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
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
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.
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.
Abstract
T cells are important in preventing severe disease from SARS-CoV-2, but scalable and field-adaptable alternatives to expert T-cell assays are needed. The interferon-gamma release assay ...QuantiFERON platform was developed to detect T-cell responses to SARS-CoV-2 from whole blood with relatively basic equipment and flexibility of processing timelines. Forty-eight participants with different infection and vaccination backgrounds were recruited. Whole blood samples were analysed using the QuantiFERON SARS-CoV-2 assay in parallel with the well-established ‘Protective Immunity from T Cells in Healthcare workers’ (PITCH) ELISpot, which can evaluate spike-specific T-cell responses. The primary aims of this cross-sectional observational cohort study were to establish if the QuantiFERON SARS-Co-V-2 assay could discern differences between specified groups and to assess the sensitivity of the assay compared with the PITCH ELISpot. The QuantiFERON SARS-CoV-2 distinguished acutely infected individuals (12–21 days post positive PCR) from naïve individuals (P < 0.0001) with 100% sensitivity and specificity for SARS-CoV-2 T cells, whilst the PITCH ELISpot had reduced sensitivity (62.5%) for the acute infection group. Sensitivity with QuantiFERON for previous infection was 12.5% (172–444 days post positive test) and was inferior to the PITCH ELISpot (75%). Although the QuantiFERON assay could discern differences between unvaccinated and vaccinated individuals (55–166 days since second vaccination), the latter also had reduced sensitivity (44.4%) compared to the PITCH ELISpot (66.6%). The QuantiFERON SARS-CoV-2 assay showed potential as a T- cell evaluation tool soon after SARS-CoV-2 infection but has lower sensitivity for use in reliable evaluation of vaccination or more distant infection.
With the exception of acute infection group, the PITCH ELISpot S1 + S2 had greater sensitivity for SARS-CoV-2 specific T-cell responses compared with the QuantiFERON SARS-CoV-2 assay tube Ag3.
Graphical Abstract
Graphical Abstract
Diagnosing drug-induced allergy, especially nonimmediate phenotypes, is challenging. Incorrect classifications have unwanted consequences.
We sought to evaluate the diagnostic utility of IFN-γ ...ELISpot and clinical parameters in predicting drug-induced nonimmediate hypersensitivity using machine learning.
The study recruited 393 patients. A positive patch test or drug provocation test (DPT) was used to define positive drug hypersensitivity. Various clinical factors were considered in developing random forest (RF) and logistic regression (LR) models. Performances were compared against the IFN-γ ELISpot-only model.
Among the 102 patients who had 164 DPTs, most patients had severe cutaneous adverse reactions (35/102, 34.3%) and maculopapular exanthems (33/102, 32.4%). Common suspected drugs were antituberculosis drugs (46/164, 28.1%) and β-lactams (42/164, 25.6%). Mean (SD) age of patients with DPT was 52.7 (20.8) years. IFN-γ ELISpot, fixed drug eruption, Naranjo categories, and nonsteroidal anti-inflammatory drugs were the most important features in all developed models. The RF and LR models had higher discriminating abilities. An IFN-γ ELISpot cutoff value of 16.0 spot-forming cells/10
PBMCs achieved 94.8% specificity and 57.1% sensitivity. Depending on clinical needs, optimal cutoff values for RF and LR models can be chosen to achieve either high specificity (0.41 for 96.1% specificity and 0.52 for 97.4% specificity, respectively) or high sensitivity (0.26 for 78.6% sensitivity and 0.37 for 71.4% sensitivity, respectively).
IFN-γ ELISpot assay was valuable in identifying culprit drugs, whether used individually or incorporated in a prediction model. Performances of RF and LR models were comparable. Additional test datasets with DPT would be helpful to validate the model further.
Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ...ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST− ELISA− ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.
The COVID-19 (coronavirus disease 2019) pandemic has spurred the development of highly effective quantitative methods for assessing the adaptive immune response to the SARS-CoV-2 (severe acute ...respiratory syndrome-related coronavirus 2) virus. In order to assess the humoral component of the immune response, various methods for detecting immunoglobulins A, M, G are widely used. ELISPOT seems to be the most accessible and effective method to assess the level of T cells that specifically respond to the SARS-CoV-2 virus antigens.
The aim
. To assess cell-mediated and humoral immunity in COVID-19 in residents of the Republic of Crimea.
