Special Issue "ELISPOT Research"

Guest Editor
Dr. Alexander E. Kalyuzhny
Neuroscience, UMN Twin Cities, 6-145 Jackson Hall, 321 Church St SE, Minneapolis, MN 55455, USA
Website: http://www.tc.umn.edu/~kalyu001/
E-Mail: kalyu001@umn.edu
Phone: +1 612 624 2991
Interests: physiology of pain; antinociceptive brainstem circuit; cellular localization, trafficking and oligomerization of opioid receptors; drugs of abuse; cytokines and cytokine receptors

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Type of Paper: Article 
Title: Identification of CD8+T Lymphocyte Epitopes from the Herpes Simplex Virus Type 2 ICP27, VP22 and VP13/14 Proteins to Facilitate Vaccine Design and Characterization 
Authors: Rebecca Platt, Tansi Khodai, Tim Townend, Helen Bright, Paul Cockle, Luis Perez-Tosar, Rob Webster, Brian Champion, Timothy Hickling and Fareed Mirza 
Affiliation: Pfizer Global Research and Development (PGRD), Sandwich laboratories, Kent, CT13-9NJ, UK; E-Mail: timothy.hickling@pfizer.com 
Abstract: CD8+ T cells play an important role in controlling HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. A panel of 44 9mer peptides from HSV-2 proteins ICP27, VP22 and VP13/14 were selected based on in silico predictions of binding to human HLA-A201 and mouse H-2Kd, Ld and Dd molecules. From these 44 peptides, nine previously uncharacterised CD8+ T cell epitopes were identified using peptide pulsed splenocytes from HSV-2 infected BALB/c mice. Furthermore, HSV-2 specific peptide sequences were able to stabilise HLA-A2 surface expression with intermediate or high affinity binding (Affinity Index ≥1.25). To demonstrate antigen specificity and functionality, peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A2+ healthy donor that produced IFN-γ following peptide stimulation. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by particle mediated epidermal delivery of DNA encoding ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilisation upon peptide stimulation. These vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. In conclusion, this study describes novel HSV-2 epitopes that elicit strong cellular responses during acute infection. Additionally, DNA vaccination induced strong CD8+ T cell responses in mice following biolistic delivery. These novel sequences may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings.

Type of Paper: Review
Title: Performance of T-cell Interferon-γ Release Assays in the Rapid Diagnosis of Tuberculosis in High Disease Burden Region
Authors: Jiali Wang, Linyun Shao, Wenhong Zhang
Affiliation: Department of Infectious Disease, Huashan Hospital; E-Mail: zhangwenhong@fudan.edu.cn
Abstract: The T-cell interferon-γ release assays (IGRAs) serve as alternatives to the tuberculin skin test (TST) in the diagnosis of tuberculosis. It is recognized that IGRAs have high specificity, even in those that have been vaccinated by bacille Calmette-Guérin (BCG). However, the sensitivity remains unsatisfying, and their diagnostic values across different areas and populations are still controversial. Here we review the evidence concerning the sensitivity and specificity of these assays in high disease burden region with expansive BCG vaccination and furthermore, try to figure out its utility and limitation in clinical scenarios and give our suggestions for prospective researches in these special regions.

Type of Paper: Article
Title: ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis
Authors: L. S. Sibley, A. D. White, A. Marriott, M. J. Dennis and S. A. Sharpe
Affiliation: Microbiological Services, Health Protection Agency, Porton Down, Wiltshire, UK; E-Mail: Sally.Sharpe@hpa.org.uk
Abstract: Tuberculosis is a global health problem. The BCG vaccine has variable efficacy (0-80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced after BCG vaccination. In the assessment of new TB vaccines, induction of an IFNγ response is used as a biomarker of successful vaccination. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials. At HPA Porton, this method is used in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by PBMCs following specific stimulation onto a membrane so individual cells can be enumerated and the frequency of responding cells determined, hence spot forming units (SFU) per 106 cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the SFU in BCG vaccinated and unvaccinated subjects were similar, although the spots were observed to be larger in vaccinated subjects. Spot size provides a measure of the quantity of cytokine produced by individual cells, which is a measurement available from AID software. The addition of spot size in combination with spot forming units was investigated in studies of BCG immunogenicity.  This additional readout was found to enhance the discriminatory power of the ELISPOT assay.

Type of Paper: Review
Title: ELISPOT Assay for Monitoring CTL Activity in Cancer Vaccine Clinical Trials
Authors: K. Dunham, S. Strobl and A. Malyguine
Affiliation: Laboratory of Cell Mediated Immunity, SAIC-Frederick, Inc., NCI at Frederick, Frederick, MD, USA; E-Mail: malyguinea@mail.nih.gov
Abstract: Immune response profiling and monitoring are the key elements in the development of new biotherapies and a variety of assays have been introduced for monitoring immune response to immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms of a tested therapeutic modality. Therefore, selection of monitoring methods for an appropriate assessment of cell-mediated cytotoxicity, representing the key mechanism of the immune responses against various pathogens and tumor, are thought to be crucial for revealing potential correlations between the clinical and immunologic responses during and after immunotherapy. Assays that can monitor both CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, showed the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B (GrB) ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity compared to the IFN-γ ELISPOT, since GrB and perforin are key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data published by other investigators regarding an application of various modifications of ELISPOT assay for monitoring CTL activity in clinical vaccine trials.

