Specific IFN-gamma Research Articles

Effectiveness of COVID-19 primary and booster vaccination in HIV-infected individuals

Effectiveness of COVID-19 primary and booster vaccination in HIV-infected individuals

Final report and long-term outcomes: Phase I trial of a HER2 intracellular plasmid-based vaccine in HER2+ advanced stage breast cancer.

2619 Background: Vaccination with the intracellular domain (ICD) of HER2 in pre-clinical models is both immunogenic and protective against the development of mammary tumors. This study (NCT00436254) was designed to examine the safety and optimal immunogenic dose of a DNA-based vaccine encoding the HER2 ICD in subjects with HER2+ breast cancer. Methods: Sixty-six patients with stage III or IV HER2 + breast cancer in remission or with stable bone only disease were enrolled into three vaccine arms: 1 (10mcg dose of plasmid), 2 (100mcg) and 3 (500mcg). Vaccines were administered i.d. monthly for three immunizations. Endpoints included safety and optimal dose. HER2 specific IFN-gamma immune responses were evaluated and DNA persistence at the vaccine site was assessed. Toxicity and clinical outcomes were followed for 10 years. Results: The majority of vaccine-related toxicity was grade 1 (89%) and grade 2 (11%) and was not significantly different between the three dose arms. All Arms developed HER2 ICD immunity after vaccination, however, patients in Arm 2 and Arm 3 had significantly better immune responses (of higher magnitude and at most time points) than patients in Arm 1 (p=0.003 and p<0.001, respectively) after adjusting for baseline factors. At 60 weeks, the number of patients who maintained the greatest fold-difference in HER2 ICD immune responses from their baseline was highest in Arm 2 (73%) when compared to Arm 1 (47%) and Arm 3 (45%). Associations between ICD responses and plasmid DNA persistence at the vaccine site were estimated via linear regression models. HER ICD immunity after the end of immunizations, relative to baseline, was significantly lower in patients with DNA persistence at week 16 compared to those without persistence (p=0.02). Patients at the highest dose demonstrated the greatest incidence of plasmid persistence (92%) as compared to 33% in Arm 1 and 10% in Arm 2. The median time of follow-up was 118.6 months (Arm 1), 99.7 months (Arm 2), and 73.5 months (Arm 3). The median OS and PFS has not been reached in any Arm and did not differ with respect to treatment arm (Log-rank p-value 0.36 for OS, and 0.63 for PFS). However, we observed a separation of Kaplan-Meier curves for OS from about 40 months and curves for PFS from about 30 months, and the separation maintained until the end of the study for Arm 2 versus Arm 1 and Arm 3. One patient in Arm 2 developed lymphocytic colitis 2.2 years from enrollment deemed possibly related to vaccination. Conclusions: An intermediate dose (100mcg) of vaccine was immunogenic and associated with persistence of immunity at 60 weeks. A randomized phase II trial of the HER2 ICD plasmid-based vaccine in the neoadjuvant setting is in development. Clinical trial information: NCT00436254.

A Critical Role for IL4 in the Neonate after Exposure to Aerosolized Ovalbumin in a Murine Model of Allergen Sensitization

Epidemiological studies have suggested that childhood exposure to allergens can lead to a Th2 response culminating in allergic asthma. We hypothesized that blocking the activity of IL4 with antibodies during the time of neonatal exposure to allergens can significantly reduce the risk of allergic sensitization at adulthood. On day 0, neonatal BALB/c mice were exposed to a 1% aerosolized ovalbumin (OVA) for 20 mins while the controls received aerosolized PBS. One group from the OVA exposed mice received 0.5 mg of anti-IL4 also on day 0. At adulthood, all groups received additional doses of aerosolized OVA. Blood samples were obtained for eosinophil and antibody analyses. In-vitro re-stimulation of lung cells for cytokine analysis was also carried out. Mice exposed to aerosolized OVA as neonates were primed for a Th2 response with elevated levels of eosinophils, OVA specific IgE and Th2 cytokines (IL4, 5, 10, 13) but no detectable levels of IFN-gamma. The PBS controls that received aerosolized OVA at adulthood (8 weeks old) were unresponsive with respect to OVA specific IgE, Th2 cytokines and IFN-gamma. A single dose of anti-IL4 when given on day 0 to neonates concurrently with aerosolized OVA prevented the dysregulated allergic response to OVA at adulthood. Furthermore, these anti-IL4 treated neonatal mice, made a normal Th2 response when primed with an irrelevant antigen such as TNP-KLH while remaining unresponsive to aerosolized doses of OVA. Treatment with anti-IL4 at infancy may have therapeutic benefits against common allergens when administered in individuals particularly, during the allergy season.

