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The COVID-19 pandemic forced the rapid development of methods to measure humoral and cellular immunity against SARS-CoV-2. The lack of a global standardized protocol and the high variability of intra- and inter-assay precision of the T-cell response made it difficult to compare T-cell assay results with those of other laboratories. The interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assay for immunogenicity evaluation was validated using naturally infected donor peripheral blood mononuclear cells, a commercially available IFN-γ ELISpot kit, and a SARS-CoV-2 specific peptide pool. Depending on anti-CD3 and peptide pool stimulation, the mean coefficients of variation (CVs) of the intra-assay precision were 19.0% and 13.4%, respectively. The mean CVs of the inter-assay precision were 26.1% and 25.4%, and the mean CVs for reproducibility were 6.7% and 15.9%, respectively. Linearity with an R-squared value between 0.98 and 0.99 was established, and the mean CVs between the lots were 17.6% and 6.6%, depending on the anti-CD3 and peptide pool stimulation, respectively. The limit of detection was 11 spot-forming counts per well. Taken together, we demonstrated that the IFN-γ ELISpot assay is feasible for evaluating SARS-CoV-2-specific cell-mediated immune function through validation based on standard operating procedures.

COVID-19 vaccine evaluations are mainly focused on antibody analyses, but there is growing interest in measuring the cellular immune responses from the researchers evaluating these vaccines. The cellular responses to several COVID-19 vaccines have been studied using the enzyme-linked immunospot (ELISPOT) assay for IFN-γ. However, the ELISPOT assay is no longer used only for research purpose and so the performance of this assay must be validated. Since the bioanalytical validation of ELISPOT-IFN-γ is essential for evaluating the method's effectiveness and establishing confidence in a vaccine's immunogenicity, the present work validates the ELISPOT-IFN-γ assay's performance in determining the frequency of IFN-γ-producing cells after stimulation with the SARS-CoV-2 spike protein. The validation was performed in peripheral blood mononuclear cells from volunteers immunized with anti-COVID-19 vaccines. According to the findings, the LOD was 17 SFU and the LLOQ was 22 SFU, which makes the method highly sensitive and suitable for evaluating low levels of cellular responses. The procedure's accuracy is confirmed by the correlation coefficients for the spike protein and anti-CD3 + , being 0.98 and 0.95, respectively. The repeatability and intermediate precision tests were confirmed to be reliable by obtaining a coefficient of variation of ≤25%. The results obtained in this validation enable the assay to be employed for studying antigen-specific cells and evaluating cellular responses to vaccines.

Drug causality assessment in severe cutaneous adverse reactions (SCARs) remains challenging. We investigated the usefulness of in-vivo drug patch tests (PT), ex-vivo interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay, and lymphocyte transformation test (LTT) in 30 SCARs patients within the past 36 months. Drug PT yielded a 20% positivity rate (n = 6), while IFN-γ ELISpot and LTT showed positive rates of 56.67% (n = 17) and 41.38% (n = 12), respectively. Combining the three tests resulted in an overall positive rate of 66.67% (n = 20) of cases. IFN-γ ELISpot offered additional positivity, especially with oxypurinol. Employing a combined diagnostic approach may enhance the chances of obtaining a positive result., (© 2024 Japanese Dermatological Association.)

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients., Competing Interests: Declaration of competing interest M.B.M., K.E.R., and I.R.T. are members of Immune Functional Diagnostics, LLC (IFDx LLC) and receive no direct financial compensation. IFDx LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. S.C·B, L.L.M., R.S.H., and the University of Florida may receive royalty income based on a technology developed by S.C.B. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. C.C.C. and the University of Cincinnati may receive royalty income based on a technology developed by C.C.C. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research., (Published by Elsevier B.V.)

Background: Within endemic regions in southern and eastern Germany, Borna disease virus 1 (BoDV-1) causes rare zoonotic spill-over infections in humans, leading to encephalitis with a high case-fatality risk. So far, intra-vitam diagnosis has mainly been based on RT-qPCR from cerebrospinal fluid (CSF) and serology, both being associated with diagnostic challenges. Whilst low RNA copy numbers in CSF limit the sensitivity of RT-qPCR from this material, seroconversion often occurs late during the course of the disease., Case Presentation: Here, we report the new case of a 40 - 50 year-old patient in whom the detection of virus-specific T cells via ELISpot corroborated the diagnosis of BoDV-1 infection. The patient showed a typical course of the disease with prodromal symptoms like fever and headaches 2.5 weeks prior to hospital admission, required mechanical ventilation from day three after hospitalisation and remained in deep coma until death ten days after admission., Results: Infection was first detected by positive RT-qPCR from a CSF sample drawn four days after admission (viral load 890 copies/mL). A positive ELISpot result was obtained from peripheral blood collected on day seven, when virus-specific IgG antibodies were not detectable in serum, possibly due to previous immune adsorption for suspected autoimmune-mediated encephalitis., Conclusion: This case demonstrates that BoDV-1 ELISpot serves as additional diagnostic tool even in the first week after hospitalisation of patients with BoDV-1 encephalitis., (© 2024. The Author(s).)

