IFN-γ Enzyme-linked Immunospot Research Articles

T cell response specificity and magnitude against SIVmac239 are not concordant in major histocompatibility complex-matched animals

BackgroundCD8+ T cell responses, restricted by major histocompatibility complex (MHC) class I molecules, are critical to controlling human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication. Previous studies have used MHC-matched siblings and monozygotic twins to evaluate genetic and stochastic influences on HIV-specific T cell responses and viral evolution. Here we used a genetically restricted population of Mauritian cynomolgus macaques (MCM) to characterize T cell responses within nine pairs of MHC-matched animals.FindingsIn MHC-matched animals, there was considerable heterogeneity in the specificity and magnitude of T cell responses detected via individual peptide gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays. These findings were further supported by full proteome pooled peptide matrix ELISPOT data collected from this cohort at 52 weeks post-infection. Interestingly, peptide regions that elicited dominant T cell responses were more commonly shared between MHC-matched MCM than peptide regions that elicited non-dominant T cell responses.ConclusionsOur findings suggest that, while some T cell responses mounted during chronic infection by MHC-matched MCM are similar, the majority of responses are highly variable. Shared responses detected in this study between MHC-matched MCM were directed against epitopes that had previously elicited relatively dominant responses in MCM with the same MHC class I haplotype, suggesting that the factors that influence dominance may influence the reproducibility of responses as well. This may be an important consideration for future T cell-based vaccines aiming to consistently and reproducibly elicit protective T cell responses.

Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4(+) T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4(+) T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.

Inhibition of Mycobacterial GrowthIn Vitrofollowing Primary but Not Secondary Vaccination with Mycobacterium bovis BCG

Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.

Identification of Promiscuous KIF20A Long Peptides Bearing Both CD4+ and CD8+ T-cell Epitopes: KIF20A-Specific CD4+ T-cell Immunity in Patients with Malignant Tumor

To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays. We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4(+) T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues. These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs.

Quantitative and Qualitative Differences in the T Cell Response to HIV in Uninfected Ugandans Exposed or Unexposed to HIV-Infected Partners

HIV-exposed and yet persistently uninfected individuals have been an intriguing, repeated observation in multiple studies, but uncertainty persists on the significance and implications of this in devising protective strategies against HIV. We carried out a cross-sectional analysis of exposed uninfected partners in a Ugandan cohort of heterosexual serodiscordant couples (37.5% antiretroviral therapy naive) comparing their T cell responses to HIV peptides with those of unexposed uninfected individuals. We used an objective definition of exposure and inclusion criteria, blinded ex vivo and cultured gamma interferon (IFN-γ) enzyme-linked immunospot assays, and multiparameter flow cytometry and intracellular cytokine staining to investigate the features of the HIV-specific response in exposed versus unexposed uninfected individuals. A response rate to HIV was detectable in unexposed uninfected (5.7%, 95% confidence interval [CI] = 3.3 to 8.1%) and, at a significantly higher level (12.5%, 95% CI = 9.7 to 15.4%, P = 0.0004), in exposed uninfected individuals. The response rate to Gag was significantly higher in exposed uninfected (10/50 [20.%]) compared to unexposed uninfected (1/35 [2.9%]) individuals (P = 0.0004). The magnitude of responses was also greater in exposed uninfected individuals but not statistically significant. The average number of peptide pools recognized was significantly higher in exposed uninfected subjects than in unexposed uninfected subjects (1.21 versus 0.47; P = 0.0106). The proportion of multifunctional responses was different in the two groups, with a higher proportion of single cytokine responses, mostly IFN-γ, in unexposed uninfected individuals compared to exposed uninfected individuals. Our findings demonstrate both quantitative and qualitative differences in T cell reactivity to HIV between HESN (HIV exposed seronegative) and HUSN (HIV unexposed seronegative) subject groups but do not discriminate as to whether they represent markers of exposure or of protection against HIV infection.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.

Abstract B60: A potential of glypican-3-derived peptide vaccine therapy against cancer.