Methods
. The study was performed on 24 volunteers: the presence of coronavirus antibodies was determined by ELISA method, and the presence of contact with coronavirus proteins – by the ELISPOT “TigraTest® SARS-CoV-2” method (Generium, Russia). For retrospective study of humoral immunity in the population, we assessed 10 000 ELISA tests (ECOlab IgM and IgG, Russia) performed in our laboratory for the period from July 2020 to January 2022.
Results
. The results show the effectiveness of using the ELISPOT method to detect latent forms of coronavirus infection. It is important to note that there is statistically significant relationship between the timing of the disease and the number of spots in both antigen panels. After vaccination against SARS-CoV-2, cell-mediated immunity lasts up to 6 months or more.
Conclusions
. As a result of the study, it was found that during 2021, the level of immunization of the population of the Republic of Crimea against COVID-19 has significantly increased; the proportion of residents who have positive IgG test has increased from 27 % to 87 %. The results of ELISPOT studies using a set of reagents for in vitro detection of blood T-lymphocytes that specifically respond to SARS-COV-2 virus antigens (“TigraTest® SARS-CoV-2”) showed that this method is more sensitive than ELISA in detecting latent diseases.
During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of ...evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit “negative” patients.
•Quantification of anti-SARS-CoV-2 memory T cell response using IFNγ-ELISpot assay was higher as compared to T-cell LPA.•The use of different peptide pools did not impact detection of anti-SARS-CoV-2 memory T cell response by IFNγ-ELISpot assay.•We used an innovative approach, the mixed Gaussian modeling, to define new thresholds of anti-Spike memory T cell response.
The ORFs of both native and codon‐optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin‐controlled gene expression system in the NZ9000 strains of Lactococcus ...lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific‐pathogen‐free C57BL/6 mice were inoculated with L lactis harboring pNZ8123‐rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme‐linked immunosorbent assay and enzyme‐linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7‐specific antibody and greatest numbers of E7‐specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV‐16 E7 specific IL‐2‐ and IFN‐γ‐secreting T cells in antigen‐stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7‐expressing tumor cell line (TC‐1). Moreover, L lactis containing pNZ8123‐HPV16‐optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.
L. lactis system is an appropriate host for the production of recombinant HPV‐16 E7 oncoprotein.
Codon optimization, along with optimization of culture condition can enhance the secretion of the rE7 protein.
Lactococcus lactis harboring pNZ8123‐HPV16‐optiE7 induced significantly higher percentages of CD4+ and CD8+ cells compared to the L lactis harboring pNZ8123‐HPV16‐E7.
Oral immunization of mice with recombinant L lactis NZ9000 expressing human papillomavirus type 16 E7 antigen can effectively stimulate both humoral and cellular immune responses.
Background: Interferon-γ (IFN-γ) secretion by T cells is a key correlate of immune protection against many pathogens including tuberculosis and the neglected tropical disease melioidosis. Clinical ...studies in tropical regions of immune responses to pathogens and vaccine monitoring studies require the collection of samples in resource-limited rural areas and subsequent shipment to central laboratories for downstream assays and long-term storage. Here, we studied the impact of two different shipping temperatures on the viability, composition and function of peripheral blood mononuclear cells (PBMC) using multi-colour flow cytometry and IFN-g enzyme-linked immunospot assay (IFN-g ELISpot), in order to provide guidance on sample shipment conditions for future clinical studies. Methods: Paired peripheral blood mononuclear cell (PBMC) samples from recovered melioidosis patients were stored in liquid nitrogen (-196°C) and then shipped from Bangkok, Thailand to Oxford, UK at either -80°C (dry ice) or -196°C (dry shipper). After thawing, cell viability and composition were assessed by flow cytometry and antigen specific responses to Burkholderia pseudomallei (BP) were measured using IFN-g ELISpot. Results: We observed modest lowering of viability in the majority of samples and a reduction in IFN-g responses to BP which correlated to a decrease of monocytes and natural killer cells in samples shipped at -80°C compared to -196°C. Despite being lower in magnitude antigen-specific responses remained detectable in the majority of samples. Conclusions: Here we demonstrate that shipment of cryopreserved PBMC at -196°C has a benefit on cell viability, recovery and T cell responses to bacterial antigens, although useful information can still be obtained from samples shipped at -80°C, thus providing important guidance for sample management in future clinical trials.
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