Abstract 2539: Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors

Abstract Wilms' tumor gene 1 (WT1) is reported to be expressed in various types of malignancies and be one of promising target antigens for cancer immunotherapy. However, the synthetic short peptide vaccines based on the WT1 sequences limit a candidate patient by its HLA restriction. In this study, we performed the clinical study for dendritic cell (DC) vaccines using peptide pools consisting mainly of 15-mer sequences with 11 amino acids overlaps of full-length WT1 protein (PepTivator® WT1). We previously reported that copulsing tumor antigen-pulsed DC with zoledronate (Zol) could efficiently enhance expansion of tumor antigen specific CD8+ T cells by activation of gamma-delta T cells. In the present study, we studied the immunological response induced by PepTivator® WT1-pulsed Zol-DC vaccines in solid tumors, and also investigated the feasibility and safety of PepTivator® WT1-pulsed Zol-DC vaccines. Five patients with WT1-positive solid tumor (ovary: 4, lung: 1) were enrolled. PBMCs were collected by leukapheresis from the patients and the adherent cell fraction was differentiated into DCs by conventional method. In this vaccine therapy, PepTivator® WT1-pulsed Zol-DC (more than 5x106 cells per injection) was administered to patients subcutaneously for six times every two weeks. In order to examine the specific immune responses to WT1 antigen, PBMCs before therapy (pre-PBMC) and PBMCs after therapy (post-PBMC) were in vitro restimulated with PepTivator® WT1-pulsed Zol-DCs for 14 days and the expanded cells were separated into CD8+ cells and CD4+ cells by MACS, and the antigen specific IFN-gamma- producing cells were detected by ELISpot assay. As a result, no serious adverse events were seen in three patients who had completed all DC vaccines (Patient ID: #1, #2 and #5). DTH reaction was observed in all three patients. During in vitro restimulation and expansion, there was more than three-fold higher expansion of T cells responding to PepTivator® WT1-pulsed Zol-DC in post-PBMC compared to pre-PBMC among two of three patients (#1, #2). In these two patients, the antigen specific IFN-gamma-producing cells were detected in CD8+ T cells from post-PBMC, but not from pre-PBMC. As for CD4+ T cells, however, the antigen specific IFN-gamma-producing cells were not detected, neither in pre-PBMC, nor in post-PBMC. We found that PepTivator® WT1-pulsed Zol-DC vaccines are feasible and safe, and can induce antigen specific CD8+ T cells. We need more extensive investigation for the immunological monitoring and clinical outcome. Citation Format: Shoko Saiwaki, Shigenori Goto, Masashi Takahara, Haruka Matsushita, Takashige Kondo, Hermann Bohnenkamp, Ryuji Maekawa, Takashi Kamigaki. Clinical prospective study of PepTivator® WT1-pulsed DC vaccines with assessment of immunological responses in solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2014-2539

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Immunogenicity comparison of the intradermal or endobronchial boosting of BCG vaccinates with Ad5-85A

Experiments in small animal models have indicated that intranasal vaccination confers a greater degree of protection against TB than other routes such as intradermal (i.d.) or intramuscular. In this work, using a prime-boost vaccination strategy, we have compared in cattle vaccinated with BCG as a priming vaccine the boosting capabilities of Ad5-85A delivered either via the endobronchial (e.b.) or i.d. route. We show that Ad5-85A delivered through either route induced comparable peripheral blood antigen specific responses, and that both i.d. and e.b. routes induced bronchioalveolar lavage cells (BALC) that produced antigen-specific IFNgamma. We also show that, regardless of the route of boosting, the kinetics of peripheral blood and BALC responses, as assessed by antigen specific IFNgamma production, are different with systemic responses being detectable earlier than mucosal responses.These results contribute to our understanding on how different vaccination strategies may affect different compartments of the immune response and in turn to the development of safer and more effective vaccines.