Abstract Introduction: Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans, which are attached to the cell surface via the glycosylphosphatidylinositol (GPI) anchor. We reported its identification as a carcinoembryonic antigen, an ideal target for anticancer immunotherapy against HCC due to its specific overexpression in HCC (80%) and its correlation with a poor prognosis. Furthermore, we identified both HLA-A24(A*24:02) and H-2Kd-restricted GPC3298-306 (EYILSLEEL), as well as HLA-A2(A*02:01)-restricted GPC3144-152 (FVGEFFTDV), as peptides that can induce GPC3-reactive cytotoxic T lymphocytes (CTLs) without inducing autoimmunity. Phase-I trial: 33 patients with advanced Hepatocellular carcinoma (HCC) were administered GPC3 peptide vaccination with dose-escalation. Peptides were emulsified with IFA and administered in liquid form by intradermal injection on days 1, 15, and 29. We collected immunological evidence, demonstrated antitumor effects, and showed the safety of the GPC3-derived peptide vaccine. One patient showed a partial response (PR) and 4 of the 19 patients with stable disease (SD) had tumor necrosis or regression that did not meet the criteria for a partial response. We also analyzed the GPC3-specific CTL frequency ex vivo by IFN-γ enzyme-linked immunospot (ELISPOT) assay. In most patients, GPC3 peptide-specific CTLs appeared in the peripheral blood. Tumor biopsies were performed in seven patients to evaluate infiltration of CD8-positive T cells by immunohistochemical staining. The results in five cases showed marked infiltration of CD8-positive T cells into the tumor after vaccination. The GPC3 peptide-specific CTL frequency in peripheral blood was correlated with overall survival in patients with HCC who received the peptide vaccination. In multivariate analysis, the frequency of GPC3-peptide-specific CTLs was the predictive factor for overall survival in this trial. These observations suggest that GPC3-derived peptide vaccines represent a novel therapy for patients with HCC, with the potential to improve overall survival. Ongoing trial: We subsequently conducted a phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for patients with HCC. Forty patients with initial HCC who had undergone surgery or radiofrequency ablation were enrolled in this phase II, open-label, single-arm trial. We did not confirm whether the tumor-infiltrating lymphocytes detected after vaccination were GPC3 peptide-specific CTLs in Phase I. Therefore, we are initiating a pilot study of liver biopsies performed before and after GPC3 peptide vaccination for advanced HCC. GPC3 is overexpressed in several malignant tumors, including ovarian clear cell carcinoma (CCC), which had a poor prognosis due to low sensitivity to conventional chemotherapy. In a phase II study in ovarian CCC patients, one patient showed PR. Development of a novel strategy: Although the peptide vaccine is a potentially attractive treatment modality, the antitumor effects of the peptide vaccine alone are not dramatic for advanced HCC. Therefore, we aim to develop combinational approaches or strong antigen-specific immunotherapies, such as adoptive cell therapy following lymphodepletion. The density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. To address this problem, we are performing intratumoral peptide injection in our ongoing study using mice. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improving antigen-specific cancer immunotherapy. Conclusion: We might show the potential of GPC3 peptide vaccine therapy in these clinical trials. Citation Format: Tetsuya Nakatsura, Toshiaki Yoshikawa, Yu Sawada. A potential of glypican-3-derived peptide vaccine therapy against cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B60.

Abstract B73: Proof of glypican-3 (GPC3) peptide specific CTLs infiltrating into tumor tissue derived from advanced HCC patient vaccinated with GPC3 peptide.

Abstract Background: Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we completed a phase I clinical trial of HLA-A2-restricted GPC3 144-152 (FVGEFFTDV) and A24-restricted GPC3 298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination was well tolerated, and indicated many immunological responses. Furthermore, GPC3-specific CTL frequency after vaccination correlated with overall survival. In addition, tumor biopsy was carried out in 7 patients to evaluate the therapeutic effect after vaccination. We evaluated the infiltration of CD8-positive T cells by immunohistochemical staining. In 5 of 7 cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination. However, we did not confirm that the tumor-infiltrating lymphocytes detected after vaccination were GPC3 peptide-specific CTLs. We are currently initiating a pilot study of liver biopsies carried out before and after GPC3 peptide vaccination for advanced HCC to determine whether tumor-infiltrating lymphocytes are indeed GPC3 peptide-specific CTLs. We tried to detect GPC3 peptide-specific CTLs in biopsy specimens from vaccinated patients using GPC3-Dextramer. Materials and Methods: The trial was designed to assess clinical response, and whether tumor-infiltrating lymphocytes with anti-tumor effect are increased indeed after GPC3 peptide vaccination for advanced HCC (UMIN-CTR number: 000005093). Vaccinations with 3.0 mg of GPC3 peptide, emulsified with incomplete Freund's adjuvant (Montanide ISA-51 VG) were carried out intradermally at 14-day intervals. Peripheral blood was obtained from each patients at times designated in the protocol (before the first vaccination and 2 weeks after each vaccination) and centrifuged using a Ficoll–Paque gradient. Immunological responses were analyzed ex vivo by IFN-γ Enzyme-linked immunospot (ELISPOT) assay using peripheral blood mononuclear cells (PBMCs). Tumor biopsy was carried out after 5-6th vaccination. The cells obtained from biopsy specimens were stained anti-CD3, anti-CD8 antibody and GPC3-Dextramer and analyzed using FACSAria cell sorter. Results: We could obtain evidence of GPC3 peptide-specific CTLs infiltrating into the HCC tumor from one case. In this case, the frequency of GPC3 peptide specific CTLs after vaccination (290 of 5 x 105 PBMCs) was larger than that before vaccination (0 of 5 x 105 PBMCs) ex vivo in IFN-γ ELISPOT assay. In flow cytometer analysis, CD3+, CD8+, GPC3-Dextramer+ cells were detected in cells obtained from a biopsy specimen of this vaccinated patient. Moreover, we established GPC3 peptide specific CTL clones from these cells by single cell sort using GPC3-Dextramer. These GPC3-Dextramer+ CTL clones showed GPC3 peptide specific IFN-γ secretion. Conclusion: We could prove that the GPC3 peptide-specific CTLs have been infiltrated into the tumor tissue after peptide vaccination. The evidence serves as a proof-of concept for GPC3 peptide vaccine therapy. Citation Format: Toshiaki Yoshikawa, Mayuko Sakai, Kazuya Ofuji, Mari Takahashi, Manami Shimomura, Yu Sawada, Daisuke Nobuoka, Tetsuya Nakatsura. Proof of glypican-3 (GPC3) peptide specific CTLs infiltrating into tumor tissue derived from advanced HCC patient vaccinated with GPC3 peptide. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B73.

The antitumor effect of human cord blood-derived dendritic cells modified by the livin α gene in lung cancer cell lines

The growth of malignant tumors is associated with mechanisms of immune escape and inhibition of apoptosis. Livin is a novel member of the inhibitors of apoptosis (IAP) protein family that inhibits cell apoptosis. Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. In this study, we used umbilical cord blood (UCB)-derived dendritic cells (DCs) infected with a recombinant adenovirus encoding the livin gene as a vaccine to activate effector cells such as cytotoxic T lymphocytes (CTLs) to recognize and kill livin-expressing cancer cells in vitro as an improved strategy for overcoming the ability of these cancer cells to escape apoptosis and antitumor immune responses. We employed interferon-γ (IFN-γ) enzyme-linked immunospot assays to confirm that our immunization strategy induced an antigen-specific reaction to livin and flow cytometric analysis of staining with Annexin V and PI to measure the cytotoxic activity of the effector cells against the livin-expressing lung cancer cell lines A549 and H460. Our results show that the recombinant adenovirus was able to promote the maturation of the UCB-derived DCs. This DC vaccine could activate antigen-specific T cells to produce IFN-γ upon recognition of livin peptide in the context of the appropriate HLA molecule. The antigen-specific T cells mediate significant cytotoxicity against the cancer cells, but are unlikely to cause an autoimmune reaction against the human bronchial epithelia cells (HBE), which do not express livin.