For the rational design of epitope-specific vaccines, identifying epitopes that can be processed and presented is essential. As algorithm-based epitope prediction is frequently discordant with actually recognized CD8 + T-cell epitopes, we developed an in vitro CD8 T-cell priming protocol to enable the identification of truly and functionally expressed HLA class I epitopes. The assay was established and validated to identify epitopes presented by hepatitis C virus (HCV)-infected cells. In vitro priming of naïve CD8 T cells was achieved by culturing unfractionated PBMCs in the presence of a specific cocktail of growth factors and cytokines, and next exposing the cells to hepatic cells expressing the NS3 protein of HCV. After a 10-day co-culture, HCV-specific T-cell responses were identified based on IFN-γ ELISpot analysis. For this, the T cells were restimulated with long synthetic peptides (SLPs) spanning the whole NS3 protein sequence allowing the identification of HCV-specificity. We demonstrated that this protocol resulted in the in vitro priming of naïve precursors to antigen-experienced T-cells specific for 11 out of 98 SLPs tested. These 11 SLPs contain 12 different HLA-A*02:01-restricted epitopes, as predicted by a combination of three epitope prediction algorithms. Furthermore, we identified responses against 3 peptides that were not predicted to contain any immunogenic HLA class I epitopes, yet showed HCV-specific responses in vitro. Separation of CD8 + and CD8 - T cells from PBMCs primed in vitro showed responses only upon restimulation with short peptides. We established an in vitro method that enables the identification of HLA class I epitopes resulting from cross-presented antigens and that can cross-prime T cells and allows the effective selection of functional immunogenic epitopes, but also less immunogenic ones, for the design of tailored therapeutic vaccines against persistent viral infections and tumor antigens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies., Methods: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10., Results: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases., Conclusions: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: M. Luppi, L. Potenza and P. Barozzi have applied for an Italian patent regarding clinical applications of the ELISpot assay for the diagnosis of Aspergillus infection [PCT: WO2008/075395A3, IT2007/000867]. M. Luppi, L. Potenza, D. Vallerini, P. Barozzi and F. Forghieri have applied for an Italian and US patent regarding clinical applications of the ELISpot assay for the diagnosis of Mucorales infection [PCT: WO2012073160 A1, IT MI20102224, US 2013/0260395]. This does not alter the authors’ adherence to all Journal policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Other authors of this manuscript have no conflicts of interest related to the study to disclose., (Copyright: © 2024 Bettelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8 + ) and helper (CD4 + ) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

