001). A strong positive correlation for IFN score I between the two sample types was found (p < 0.0001). Patients with severe disease (requiring intensive care) had lower IFN score I than those with milder disease, in both blood (p = 0.0005) and nasal swabs (p = 0.0078). IFN score III was down-regulated in bronchiolitis patients compared to HC in blood samples (p < 0.0001), while in nasal swabs, this down-regulation was evident only at discharge (p < 0.0001 vs admission; p = 0.0546 vs HC). This study enhances understanding of IFN signature dynamics in RSV bronchiolitis, emphasizing the importance of sample type and signature specificity, and suggests their potential use as prognostic tools to improve patient management.
{"title":"Peripheral blood and nasal swabs Type I and III IFNs signature in RSV positive infant with bronchiolitis.","authors":"Francesco Savino, Paola Montanari, Maddalena Dini, Anna Pau, Lorenzo Ferroglio, Sara Burdisso, Cristina Calvi, Anna Clemente, Stefano Gambarino, Ilaria Galliano, Massimiliano Bergallo","doi":"10.1016/j.jim.2025.113918","DOIUrl":"10.1016/j.jim.2025.113918","url":null,"abstract":"<p><p>Bronchiolitis is an acute lower respiratory tract infection mainly affecting children under 24 months, primarily caused by Respiratory Syncytial Virus (RSV). Interferons (IFNs), cytokines central to innate and adaptive immunity, are secreted in response to viral infections. In RSV bronchiolitis, Type I and III IFN Signatures modulate antiviral responses and impact disease severity. This study aimed to evaluate the correlation between Type I and III IFNs transcriptional signatures in blood and nasal swabs with clinical presentation. We analyzed transcription levels of Type I and III IFN Signatures using a PCR real-time TaqMan assay in blood and nasal swabs from children hospitalized for RSV bronchiolitis and from healthy controls (HC). Interferon score I was significantly higher in bronchiolitis patients at admission than at discharge and compared to HC, in both blood and nasal swabs (p < 0.0001). A strong positive correlation for IFN score I between the two sample types was found (p < 0.0001). Patients with severe disease (requiring intensive care) had lower IFN score I than those with milder disease, in both blood (p = 0.0005) and nasal swabs (p = 0.0078). IFN score III was down-regulated in bronchiolitis patients compared to HC in blood samples (p < 0.0001), while in nasal swabs, this down-regulation was evident only at discharge (p < 0.0001 vs admission; p = 0.0546 vs HC). This study enhances understanding of IFN signature dynamics in RSV bronchiolitis, emphasizing the importance of sample type and signature specificity, and suggests their potential use as prognostic tools to improve patient management.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113918"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-08-05DOI: 10.1016/j.jim.2025.113921
Mojca Lunder, Jernej Luzar, Tim Ključevšek, Aleš Berlec, Borut Štrukelj, Maja Skerbinjek Kavalar, Ana Koren, Peter Korošec
The prevalence of peanut allergy has been steadily increasing over the last decades. Among food allergies it shows the highest severity and is potentially fatal. Allergen Ara h 2 stands out as the main elicitor of allergic reactions. Research in the field of peanut immunotherapy still strives to improve the significant drawbacks of currently approved therapeutics. In this study we tested three epitope-like peptides as epitope-specific treatment. The epitope-like peptides (mimotopes) of major peanut allergen Ara h 2 were expressed as fusions with scaffold protein - lactococcal peptidoglycan binding domain of AcmA. Mimotope-decorated L. lactis and mimotope-AcmA fusion proteins were characterized. The mimotopes retained the ability to bind IgE from peanut-allergic patients' sera. They showed low basophil activation in peanut-allergic patients, except for mimotope DHPRYGP fused to AcmA suggesting its allergenic activity was partially preserved due to the highest similarity with Ara h 2 epitope. Mimotopes fused to AcmA can elicit T cell response; the increased secretion of IFN-γ suggests the shift of T cell immune response to non-allergic type 1. The use of recombinant L.lactis as a delivery system for surface displayed mimotopes represents a promising strategy that would enable delivery and expression of mimotopes on mucosal surfaces and at the same time beneficial immunomodulating properties.
