IFN-γ ELISPOT Assay Research Articles

Identification of novel hepatitis B virus therapeutic vaccine candidates derived from polymerase protein.

Hepatitis B virus (HBV) infection is a worldwide health problem with high morbidity and mortality rates. The therapeutic vaccine is a promising method of treatment, and HBV polymerase plays a vital role in viral replication. Therefore, a therapeutic vaccine that binds to HBV DNA polymerase may control HBV infection. We predicted and selected epitopes of polymerase using online databases and analysis software. We then performed molecular docking and peptide binding assays to evaluate the binding energies and affinities between polymerase epitopes and the HLA-A0201 molecule. Finally, we induced T cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors using each epitope and quantified the functions of epitope-specific T cells by IFN-γELISPOT assay, T2 cell cytotoxicity assay, HepG2.2.15 cell cytotoxicity assay and HBV gene expression assays. Four epitopes (RVTGGVFLV, GLLGFAAPF, LLDDEAGPL and YMDDVVLGA) had low binding energy and two epitopes (RVTGGVFLV and GLLGFAAPF) had a high binding affinity. The T cells stimulated by two epitopes (GLLGFAAPF and HLYSHPIIL) had a greater ability to induce immune response and suppress HBV. The HBV DNA polymerase epitopes identified in this study are promising targets for designing an epitope-based therapeutic vaccine against HBV.

Complete rejection of large established breast cancer by local immunochemotherapy with T cell activation against neoantigens.

Cancer immunotherapies, including immune checkpoint blockage and adoptive transfer of CAR-T cells, have achieved historical successes for many kinds of malignancy. However, a minority of patients survive long term over 5years without relapse, perhaps owing to tumor heterogeneity and potent immunosuppression in the tumor microenvironment. Here, using an established mouse tumor model of triple-negative 4T1 breast cancer, we show that local immunochemotherapy triggers powerful local and systemic antitumor immunity. Paraneoplastic injection of CpG, α-OX40, and anthracycline completely eliminated both local and distant large established 4T1 breast cancer without obvious relapse. Analysis of the immune cells at tumor tissues, draining lymph nodes, and spleens revealed that the local treatment increased the infiltration of CD4+ and CD8+ T cells in all three tissues and inhibited the accumulation of myeloid-derived suppressor cells in the spleen in a delayed response. Most importantly, this treatment triggered systemic T cell response against 4T1 tumors and some of their neoantigen epitopes as detected by IFN-γ ELISpot and intracellular cytokine assays in splenocytes. Furthermore, T cells showed specific cytotoxic activity against 4T1 tumor cells in vitro. In general, this local immunochemotherapy provides a new approach to target highly diverse neoantigens in various types of cancers without complicated and expensive antigen identification via next-generation sequencing.

A Placebo-Controlled, Double-Blind Randomized (Phase IIB) Trial of Oral Administration with HPV16 E7-Expressing Lactobacillus, GLBL101c, for the Treatment of Cervical Intraepithelial Neoplasia Grade 2 (CIN2).

Cervical intraepithelial neoplasia (CIN), a precursor lesion to cervical cancer, is caused by high-risk human papillomavirus (HPV); high-grade CIN lesions (CIN2-3) are precancerous and require treatment. No globally approved therapy is available for CIN2-3 treatment. This study is a placebo-controlled randomized clinical trial of GLBL101c treatment for CIN2 in 40 patients with HPV16-positive CIN2 who were 1:1 randomized to receive GLBL101c (1 g/daily) or placebo for 5 days at 1, 2, 4, and 8 weeks. No differences were noted between the GLBL101c and placebo groups for patient background and adverse events. Moreover, no statistically significant difference was noted between the two groups at the primary endpoint, pathological regression after 16 weeks of the first oral dose; however, only in the GLBL101c group, two patients had complete regression (CR; regression to normal within 16 weeks). IFNγ production was significantly correlated with the number of spots identified by the interferon gamma enzyme-linked immunospot (IFNγ-ELISPOT) assay using cervical lymphocytes (CxLs) or peripheral blood mononuclear cells. In the two cases of CR, E7-specific Th1 immune responses were observed at week 16. Therefore, we concluded as a novel Lactobacillus-based vaccine with stronger immunogenicity than GLBL101c should be developed.

T-Cell and Antibody Responses to First BNT162b2 Vaccine Dose in Previously SARS-CoV-2-Infected and Infection-Naive UK Healthcare Workers: A Multicentre, Prospective, Observational Cohort Study

Background: Following a single dose of BNT162b2 mRNA vaccine, higher antibody titres are observed following prior SARS-CoV-2 infection than in infection-naive individuals, but T-cell responses are less well defined.Methods: We sampled healthcare workers (HCWs) enrolled in the UK PITCH study, before and after BNT162b2 mRNA vaccination. We measured spike-specific antibody, and quantified T-cell responses by IFNγ ELISpot assay and intracellular staining of peripheral blood mononuclear cells (PBMC), comparing SARS-CoV-2-naïve individuals to those with prior infection.Findings: HCWs aged 22 to 71 years received one (n=216) or two (n=21) vaccine doses. After a single dose, the spike-specific T-cell response was six-fold higher in previously-infected vs. naive individuals (median 340 vs. 58 SFU/106 PBMC, p6 PBMCs, p=0.65; cryopreserved PBMC, n=117). Anti-spike IgG levels following a single dose in those previously infected (median 512.9 antibody units/ml (AU/ml)) were 6.8-fold higher vs. naïve individuals following one dose (median 75.0 AU/ml, pin vitro neutralisation of the B.1.351 variant of concern compared to naive individuals.Interpretation: Following a single BNT162b2 dose, HCWs with a prior history of SARS-CoV-2 infection have significantly higher T-cell and antibody responses than naive individuals.Funding UK Department of Health and Social Care and UK Coronavirus Immunology Consortium.Funding: UK Department of Health and Social Care and UK Coronavirus Immunology Consortium.Conflict of Interest: D.W.E. declares lecture fees from Gilead, outside the submitted work. The other authors have no conflicts of interest to declare.Ethical Approval: PITCH was recognised as a sub-study of SIREN on 2 December 2020, which was approved by the Berkshire Research Ethics Committee, Health Research 250 Authority (IRAS ID 284460, REC reference 20/SC/0230).

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BackgroundProtection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising.MethodsThis study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection.ResultsIFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry.ConclusionsThis study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Longitudinal Assessment of Anti-SARS-CoV-2 Immune Responses for Six Months Based on the Clinical Severity of COVID-19

Background: There is insufficient data on the longevity of immunity acquired following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19).Methods: Convalescent blood was collected prospectively from 97 patients who had recovered from COVID-19. The level of humoral immunity to SARS-CoV-2 was measured using the anti-SARS-CoV-2 nucleocapsid immunoglobulin G (IgG) assay and the plaque reduction neutralization test with sera collected at three periods: within 8 weeks, at 9-20 weeks, and at 22-27 weeks after diagnosis. IFN-γ ELISpot assays were conducted using overlapping peptides covering spike, nucleocapsid, and membrane proteins of SARS-CoV-2.Findings: The study population comprised asymptomatic (n=14), symptomatic/non-pneumonic (n=42), and pneumonic (n=41) patients. The anti-SARS-CoV-2 IgG and neutralizing antibody (NAb) titers lasted until six months after diagnosis, with positivity rates of 66·7% and 86·9%, respectively. The level of humoral immunity at six months after diagnosis were significantly higher in the pneumonic group than in the symptomatic/non-pneumonic group. Older age, prolonged viral shedding and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. SARS-CoV-2 specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity.Interpretation: Most (> 85%) patients carries NAb until six months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.Funding Statement: This study was supported by a grant of the Korea National Institute of Health, Korea Disease Control and Prevention Agency (project number: 2020-ER5314-00), Korea University Guro Hospital (Korea research-driven hospital) (No. O2001061), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0452).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The study protocol was approved by the Institutional Review Board of the Korea University Guro Hospital (approval number: 2020GR0130). All participants provided written informed consent.

A Phase 2 Randomized Placebo-Controlled Adjuvant Trial of GI-4000, a Recombinant Yeast Expressing Mutated RAS Proteins in Patients with Resected Pancreas Cancer.