Background: T cell immunity plays a pivotal role in mitigating the severity of coronavirus disease 2019 (COVID-19). Therefore, reliable functional T cell assays are required to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell immunity in specific patient populations., Methods: We recruited a cohort of 23 healthcare workers who received their bivalent Omicron BA.1 / ancestral mRNA booster vaccination or were infected with the Omicron variant at a median of 144 days and 227 days before blood collection, respectively. In this cohort, we compared the performances of two widely utilized commercial SARS-CoV-2 interferon-gamma release assays (IGRAs), i.e., QuantiFERON SARS-CoV-2 and T-SPOT.COVID, and an in-house designed Omicron enzyme-linked immunospot (ELISpot)., Results: The QuantiFERON SARS-CoV-2 and T-SPOT.COVID assays detected SARS-CoV-2 spike-specific T cells in 34.8 % and 21.7 % of participants, respectively. Moreover, our in-house designed ELISpot that included Omicron BA.4 and BA.5 full-spike peptides detected T cell responses in 47.8 % of participants and was strongly associated with the T-SPOT.COVID., Conclusion: The evaluation of SARS-CoV-2 T cell immunity using commercially accessible assays may yield disparate outcomes as results from different assays are not directly comparable. A specific Omicron ELISpot should be considered to assess Omicron-specific T cell immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In the monitoring of human Toxoplasma gondii infection, it is crucial to confirm the development of a specific Th1/Th17 immune response memory. The use of a simple, specific, and sensitive assay to follow the T-cell activation is thus required. Current protocols are not always specific as stimulation with peptides is Human Leukocyte Antigen (HLA)-dependent, while stimulation with total-lysis antigens tends to stimulate seronegative donors resulting to false positives. Here, an improved ELISPOT protocol is reported, using peripheral blood mononuclear cells (PBMC) of T.gondii-infected donors, incubated with the inactivated parasite. The results showed that, contrary to standard protocols, a pre-incubation step at high cell density in presence of the inactivated parasite allowed a specific Th1/Th17 response with the secretion of IFN-γ, IL-2, IL-12 and IL-17 cytokines. This protocol allows to evaluate precisely the immune response after a T.gondii infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fasquelle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool ( p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8 + T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8 + T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8 + T cells., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Enzyme-Linked Immunosorbent Spot assay (ELISpot) is an immunoassay used to quantify individual protein-specific secreting cells. Proteins secreted by cells cultured in ELISpot plates (96- or 8-well format) bind to a specific antigen bound to a PVDF membrane at the bottom of the well. A detection antibody followed by an enzymatic reaction is used to identify secreted protein bound to the membrane coated antigen. This reaction results in distinct "spots" on the membrane corresponding to individual protein secreting cells. While the design is similar to an ELISA, ELISpots quantify the number and relative amount of secreted protein on a single cell level, as opposed to an ELISA that reveals the concentration of secreted proteins from a population of cells. The sensitivity, robustness, and diversity of different antigens used by ELISpots have led to an array of research applications such as measuring cytokines from cytotoxic T cells in cancer and quantifying antibody specificity from B cells following vaccinations. Improvements have been made to assays measuring cytokines and antibodies on a single cell basis, such as intracellular flow cytometry. Yet the ability of an ELISpot to evaluate the quantity and quality of protein secretion on an individual cell basis remains unmatched. Here, we describe the use of a modified ELISpot assay to detect antigen-specific memory B cells in the setting of a viral infection and autoimmunity., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Antigen-specific B-cell ELISPOT and multicolor FluoroSpot assays, in which the membrane-bound antigen itself serves as the capture reagent for the antibodies that B cells secrete, inherently result in a broad range of spot sizes and intensities. The diversity of secretory footprint morphologies reflects the polyclonal nature of the antigen-specific B cell repertoire, with individual antibody-secreting B cells in the test sample differing in their affinity for the antigen, fine epitope specificity, and activation/secretion kinetics. To account for these heterogeneous spot morphologies, and to eliminate the need for setting up subjective counting parameters well-by-well, CTL introduces here its cutting-edge deep learning-based IntelliCount ™ algorithm within the ImmunoSpot ® Studio Software Suite, which integrates CTL's proprietary deep neural network. Here, we report detailed analyses of spots with a broad range of morphologies that were challenging to analyze using standard parameter-based counting approaches. IntelliCount ™ , especially in conjunction with high dynamic range (HDR) imaging, permits the extraction of accurate, high-content information of such spots, as required for assessing the affinity distribution of an antigen-specific memory B-cell repertoire ex vivo. IntelliCount ™ also extends the range in which the number of antibody-secreting B cells plated and spots detected follow a linear function; that is, in which the frequencies of antigen-specific B cells can be accurately established. Introducing high-content analysis of secretory footprints in B-cell ELISPOT/FluoroSpot assays, therefore, fundamentally enhances the depth in which an antigen-specific B-cell repertoire can be studied using freshly isolated or cryopreserved primary cell material, such as peripheral blood mononuclear cells., (© 2024. The Author(s).)

Enzyme-linked immunospot (ELISPOT) is one of the most important methods to measure the number of specific cells by detecting protein secretion at a single-cell level. However, traditional ELISPOT based on enzyme-substrate color development can only detect one target. Therefore, scientists developed multiple-target ELISPOT based on enzyme-substrate coloring. Besides, FluoroSPOT that can detect 2-4 fluorescent signals are developed. Nevertheless, the maximum detection targets of multiple-target ELISPOT and FluoroSPOT are around 4, and the signal amplification system can be further optimized. Fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification, was developed to detect multiple targets simultaneously. In this method, multiple targets can be detected in one round and multiple rounds of detection can be conducted, and thus a large number of targets can be detected. Besides, signal amplification is achieved by DNA complementary pairing and modular orthogonal DNA concatemers, and thus cells secreting limited amounts of proteins can be detected. According to the studies, FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable by ELISPOT. Furthermore, FOLISPOT can be utilized to detect more than 6 targets, by allowing sequential detection of multiple targets in one round and sequential detection in multiple rounds., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Virus-specific T cells are critical to mediating viral control; however, Hepatitis B virus (HBV)-specific T cells among chronic Hepatitis B (CHB) patients are functionally exhausted. The inability to consistently measure the ex vivo functionality of HBV-specific T cells has prevented meaningful analysis during antiviral events such as HBeAg seroconversion, hepatic flares, and HBsAg loss. We optimized the traditional IFN-γ ELISpot assay to measure total ex vivo HBV-specific T cell frequencies using CHB PBMCs stimulated with HBV overlapping peptide (OLP) pools. This was then further adapted to assess individual antigen specificity (core, envelop, polymerase, X) and multifunctional HBV-specific T cells using a 3-analyte FluoroSpot assay. This protocol encompasses two major components: (1) PBMC handling/stimulation and (2) assay plate preparation and spot development. By performing this assay, ex vivo CHB patient T cell responses could be assessed longitudinally during immunotherapy or other important clinical events., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ELISpot assay has a solid place in the immune monitoring field for over 40 years. It is an assay that can assess the function of single immune cells in a straightforward and easy-to-learn approach. Its use in basic research, translational, and clinical work has been documented in countless publications. Harmonization guidelines and invaluable tools for optimal assay performance and evaluation exist. However, the validation of an established ELISpot protocol has been left to diverse opinions about how to interpret and tackle typical validation parameters. This chapter addresses important considerations for ELISpot validation, including the interpretations of validation parameters for a meaningful description of assay performance., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest., Competing Interests: Declaration of Competing Interest FD, DDT, CM, PP, CS, GF, IF, FV, IR, GP, MM and AM are employed by VisMederi Srl; EM is founder and Chief Scientific Officer of VisMederi Srl and VisMederi Research Srl; IM is employed by University of Siena., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