在过去的几十年里，花生过敏的发病率一直在稳步上升。在食物过敏中，它的严重程度最高，可能致命。过敏原a2是引起过敏反应的主要刺激物。花生免疫治疗领域的研究仍在努力改进目前批准的治疗方法的显著缺陷。在这项研究中，我们测试了三种表位样肽作为表位特异性治疗。花生主要过敏原Ara h 2的表位样肽（mimotopes）与支架蛋白-乳球菌肽聚糖结合域AcmA融合表达。对mimotope-装饰乳杆菌和mimotope-AcmA融合蛋白进行了表征。这些基因基保留了结合花生过敏患者血清中IgE的能力。在花生过敏患者中，除了与AcmA融合的mimotope DHPRYGP外，它们显示出较低的嗜碱性粒细胞活化，这表明由于与Ara h2表位的高度相似性，其致敏活性部分保留。与AcmA融合的模位可以引起T细胞反应；IFN-γ分泌增加提示T细胞免疫反应向非过敏性1型转移。利用重组乳杆菌作为表面显示的mimotopes的递送系统代表了一种很有前途的策略，可以在粘膜表面递送和表达mimotopes，同时具有有益的免疫调节特性。
{"title":"Lactococcus lactis as a delivery system for surface displayed mimotopes of major peanut allergen Ara h 2.","authors":"Mojca Lunder, Jernej Luzar, Tim Ključevšek, Aleš Berlec, Borut Štrukelj, Maja Skerbinjek Kavalar, Ana Koren, Peter Korošec","doi":"10.1016/j.jim.2025.113921","DOIUrl":"10.1016/j.jim.2025.113921","url":null,"abstract":"<p><p>The prevalence of peanut allergy has been steadily increasing over the last decades. Among food allergies it shows the highest severity and is potentially fatal. Allergen Ara h 2 stands out as the main elicitor of allergic reactions. Research in the field of peanut immunotherapy still strives to improve the significant drawbacks of currently approved therapeutics. In this study we tested three epitope-like peptides as epitope-specific treatment. The epitope-like peptides (mimotopes) of major peanut allergen Ara h 2 were expressed as fusions with scaffold protein - lactococcal peptidoglycan binding domain of AcmA. Mimotope-decorated L. lactis and mimotope-AcmA fusion proteins were characterized. The mimotopes retained the ability to bind IgE from peanut-allergic patients' sera. They showed low basophil activation in peanut-allergic patients, except for mimotope DHPRYGP fused to AcmA suggesting its allergenic activity was partially preserved due to the highest similarity with Ara h 2 epitope. Mimotopes fused to AcmA can elicit T cell response; the increased secretion of IFN-γ suggests the shift of T cell immune response to non-allergic type 1. The use of recombinant L.lactis as a delivery system for surface displayed mimotopes represents a promising strategy that would enable delivery and expression of mimotopes on mucosal surfaces and at the same time beneficial immunomodulating properties.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113921"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144799349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-28DOI: 10.1016/j.jim.2025.113969
Danyang Zhang , Yourong Yang , Zhen Zhang , Junxian Zhang , Xueqiong Wu , Yan Liang
Background
Mycobacterium tuberculosis (MTB) Ag85A has become a component of multiple new tuberculosis vaccines. It is necessary to evaluate the immunogenicity, biological distribution, and safety of ag85a plasmid DNA (pDNA) to lay the foundation for the design of new vaccines.
Method
Chronic toxicity test: cynomolgus monkeys were injected intramuscularly with different doses of ag85a pDNA, and the vaccine absorption kinetics, tissue distribution, and toxicity were observed. Their immune function was evaluated. Acute toxicity test: Mice were injected intramuscularly 0.5 ml saline, and injected intramuscularly and intravenously 0.5 mg/0.5 ml ag85a pDNAs, respectively. The toxicity and death of the mice were observed continuously for 14 days. Allergic test: Guinea pigs were intraperitoneally injected with different doses of ag85a pDNA. After stimulation, the allergic reaction and its severity were observed.