Purpose: GI-4000, a series of recombinant yeast expressing four different mutated RAS proteins, was evaluated in subjects with resected ras-mutated pancreas cancer.Methods: Subjects (n = 176) received GI-4000 or placebo plus gemcitabine. Subjects' tumors were genotyped to identify which matched GI-4000 product to administer. Immune responses were measured by interferon-γ (IFNγ) ELISpot assay and by regulatory T cell (Treg) frequencies on treatment. Pretreatment plasma was retrospectively analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry for proteomic signatures predictive of GI-4000 responsiveness.Results: GI-4000 was well tolerated, with comparable safety findings between treatment groups. The GI-4000 group showed a similar pattern of median recurrence-free and overall survival (OS) compared with placebo. For the prospectively defined and stratified R1 resection subgroup, there was a trend in 1 year OS (72% vs. 56%), an improvement in OS (523.5 vs. 443.5 days [hazard ratio (HR) = 1.06 [confidence interval (CI): 0.53–2.13], p = 0.872), and increased frequency of immune responders (40% vs. 8%; p = 0.062) for GI-4000 versus placebo and a 159-day improvement in OS for R1 GI-4000 immune responders versus placebo (p = 0.810). For R0 resection subjects, no increases in IFNγ responses in GI-4000–treated subjects were observed. A higher frequency of R0/R1 subjects with a reduction in Tregs (CD4+/CD45RA+/Foxp3low) was observed in GI-4000–treated subjects versus placebo (p = 0.033). A proteomic signature was identified that predicted response to GI-4000/gemcitabine regardless of resection status.Conclusion: These results justify continued investigation of GI-4000 in studies stratified for likely responders or in combination with immune check-point inhibitors or other immunomodulators, which may provide optimal reactivation of antitumor immunity.ClinicalTrials.gov Number: NCT00300950.

Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

Peptide Vaccination against Cytomegalovirus Induces Specific T Cell Response in Responses in CMV Seronegative End-Stage Renal Disease Patients.

Introduction: Cytomegalovirus (CMV) reactivation occurs in seronegative patients after solid organ transplantation (SOT) particularly from seropositive donors and can be lethal. Generation of CMV-specific T cells helps to prevent CMV reactivation. Therefore, we initiated a clinical phase I CMVpp65 peptide vaccination trial for seronegative end-stage renal disease patients waiting for kidney transplantation. Methods: The highly immunogenic nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) in a water-in-oil emulsion (Montanide™) plus imiquimod (Aldara™) as an adjuvant was administered subcutaneously four times biweekly. Clinical course as well as immunological responses were monitored using IFN-γ ELISpot assays and flow cytometry for CMV-specific CD8+ T cells. Results: Peptide vaccination was well tolerated, and no drug-related serious adverse events were detected except for Grade I–II local skin reactions. Five of the 10 patients (50%) mounted any immune response (responders) and 40% of the patients presented CMV-specific CD8+ T cell responses elicited by these prophylactic vaccinations. No responders experienced CMV reactivation in the 18 months post-transplantation, while all non-responders reactivated. Conclusion: CMVpp65 peptide vaccination was safe, well tolerated, and clinically encouraging in seronegative end-stage renal disease patients waiting for kidney transplantation. Further studies with larger patient cohorts are planned.

Dose Dependent Antimicrobial Cellular Cytotoxicity – Implications for ex vivo diagnostics

Introduction: Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity. Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above. Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32-100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested. Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Comparison of Three Cellular Assays to Predict the Course of CMV Infection in Liver Transplant Recipients

To estimate protection from cytomegalovirus (CMV) replication after solid organ transplantation, CMV serology has been considered insufficient and thus CMV immunity is increasingly assessed by cellular in vitro methods. We compared two commercially available IFN-γ ELISpot assays (T-Track CMV and T-SPOT.CMV) and an IFN-γ ELISA (QuantiFERON-CMV). Currently, there is no study comparing these three assays. The assays were performed in 56 liver transplant recipients at the end of antiviral prophylaxis and one month thereafter. In CMV high- or intermediate-risk patients the two ELISpot assays showed significant correlation (p < 0.0001, r > 0.6) but the correlation of the ELISpot assays with QuantiFERON-CMV was weaker. Results of both ELISpot assays were similarly predictive of protection from CMV-DNAemia ≥500 copies/mL [CMV pp65 T-SPOT.CMV at the end of prophylaxis: area under curve (AUC) = 0.744, cut-off 142 spot forming units (SFU), sensitivity set to 100%, specificity 46%; CMV IE-1 T-Track CMV at month 1: AUC = 0.762, cut-off 3.5 SFU, sensitivity set to 100%, specificity 59%]. The QuantiFERON-CMV assay was inferior, reaching a specificity of 23% when setting the sensitivity to 100%. In conclusion, both CMV-specific ELISpot assays appear suitable to assess protection from CMV infection/reactivation in liver transplant recipients.

