Interferon-gamma Enzyme-linked Immunospot Assay Research Articles

Cell-Mediated Immunity (CMI) for SARS-CoV-2 Infection Among the General Population of North India: A Cross-Sectional Analysis From a Sub-sample of a Large Sero-Epidemiological Study.

Background Cell-mediated immunity (CMI), or specifically T-cell-mediated immunity, is proven to remain largely preserved against the variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including Omicron. The persistence of cell-mediated immune response in individuals longitudinally followed up for an extended period remains largely unelucidated. To address this, the current study was planned to study whether the effect of cell-mediated immunity persists after an extended period of convalescence or vaccination. Methods Whole blood specimens of 150 selected participants were collected and tested for Anti-SARS-CoV-2 Interferon-gamma (IFN-γ) response. Ex vivo SARS-CoV-2-specific interferon-gamma Enzyme-linked Immunospot (IFN-γ ELISpot) assay was carried out to determine the levels of virus-specific IFN-γproducing cells in individual samples. Findings Out of all the samples tested for anti-SARS-CoV-2 T-cell-mediated IFN-γ response, 78.4% of samples were positive. The median (interquartile range) spots forming units (SFU) per million levels of SARS-CoV-2-specific IFN-γ producing cells of the vaccinated and diagnosed participants was 336 (138-474) while those who were vaccinated but did not have the disease diagnosis was 18 (0-102); the difference between the groups was statistically significant. Since almost all the participants were vaccinated, a similar pattern of significance was observed when the diagnosedand the never-diagnosed participants were compared, irrespective of their vaccination status. Interpretations Cell-mediated immunity against SARS-CoV-2 persisted, irrespective of age and sex of the participant, for more than six months of previous exposure. Participants who had a history of diagnosed COVID-19 infection had better T-cell response compared to those who had never been diagnosed, in spite of being vaccinated.

Longitudinal Analysis of SARS-CoV-2-Specific Cellular and Humoral Immune Responses and Breakthrough Infection following BNT162b2/BNT162b2/BNT162b2 and ChAdOx1/ChAdOx1/BNT162b2 Vaccination: A Prospective Cohort in Naive Healthcare Workers.

Assessing immune responses post-SARS-CoV-2 vaccination is crucial for optimizing vaccine strategies. This prospective study aims to evaluate immune responses and breakthrough infection in 235 infection-naïve healthcare workers up to 13-15 months after initial vaccination in two vaccine groups (108 BNT/BNT/BNT and 127 ChAd/ChAd/BNT). Immune responses were assessed using the interferon-gamma enzyme-linked immunospot (ELISPOT) assay, total immunoglobulin, and neutralizing activity through surrogate virus neutralization test at nine different time points. Both groups exhibited peak responses one to two months after the second or third dose, followed by gradual declines over six months. Notably, the ChAd group exhibited a gradual increase in ELISPOT results, but their antibody levels declined more rapidly after reaching peak response compared to the BNT group. Six months after the third dose, both groups had substantial cellular responses, with superior humoral responses in the BNT group (p < 0.05). As many as 55 breakthrough infection participants displayed higher neutralization activities against Omicron variants, but similar cellular responses compared to 127 infection-naïve individuals, suggesting cross-immunity. Distinct neutralization classifications (<30%, >80% inhibition) correlated with different ELISPOT results. Our study reveals diverse immune response patterns based on vaccine strategies and breakthrough infections, emphasizing the importance of understanding these dynamics for optimized vaccination decisions.

Response to Chemoimmunotherapy Is Associated With Expansion of Systemic Antitumor CD4 + Th1 Response in Metastatic Non-Small Cell Lung Cancer.

Limited data have reported the evolution of antitumor immune responses under chemoimmunotherapy (chemo-IO) in patients with metastatic non-small cell lung cancer. In this concise study, we performed dynamic monitoring of antitumor CD4 + T helper 1 (Th1) response in peripheral blood from 12 patients receiving a first-line chemo-IO. Tumor-reactive CD4 + Th1 cells were assessed within blood lymphocytes using interferon-gamma enzyme-linked immunospot assay to detect telomerase (TERT)-specific T cells at baseline, 3 and 12 months after treatment. An induction of circulating anti-TERT CD4 + Th1 response were found in 6 of 12 patients at 3 months after chemo-IO. In contrast, 3 patients had a substantial decrease in their preexisting response and 3 remained nonimmune responders. Among patients with chemo-IO-induced immune response, half achieved an objective clinical response and had long-lasting circulating anti-TERT CD4 + Th1 cells detected for at least 1 year. In contrast, no objective response was documented in nonimmune responders and a link between the loss of anti-TERT CD4 + Th1 responses were observed in patients with progressive disease. This preliminary work supports a relationship between the efficacy of combinatorial chemo-IO and circulating anti-TERT CD4 + Th1 responses and highlights the interest to implement blood-based monitoring of tumor-reactive CD4 + T cells that could be additional help for patient management.

Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium.

Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.

Novel peptide-based vaccine targeting heat shock protein 90 induces effective antitumor immunity in a HER2+ breast cancer murine model

BackgroundHeat shock protein 90 (HSP90) is a protein chaperone for most of the important signal transduction pathways in human epidermal growth factor receptor 2-positive (HER2+) breast cancer, including human epidermal...

Machine learning approach for culprit drug identification based on drug-specific interferon-gamma releasing cells and clinical parameters in non-immediate drug hypersensitivity

This study aims to evaluate diagnostic utility for interferon-gamma enzyme-linked immunospot assay (ELISpot) and clinical parameters against drug provocation test (DPT) in predicting drug-induced non-immediate hypersensitivity using a machine learning approach.

Antiviral T-Cell Frequencies in a Healthy Population: Reference Values for Evaluating Antiviral Immune Cell Profiles in Immunocompromised Patients

Viral infections and reactivations are major causes of morbidity and mortality after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) as well as in patients with immunodeficiencies. Latent herpesviruses (e.g., cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6), lytic viruses (e.g., adenovirus), and polyomaviruses (e.g., BK virus, JC virus) can cause severe complications. Antiviral drugs form the mainstay of treatment for viral infections and reactivations after transplantation, but they have side effects and cannot achieve complete viral clearance without prior reconstitution of functional antiviral T-cell immunity. The aim of this study was to establish normal ranges for virus-specific T-cell (VST) frequencies in healthy donors. Such data are needed for better interpretation of VST frequencies observed in immunocompromised patients. Therefore, we measured the frequencies of VSTs against 23 viral protein-derived peptide pools from 11 clinically relevant human viruses in blood from healthy donors (n = 151). Specifically, we determined the VST frequencies by interferon-gamma enzyme-linked immunospot assay and classified their distribution according to age and gender to allow for a more specific evaluation and prediction of antiviral immune responses. The reference values established here provide an invaluable tool for immune response evaluation, intensity of therapeutic drugs and treatment decision-making in immunosuppressed patients. This data should make an important contribution to improving the assessment of immune responses in immunocompromised patients.

Performance of Xpert MTB/RIF Ultra for the Diagnosis of Pulmonary Tuberculosis Using Bronchoalveolar Lavage Samples in People Living with HIV/AIDS (PLWHA) in China: A Prospective Study.

BackgroundSputum is commonly used for the diagnostic testing of pulmonary tuberculosis (PTB), but people living with HIV/AIDS (PLWHA) usually have little sputum. Moreover, the automated molecular test, Xpert MTB/RIF assay (Xpert), has a low sensitivity in PLWHA. We aimed to estimate the performance of Xpert Ultra on the detection of Mycobacterium tuberculosis (MTB) using bronchoalveolar lavage (BAL).MethodsFrom February 5, 2018 to March 30, 2019, a total of 99 PLWHA with suspected PTB at the Third People’s Hospital of Shenzhen, China, were recruited. The information on demographics and medical history, blood MTB antigen-specific interferon gamma enzyme-linked immunospot assay (T-SPOT.TB), T lymphocyte subsets, and plasma HIV RNA load were collected. Computed tomography (CT) and flexible bronchoscopy were performed, and BAL and blood samples were collected. Testing of acid-fast bacilli (AFB), tuberculosis real-time fluorescence quantitative PCR (TBDNA), Ultra, Xpert, and MTB culture were conducted.ResultsCompared to BAL MTB culture for tuberculosis diagnosis, Ultra, Xpert, T-SPOT.TB, TBDNA and AFB smear had the sensitivity of 0.96 (24/25), 0.80 (20/25), 0.84 (21/25), 0.44 (11/25), and 0.12 (3/25), respectively; and the specificity of 0.92 (68/74), 0.96 (71/74), 0.93 (69/74), 0.96 (71/74), and 0.99 (73/74), respectively. Our study found that the sensitivity of Ultra was higher than that of culture and Xpert (AUC 0.92, 0.86 and 0.84, respectively). The results also indicated that PLWHA with CD4 <200 cells/mm3 had reduced both sensitivity (from 1.00 and 0.86 to 0.94 and 0.78, respectively) and specificity (from 0.96 and 1.00 to 0.90 and 0.41, respectively) of Ultra and Xpert for the diagnosis of PTB.DiscussionOur data supported an increased sensitivity of Ultra compared to that of Xpert on BAL samples of PLWHA, regardless of the CD4 counts and reference diagnosis standards.

Value of gamma interferon enzyme-linked immunospot assay in the diagnosis of peritoneal dialysis-associated tuberculous peritonitis

BackgroundTuberculous peritonitis is the most common form of extrapulmonary tuberculosis infection in peritoneal dialysis patients. However, diagnosing tuberculous peritonitis quickly and early has always been a challenge for nephrologists. Mycobacterium tuberculosis antigen-specific gamma interferon enzyme-linked immunospot (IFN-γ ELISPOT) assay has been widely used in the clinical diagnosis of tuberculous pleurisy and peritonitis, but its use has not been reported for uremia.MethodsThis study mainly verified the feasibility of using the M. tuberculosis antigen-specific IFN-γ ELISPOT assay in the diagnosis of continuous ambulatory peritoneal dialysis (CAPD) patients with tuberculous peritonitis. Taking M. tuberculosis culture as the gold standard, the IFN-γ ELISPOT assay was used to analyze peripheral blood and peritoneal dialysis fluid of patients, and the receiver operating characteristic (ROC) curves in patients with tuberculous peritonitis (TBP) or non-tuberculous peritonitis (NTBP) were analyzed.ResultsThe area under the receiver operating characteristic curve (AUC) was 0.927 (95% CI 0.816–1.000, P = 0.001) for the ELISPOT assay with peritoneal fluid mononuclear cells (PFMC), which was higher than that for the ELISPOT assay with peripheral blood mononuclear cells (PBMC) (0.825, 95% CI 0.6490–1.000, P = 0.011). The cutoff value for the diagnosis of TBP was 40 spot-forming cells (SFCs)/2 × 105 for the ELISPOT with PBMC, with a sensitivity of 55.6%, a specificity of 92.3%, and a diagnostic efficiency of 77.3%. The cutoff value for the diagnosis of TBP was 100 SFCs/2 × 105 for the ELISPOT on PFMC, with a sensitivity, specificity, and diagnostic efficiency 77.8%, 84.6%, and 81.8%, respectively. Parallel and serial testing algorithms appeared more accurate than single ELISPOT assays with PBMC, but ELISPOT assays with PFMC.ConclusionsThe IFN-γ release test can be used for the early diagnosis of CAPD-related TBP; compared with peripheral blood, peritoneal fluid may be a more effective and accurate medium to diagnose CAPD complicated with tuberculous peritonitis.

Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates.

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.

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.

Immune response to varicella-zoster virus before and after renal transplantation

BackgroundHerpes zoster (HZ) risk is high in renal transplant recipients. Vaccination prior to transplantation may provide a useful strategy for the prevention of HZ in the posttranplantation period. However, it is not known whether immunity to varicella-zoster virus (VZV) is affected due to treatment surrounding transplantation. MethodsBoth humoral and cellular immunity to VZV were determined prior to and 2–3 years after renal transplantation in 60 adult patients, and 62 matched healthy controls. VZV-specific cellular immunity was measured by an interferon gamma (IFNγ) enzyme-linked immunospot (ELISpot) assay and by analyzing T-cell functionality using flowcytometry. VZV-IgG levels were measured using an in-house glycoprotein enzyme-linked immunosorbent assay (gpELISA). ResultsUsing paired analysis, it was determined that numbers of IFNγ-producing cells did not change after transplantation, but were significantly lower in transplant recipients after transplantation than in controls (p = 0.028). Patients in whom the post-transplant period was complicated by rejection or any acute infection (excluding HZ) had a lower number of IFNγ-producing cells than patients who did not. VZV IgG levels did not differ from controls, but a significant decrease was observed after transplantation (p < 0.0001). ConclusionsVZV-specific cellular immunity, which is essential in the prevention of HZ, did not markedly change in patients following renal transplantation. This suggests that preventive vaccination before transplantation may be beneficial. Our results extend knowledge on VZV immunity after transplantation, vital when considering strategies for the prevention of HZ in these patients.

P1217VALUE OF MYCOBACTERIUM TUBERCULOSIS SPECIFY ELISOPT IN THE DIAGNOSIS OF PERITONEAL DIALYSIS-ASSOCIATED TUBERCULOUS PERITONITIS PATIENTS

Abstract Background and Aims Patients with ESRD undergoing chronic dialysis are 6 to 25 times more likely to develop tuberculosis (TB) than the general population. Tuberculous peritonitis is the most common form of tuberculosis infection in peritoneal dialysis patients. However, diagnosing active TB infection can be challenging, early and rapid diagnosis of tuberculous peritonitis has always been a challenge for nephrologists. M.tuberculosis antigen-specific IFN-γ production by peripheral blood mononuclear cells (PBMC) was determined by using an in-house ELISPOT assay by Institute of Hepatology of Shenzhen Third Hospital.In this study, we evaluated diagnostic performance of an M.tuberculosis antigen-specific gamma interferon enzyme-linked immunospot (ELISPOT) assay in continuous ambulatory peritoneal dialysis (CAPD) patients with tuberculous peritonitis. Method There were nine patients (1.29%) with tuberculous peritonitis among 700 CAPD patients. M. tuberculosis accounted for 2.84 % of all peritonitis.IFN-γ response in peripheral blood and peritoneal fluid were determined in 9 patients with tuberculous peritonitis(TBP),at the same time, 13 cases diagnosed with non-tuberculous peritonitis(NTBP) were enrolled.All patients were examined for tuberculosis-specific IFN-γ ELISPOT in peripheral blood and peritoneal dialysis fluid. Results 22 patients were recruited into this study, 9 were classified with confirmed tuberculosis peritonitis, 13 patients with bacterial or fungal peritonitis were enrolled as controls during the same period, in this patients, 3 were caused by Gram positive bacteria, 3 were caused by Gram negative bacteria, 3 were caused by fungus, 4 patients were negative athogenic bacteria culture .The AUC of ROC curve was 0.927 (95%CI:0.816–1.000,P=0.001) for ELISPOT on PFMC, which was higher than that of ELISPOT on PBMC (0.825,95%CI: 0.6490–1.000,P=0.011).When the cutoff value for the diagnosis of TBP was 40.5 SFCs/2 × 105 for ELISPOT on PBMC, with a sensitivity of 55.6%, a specificity of 92.3%,a diagnostic efficiency of 77.3%,The positive predictive value was83.3%, with negative predictive value of 75%,positive likelihood ratio of 7.2, and negative likelihood ratio of 0.48.The sensitivity, specificity,diagnostic efficiency,PPV, NPV, LR+, and LR- of the ELISPOT assay for the diagnosis of active TBP when performed on the PFMC were 77.8%, 84.6%, 81.8%, 77.8%, 84.6%, 5.05 and 0.26, respectively.Receiver operating characteristic(ROC) curves were used to estimate the diagnostic values of Elispot on PFMC and PBMC in 22 patients with confirmed TBP or NTBP. Conclusion IFN-γ release test can be used for early diagnosis of CAPD-related TBP; compared with peripheral blood, peritoneal dialysate may be a more effective and accurate way to diagnose CAPD complicated with tuberculous peritonitis.

Treatment of an aggressive orthotopic murine glioblastoma model with combination checkpoint blockade and a multivalent neoantigen vaccine.

Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

A randomized open-label trial of 2-dose or 3-dose pre-exposure rabies prophylaxis among Thai children

BackgroundWorld Health Organization changed the recommendation for pre-exposure rabies prophylaxis from 3-dose to 2-dose regimen in 2018. Given limited data of 2-dose regimens in pediatric population, this study aimed to compare the immunogenicity between 2-dose and 3-dose pre-exposure rabies immunization. MethodsThis study was conducted among healthy children aged 2–12 years. They were randomized to 2-dose vaccination (2D) on days 0 and 28 or 3-dose vaccination (3D) on days 0, 7, and 28. Purified Vero cell rabies vaccine (PVRV-Verorab™) was administered intramuscularly. Rabies virus neutralizing antibody (RVNA) titers were measured at 3 time points: 14-day after complete vaccination, 1-year pre-booster vaccination, and 7-day post-booster dose to mimic scenario of rabies exposure. RVNA titers ≥0.5 IU/ml were considered adequate antibody. T cell specific response to rabies vaccine antigen was measured using the interferon-gamma enzyme linked immunospot assay. ResultsFrom September to October 2017, 107 participants (51% males), 78 in 2D group and 29 in 3D group were enrolled. Median age was 5.8 years (IQR 4.4–7.3). All participants had RVNA titers ≥0.5 IU/ml after primary vaccination [GMT 2D: 18.6 (95%CI 15.9–21.8) and 3D: 16.3 (95%CI 13.2–20.1 IU/ml), p = 0.35]. At 1-year prior to receiving the booster, only 80% of the children in 2D group maintained RVNA titers ≥0.5 IU/ml compared to 100% of the children in 3D group (p = 0.01). However, all participants in both groups had RVNA ≥0.5 IU/ml at 7-day post booster vaccination [GMT 2D: 20.9 (95%CI 17.4–25.3) and 3D: 22.2 (95%CI 15.8–31.4) IU/ml (P = 0.75)]. The median number of IFN-γ secreting cells at 7-day post-booster dose was 98 and 128 SFCs per 106 PBMCs in the 2D and 3D groups, respectively (P = 0.30). ConclusionsTwo-dose primary rabies immunization provided adequate antibody at post primary vaccination and post booster. The results support 2-dose regimen of pre-exposure rabies immunization in the pediatric population.

Molecular docking predictions of fragrance binding to human leukocyte antigen molecules.

Over 4000 small chemicals have been identified as allergens capable of inducing skin sensitization. Many sensitizers are hypothesized to act as haptens producing novel antigens, which can be presented to T cells by human leukocyte antigens (HLAs). Recent studies suggest that some chemical allergens use hapten-independent mechanisms. To determine whether molecular docking can identify HLA molecules that bind skin-sensitizing chemical allergens. Structural models of HLA molecules were used as the basis for molecular docking of 22 chemical allergens. Allergens predicted to bind HLA-B*57:01 were tested for their ability to stimulate T cells by the use of proliferation and interferon-gamma enzyme-linked immunospot assays. Chemical allergens that did not satisfy the criteria for hapten activity in vitro were predicted to bind more strongly to common HLA isoforms than those with known hapten activity. HLA-B*57:01, which is an HLA allele required for drug hypersensitivity reactions, was predicted to bind several allergens, including benzyl benzoate, benzyl cinnamate, and benzyl salicylate. In in vitro T cell stimulation assays, benzyl salicylate and benzyl cinnamate were found to stimulate T cell responses from HLA-B*57:01 carriers. These data suggest that small-molecule skin sensitizers have the potential to interact with HLA, and show that T cell-based in vitro assays may be used to evaluate the immunogenicity of skin-sensitizing chemicals.

Hypersensitivity reactions to antituberculosis drugs confirmed by interferon gamma enzyme-linked Immunospot assay

Hypersensitivity reactions to antituberculosis drugs confirmed by interferon gamma enzyme-linked Immunospot assay

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

Therapy of Patient with Recurrent HCC After Liver Transplantation with T-cells transiently expressing HBV-specific TCR

Background Hepatitis B virus (HBV) antigen expression frequently occurs in HBV-related hepatocellular carcinoma (HCC). We are performing a phase 1 trial to evaluate the safety and efficacy of activated autologous T cells, that express transiently an HBsAg/HLA-class I restricted specific T cell receptor (HBV/TCR-T cell) trough mRNA TCR electroporation. Here we report the first case of our trial. METHODS: The patient was given an intravenous injection of 4 doses of escalating HBV/TCR-T cell (1x104, 1x105, 1x106, and 5x106/kg) in the first treatment cycle, followed by a second treatment cycle of 4 doses of 5x106/kg one month later. The patient was evaluated every 8 weeks by radiologic imaging. Patients’ immune responses were monitored using an interferon-gamma enzyme-linked immunospot assay. Results The patient was a 41-year-old man with HBV associated HCC and undergone liver transplant at Jan 22nd, 2016. HCC relapse at the T12 thoracic vertebra was found 10 months after liver transplant, followed by a vertebral metastasis resection and radiotherapy. At 3 months after the resection, a new T9 metastasis was detected and treated with heavy ion radiotherapy once again. However, the serum α-fetoprotein (AFP) was increased from 167 to 2536 ng/ml and the tumor diameter increased from 3.6 to 5.8 cm within 4 months during the treatment. Then the first cycle of the HBV/TCR-T cell was given with serum HBV-DNA detected negative. The serum AFP drop from 2629 to 488 ng/ml after the first treatment cycle. However, the AFP was then gradually increased to 821 ng/ml after the second treatment cycle. HBV-DNA coding for HBV antigen was detected on the primary HCC. mRNA TCR-redirected T cells infusion was associated with a reduction in AFP levels without exacerbation of liver inflammation or other toxicity. During the HBV/TCR-T cell therapy, the metastasis tumor diameter was not increased without new lesions detected, which mean the disease stabilized in this case according to the Response Evaluation Criteria In Solid Tumors criteria. This clinical response continued for 5 months after the initiation of HBV/TCR-T cell therapy. We did not observe any serious adverse reactions to the immunotherapy, which was well tolerated. Conclusion We provide initial evidence of the safety and antitumor activity of autologous TCR-redirected immunotherapy in patients with recurrent hepatitis B-associated HCC after liver transplantation.

Abstract 5636: Identification of bladder cancer-associated cancer-testis antigens-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes

Abstract DEP domain containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1) are human cancer testis antigens that are frequently overexpressed in urinary bladder cancer. The growth of bladder cancer cells was significantly inhibited by knockdown of DEPDC1 or MPHOSPH1 expression using small interfering RNAs; thus, DEPDC1 and MPHOSPH1 are essential for tumor growth and survival, indicating the clinical significance of these antigens as targets for antigen-specific cancer immunotherapy. Subsequent reports further identified highly immunogenic DEPDC1- and MPHOSPH1-derived short peptides (SPs) that can induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) isolated from patients with bladder cancer. These DEPDC1- and MPHOSPH1-derived SPs vaccine administered to patients with progressive bladder cancer was well tolerated and effectively activated SPs-specific CTLs in vivo, and the better CTL induction was associated with longer survival of patients in a recent phase I and II clinical trial (Obara W et al. Ann. Oncol. 28: 798, 2017). Here, we aimed to identify long peptides (LPs) derived from DEPDC1 and MPHOSPH1 that induced both T-helper (Th) cells and tumor-reactive CTLs. Stimulation of PBMCs isolated from healthy donors with the synthetic DEPDC1- and MPHOSPH1-LPs predicted to bind to promiscuous human HLA class II molecules by a computer algorithm induced specific CD4-positive T cells as revealed by interferon-gamma enzyme-linked immunospot assays. Three of six LPs encompassed SPs recognized by either HLA-A2- or -A24-restricted CTLs, and all six LPs stimulated DEPDC1- or MPHOSPH1-specific Th cells restricted by promiscuous and frequently observed HLA class II molecules in the Japanese population. Some LPs are naturally processed from the proteins in DCs, and the capacity of these LPs to cross-prime SP-specific CTLs was confirmed in vivo using HLA-A2 or -A24 transgenic mice. The LP-specific and HLA class II-restricted T-cell responses were also observed in PBMCs isolated from patients with bladder cancer. Repeated stimulation of PBMCs with DEPDC1-LPs and MPHOSPH1-LPs yielded clonal Th cells expressing specific T-cell receptor (TCR)-alpha and beta genes. These DEPDC1- or MPHOSPH1-derived LPs may have applications in immunotherapy in patients with bladder cancer, and the TCR genes identified may be useful for monitoring of Th cells specific to LPs in vivo. [ This research was supported by a MEXT Grant-in-Aid for Scientific Research on Innovative Areas 22133005; JSPS KAKENHI 23650609, 24300334, 15H04311 and 16H06498 for Scientific Research on Innovative Area "Neo-self"; the Project for Cancer Research And Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and Development, AMED; and OncoTherapy Science, Inc.. ] Citation Format: Yasuharu Nishimura, Miki Tsuruta, Shohei Ueda, Poh Yin Yew, Isao Fukuda, Sachiko Yoshimura, Hiroyuki Kishi, Hiroshi Hamana, Masatoshi Hirayama, Junji Yatsuda, Atsushi Irie, Satoru Senju, Eiji Yuba, Tomomi Kamba, Masatoshi Eto, Hideki Nakayama. Identification of bladder cancer-associated cancer-testis antigens-derived long peptides encompassing both CTL and promiscuous HLA class II-restricted Th cell epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5636.