Results
Chronic and acute toxicity tests demonstrated that ag85a pDNA injections caused no clinical symptoms or tissue damage. Repeated intramuscular injections in cynomolgus monkeys enhanced specific Th1 immune responses, with pDNA rapidly entering the bloodstream and its concentration positively correlating with dosage. After 8 weeks, ag85a gene was detected only in muscles, myocardium, iliac lymph nodes, and blood. Guinea pig allergy tests showed no weight changes or allergic reactions, even after multiple sensitizations.
Conclusions
The ag85a pDNA showed good safety in cynomolgus monkeys, mice, and guinea pigs, and induced high levels of antibodies and T-cell responses, making it a candidate antigen for the construction of a new tuberculosis vaccine.
{"title":"Immunogenicity, biodistribution, and toxicology evaluation of Mycobacterium tuberculosis ag85a plasmid DNA in cynomolgus monkeys, mice and guinea pigs","authors":"Danyang Zhang , Yourong Yang , Zhen Zhang , Junxian Zhang , Xueqiong Wu , Yan Liang","doi":"10.1016/j.jim.2025.113969","DOIUrl":"10.1016/j.jim.2025.113969","url":null,"abstract":"<div><h3>Background</h3><div><em>Mycobacterium tuberculosis</em> (MTB) Ag85A has become a component of multiple new tuberculosis vaccines. It is necessary to evaluate the immunogenicity, biological distribution, and safety of <em>ag85a</em> plasmid DNA (pDNA) to lay the foundation for the design of new vaccines.</div></div><div><h3>Method</h3><div>Chronic toxicity test: cynomolgus monkeys were injected intramuscularly with different doses of <em>ag85a</em> pDNA, and the vaccine absorption kinetics, tissue distribution, and toxicity were observed. Their immune function was evaluated. Acute toxicity test: Mice were injected intramuscularly 0.5 ml saline, and injected intramuscularly and intravenously 0.5 mg/0.5 ml <em>ag85a</em> pDNAs, respectively. The toxicity and death of the mice were observed continuously for 14 days. Allergic test: Guinea pigs were intraperitoneally injected with different doses of <em>ag85a</em> pDNA. After stimulation, the allergic reaction and its severity were observed.</div></div><div><h3>Results</h3><div>Chronic and acute toxicity tests demonstrated that <em>ag85a</em> pDNA injections caused no clinical symptoms or tissue damage. Repeated intramuscular injections in cynomolgus monkeys enhanced specific Th1 immune responses, with pDNA rapidly entering the bloodstream and its concentration positively correlating with dosage. After 8 weeks, <em>ag85a</em> gene was detected only in muscles, myocardium, iliac lymph nodes, and blood. Guinea pig allergy tests showed no weight changes or allergic reactions, even after multiple sensitizations.</div></div><div><h3>Conclusions</h3><div>The <em>ag85a</em> pDNA showed good safety in cynomolgus monkeys, mice, and guinea pigs, and induced high levels of antibodies and T-cell responses, making it a candidate antigen for the construction of a new tuberculosis vaccine.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113969"},"PeriodicalIF":1.6,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144957000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-28DOI: 10.1016/j.jim.2025.113970
Ryan Sinapius, Jeremy Fisher, Christopher Manning, Amrik Singh
Background
Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of cell-based immunotherapy. To expand CAR-T therapies into additional disease indications, identification of novel tumor antigens and grafting of CARs on other types of immune cells, such as macrophages and natural killer (NK) cells are being pursued. Therefore, as this treatment modality continues to evolve, there is a need for highly specific detection reagents to interrogate CAR surface expression.
Methods/Objectives
This study uses flow cytometry to compare the ability of various commercially available CAR detection reagents' to detect CAR constructs containing distinct hinges, linker sequences, and scFv designs. Furthermore, interrogation off-target interactions were performed within a PBMC matrix.