Type of Paper: Review
Title: Enzyme Linked Immuno-spot; a Useful Tool in the Search for Elusive Immune Markers in Common Pediatric Immunological Diseases
Authors: Maria Faresjö
Affiliation: The Biomedical platform, Department of Natural Science and Biomedicine, School of Health Sciences, Jönköping University and County Hospital, Ryhov, Jönköping, Sweden; E-Mail: maria.faresjo@hhj.hj.se
Abstract: In order to provide better therapy we strives to increase our knowledge how the immune system behaves and communicates in common pediatric immunological diseases e g allergic disease, type 1 diabetes and celiac disease. However, dealing with pediatric diseases, where study subjects are almost exclusively children, blood volumes available for immunological studies are limited and samples given must be carefully handled and used to its full extent. Detection of single immune markers can be easily detected by traditionally Enzyme Linked Immunosorbent Assay (ELISA) whereas multiplex detection of immune markers can be detected by either fluorochrome technique (Luminex) or protein microarray. These techniques however are sometimes not sensitive enough to detect low-secreted immune markers in limited sample sizes. This makes a strong case for the use of Enzyme Linked Immuno-spot (ELISPOT), a sensitive and reliable immunological method, for studies of immune markers at the single cell level in order to pin-point elusive immune markers in common pediatric immunological diseases.

Type of Paper: Article
Title: ELISPOT assay as a biomarker for acute graft-versus-host disease and concurrent infections
Authors: Masahiro Hirayama and Eiichi Azuma *
Affiliation: Department of Pediatrics and Cell Transplantation, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; E-Mail: e-azuma@clin.medic.mie-u.ac.jp
Abstract: Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Infections may coexist and in certain circumstances aggravate aGVHD. It was described that type 1 as well as type 2 cytokines are important mediators of acute GVHD. We measured spot-forming cells (SFCs) for interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-12, IL-13, IL-17, and IL-22 in peripheral blood from 80 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation by using enzyme-linked immunospot (ELISPOT) assay that reflects ongoing immune status in vivo. A serial monitoring showed that both type 1 and type 2 cytokine SFCs were correlated with aGVHD activity. The numbers of IFN-γ and IL-4 SFCs in patients with grade II-IV aGVHD were significantly higher than those in patients with grade 0-I aGVHD. Elevation of IFN-γ SFCs was correlated with clinically significant aGVHD but not with infection itself, e.g., cytomegalovirus infection. Evaluating cytokine SFCs is a clinically relevant biomarker for the diagnostic and therapeutic evaluation of aGVHD and concurrent infections.
Keywords: ELISPOT, acute GVHD, HSCT, IFN-γ, Biomarker

Type of Paper: Article
Title: Development and Optimization of a Gamma Interferon Biomarker Assay for Quantitation of Cellular Immune Responses to Measles Antigens in Non-Human Primates
Authors: V. Jawa, M. Deshpande, D. Wullner, L. Zhou and N. Chirmule
Affiliation: Clinical Immunology, Medical Sciences, Amgen, Inc.; E-Mail: vjawa01@amgen.com
Abstract: We have developed and optimized an in vitro functional assay using whole blood derived peripheral blood derived mononuclear cells (PBMC) from non-human primates. The assay was used to quantitate measles-specific T cell responses. For the qualification experiments, PBMC from 36 Chinese cynomolgus monkeys immunized with measles vaccine were harvested and frozen in liquid nitrogen. These cells were tested for reactivity to a measles peptide cocktail, using an IFN-ELISPOT assay and a Luminex based cytokine secretion assay. The optimization and qualification experiments describe: (i) evaluation of number of
cells to be plated in culture, (ii) pre-resting of cells before plating, (iii) viability of cells, (iv) impact of costimulatory cytokines (IL2, IL7, and IL15) on antigen-specific responses. An assay specific cut point and a stimulation index was calculated to distinguish responders from non-responders. Using this qualified assay, measles-specific T cell responses were evaluated in 56 monkeys. These assays may provide an assessment of antigen-specific T cell function in vivo, and has various applications, such as evaluation of immune competence status of animals and immunomodulatory effects of proteins on immune cells.

Type of paper: Review
Title: The Role of ELISPOT Assays in Risk Assessment Pre- and Post-Kidney Transplantation
Authors: Jennifer R Zitzner, Joe Leventhal and Anat Tambur
Affiliation: Comprehensive Transplant Center, Northwestern University, Chicago Illinois, USA; E-Mail: a-tambur@northwestern.edu
Abstract: Immunologic risk in kidney transplantation is typically minimized by selecting donor and recipient pairs with the least number of human leukocyte antigen (HLA) mismatches as well as the absence or limited presence of donor specific antibodies. However, numerous other factors may play a role in the success of transplant outcome and patient health. The use of T-cell and B-cell allospecific ELISPOT assays have helped elucidate additional factors in post-transplant outcome. In this review we will evaluate numerous uses of ELISPOT assays in the ability to assess the pre- and post-transplant immunologic risk of rejection episodes, graft survival and recipient viral susceptibility as well as the utility of ELISPOT assays in monitoring tolerance and withdrawal of immunosuppressive medications following kidney transplantation.

Type of Paper: Article
Title: Characterization of Spontaneous Immune Responses Against long Peptides Derived from Bcl-X(L) in Cancer Patients using ELISPOT
Author: Stine Kiaer Larsen, Inge Marie Svane, Per thor Straten and Mads Hald Andersen *
Affiliation: Center for Cancer Immune Therapy (CCIT), Department of Hematology, Copenhagen University Hospital, Herlev, Herlev Ringvej 75, DK-2730 Herlev, Denmark; E-Mail: mahaan03@heh.regionh.dk
Abstract: In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9-10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing PMBC for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC from melanoma patients for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126-188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20-23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the INF-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides.