A Whole Blood Enzyme-Linked Immunospot Assay for Functional Immune Endotyping of Septic Patients.

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.

Validation of an IFN-gamma ELISpot assay to measure cellular immune responses against viral antigens in non-human primates.

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.

A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay.

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.

Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2-Specific IgG.

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.

Establishment of a rapid ELISPOT assay for influenza virus titration and neutralizing antibody detection.

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 (R2 = 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 (R2 = 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.

ELISPOT assay of interferon-γ secretion for evaluating human cytomegalovirus reactivation risk in allo-HSCT recipients.

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.

SARS-CoV-2-specific humoral and cellular immunity in two renal transplants and two hemodialysis patients treated with convalescent plasma.

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.

Prospective comparison of two enzyme-linked immunosorbent spot assays for the diagnosis of Lyme neuroborreliosis.

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.

Phase 1 Clinical Trial of a Conditionally Replication-Defective Human Cytomegalovirus (CMV) Vaccine in CMV-Seronegative Subjects.

BACKGROUND: A conditionally replication-defective human cytomegalovirus (CMV) vaccine (V160) derived from AD169 and genetically engineered to express CMV pentameric complex (gH/gL/pUL128/pUL130/pUL131) was developed and evaluated for phase 1 vaccine safety and immunogenicity in CMV-seronegative and CMV-seropositive adults. METHODS: Subjects received 3 doses of V160 or placebo on day 1, month 1, and month 6. Four vaccine dose levels, formulated with or without aluminum phosphate adjuvant, were evaluated. Injection-site and systemic adverse events (AEs) and vaccine viral shedding were monitored. CMV-specific cellular and humoral responses were measured by interferon-gamma ELISPOT and virus neutralization assay up to 12 months after last dose. RESULTS: V160 was generally well-tolerated, with no serious AEs observed. Transient, mild-to-moderate injection-site and systemic AEs were reported more frequently in vaccinated subjects than placebo. Vaccine viral shedding was not detected in any subject, confirming the nonreplicating feature of V160. Robust neutralizing antibody titers were elicited and maintained through 12 months postvaccination. Cellular responses to structural and nonstructural viral proteins were observed, indicating de novo expression of viral genes postvaccination. CONCLUSIONS: V160 displayed an acceptable safety profile. Levels of neutralizing antibodies and T-cell responses in CMV-seronegative subjects were within ranges observed following natural CMV infection. CLINICAL TRIAL REGISTRATION: . NCT01986010.

Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders.

T cell responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI) due to graft-versus-leukemia effect. For better characterization of the T cell responses, we assessed frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells using ELISpot and pMHC multimer assays and analyzed the frequency of regulatory T cells (Treg) as well as cytokine profiles before/after DLI. The data were correlated to the clinical course of patients. Significantly more LAA-derived T cell epitopes (p = 0.02) were recognized in clinical responders (R) when compared to nonresponders (NR). In addition, pMHC multimer-based flow cytometry showed a significantly higher frequency of LAA-specific T cells in R versus NR. The frequency of Treg in R decreased significantly (p = 0.008) while keeping stable in NR. No differences in T cell subset analysis before/after DLI were revealed. Clinical responders were correlated to specific immune responses and all clinical responders showed an increase of specific immune responses after DLI. Cytokine assays using enzyme-linked immunosorbent assay showed a significant increase of IL-4 after DLI. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. Moreover, this study suggests that enhanced LAA diversity in T cell responses as well as decreasing numbers of Treg contribute to clinical outcome of patients treated with DLI.

Natural T cell autoreactivity to melanoma antigens: clonally expanded melanoma-antigen specific CD8 + memory T cells can be detected in healthy humans.