ELISPOT and FluoroSpot assays, collectively called ImmunoSpot assays, permit to reliable detection of rare antigen-specific T cells in freshly isolated cell material, such as peripheral blood mononuclear cells (PBMC). Establishing their frequency within all PBMC permits to assess the magnitude of antigen-specific T-cell immunity; the simultaneous measurement of their cytokine signatures reveals these T-cells' lineage and effector functions, that is, the quality of T-cell-mediated immunity. Because of their unparalleled sensitivity, ease of implementation, robustness, and frugality in PBMC utilization, T-cell ImmunoSpot assays are increasingly becoming part of the standard immune monitoring repertoire. For regulated workflows, stringent audit trails of the data generated are a requirement. While this has been fully accomplished for the analysis of T-cell ImmunoSpot assay results, such are missing for the wet laboratory implementation of the actual test performed. Here we introduce a solution for enhancing and verifying the error-free implementation of T-cell ImmunoSpot assays., (© 2024. The Author(s).)

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51 Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51 Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-ɣ (IFN-ɣ) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-ɣ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners. The introduction of the IFN-ɣ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-ɣ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable. In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-ɣ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use., Competing Interests: Declaration of Competing Interest Authors EM and EG are employed by VisMederi srl. MJ and BS are employed by GlaxoSmithKline. SB and AP are employed by Sanofi and may hold stock.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Cell and gene therapies have demonstrated impressive therapeutic efficacy in various human diseases. Nevertheless, cellular immune response directed against these therapeutic agents is an obstacle for achieving long-lasting clinical efficacy. Therefore, it is crucial to develop robust assays to accurately monitor cellular immunogenicity towards these therapies. Enzyme-linked immunospot (ELISpot) assay is one of the primarily used methods for measuring cellular immune response in clinical programs, which requires isolation of the peripheral blood mononuclear cells (PBMCs). The quality of this clinical material is one of the most critical factors that impact the robust assessment of cellular immune responses. The optimal blood sample processing conditions, however, remain poorly understood. In this study, we examined the impact of blood sample processing time on the performance characteristics of ELISpot to measure antigen-specific cellular responses. Blood samples that were processed after overnight delay resulted in a loss of ELISpot signals. We subsequently optimized several parameters of sample processing, and successfully recovered ELISpot signals for the blood samples that are processed within 32 h. Furthermore, several mitigation strategies were employed that would potentially address the impact of granulocyte contamination on detection of antigen-specific cellular responses. Our investigation provides an extension of sample processing window for clinical studies and is significant for resolving the logistical challenge of whole blood sample shipment for timely PBMC preparation in cell/gene therapy clinical studies., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Inborn errors of immunity (IEI) that present with recurrent infections are largely due to antibody (Ab) deficiencies. Therefore, assessment of the B-cell and Ab compartment is a major part of immunologic evaluation. Here we provide an overview about cellular assays used to study B-cell function and focus on lymphocyte proliferation assay (LPA), opsonophagocytic assay (OPA), and the Enzyme-linked Immunosorbent Spot Assay (ELISPOT) including clinical applications and limitations of these techniques. LPAs assess ex-vivo cell proliferation in response to various stimuli. Clinically available LPAs utilize peripheral blood mononuclear cells and mostly assess T-cell proliferation with pokeweed mitogen considered the most B-cell specific stimulus. In the research setting, isolating B cells or using more B-cell specific stimuli such as CD40L with IL-4/IL-21 or the TLR9 ligand CpG can more specifically capture the proliferative ability of B cells. OPAs are functional in-vitro killing assays used to evaluate the ability of IgG Ab to induce phagocytosis applied when assessing the potency of vaccine candidates or along with avidity assays to evaluate the quality of secreted IgG. The B-cell ELISPOT assesses Ab production at a cellular level and can characterize the Ab response of particular B-cell subtypes. It can be used in patients on IgG therapy by capturing specific Abs produced by individual B cells, which is not affected by exogenous IgG from plasma donors, and when assessing the vaccine response in patients on immunomodulatory drugs that can affect memory B-cell function. Emerging approaches that are only available in research settings are also briefly introduced., Competing Interests: Declaration of Competing Interest No., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today's evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection., Competing Interests: Authors EM and EG are employed by VisMederi srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Waerlop, Leroux-Roels, Lambe, Bellamy, Medaglini, Pettini, Cox, Trieu, Davies, Bredholt, Montomoli, Gianchecchi and Clement.)