HLA-B7–Restricted Islet Epitopes Are Differentially Recognized in Type 1 Diabetic Children and Adults and Form Weak Peptide-HLA Complexes

The cartography of β-cell epitopes targeted by CD8+ T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify β-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7–transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD530–538; 44% T cell–positive patients) or adults (GAD311–320; 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2–restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on β-cell–specific (HLA-B7 tetramer–positive) T cells revealed uniform IFN-γ and transforming growth factor-β (TGF-β) mRNA expression, different from HLA-A2–restricted T cells. We conclude that HLA-B7–restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ+TGF-β+CD8+ T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7+ individuals.

HIV control through a single nucleotide on the HLA-B locus.

Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells, and strong linkage disequilibrium between HLA class I alleles complicates the discrimination of individual HLA allelic effects from those of other HLA and non-HLA alleles on the same haplotype. Here, we exploit an experiment of nature involving two recently diverged HLA alleles, HLA-B*42:01 and HLA-B*42:02, which differ by only a single amino acid. Crucially, they occur primarily on identical HLA class I haplotypes and, as Bw6 alleles, do not act as NK cell ligands and are therefore largely unconfounded by other genetic factors. We show that in an outbred cohort (n = 2,093) of HIV C-clade-infected individuals, a single amino acid change at position 9 of the HLA-B molecule critically affects peptide binding and significantly alters the cytotoxic T lymphocyte (CTL) epitopes targeted, measured directly ex vivo by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay (P = 2 × 10(-10)) and functionally through CTL escape mutation (P = 2 × 10(-8)). HLA-B*42:01, which presents multiple Gag epitopes, is associated with a 0.52 log(10) lower viral-load set point than HLA-B*42:02 (P = 0.02), which presents no p24 Gag epitopes. The magnitude of this effect from a single amino acid difference in the HLA-A*30:01/B*42/Cw*17:01 haplotype is equivalent to 75% of that of HLA-B*57:03, the most protective HLA class I allele in this population. This naturally controlled experiment represents perhaps the clearest demonstration of the direct impact of a particular HIV-specific CTL on disease control.

Combined Immunization of Mice with DNA, rMVA and rAd5 Expressing HIV-1 Structural Genes from Different Subtypes

The objective of present paper is to study the immunogenicity of combinations of multiple vector vaccines expressing HIV-1 structural genes from different subtypes. Mice were vaccinated with DNA (B'/C) and rMVA (B'/C) vaccines expressing B'/C recombinant subtype gag-pol and env genes, DNA (B') and rAd5 (B') vaccines expressing subtype B' gag gene with different combination schemes. HIV-1 Gag-specific cellular immune responses and P24- specific IgG levels were analyzed by IFN-γ enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) respectively. ELISPOT results indicated that the Gag-specific cellular immune responses induced by combination of three vaccines were much higher than that induced by combination of two vaccines. Among the groups of mice immunized with two vaccines, the groups with rAd5 booster elicited higher cellular immune responses compared with the groups with rMVA booster. All the test groups of three vaccines in combination could induce similar level of cellular immune responses, which did not correlate with the immunization order. ELISA results showed that p24- specific IgG induced by combination of three vaccines were much higher than that induced by combination of two vaccines. It indicates that the combination scheme of multiple vector vaccines maybe a promising AIDS vaccine strategy.

Varicella-Zoster Virus-Specific Enzyme-Linked Immunospot Assay Responses and Zoster-Associated Pain in Herpes Zoster Subjects