We used four-color ImmunoSpot® assays, in conjunction with peptide pools that cover the sequence of tyrosinase (Tyr), melanoma-associated antigen A3 (MAGE-A3), melanocyte antigen/melanoma antigen recognized by T cells 1 (Melan-A/MART-1), glycoprotein 100 (gp100), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) to characterize the melanoma antigen (MA)-specific CD8 + cell repertoire in PBMC of 40 healthy human donors (HD). Tyr triggered interferon gamma (IFN-γ)-secreting CD8 + T cells in 25% of HD within 24 h of antigen stimulation ex vivo. MAGE-A3, Melan-A/MART-1, and gp100 also induced recall responses in 10%, 7.5%, and 2.5% of HD, respectively. At this time point, these CD8 + T cells did not yet produce GzB (granzyme B). However, they engaged in GzB production after 72 h of antigen stimulation. By this 72-h time point, 57.5% of the HD responded to at least one, and typically several, of the MA. A closer characterization of the Tyr-specific CD8 + T cell repertoire indicated that it was low-affinity, and to primarily entail a stem cell-like subpopulation. Collectively, our data reveal pre-existing endogenous T cell immunity against melanoma antigens in healthy donors, and analogous to natural autoantibodies, we have termed this "natural T cell autoreactivity".

Exploring Frequencies of Circulating Specific Th17 Cells against Myeloperoxidase and Proteinase 3 in ANCA Associated Vasculitis.

: Background: The role of the T helper 17 (Th17) cell subset in anti-neutrophil cytoplasm antibodies (ANCA) associated vasculitis (AAV) is controversial. We hypothesized that a specific Th17 response to myeloperoxidase (MPO) or proteinase 3 (PR3) is detectable in AAV patients and is different among the disease phases. METHODS: We analyzed 43 AAV patients with renal involvement (21 acute and 22 remission patients), and 12 healthy controls. Peripheral blood mononuclear cells (PBMCs) were cultured with PR3/MPO over 48 h. Thereafter, frequencies of MPO/PR3-specific Th17 cells were assessed using an enzyme-linked immunosorbent spot (ELISpot) assay. Supernatant IL-17 concentration was quantified using ELISA. Finally, specific Th17 response after depletion of T regulatory lymphocytes (T-regs) in some remission patients was compared to the non T-reg-depleted response. RESULTS: Specific Th17 cell number was higher in acute patients compared to remission (p = 0.004). Specific Th17 cell number performed well in the disease activity detection (ROC curve area under the curve (AUC) = 0.87; p = 0.0001) with an optimal cut-off of 6 spots/million. Patients above this cut-off showed higher serum creatinine (p = 0.004), C-reactive protein (CRP) (p = 0.001) and ANCA titer (p = 0.032). Supernatant IL-17 concentration was higher in acute patients compared to remission (p = 0.035) and did not normalize to healthy control levels (p = 0.01). CONCLUSIONS: A specific Th17 cell response is present in AAV patients. This response is more pronounced in the acute phase, but persists in remission.

Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation.

BACKGROUND: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21. RESULTS: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined. CONCLUSIONS: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-MicroRNA-122, RG-101.

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing. CONCLUSION: Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).

The Cytomegalovirus-Specific IL-21 ELISpot Correlates with Allograft Function of Kidney Transplant Recipients.

In kidney transplant recipients, the cytomegalovirus (CMV) is frequently causing infection/reactivation and can trigger allograft rejection. To assess the risk of reactivation, the cellular immune response against CMV is increasingly assessed by cellular in vitro methods, such as the interferon (IFN)-γ ELISpot. In the current study we compared the IFN-γ ELISpot with our newly established CMV-specific ELISpot assays determining IL-17A, IL-21, IL-22, granzyme B, and perforin and correlated the results with flow cytometric data and clinical parameters. In 77 kidney transplant recipients, the highest frequency was observed for CMV pp65-specific cells secreting IFN-γ, followed by cells secreting IL-21 (62.9 and 23.2 Δ spot forming cells/105 cells). We observed a positive correlation between the percentage of CMV-specific CD3+ CD4+ CD154+ cells and results of the CMV-specific IL-21 ELISpot (p = 0.002). Results of the CMV pp65-specific IL-21 ELISpot correlated negatively with kidney function (estimated glomerular filtration rate, p = 0.006) and were significantly higher in women (p = 0.005). IL-21, a cytokine involved in aging that is secreted by activated CD4+ T cells, may also impact on allograft function. Thus, the CMV-specific IL-21 ELISpot could become a new tool to assess if CMV seropositivity represents a hazard for the graft.