Results
All antibody-based CAR detection reagents demonstrated specificity and robust signal in stable CAR-Jurkat cell lines and primary human CAR-T cells. Protein L only detected anti-CD20 (Leu16) scFv-based CAR; off-target non-CAR staining was observed in non-transduced human PBMCs. Detection using recombinant protein antigen provides clean results in single-stain samples for CD19 and BMCA proteins, though CD20 did not produce positive staining in anti-CD20 CARs. Additionally, in live human PBMCs, false-positive signals may be observed for antibody conjugates due to the binding of human Fc receptors to IgG.
Conclusions
CAR idiotype antibodies, recombinant antigens, and anti-linker antibodies were able to provide specific and robust detection of CAR-expressing cells. However, the utility of both idiotype antibodies and recombinant antigens is limited to CARs targeting specific antigens. The anti-linker antibodies enable universal and broad detection of CARs independent of target antigens.
{"title":"CAR binding efficacy and off-target interactions for common CAR detection reagents","authors":"Ryan Sinapius, Jeremy Fisher, Christopher Manning, Amrik Singh","doi":"10.1016/j.jim.2025.113970","DOIUrl":"10.1016/j.jim.2025.113970","url":null,"abstract":"<div><h3>Background</h3><div>Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of cell-based immunotherapy. To expand CAR-T therapies into additional disease indications, identification of novel tumor antigens and grafting of CARs on other types of immune cells, such as macrophages and natural killer (NK) cells are being pursued. Therefore, as this treatment modality continues to evolve, there is a need for highly specific detection reagents to interrogate CAR surface expression.</div></div><div><h3>Methods/Objectives</h3><div>This study uses flow cytometry to compare the ability of various commercially available CAR detection reagents' to detect CAR constructs containing distinct hinges, linker sequences, and scFv designs. Furthermore, interrogation off-target interactions were performed within a PBMC matrix.</div></div><div><h3>Results</h3><div>All antibody-based CAR detection reagents demonstrated specificity and robust signal in stable CAR-Jurkat cell lines and primary human CAR-T cells. Protein L only detected anti-CD20 (Leu16) scFv-based CAR; off-target non-CAR staining was observed in non-transduced human PBMCs. Detection using recombinant protein antigen provides clean results in single-stain samples for CD19 and BMCA proteins, though CD20 did not produce positive staining in anti-CD20 CARs. Additionally, in live human PBMCs, false-positive signals may be observed for antibody conjugates due to the binding of human Fc receptors to IgG.</div></div><div><h3>Conclusions</h3><div>CAR idiotype antibodies, recombinant antigens, and anti-linker antibodies were able to provide specific and robust detection of CAR-expressing cells. However, the utility of both idiotype antibodies and recombinant antigens is limited to CARs targeting specific antigens. The anti-linker antibodies enable universal and broad detection of CARs independent of target antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113970"},"PeriodicalIF":1.6,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144932472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-25DOI: 10.1016/j.jim.2025.113968
Hendrika W. Grievink , Coen Breedveld , John Öhd , Mark Schoonderwoerd , Hjalmar P. Permentier , Amanda C. Foks , Ilze Bot , Elsa Neubert , Matthijs Moerland
Neutrophils are an emerging target for therapeutical intervention in both autoimmune diseases as well as cancer. Since healthy humans lack constitutive neutrophil activation, induction of neutrophil activation is necessary to evaluate investigational compounds and can be achieved via intravenous administration of lipopolysaccharides (LPS). Furthermore, LPS stimulation can be performed ex vivo during clinical trials, and in vitro for pre-clinical analysis. Therefore, we aimed to provide a time course of neutrophil responses after in vivo LPS administration using samples from human endotoxemia trials and compared this to in vitro LPS stimulated whole blood cultures. We performed shotgun proteomics on in vivo stimulated neutrophils, and measured neutrophil activation by flow cytometry using CD11b and CD62L as activation markers and elastase, MPO, LL37 and nucleosome levels as degranulation and NETosis markers. Neutrophil numbers rapidly increased after LPS administration. In line, we found significant increases in neutrophil activation and degranulation markers both in vitro as well as in vivo, which all returned to baseline within 24 h. Degranulation proteins and NETosis related nucleosomes rapidly increased after LPS administration (1 h after exposure) in vivo, while higher concentrations of LPS were necessary in vitro. Lastly, shotgun proteomics revealed little but significant differences in the neutrophil proteome after in vivo LPS administration, pointing to degranulation after LPS stimulation. Both, the in vitro whole blood LPS stimulation assay and the human endotoxemia model, could be valuable tools for evaluation of the effects of future drugs modulating neutrophil responses during preclinical and clinical development.
{"title":"In vitro and in vivo lipopolysaccharide-driven activation of human neutrophils in healthy volunteers as a tool for clinical drug development","authors":"Hendrika W. Grievink , Coen Breedveld , John Öhd , Mark Schoonderwoerd , Hjalmar P. Permentier , Amanda C. Foks , Ilze Bot , Elsa Neubert , Matthijs Moerland","doi":"10.1016/j.jim.2025.113968","DOIUrl":"10.1016/j.jim.2025.113968","url":null,"abstract":"<div><div>Neutrophils are an emerging target for therapeutical intervention in both autoimmune diseases as well as cancer. Since healthy humans lack constitutive neutrophil activation, induction of neutrophil activation is necessary to evaluate investigational compounds and can be achieved <em>via</em> intravenous administration of lipopolysaccharides (LPS). Furthermore, LPS stimulation can be performed <em>ex vivo</em> during clinical trials, and <em>in vitro</em> for pre-clinical analysis. Therefore, we aimed to provide a time course of neutrophil responses after <em>in vivo</em> LPS administration using samples from human endotoxemia trials and compared this to <em>in vitro</em> LPS stimulated whole blood cultures. We performed shotgun proteomics on <em>in vivo</em> stimulated neutrophils, and measured neutrophil activation by flow cytometry using CD11b and CD62L as activation markers and elastase, MPO, LL37 and nucleosome levels as degranulation and NETosis markers. Neutrophil numbers rapidly increased after LPS administration. In line, we found significant increases in neutrophil activation and degranulation markers both <em>in vitro</em> as well as <em>in vivo</em>, which all returned to baseline within 24 h. Degranulation proteins and NETosis related nucleosomes rapidly increased after LPS administration (1 h after exposure) <em>in vivo</em>, while higher concentrations of LPS were necessary <em>in vitro</em>. Lastly, shotgun proteomics revealed little but significant differences in the neutrophil proteome after <em>in vivo</em> LPS administration, pointing to degranulation after LPS stimulation. Both, the <em>in vitro</em> whole blood LPS stimulation assay and the human endotoxemia model, could be valuable tools for evaluation of the effects of future drugs modulating neutrophil responses during preclinical and clinical development.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113968"},"PeriodicalIF":1.6,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144934090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-12DOI: 10.1016/j.jim.2025.113920
Elin Manell , M. Esad Gunes , Philip Jordache , Satyajit Patwardhan , Julie Hong , David Sachs , Joshua Weiner
Skin and vascularized composite allografts (VCA) containing skin are transplanted to restore form and function of tissues after major injuries. Skin has long been recognized as being particularly immunogenic, causing high risk of rejection and immune sensitization. Due to skin-specific antigens, donor skin is often rejected even in animals that are tolerant of the remaining donor tissue. To study the reaction of lymphocyte subsets against these minor and/or tissue-specific skin antigens in swine made tolerant to allogeneic donors through hematopoietic stem cell transplants (HSCT), we developed a skin-adapted variation of the mixed lymphocyte reaction (MLR). We processed porcine skin into single cell suspensions to be used as stimulators. Peripheral blood mononuclear cells were used as responders. We first optimized the concentrations of skin stimulators to achieve T cell proliferation with minimal self-background reactivity. The assay was then tested in two pigs that had received combined VCA/HSCT. The first pig had not rejected any part of the VCA, and the second pig was actively rejecting the epidermis of the VCA at the time of the assay. Despite lack of anti-donor MLR responses against donor lymphocytes in the peripheral blood in either animal, the second pig demonstrated a specific response against donor skin cells. Our results suggest that the assay will be useful to study recipient sensitization against skin antigens, even in otherwise tolerant animals. This assay may have both diagnostic and therapeutic implications for immune responses specific to the skin.