Enzyme-linked immunosorbent assay (ELISA) is one of the most common methods in biological studies, and enzyme-linked immunospot (ELISpot) is a method to measure specific cell numbers by detecting protein secretion at a single-cell level. However, these two current methods can only detect one signal at one time and the sensitivity is not high enough to test low-concentration samples, which are major shortcomings in systematically analyzing the samples of interest. Herein, we demonstrated fluorescence-based oligo-linked immunosorbent assay (FOLISA) and fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification. Signal amplification and simultaneous multiple-target detection were achieved by DNA complementary pairing and modular orthogonal DNA concatemers. By comparing FOLISA with traditional ELISA and comparing FOLISPOT with traditional ELISPOT, we found that the detection sensitivities of FOLISA and FOLISPOT are much higher than those of traditional ELISA and ELISPOT. The detection limit of ELISA is around 3 pg/mL, and the detection limit of FOLISA is below 0.06 pg/mL. FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable for ELISPOT. Furthermore, FOLISA and FOLISPOT allowed sequential detection of multiple targets by using a single dye or multiple dyes in one round and sequential detection in multiple rounds. Thus, FOLISA and FOLISPOT enabled simultaneous detection of a large number of targets, significantly improved the detection sensitivity, and overcame the shortcomings of ELISA and ELISPOT. Overall, FOLISA and FOLISPOT presented effective and general platforms for rapid and multiplexed detection of antigens or antibodies with high sensitivity, either in laboratory tests or potentially in clinic tests.

Adeno-Associated Virus (AAV)-based gene therapy vectors are in development for many inherited human disorders. In nonclinical studies, cellular immune responses mediated by cytotoxic T cells may target vector-transduced cells, which could impact safety and efficacy. Here, we describe the bioanalytical validation of an interferon-gamma (IFN-γ)-based Enzyme-Linked Immunospot (ELISpot) assay for measuring T cell responses against viral antigens in cynomolgus monkeys. Since ELISpots performed with antigen-derived peptides offer a universal assay format, method performance characteristics were validated using widely available peripheral blood mononuclear cells (PBMCs) responsive to cytomegalovirus peptides. The limit of detection and confirmatory cut point were established using statistical methods; precision, specificity, and linearity were confirmed. Monkey PBMCs from an AAV5 gene therapy study were then analyzed, using peptide pools spanning the vector capsid and transgene product. AAV5-specific T cell responses were detected only in 2 of 18 monkeys at Day 28, but not at Day 13 and 56 after vector administration, with no correlation to liver enzyme elevations or transgene expression levels. No transgene product-specific T cell responses occurred. In conclusion, while viral peptide-specific IFN-γ ELISpots can be successfully validated for monkey PBMCs, monitoring peripheral T cell responses in non-clinical AAV5 gene therapy studies was of limited value to interpret safety or efficacy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature.)

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4 + T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hyun, Lee, Jo, Baek, Kim, Sohn and Kim.)

Formation of neutralizing antibodies and cellular immune response with repeat adeno-associated virus (AAV) gene therapy dosing are critical concerns in translational, large animal studies. The enzyme-linked immunospot/immunosorbent spot (ELISpot) assay introduced a way to track B- and/or T-cell response to therapy over time at a protein level. We describe the protocol for this assay looking at relative interferon (IFN)-γ secretion in pre- and post-AAV injections in a pig model., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The differential diagnosis between tuberculous meningitis (TBM) and bacterial meningitis (BM) remains challenging in clinical practice. This study aimed to establish a diagnostic model that could accurately distinguish TBM from BM., Methods: Patients with TBM or BM were recruited between January 2017 and January 2021 at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort). The detection for indicators involved in cerebrospinal fluid (CSF) and T-SPOT assay were performed simultaneously. Multivariate logistic regression was used to create a diagnostic model., Results: A total of 174 patients (76 TBM and 98 BM) and another 105 cases (39 TBM and 66 BM) were enrolled from Qiaokou cohort and Caidian cohort, respectively. Significantly higher level of CSF lymphocyte proportion while significantly lower levels of CSF chlorine, nucleated cell count, and neutrophil proportion were observed in TBM group when comparing with those in BM group. However, receiver operating characteristic (ROC) curve analysis showed that the areas under the ROC curve (AUCs) produced by these indicators were all under 0.8. Meanwhile, tuberculosis-specific antigen/phytohemagglutinin (TBAg/PHA) ratio yielded an AUC of 0.889 (95% CI, 0.840-0.938) in distinguishing TBM from BM, with a sensitivity of 68.42% (95% CI, 57.30%-77.77%) and a specificity of 92.86% (95% CI, 85.98%-96.50%) when a cutoff value of 0.163 was used. Consequently, we successfully established a diagnostic model based on the combination of TBAg/PHA ratio, CSF chlorine, CSF nucleated cell count, and CSF lymphocyte proportion for discrimination between TBM and BM. The established model showed good performance in differentiating TBM from BM (AUC: 0.949; 95% CI, 0.921-0.978), with 81.58% (95% CI, 71.42%-88.70%) sensitivity and 91.84% (95% CI, 84.71%-95.81%) specificity. The performance of the diagnostic model obtained in Qiaokou cohort was further validated in Caidian cohort. The diagnostic model in Caidian cohort produced an AUC of 0.923 (95% CI, 0.867-0.980) with 79.49% (95% CI, 64.47%-89.22%) sensitivity and 90.91% (95% CI, 81.55%-95.77%) specificity., Conclusions: The diagnostic model established based on the combination of four indicators had excellent utility in the discrimination between TBM and BM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Luo, Xue, Lin, Mao, Tang, Song, Liu, Wu, Liu, Zhou, Xu, Xiong, Wang, Yuan, Gan, Sun and Wang.)