T-Cell and Antibody Responses to First BNT162b2 Vaccine Dose in Previously SARS-CoV-2-Infected and Infection-Naive UK Healthcare Workers: A Multicentre, Prospective, Observational Cohort Study

Background: Following a single dose of BNT162b2 mRNA vaccine, higher antibody titres are observed following prior SARS-CoV-2 infection than in infection-naive individuals, but T-cell responses are less well defined. Methods: We sampled healthcare workers (HCWs) enrolled in the UK PITCH study, before and after BNT162b2 mRNA vaccination. We measured spike-specific antibody, and quantified T-cell responses by IFNγ ELISpot assay and intracellular staining of peripheral blood mononuclear cells (PBMC), comparing SARS-CoV-2-naïve individuals to those with prior infection. Findings: HCWs aged 22 to 71 years received one (n=216) or two (n=21) vaccine doses. After a single dose, the spike-specific T-cell response was six-fold higher in previously-infected vs. naive individuals (median 340 vs. 58 SFU/10 6 PBMC, p in vitro neutralisation of the B.1.351 variant of concern compared to naive individuals. Interpretation: Following a single BNT162b2 dose, HCWs with a prior history of SARS-CoV-2 infection have significantly higher T-cell and antibody responses than naive individuals. Funding: UK Department of Health and Social Care and UK Coronavirus Immunology Consortium.Declaration of Interests: None to declare. Ethics Approval Statement: PITCH was recognised as a sub-study of SIREN on 2 December 2020, which was approved by the Berkshire Research Ethics Committee, Health Research 250 Authority (IRAS ID 284460, REC reference 20/SC/0230). Some participants were recruited under aligned REC-approved study protocols (Appendix). The study was conducted in compliance with the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines.

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.

CTL ELISPOT Assay and T Cell Detection.

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.

T-Cell Response in Dermatitis Herpetiformis: May Epidermal Transglutaminase Play a Role in Predicting Clinical Relapse?

We read with interest the paper by Kalliokoski et al. (2020) investigating whether PBMCs collected from patients with dermatitis herpetiformis (DH) undergoing a 3-day oral gluten challenge react to gluten-derived gliadin in the enzyme-linked immunospot (ELISpot) assay similarly to those from patients with celiac disease (CD). Interestingly, the authors found that on stimulation with tissue transglutaminase (TG)-gliadin or either of the gliadin peptides, 7 of the 15 patients (47%) with DH and 15 of the 18 patients (83%) with CD were considered responders in the IFN-γ ELISpot assay.

Age and frailty in COVID-19 vaccine development

Age and frailty in COVID-19 vaccine development

Identification of Novel Yellow Fever Class II Epitopes in YF-17D Vaccinees.

Yellow fever virus (YFV) is a mosquito-borne member of the genus flavivirus, including other important human-pathogenic viruses, such as dengue, Japanese encephalitis, and Zika. Herein, we report identifying 129 YFV Class II epitopes in donors vaccinated with the live attenuated YFV vaccine (YFV-17D). A total of 1156 peptides predicted to bind 17 different common HLA-DRB1 allelic variants were tested using IFNγ ELISPOT assays in vitro re-stimulated peripheral blood mononuclear cells from twenty-six vaccinees. Overall, we detected responses against 215 YFV epitopes. We found that the capsid and envelope proteins, as well as the non-structural (NS) proteins NS3 and NS5, were the most targeted proteins by CD4+ T cells from YF-VAX vaccinated donors. In addition, we designed and validated by flow cytometry a CD4+ mega pool (MP) composed of structural and non-structural epitopes in an independent cohort of vaccinated donors. Overall, this study provides a comprehensive prediction and validation of YFV epitopes in a cohort of YF-17D vaccinated individuals. With the design of a CD4 epitope MP, we further provide a useful tool to detect ex vivo responses of YFV-specific CD4 T cells in small sample volumes.

Transgenic CD8αβ co-receptor rescues endogenous TCR function in TCR-transgenic virus-specific T cells

BackgroundGenetically engineered virus-specific T cells (VSTs) are a platform for adoptive cell therapy after allogeneic hematopoietic stem cell transplantation. However, redirection to a tumor-associated antigen by the introduction of a...