{"title":"A novel variation of the mixed lymphocyte reaction for measuring T cell responses to skin-specific antigens of pigs","authors":"Elin Manell , M. Esad Gunes , Philip Jordache , Satyajit Patwardhan , Julie Hong , David Sachs , Joshua Weiner","doi":"10.1016/j.jim.2025.113920","DOIUrl":"10.1016/j.jim.2025.113920","url":null,"abstract":"<div><div>Skin and vascularized composite allografts (VCA) containing skin are transplanted to restore form and function of tissues after major injuries. Skin has long been recognized as being particularly immunogenic, causing high risk of rejection and immune sensitization. Due to skin-specific antigens, donor skin is often rejected even in animals that are tolerant of the remaining donor tissue. To study the reaction of lymphocyte subsets against these minor and/or tissue-specific skin antigens in swine made tolerant to allogeneic donors through hematopoietic stem cell transplants (HSCT), we developed a skin-adapted variation of the mixed lymphocyte reaction (MLR). We processed porcine skin into single cell suspensions to be used as stimulators. Peripheral blood mononuclear cells were used as responders. We first optimized the concentrations of skin stimulators to achieve T cell proliferation with minimal self-background reactivity. The assay was then tested in two pigs that had received combined VCA/HSCT. The first pig had not rejected any part of the VCA, and the second pig was actively rejecting the epidermis of the VCA at the time of the assay. Despite lack of anti-donor MLR responses against donor lymphocytes in the peripheral blood in either animal, the second pig demonstrated a specific response against donor skin cells. Our results suggest that the assay will be useful to study recipient sensitization against skin antigens, even in otherwise tolerant animals. This assay may have both diagnostic and therapeutic implications for immune responses specific to the skin.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113920"},"PeriodicalIF":1.6,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Antibody-dependent enhancement (ADE) of infection is a concerning phenomenon in SARS-CoV-2 infection, since it may develop disease severity. Although ADE has been demonstrated in animal models, the pathogenic mechanism has not been fully elucidated. The present study aimed to develop a simple assay system for detecting SARS-CoV-2 ADE activity in any antibody specimen. Vero E6/TMPRSS2/FcγRIIA, a Vero E6 cell strain expressing TMPRSS2 and FcγRIIA, was established. Seventeen plasma samples from convalescent patients and seven commercial antibodies were used as antibody specimens. Vero E6/TMPRSS2/FcγRIIA cells, infected with SARS-CoV-2 in the presence of antibody, were shown to exhibit ADE activity, with the plaque number increasing dramatically compared with the control in the absence of antibody specimens. Most plasmas displayed both ADE and neutralizing activities. Furthermore, 6 commercial antibodies recognizing structural proteins (membrane, nucleocapsid, envelope, and spike [transmembrane and cytoplasmic domains] proteins and non-structural protein ORF7) showed no potential ADE activity, while a commercial antibody recognizing spike RBD displayed ADE activity. These results indicate that antibodies possessing neutralizing and/or enhancing activity might be generated by either viral infection or vaccination. The present ADE assay may help to evaluate the risk of disease severity for individuals upon future reinfection, vaccination and/or immunotherapy.