Varicella-zoster virus (VZV)-specific cell-mediated immunity (CMI) responses were compared over time following an episode of herpes zoster (HZ) with those of age-, race-, and gender-matched healthy controls (HC) without HZ, using a validated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. The zoster brief-pain inventory (ZBPI) was used to assess zoster-associated pain. HZ patients (n = 140) had significantly higher IFN-γ ELISPOT responses to VZV antigen than did HC (n = 140). ELISPOT geometric mean count (GMC) responses (with 95% confidence intervals [CI]) for subjects who presented within 72 h were as follows: for HZ patients ≥ 60 years of age, at day 0 the GMC was 110 and at week 2 the GMC was 235; for HZ patients 21 to 59 years of age, at day 0 the GMC was 111 and at week 2 the GMC was 198; for HC ≥ 60 years of age, at day 0 the GMC was 19 and at week 2 the GMC was 18; and for HC 21 to 59 years of age, at day 0 the GMC was 59 and at week 2 the GMC was 56. The mean pain score (95% CI) across age groups at 1 week postrash (n = 106) was 6.0 (5.5, 6.5) and at 2 weeks postrash (n = 119) was 3.5 (2.9, 4.0). The percentage of HZ patients with substantial pain (score ≥ 3) at 6 weeks postrash increased with age from 8% for patients 21 to 49 years of age to 16% for patients 50 to 59 years of age to 22% for patients ≥ 60 years of age. The VZV-specific CMI response was substantially boosted by an episode of HZ, as measured by ELISPOT results. Older adults had lower VZV-specific cellular immunity than younger subjects at baseline, but the boosting effect of HZ was substantial for all age groups. HZ patients experienced considerable zoster-associated acute (1 to 2 weeks after rash) pain across age groups, while chronic pain increased with age.

Corticosteroid-modulated Immune Activation in the Tuberculosis Immune Reconstitution Inflammatory Syndrome

HIV-tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an immunopathological reaction to mycobacterial antigens induced by antiretroviral therapy. Prednisone reduces morbidity in TB-IRIS, but the mechanisms are unclear. To determine the effect of prednisone on the inflammatory response in TB-IRIS (antigen-specific effector T cells, cytokines, and chemokines). Blood was taken from participants in a randomized placebo-controlled trial of prednisone for TB-IRIS, at 0, 2, and 4 weeks. Participants received prednisone at a dosage of 1.5 mg/kg/day for 2 weeks followed by 0.75 mg/kg/day for 2 weeks, or placebo at identical dosages. Analyses included IFN-γ enzyme-linked immunospot (ELISPOT), reverse transcription-polymerase chain reaction on peripheral blood mononuclear cells after restimulation with heat-killed Mycobacterium tuberculosis, Luminex multiplex cytokine analysis of corresponding tissue culture supernatants, and Luminex multiplex cytokine analysis of serum. Fifty-eight participants with TB-IRIS (31 receiving prednisone, 27 receiving placebo) were included. In serum, significant decreases in IL-6, IL-10, IL-12 p40, tumor necrosis factor-α, IFN-γ, and IFN-γ-induced protein-10 concentrations during prednisone, but not placebo, treatment were observed. No differences in ELISPOT responses comparing prednisone and placebo groups were shown in response to ESAT-6 (early secreted antigen target-6), Acr1, Acr2, 38-kD antigen, or heat-killed H37Rv M. tuberculosis. Purified protein derivative ELISPOT responses increased over 4 weeks in the prednisone group and decreased in the placebo group (P = 0.007). The beneficial effects of prednisone in TB-IRIS appear to be mediated via suppression of predominantly proinflammatory cytokine responses of innate immune origin, not via a reduction of the numbers of antigen-specific T cells in peripheral blood.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL) Activity in Cancer Vaccine Clinical Trials.

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (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, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

Abstract LB-135: A pilot study of peptide-based vaccines in combination with poly ICLC in patients with WHO grade 2 low-grade glioma