Human cytomegalovirus (HCMV) is a common cause of significant morbidity and mortality in transplant recipients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We evaluated interferon-γ (IFN-γ) secretion by HCMV NLV-specific CD8 + T cells in HCMV-reactivated allo-HSCT recipients using an enzyme-linked immunospot (ELISPOT) assay at 3 months post-transplantation. Blood samples from 47 recipients were tested for HCMV DNAemia, HCMV pp65 antigenemia, and anti-HCMV immunoglobulins (IgG/IgM) over 3 months post-transplantation. Of the 47 transplant recipients, 26 were HLA-A*02 positive and 21 were HLA-A*02 negative. The results were essentially consistent between the 47 transplant recipients and the HLA-A*02-positive recipients. HCMV DNAemia was not linearly correlated with IFN-γ spot-forming cells (SFCs) counts; IFN-γ SFCs counts did not differ significantly between the HCMV DNAemia-positive and -negative groups, whereas the HCMV-DNA virus loads were inversely correlated with the IFN-γ SFCs counts. HCMV pp65 antigenemia was not linearly correlated with IFN-γ SFCs counts; IFN-γ SFCs counts in the HCMV pp65 antigenemia-positive and -negative groups were similar. More IFN-γ SFCs counts were detected in transplant recipients with high anti-HCMV-IgG antibody titers than in those with low anti-HCMV-IgG titers pre-transplantation in the 47 recipients. Anti-HCMV-IgG antibody titers were positively linearly correlated with IFN-γ SFCs counts in HLA-A*02-positive recipients. The HCMV infection indicators used to monitor HCMV reactivation had different values in transplant recipients. The use of the IFN-γ SFCs counts measured by ELISPOT to evaluate the risk of HCMV reactivation needs further study., (© 2021 Wiley Periodicals LLC.)

Acute rejection remains an important problem after kidney transplantation. Enzyme-linked immunosorbent spot (ELISPOT) assay has been investigated extensively and has shown promising results as a predictor of allograft rejection. The objective of this study was to systematically review and analyze the predictive value of the donor-specific ELISPOT assay to identify recipients at risk for acute rejection. Electronic databases were searched for studies reporting donor-specific ELISPOT and kidney transplantation outcomes. Odds ratio (OR) for acute rejection was calculated, along with standardized mean difference (SMD) of cytokine producing-cells between recipients with and without acute rejection. Pooled estimates were calculated using random-effect models. The positive ELISPOT cutoff frequencies were extracted from each study. From 665 articles found, 32 studies were included in the meta-analysis. IFN-γ was the most investigated cytokine (30 out of 32 studies). Patients with positive pre-transplantation donor-reactive IFN-γ ELISPOT had an OR of 3.3 for acute rejection (95%-CI 2.1 to 5.1), and OR of 6.8 (95%-CI 2.5 to 18.9) for post-transplantation ELISPOT. Recipients with rejection had significantly higher frequencies of pre- and post-transplantation cytokine producing-cells (SMD 0.47, 95%-CI 0.07 to 0.87 and SMD 3.68, 95%-CI 1.04 to 6.32, respectively). Pre-transplantation ELISPOT had a positive predictive value of 43% and a negative predictive value of 81% for acute rejection. A positive ELISPOT result was associated with a lower estimated glomerular filtration rate (SMD -0.59, 95%-CI -0.83 to -0.34). In conclusion, patients with a high frequency of donor-reactive IFN-γ ELISPOT are at higher risk for acute rejection. The donor-specific IFN-γ ELISPOT assay can serve as an immune-monitoring tool in kidney transplantation., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aim: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before., Materials and Methods: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated., Results: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes., Conclusion: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4 + T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic ( n  = 18) and antiretroviral therapy (ART)-treated subjects ( n  = 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. IMPORTANCE Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.

Seasonal influenza is an acute respiratory infection causing around 500 000 global deaths annually. There is an unmet medical need to develop more effective antiviral drugs and vaccines against influenza infection. A rapid, accurate, high-throughput titration assay for influenza virus particles or neutralizing antibodies would be extremely useful in these research fields. However, commonly used methods such as tissue culture infective dose and plaque-forming units (PFU) for virus particle quantification, and the plaque reduction neutralization test (PRNT) for antibody determination are time-consuming, laborious, and have limited accuracy. In this study, we developed an efficient assay based on the enzyme-linked immunospot (ELISPOT) technique for the influenza virus and neutralizing antibody titration. Two broad-spectrum antibodies recognizing the nucleoproteins of influenza A and B viruses were used in the assay to broadly and highly sensitively detect influenza virus-infected cells at 16 hours postinfection. An optimized cell culture medium with no tosyl phenylalanyl chloromethyl ketone trypsin and high dose oseltamivir acid was used to improve quantitation accuracy. This ELISPOT assay displayed a good correlation (R 2  = 0.9851) with the PFU assay when used to titrate 30 influenza virus isolates. The assay was also applied to measure influenza-neutralizing antibodies in 40 human sera samples, showing a good correlation (R 2  = 0.9965) with the PRNT assay. This ELISPOT titration assay is a rapid, accurate, high-throughput assay for quantification of influenza virus and neutralizing antibodies, and provides a powerful tool for research into and development of drugs and vaccines against influenza., (© 2020 Wiley Periodicals LLC.)

When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten recovery, is contraindicated. Antiviral treatment with convalescent plasma (CP) could be an alternative treatment option. In this case report, we present two kidney transplant recipients and two hemodialysis patients who were infected with SARS-CoV-2 and received CP. Antibodies against the receptor-binding domain in the S1 subunit of the SARS-CoV-2 spike protein were determined sequentially by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and neutralization assay and specific cellular responses by interferon-gamma ELISpot. Before treatment, in both kidney transplant recipients and one hemodialysis patient antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers (≤1:40). ELISpot responses in the four patients were either weak or absent. After CP treatment, we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific cellular responses. After intermittent clinical improvement, one kidney transplant recipient again developed typical symptoms on Day 12 after treatment and received a second cycle of CP treatment. Altogether, three patients clinically improved and could be discharged from the hospital. However, one 83-year-old multimorbid patient deceased. Our data suggest that the success of CP therapy may only be temporary in patients with chronic kidney disease; which requires close monitoring of viral load and antiviral immunity and possibly an adaptation of the treatment regimen., (© 2021 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-1 19 , MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria., Competing Interests: PJ, CSm, and NA are employed by Mabtech AB, Sweden. Several of the reagents and the FluoroSpot reader system used in this study are produced by Mabtech. Mabtech has no influence on the content of this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jahnmatz, Sundling, Yman, Widman, Asghar, Sondén, Stenström, Smedman, Ndungu, Ahlborg and Färnert.)

Hepatitis B virus (HBV)-specific T cells play a critical role in determining the outcome of HBV infection. However, T cell response induced by predominant Ag in chronic infection is hardly detectable owing to the lack of a suitable assay. We herein established an optimized method to enumerate HBV-specific T cells and assessed the association between HBV surface Ag (HBsAg) and HBV DNA. Sixty chronic HBV infection patients were enrolled. HBV-specific T cells were expanded by using overlapping peptide pools covering the entire sequence of HBV genotypes B and C. IFN-γ-producing HBV-specific T cells were detected by a cultured enzyme-linked immunospot (ELISPOT) assay, ex vivo ELISPOT assay, or flow cytometry staining. The association between HBV-specific T cells and serum levels of HBsAg and HBV DNA were analyzed. Cultured ELISPOT assay had a higher sensitivity than ex vivo ELISPOT in the detection of HBV-specific T cells. Moreover, consistent results were acquired by flow cytometry analysis and cultured ELISPOT assay, but the latter required only a limited number of cells for detection. Interestingly, HBV core peptide pool induced a robust HBV-specific T cell response in patients with lower levels of HBV DNA and HBsAg. Specifically, the frequency of HBV core Ag-specific IFN-γ + spot-forming cells was inversely correlated with serum levels of HBV DNA and HBsAg. An optimized cultured ELISPOT assay reveals the association between HBV core Ag-induced T cell response and HBV control; this method may favor the investigation of HBV-specific T cell in chronic HBV infection., (©2020 Society for Leukocyte Biology.)