{"title":"Development of in vitro plaque-based assay for assessment of antibody-dependent enhancement in SARS-CoV-2 infection","authors":"Pimploy Rattanaamnuaychai , Surachet Benjathummarak , Pannamthip Pitaksajjakul , Pongrama Ramasoota , Yong Poovorawan , Hiroto Mizushima , Masashi Tatsumi , Yoshiharu Matsuura , Atsushi Yamanaka","doi":"10.1016/j.jim.2025.113919","DOIUrl":"10.1016/j.jim.2025.113919","url":null,"abstract":"<div><div>Antibody-dependent enhancement (ADE) of infection is a concerning phenomenon in SARS-CoV-2 infection, since it may develop disease severity. Although ADE has been demonstrated in animal models, the pathogenic mechanism has not been fully elucidated. The present study aimed to develop a simple assay system for detecting SARS-CoV-2 ADE activity in any antibody specimen. Vero E6/TMPRSS2/FcγRIIA, a Vero E6 cell strain expressing TMPRSS2 and FcγRIIA, was established. Seventeen plasma samples from convalescent patients and seven commercial antibodies were used as antibody specimens. Vero E6/TMPRSS2/FcγRIIA cells, infected with SARS-CoV-2 in the presence of antibody, were shown to exhibit ADE activity, with the plaque number increasing dramatically compared with the control in the absence of antibody specimens. Most plasmas displayed both ADE and neutralizing activities. Furthermore, 6 commercial antibodies recognizing structural proteins (membrane, nucleocapsid, envelope, and spike [transmembrane and cytoplasmic domains] proteins and non-structural protein ORF7) showed no potential ADE activity, while a commercial antibody recognizing spike RBD displayed ADE activity. These results indicate that antibodies possessing neutralizing and/or enhancing activity might be generated by either viral infection or vaccination. The present ADE assay may help to evaluate the risk of disease severity for individuals upon future reinfection, vaccination and/or immunotherapy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113919"},"PeriodicalIF":1.6,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144781255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-07-28DOI: 10.1016/j.jim.2025.113917
Lisa Dyleski , Ying Wang , Elena Seletskaia , Lee Walus , Teresa Caiazzo , Alison Joyce , Daniel Baltrukonis , Christopher Lepsy
Polyethylene glycol (PEG) has been widely used in household products from skin care and cosmetics, to baby wipes and cleaners, as well as protein biotherapeutics. More recently, PEG has become a critical component of lipid nanoparticles (e.g., LNP or Accurin platforms) used to deliver small molecule and ribonucleic acid (RNA) therapeutics. A potential concern with therapeutics containing PEG is that the PEG moiety can induce anti-PEG antibodies that may impact patient pharmacokinetics (PK), safety and/or efficacy of the drug ((Ju et al., 2023), (Warren et al., 2021)). The accurate assessment of anti-PEG antibodies may be needed and their relationship to clinical PK, safety and efficacy mandates reliable bioanalytical methods with suitable specificity and sensitivity with characterization at the isotype level. Here we report the development of a novel multiplexed anti-PEG antibody sandwich-immunoassay, using an electrochemiluminescence (ECL) format to simultaneously detect major immunoglobulin (Ig) isotypes (IgM, IgG and IgE) of anti-PEG antibodies in human serum. As the initial step in the platform application's proof-of-concept (POC), we demonstrated that this first-ever multiplexed anti-PEG isotyping assay is highly specific with suitable sensitivities across all major immunoglobulin isotypes. Our holistic approach first focused on establishing critical assay components required for the multiplex format, followed by implementation of a sample pre-treatment procedure to optimize specificity and sensitivity. We then addressed non-specific matrix interference in the presence of potentially confounding factors, such as the high prevalence of pre-existing anti-PEG antibodies in human serum. This approach resulted in a specific and sensitive multiplex assay. Based on preliminary evaluation, the estimated assay sensitivities met regulatory expectations for clinical ADA assays (≤ 100 ng/mL). Notably, the estimated assay sensitivity for IgE isotypes of anti-PEG antibodies was ≤25 ng/mL, as determined using an IgE isotype specific anti-PEG antibody positive control.
{"title":"Development of a novel anti-PEG antibody assay enabling investigation of potential immunogenicity triggered by the PEG moiety of biotherapeutics","authors":"Lisa Dyleski , Ying Wang , Elena Seletskaia , Lee Walus , Teresa Caiazzo , Alison Joyce , Daniel Baltrukonis , Christopher Lepsy","doi":"10.1016/j.jim.2025.113917","DOIUrl":"10.1016/j.jim.2025.113917","url":null,"abstract":"<div><div>Polyethylene glycol (PEG) has been widely used in household products from skin care and cosmetics, to baby wipes and cleaners, as well as protein biotherapeutics. More recently, PEG has become a critical component of lipid nanoparticles (e.g., LNP or Accurin platforms) used to deliver small molecule and ribonucleic acid (RNA) therapeutics. A potential concern with therapeutics containing PEG is that the PEG moiety can induce anti-PEG antibodies that may impact patient pharmacokinetics (PK), safety and/or efficacy of the drug ((<span><span>Ju et al., 2023</span></span>), (<span><span>Warren et al., 2021</span></span>)). The accurate assessment of anti-PEG antibodies may be needed and their relationship to clinical PK, safety and efficacy mandates reliable bioanalytical methods with suitable specificity and sensitivity with characterization at the isotype level. Here we report the development of a novel multiplexed anti-PEG antibody sandwich-immunoassay, using an electrochemiluminescence (ECL) format to simultaneously detect major immunoglobulin (Ig) isotypes (IgM, IgG and IgE) of anti-PEG antibodies in human serum. As the initial step in the platform application's proof-of-concept (POC), we demonstrated that this first-ever multiplexed anti-PEG isotyping assay is highly specific with suitable sensitivities across all major immunoglobulin isotypes. Our holistic approach first focused on establishing critical assay components required for the multiplex format, followed by implementation of a sample pre-treatment procedure to optimize specificity and sensitivity. We then addressed non-specific matrix interference in the presence of potentially confounding factors, such as the high prevalence of pre-existing anti-PEG antibodies in human serum. This approach resulted in a specific and sensitive multiplex assay. Based on preliminary evaluation, the estimated assay sensitivities met regulatory expectations for clinical ADA assays (≤ 100 ng/mL). Notably, the estimated assay sensitivity for IgE isotypes of anti-PEG antibodies was ≤25 ng/mL, as determined using an IgE isotype specific anti-PEG antibody positive control.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113917"},"PeriodicalIF":1.6,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144753477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-07-19DOI: 10.1016/j.jim.2025.113906
Hope Mataramvura, Patience Ncube, Kerina Duri
Background
Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.
Methods
Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.
Results
We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8–6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5–8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (p = 0.092). Median TPP was 90 min (IQR:64.8–112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 106 cells/ml) (p = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (r = −0.35, p < 0.001; r = −0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/− NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3–22.2)] than in females [23.8 % (IQR:14.3–29.9)] (p = 0.018).
Conclusion
HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.
{"title":"From phlebotomy to peripheral blood mononuclear cell isolation; effect of time and early-life HIV/ART exposure on cell yield and viability in paediatric samples in a high HIV prevalence setting","authors":"Hope Mataramvura, Patience Ncube, Kerina Duri","doi":"10.1016/j.jim.2025.113906","DOIUrl":"10.1016/j.jim.2025.113906","url":null,"abstract":"<div><h3>Background</h3><div>Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.</div></div><div><h3>Methods</h3><div>Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.</div></div><div><h3>Results</h3><div>We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8–6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5–8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (<em>p</em> = 0.092). Median TPP was 90 min (IQR:64.8–112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 10<sup>6</sup> cells/ml) (<em>p</em> = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (<em>r</em> = −0.35, <em>p</em> < 0.001; <em>r</em> = −0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/− NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3–22.2)] than in females [23.8 % (IQR:14.3–29.9)] (<em>p</em> = 0.018).</div></div><div><h3>Conclusion</h3><div>HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113906"},"PeriodicalIF":1.6,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144682719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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