432 A NOVEL TUMOR-ASSOCIATED ANTIGEN, DNAJB8 IS HIGHLY EXPRESSED IN CANCER STEM-LIKE CELLS/CANCER-INITIATING CELLS OF RENAL CELL CARCINOMA, AND IS POTENT TARGET FOR IMMUNOTHERAPY

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2012432 A NOVEL TUMOR-ASSOCIATED ANTIGEN, DNAJB8 IS HIGHLY EXPRESSED IN CANCER STEM-LIKE CELLS/CANCER-INITIATING CELLS OF RENAL CELL CARCINOMA, AND IS POTENT TARGET FOR IMMUNOTHERAPY Satoshi Nishizawa, Yoshihiko Hirohashi, Toshihiko Torigoe, Nagahide Matsumura, Takeshi Inagaki, Yasuo Kohjimoto, Noriyuki Sato, and Isao Hara Satoshi NishizawaSatoshi Nishizawa Wakayama, Japan More articles by this author , Yoshihiko HirohashiYoshihiko Hirohashi Sapporo, Japan More articles by this author , Toshihiko TorigoeToshihiko Torigoe Sapporo, Japan More articles by this author , Nagahide MatsumuraNagahide Matsumura Wakayama, Japan More articles by this author , Takeshi InagakiTakeshi Inagaki Wakayama, Japan More articles by this author , Yasuo KohjimotoYasuo Kohjimoto Wakayama, Japan More articles by this author , Noriyuki SatoNoriyuki Sato Sapporo, Japan More articles by this author , and Isao HaraIsao Hara Wakayama, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cancers are supposed to contain a small population of highly tumorigenic cells expressing stem cell-like markers, called Cancer stem-like cells/Cancer-initiating cells (CSCs/CICs). We found that tumor-associated antigen, DNAJB8 was highly expressed in CSCs/CICs. We evaluated usefulness of CSCs/CICs-targeted immunotherapy. METHODS CSCs/CICs in mouse renal cell cancer cell line (RenCa) were isolated as side population (SP) cells with Hoechst33342 staining. The expression of DNAJB8 was assessed by flowcytometric analysis in SP and main population (MP). The expression of DNAJB8 was enhanced by gene transduction. DNAJB8 immunization was done with subcutaneous inoculation of DNA plasmid vaccine including DNAJB8 cDNA. Cytotoxic T lymphocytes (CTL) induced by immunization was evaluated by IFN-gamma Elispot assay. In vivo anti-tumor effect of DNAJB8 vaccination was assessed by inoculation of RenCa into immunized Balb/c mice. RESULTS SP cells of RenCa exhibited stem cell-like features and higher tumorigenicity than MP cells, suggesting enriched with CSCs/CICs population. DNAJB8 was preferentially expressed in SP cells compared with MP cells. The enhanced expression of DNAJB8 by gene transduction into RenCa significantly increased the population of SP and greater tumorigenicity in vivo. DNA plasmid vaccine coding DNAJB8 CTL induced specific IFN-gamma secretion of CD8+ T cells. DNAJB8 immunization by plasmid DNA significantly inhibited the tumor growth comparing with other type of tumor antigen, Survivin, which was expressed in both SP and MP cells equivalently (fig). CONCLUSIONS DNAJB8 is highly expressed in CSCs/CICs of RenCa. DNAJB8 might be a powerful target for immunotherapy against CSCs/CICs. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e177 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Satoshi Nishizawa Wakayama, Japan More articles by this author Yoshihiko Hirohashi Sapporo, Japan More articles by this author Toshihiko Torigoe Sapporo, Japan More articles by this author Nagahide Matsumura Wakayama, Japan More articles by this author Takeshi Inagaki Wakayama, Japan More articles by this author Yasuo Kohjimoto Wakayama, Japan More articles by this author Noriyuki Sato Sapporo, Japan More articles by this author Isao Hara Wakayama, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Tuberculin Skin Test (TST) Is Affected by Recent BCG Vaccination but Not by Exposure to Non-Tuberculosis Mycobacteria (NTM) during Early Life