Evaluation of IFN-γ Enzyme-linked Immunospot Assay (ELISPOT) as a First-line Test in the Diagnosis of Non-Immediate Hypersensitivity to Amoxicillin and Penicillin.

The current diagnostic algorithm for beta-lactam allergy is based on skin and provocation tests, both of which carry a certain risk of inducing hypersensitivity reactions. Thus, non-invasive in vitro tests reliable enough to replace skin and provocation tests at least in a portion of patients are desirable. We aimed to verify the utility of IFN-γ ELISPOT as a first-line test in patients with suspected non-immediate hypersensitivity reaction to amoxicillin (AMX) and penicillin (PNC). The prospective observational study included 24 patients with recent, suspected non-immediate hypersensitivity reaction to AMX or PNC and 6 recently-exposed healthy subjects. In vitro tests were performed in all patients and healthy subjects: a) IFN-γ ELISPOT with PNC, AMX and amoxicillin plus clavulanic acid (AMX-CL); b) penicillin specific IgE; c) basophil activation test (BAT). Skin and provocation tests followed only in certain patients. IFN-γ ELISPOT results with PNC and AMX stimulation did not differ from the unstimulated condition. The highest IFN-γ responses to AMX-CL were close to previously published criteria in three patients; one of which had true hypersensitivity according to drug provocation tests. Five patients with confirmed hypersensitivity by skin tests showed no response to the culprit antibiotic on IFN-γ ELISPOT assay. Our results did not support the utility of IFN-γ ELISPOT in the diagnosis of mild, non-immediate hypersensitivity to amoxicillin and penicillin.

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

BACKGROUND: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions. METHODS: Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay. RESULTS: Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3-CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive. CONCLUSION: The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection.

The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Detection of long term cellular immune response to Japanese encephalitis vaccination using IFN-γ ELIspot assay.

Vaccine is the most effective preventive measure against Japanese Encephalitis infection. Role of IFN-γ expressing T cells for JE virus clearance has been described as a part of cellular immunity. Vaccine induced immunity also involve the cellular immune response, therefore the study was aimed to observe induction and persistence of IFN-γ expressing T cells by IFN-γ ELISpot assay. The cell count increased significantly after 28 (P < 0.0001) days post vaccination, and remained higher at all time points (day 28, day 180, day 360) when compared with prevaccination. This study will be helpful for designing future vaccination strategy and improving vaccine efficacy.

Generation of antigen-specific cytotoxic T lymphocytes with activated B cells.

BACKGROUND AIMS: Dendritic cells are well known as the most potent antigen-presenting cells. Nonetheless, their use in immunotherapy has been limited by the time-consuming and laborious steps involved in their generation in vitro. Therefore, much attention has been placed on alternative antigen-presenting cells that are relatively more convenient to manipulate. METHODS: In this study, the efficacy of B cells as antigen-presenting cells, compared with dendritic cells, in the induction of cytotoxic T lymphocytes against cytomegalovirus-specific antigens was evaluated. B cells were isolated from the peripheral blood mononuclear cells of healthy individuals, loaded with α-galactosylceramide for activation, and nucleofected with cytomegalovirus-antigen coding plasmid DNA. Antigen-nucleofected B cells or dendritic cells were cocultured with T cells for 14 days in vitro. RESULTS: The proliferation of cytotoxic T lymphocytes induced by B cells was similar to that of those induced by dendritic cells. Additionally, the immunogenicity of both sets of cytotoxic T lymphocytes was similar not only in interferon-γ enzyme-linked immunospot assays but also in cytotoxicity assays. DISCUSSION: These observations suggest that α-galactosylceramide-loaded B cells could be used as antigen-presenting cells as an alternative to dendritic cells. Using B cells has several benefits, including cost-effectiveness and being both less time-consuming and less labor-intensive.