Abstract Introduction: Adult patients with WHO grade 2 low-grade glioma (LGG) have a significant risk of tumor progression despite treatment with surgery or surgery followed by radiation therapy (RT) and /or chemotherapy, and most patients eventually die of the disease. High-risk subsets of these LGG patients display astrocytoma or oligoastrocytoma histology plus any one of the following conditions: 1) age ≥40 with any extent resection; 2) age 18-39 with incomplete resection; or 3) age 18-39 with neurosurgeon-defined gross total resection with tumor size ≥ 4 cm in diameter. These patients have as high as an 89% risk of recurrence by 5 years following surgery. Methods: Based on encouraging data from a phase I vaccine study targeting multiple glioma-associated antigen (GAA) epitopes in adult high-grade glioma (HGG) patients, we initiated a bi-institutional pilot study of subcutaneous vaccinations of subcutaneous vaccinations with synthetic peptides for GAA epitopes emulsified in Montanide-ISA-51 every 3 weeks for 8 courses, and intramuscular administration of poly-ICLC in HLA-A2+ patients with: newly diagnosed high-risk LGG without prior RT (Cohort 1); newly diagnosed high-risk LGG with prior RT (Cohort 2), or recurrent LGG (Cohort 3). Primary endpoints were safety and CD8+ T-cell responses against vaccine-targeted GAAs, assessed by Enzyme-Linked Immuno-SPOT (ELISPOT) assays. Treatment response was evaluated clinically and by MR imaging. Targeted GAAs were EphA2, interleukin (IL)-13 receptor-α2, survivin, and WT1. As these GAAs are expressed not only in a subset of LGG cells but even at higher levels in HGG cells, our vaccine approach may offer both immunotherapeutic and immunoprophylactic potential to reduce the risk of tumor recurrence. Results: To date, 13, 1 and 10 patients have been enrolled in Cohorts 1, 2 and 3, respectively. No dose-limiting non-CNS toxicity has been encountered except for one case with Grade 3 fever (Cohort 1). ELISPOT assays, completed in 7 and 1 patients in Cohorts 1 and 2, respectively, demonstrated robust and sustained interferon (IFN)-γ (type-1) responses against at least 3 of the GAA epitopes in all cases, while IL-5 (type-2) responses were absent or transient in all cases. The magnitude of the IFN-γ ELISPOT responses in the current study was significantly higher than that observed in our previous phase I/II study in HGG patients (Okada H et al. 2011). Although evaluation of progression-free survival would require a longer observation period, among 9, 1 and 7 patients who completed the 8 vaccinations spanning 24 weeks, 6, 1 and 3 patients in Cohorts 1, 2 and 3, respectively, are currently with stable disease. Conclusion: Our preliminary results demonstrate that the regimen in these patients is well tolerated, and induces robust type-1 anti-GAA T-cell responses. These data suggest that patients with LGG are suitable for vaccine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-135. doi:1538-7445.AM2012-LB-135

Abstract 5371: Immune correlates of survival in advanced hepatocellular carcinoma patients treated with glypican-3-derived peptide vaccine: Results from phase I trial

Abstract Introduction: Glypican-3 (GPC3), a carcinoembryonic antigen, is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC), because it is overexpressed specifically in HCC and correlates to a poor prognosis. In a preclinical study using a mouse model, we identified HLA-A24:02-restricted GPC3298-306 (EYILSLEEL) and HLA-A02:01-restricted GPC3144-152 (FVGEFFTDV) peptides, both of which can induce GPC3-reactive cytotoxic T-lymphocytes (CTLs) without inducing autoimmunity. On the basis of these results, we conducted a clinical trial of GPC3-derived peptide vaccine for advanced HCC patients to evaluate the vaccine's safety, tolerability, and immunological and clinical responses. Methods: Thirty-three advanced HCC patients were enrolled in this non-randomized, open-label, dose escalation phase I trial. GPC3-derived peptide was administered in a liquid form emulsified with incomplete Freund's adjuvant, and intradermally injected on days 1, 15, and 29. PBMCs, obtained from all patients before and after vaccination, were examined by ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay. The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by ex vivo IFN-γ ELISPOT assay, and clinical outcomes, as measured by tumor response, time to tumor progression, and overall survival (OS). Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 18 patients showed stable disease 2 months after initiation of treatment. Four patients, of which 18 had stable disease, had tumor regression or necrosis not meeting the criteria for partial response. The median time to tumor progression was 3.4 months (95% CI, 2.1 to 4.6). The median OS was 9.0 months (95% CI, 8.0 to 10.0). Tumor biopsy was performed with adequate informed consent in 7 patients for the evaluation of the therapeutic effect after vaccination. In 5 out of 7 cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination. GPC3 peptide vaccine could induce the GPC3-specific CTL response in 30 of 33 patients by ex vivo IFN-γ ELISPOT assay, and the GPC3-specific CTL frequency after vaccination correlated with overall survival. The analysis of all 33 patients showed that the median OS was 12.2 months (95% CI, 6.5 to 18.0) in patients with high frequency of GPC3-specfic CTL, compared with 8.5 months (95% CI, 3.7 to 13.1) in those with low frequency of GPC3-specfic CTL (p = 0.033). Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. Furthermore, this study shows that peptide-specific CTL frequency can be a predictive marker for overall survival in HCC patients receiving peptide vaccination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5371. doi:1538-7445.AM2012-5371

Hepatitis C Virus-Multispecific T-Cell Responses without Viremia or Seroconversion among Egyptian Health Care Workers at High Risk of Infection

Hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) has been reported among exposed individuals without viremia or seroconversion. Limited data are available regarding CMI among at-risk, seronegative, aviremic Egyptian health care workers (HCW), where HCV genotype 4 predominates. We investigated CMI responses among HCW at the National Liver Institute, where over 85% of the patients are HCV infected. We quantified HCV-specific CMI in 52 seronegative aviremic Egyptian HCW using a gamma interferon (IFN-γ) enzyme-linked immunospot assay in response to 7 HCV genotype 4a overlapping 15-mer peptide pools covering most of the viral genome. A positive HCV-specific IFN-γ response was detected in 29 of 52 HCW (55.8%), where 21 (40.4%) had a positive response for two to seven HCV pools and 8 (15.4%) responded to only one pool. The average numbers of IFN-γ total spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) (± standard error of the mean [SEM]) in the 29 responding and 23 nonresponding HCW were 842 ± 141 and 64 ± 15, respectively (P < 0.001). Flow cytometry indicated that both CD4(+) and CD4(-) T cells produced IFN-γ. In summary, more than half of Egyptian HCW demonstrated strong HCV multispecific CMI without viremia or seroconversion, suggesting possible clearance of low HCV exposure(s). These data suggest that detecting anti-HCV and viremia to determine past exposure to HCV can lead to an underestimation of the true disease exposure and that CMI response may contribute to the low degree of chronic HCV infection in these HCW. These findings could have strong implications for planning vaccine studies among populations with a high HCV exposure rate. Further studies are needed to determine whether these responses are protective.

Use of Interferon-&amp;gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected(1). A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)(2) in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 10(5) cells per well typically used in ELISPOT assays(1,3), 1,000 T-cell clone cells in the presence of 1 x 10(5) autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes(4).

Hepatitis C virus‐specific immune responses in noninjecting drug users

Noninjection drug use, although recognized as an emerging risk factor for acquisition of other blood-born pathogens, is still unconfirmed as a route of hepatitis C virus (HCV) transmission. Our goal was to measure HCV exposure and prevalence in noninjection drug users (NIDUs). Fifty-seven NIDUs were screened by extensive questionnaire to exclude prior injection drug use and evaluated for HCV-specific serologic and cellular immune responses. HCV-specific T-cell responses were measured using interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay with overlapping HCV peptides covering the entire HCV genome. Fifteen individuals who never used illicit drugs served as negative controls. Eleven people with no history of injecting drug use (19.3%) were HCV seropositive: seven with chronic HCV infection and four with previously resolved infection. Of 51 NIDUs with ELISpot results, HCV-specific cellular immunity was detected in 5 (9.8%). These responses were relatively weak and narrow. We did not find significant associations between HCV-specific immune responses and noninjection drug use practices. Subjects with HCV-specific immunity, however, were significantly more likely to have bought sex in the past 6 months, to have had more casual partners of the opposite sex in the last 6 months, and those partners were more likely to have ever injected drugs compared to subjects without HCV-specific immunity. In summary, we found serologic or cellular HCV-specific immune responses in 27.5% of NIDUs. Our results suggest that sexual behaviour associated with noninjection drug use might be a risk factor for HCV acquisition. Additional studies are needed to precisely determine the practices that lead to HCV exposure among this population.