The tuberculin skin test (TST) is widely used in TB clinics to aid Mycobacterium tuberculosis (M.tb) diagnosis, but the definition and the significance of a positive test in very young children is still unclear. This study compared the TST in Gambian children at 4½ months of age who either received BCG vaccination at birth (Group 1) or were BCG naïve (Group 2) in order to examine the role of BCG vaccination and/or exposure to environmental mycobacteria in TST reactivity at this age. Nearly half of the BCG vaccinated children had a positive TST (≥5 mm) whereas all the BCG naïve children were non-reactive, confirming that recent BCG vaccination affects TST reactivity. The BCG naïve children demonstrated in vitro PPD responses in peripheral blood in the absence of TST reactivity, supporting exposure to and priming by environmental mycobacterial antigens. Group 2 were then vaccinated at 4½ months of age and a repeat TST was performed at 20–28 months of age. Positive reactivity (≥5 mm) was evident in 11.1% and 12.5% infants from Group 1 and Group 2 respectively suggesting that the timing of BCG vaccination had little effect by this age. We further assessed for immune correlates in peripheral blood at 4½ months of age. Mycobacterial specific IFNγ responses were greater in TST responders than in non-responders, although the size of induration did not correlate with IFNγ. However the IFNγ: IL-10 ratio positively correlated with TST induration suggesting that the relationship between PPD induced IFNγ and IL-10 in the peripheral blood may be important in controlling TST reactivity. Collectively these data provide further insights into how the TST is regulated in early life, and how a positive response might be interpreted.

Specific T-cell response induced in vivo by dendritic cells loaded with breast tumor cells antigen

Objective To explore the potential of autologous dendritic cells (DCs) loaded with breast tumor cells antigen in inducing tumor-specific T cells response in vivo. Methods Fresh tumor samples from patients with breast cancer were collected and made into apoptotic heat shock tumor cells. Peripheral blood mononuclear cells (PBMCs) were isolated. DCs were induced from monocytes with granulocytemacrophage colony-stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) combination for 5 days. DCs were co-cultured with autologous apoptotic heat shock tumor cells for 48 h. Twenty-four patients with breast cancers received four vaccinations of auto-DCs at 1 st, 2nd, 4th, and 6th week with cell number from 4×106 to 6×106. Results The vaccinations were well tolerated, and most patients experienced improved quality of life after DC immunization. Cytokine levels including IL-2, IL-12, TNF-α and IFN-γ in sera were obviously increased after DC vaccination [(68. 5±12. 4), (118. 2±31. 5), (78. 3±11.5) and (92. 6±14. 9) ng/L respectively] as compared with those before vaccinations [(33. 8±7. 2), (48. 5±10. 9), (18. 7±5. 3 ) and (20. 5±6. 3) ng/L respectively]. Both antigen specific delayed type hypersensitivity (DTH) reaction and the frequency of antigen specific IFN-gamma+CD8+T lymphocytes were examined after 4 times of DC vaccinations. The DTH tests were positive in 7 out of 10 patients. The frequency of IFN-γ+CD8+T cells was enhanced in 4 out of 10 patients. One out of 24 patients experienced a progressive disease, while the others showed no progress during a follow-up period of 34 months. Conclusion Autologous DCs loaded with heat-shocked apoptotic breast cancer cells antigen elicit specific anti-tumor T cell immune responses companying with high levels of Thl type cytokine secretion. DCs vaccination after surgery may be a novel and effective combination tool for tumor treatment. Key words: Breast carcinoma; Dendritic cells; Immunotherapy

Impaired Innate Immunity in Tlr4−/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/ − or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.

Generation of IFN-γ-producing cells that recognize the major piroplasm surface protein in Theileria orientalis-infected bovines