Abstract 3292: Neo-antigen specific T cell responses can inform cancer therapy

Abstract Objective: Spontaneous neo-antigen specific immune responses are rare in animals and humans. Our AB1 model, one of the few induced by a natural human carcinogen, has several. Given that current immunotherapies rely on unleashing responses to existing tumour neo-antigens, we evaluated the capacity of such responses to inform subsequent therapies, determining if these responses are: a) more sensitive than imaging to detect tumor recurrence after surgical resection b) able to predict responses to immune checkpoint blockade (ICPB) therapy c) able to provide evidence of the immunogenicity of chemotherapy. Methods: To examine this, we interrogated the immune response to tumour neo-antigens in BALB/c mice bearing the AB1-HA tumor model.AB1-HA whole exome sequencing and RNAseq data were analysed and the MHC-I binding affinity of mutated sequences were predicted and expression confirmed with RNA-seq data. A total of 180 neo-antigens were screened, in addition to the responses to the known neo-antigens UNC45a and UQCRC2, spontaneous immune responses to two novel neo-antigens, VMN2R67 and ELP2 were found in tumour bearing mice. a) To determine if responses to these neo-antigens detected post-resection tumor recurrence tumours were surgically resected then growth of IV luciferase transfected cells determined sequentially by an In Vivo Imaging System (IVIS) and imaged for peak bioluminescence in a Lumina II Imager (Figure), PET-CT (IV 15-FDG at 7-15MBq). For neo-antigen specific T cell detection, IFNg ELISPOT assays were performed. b) To determine if pre-treatment neo-antigen responses are able to predict responses to ICPB, we utilised a version of the dual-tumor model developed by Lesterhuis et al, enabling pre-treatment neo-antigen responses to ICPB blockade (anti-CTLA-4 + anti-GITR) compared in subsequent responders versus non-responders. c) To determine if a known immunogenic chemotherapy (gemcitabine) could boost the efficacy of vaccines designed based on spontaneous neo-antigen reactivity, we treated tumour bearing mice with gemcitabine ± a vaccine containing the neoantigen peptides Unc45a, Uq2, Vmn2r67, and Elp2 with Poly I:C. Results: a) A combination of 3 neo-antigen specific T cell responses detected metastases detection prior to IVIS or PET-CT (86±11 SFU/105 cells for the recurrence group compared to 17±5 SFU/105 cells in the non-recurrence group, p&amp;lt;0.0001).b) Pre-treatment dLN UNC45a response was higher in the responders (24 ± 14 SFU/105 vs 5 ± 2 SFU/105, p = 0.0004). Thus pretreatment responses to some neo-antigens could strongly predict response to ICPB therapy.c)Gemcitabine + vaccine delayed tumour growth compared to either treatment or adjuvant alone (p&amp;lt;0.05). Conclusions: Neo-antigen specific T cell responses increases sensitivity of recurrence/metastasis detection, predict ICPB therapy outcome and inform vaccine design for synergy with immunogenic chemotherapy. Citation Format: Bruce W. Robinson, Vanessa Fear, Catherine Forbes, Shaokang Ma, Jonathan Chee, Sam Neeve, Scott Fisher, Ian Dick, Anna Nowak, Nandini Makwana, Jessica Boulter, Jenette Creaney, Alec Redwood. Neo-antigen specific T cell responses can inform cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3292.

Abstract 2219: OnxoVEXmGM-CSF promotes systemic response to PD-L1 /PD-1 blockade in multiple mouse syngeneic tumor models

Abstract Inhibition of the immune-checkpoint axis that involves PD-L1/PD-1 has improved responses and outcomes for certain cancer patients, but there remains unmet need as only a fraction of patients respond to these therapies. Talimogene laherparepvec (T-VEC) is an FDA-approved, first-in-class oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce granulocyte-macrophage stimulating factor (GM-CSF) to enhance a tumor-antigen specific adaptive immune response. Intratumoral treatment with T-VEC has been shown to promote an inflammatory tumor microenvironment both preclinically and clinically. Combining T-VEC with immune checkpoint inhibitors is of high interest to potentially broaden and deepen the clinical responses in checkpoint-resistant tumors. In the current study, we evaluated the efficacy of OncoVEXmGM-CSF (an HSV modified like T-VEC, except that it produces murine GM-CSF) in combination with anti-PD-1/PD-L1 treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used, where only one tumor was treated with OncoVEXmGM-CSF (representing the local oncolytic effect), allowing the evaluation of systemic abscopal effect in the uninjected contralateral tumors. OncoVEXmGM-CSF in combination with anti-PD-L1/PD-1 enhanced tumor growth inhibition in both treated and contralateral tumors in the A20 and MC38 syngeneic models and overcame anti-PD-L1 resistance in the CT26 model. The combination treatment also led to a significant increase in median overall survival compared to either agent as a monotherapy. In addition, tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell response was measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. OncoVEXmGM-CSF induced and /or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that OncoVEXmGM-CSF treatment can cause direct tumor lysis along with the potentiation of an adaptive, systemic T cell-mediated anti-tumor immune response that can be enhanced by the addition of PD-L1/PD-1. Our study provides a strong rationale for the ongoing clinical evaluation of the combination of T-VEC and PD1-/PD-L1-targeted therapy in cancer patients. Citation Format: Keegan Cooke, Juan Estrada, Petia Mitchell, Jinghiu Zhan, Pedro J. Beltran, Jing Qing, Jude Canon. OnxoVEXmGM-CSF promotes systemic response to PD-L1 /PD-1 blockade in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2219.