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8 + CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The enzyme-linked immune absorbent spot (ELISpot) assay allows for the quantification of the number of cells producing a particular secreted analyte. As T lymphocytes secrete cytokines such as interferon (IFN)-γ upon binding of the T cell receptor with its cognate antigen epitope, IFN-γ ELISpot allows for the measurement of antigen-specific T cells in an immune sample. Immune cells are isolated from the vaccinated subject and incubated with the epitope/antigen of interest on polyvinylidene difluoride (PVDF)-lined microplates precoated with a capture antibody to IFN-γ. Cytokine spots are then detected utilizing an IFN-γ-specific detection antibody and an enzyme-linked conjugate. Here, we describe the quantification of OVA-specific CD8 and CD4 T cells from mouse splenocytes to measure vaccine-induced cellular responses.

Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells Basic Protocol 2: ELISpot assay for enumeration of memory B cell-derived antibody-secreting cells., (© 2020 Wiley Periodicals LLC.)

The diagnosis of tuberculous pericarditis (TBP) remains challenging. This prospective study evaluated the diagnostic value of Xpert MTB/RIF (Xpert) and T-SPOT.TB and adenosine deaminase (ADA) for TBP in a high burden setting. A total of 123 HIV-negative patients with suspected TBP were enrolled at a tertiary referral hospital in China. Pericardial fluids were collected and subjected to the three rapid tests, and the results were compared with the final confirmed diagnosis. Of 105 patients in the final analysis, 39 (37.1%) were microbiologically, histopathologically or clinically diagnosed with TBP. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio (DOR) for Xpert were 66.7%, 98.5%, 96.3%, 83.3%, 44.0, 0.338, and 130.0, respectively, compared to 92.3%, 87.9%, 81.8%, 95.1%, 7.6, 0.088, and 87.0, respectively, for T-SPOT.TB, and 82.1%, 92.4%, 86.5%, 89.7%, 10.8, 0.194, and 55.8, respectively, for ADA (≥ 40 U/L). ROC curve analysis revealed a cut-off point of 48.5 spot-forming cells per million pericardial effusion mononuclear cells for T-SPOT.TB, which had a DOR value of 183.8, while a cut-off point of 41.5 U/L for ADA had a DOR value of 70.9. Xpert (Step 1: rule-in) followed by T-SPOT.TB [cut-off point] (Step 2: rule-out) showed the highest DOR value of 252.0, with only 5.7% (6/105) of patients misdiagnosed. The two-step algorithm consisting of Xpert and T-SPOT.TB could offer rapid and accurate diagnosis of TBP.

Despite the strong demand for orally-delivered fish vaccines and the deficient response of those currently available in the market, little is known about how teleost B cells differentiate to antibody secreting cells (ASCs) in response to antigens delivered to the intestinal mucosa. To fill this gap, in the current study, we have studied the dynamics of B cell differentiation in spleen and kidney of rainbow trout (Oncorhynchus mykiss) anally immunized with antigens catalogued in mammals as thymus dependent (TD) or thymus-independent (TI). Our results show that, in the absence of additional adjuvants, rainbow trout preferentially responded to a model TI antigen such as TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The anal administration of TNP-LPS elicited TNP-specific serum antibodies, and a significant increase in the number of total and TNP-specific ASCs in both spleen and kidney, being the kidney the site where most ASCs are found at later time points. In the spleen, a proliferative response of both IgM + B and T cells was also clearly visible, while the proliferative response was weaker in the kidney. Finally, TNP-LPS also provoked a transcriptional regulation of some immune genes in the spleen and the intestine, including a decreased transcription of foxp3a and foxp3b in intestine that suggests a breach in tolerogenic responses in response to TI stimulation. These results contribute to a better understanding of how intestinal immunity is regulated in teleost and will aid in the future design of effective oral strategies for aquaculture., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Commercial cellular tests are used to diagnose Lyme borreliosis (LB), but studies on their clinical validation are lacking. This study evaluated the utility of an in-house and a commercial enzyme-linked immunosorbent spot (ELISpot) assay for the diagnosis of Lyme neuroborreliosis (LNB). Prospectively, peripheral blood mononuclear cells (PBMCs) were isolated from patients and controls and analysed using an in-house Borrelia ELISpot assay and the commercial LymeSpot assay. B. burgdorferi B31 whole cell lysate and a mixture of outer surface proteins were used to stimulate the PBMCs and the numbers of interferon-gamma-secreting T cells were measured. Results were evaluated using receiver operating characteristic (ROC) curve analysis. Eighteen active and 12 treated LNB patients, 10 healthy individuals treated for an early (mostly cutaneous) manifestation of LB in the past and 47 untreated healthy individuals were included. Both assays showed a poor diagnostic performance with sensitivities, specificities, positive and negative predictive values ranging from 44.4-66.7%, 42.0-72.5%, 21.8-33.3% and 80.5-87.0%, respectively. The LymeSpot assay performed equally poorly when the calculation method of the manufacturer was used. Both the in-house and the LymeSpot assay are unable to diagnose active LNB or to monitor antibiotic treatment success., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)