Theileriosis is a tick-borne protozoan disease caused by Theileria species. The Theileria species are classified into two groups depending on the cell type in which they proliferate and the clinical symptoms. The first group consists of lymphoproliferative Theileria species (T. parva and T. annulata), which mainly proliferate in lymphocytes, causing uncontrolled lymphocyte proliferation. The other group consists of a nonlymphoproliferative Theileria species (T. orientalis, also known as T. sergenti) that proliferates in erythrocytes and causes hemolytic anemia. Based on reports of generation of antigen-specific CD4(+) and CD8(+) T cells in lymphoproliferative theileriosis, we investigated whether T cells specific to the T. orientalis antigen are present in the nonlymphoproliferative form of the disease. In this study, we developed a new assay based on an enzyme-linked immunospot (ELISpot) to detect interferon-gamma (IFN-gamma)- and interleukin-10 (IL-10)-secreting cells in a series of cryogenically preserved bovine peripheral blood mononuclear cells (PBMCs). We first determined that IFN-gamma- and IL-10-secreting T cells were present in PBMCs by stimulating them with phytohemagglutinin L (PHA-L=red kidney bean lectin L, known as T cell stimulator), and then determined whether T. orientalis-specific T cells are present in T. orientalis-infected bovines. Peptides derived from T. orientalis major piroplasm surface protein (MPSP) were used as a T. orientalis-specific stimulator in the ELISpot assay, and peptides from glycoprotein B (gB) of the bovine herpes virus-1 (BHV-1) were used as a BHV-1-specific stimulator as a control for monitoring the immune response. Compared with results obtained using the BHV-1 (gB peptides)-specific IFN-gamma ELISpot assay to assess BHV-1-immunized Holsteins, prominent T. orientalis MPSP peptide-specific IFN-gamma and IL-10 positive spots were detected in T. orientalis-infected Holsteins but weak positive responses were exhibited by T. orientalis-infected Angus and Japanese Black cattle. As far as we are aware, this is the first report to show direct evidence of the presence of T. orientalis-specific T cells in T. orientalis-infected bovines using an antigen-specific ELISpot assay system and that T. orientalis-specific, IFN-gamma- and IL-10-producing T cells are produced in T. orientalis-infected Holsteins.

T cells co-producing Mycobacterium tuberculosis-specific type 1 cytokines for the diagnosis of latent tuberculosis

Patients treated with tumor necrosis factor (TNF)-alpha-antagonizing medication are at increased risk of developing active tuberculosis (TB), brought about mainly by reactivation of latent infection. Thus, screening for latent TB infection (LTBI) prior to administration of anti-TNF-alpha-therapy is required. For a long time, the tuberculin skin test (TST) was the only means of diagnosing LTBI, however, interferon-gamma-release assays (IGRAs), are promising new tools. Fifty two patients with dermatological disorders were included prior to implementation of anti-TNF-alpha therapy. Mycobacterium tuberculosis (MTB)-specific cytokine production, including interferon (IFN)-gamma, TNF-alpha, interleukin (IL)-2 and IL-10, was measured in CD4+ and CD8+ T cells by cytokine flow cytometry following stimulation of peripheral blood mononuclear cells (PBMC) with purified protein derivative (PPD) and early secretion antigenic target (ESAT)-6. Simultaneously, a TST was administered and 11 were TST-positive. Generally, MTB-specific IFN-gamma produced by CD4+ T cells correlated well with TST results. CD4+ T cells co-producing specific IFN-gamma and TNF-alpha after ESAT-6 stimulation showed the highest overall agreement with the TST (Kappa [kappa] = 0.87). Each single cytokine displayed individual patterns, the expression of IFN-gamma, however, showed the highest concordance with the TST (kappa = 0.82). This suggests that the enumeration of MTB-specific CD4+ T cells might introduce greater specificity for the diagnosis of latent TB, compared to the TST.

Similar cellular responses after treatment with either praziquantel or oxamniquine in Schistosoma mansoni infection.

The effect of treatment with either oxamniquine or praziquantel on S.mansoni specific IFN-gamma, IL-4, IL-5 and IL-10 was compared on PBMC which were collected pretreatment, 6 and 18 weeks post treatment. Using sandwich ELISA on the supernatants harvested from the PBMC stimulation by crude S. mansoni SEA and SWAP antigens after 5 days the levels of PBMC proliferation and cytokine production were similar according to treatment with either praziquantel or oxamniquine. Before treatment, infected groups showed low ratios, of IL-4:IFN-gamma, IL-5:IFNgamma and IL-10:IFN-gamma, indicating that IFN-gamma was high in the infected individuals. The general increase in immuno-modulation was observed post-treatment with elevated immune reactivity and cytokine production in both treatment groups. Treatment induced significant increases in levels of IL-4 (p < 0.05), IL-5 (p < 0.0001) and IL-10 (p < 0.05) cytokines 6 and 18 weeks after treatment. There were no significant differences in the increase in IL-4, IL-5 and IL-10 between children treated with praziquantel or oxamniquine. Pre-treatment IFN-gamma and IL-5 levels were positively correlated with infection (p < 0.001), while post treatment IL-4 cytokine levels were negatively correlated with baseline infection status (p < 0.001). The results suggest that treatment-induced immune responses are similar for both common anti-schistosome drugs praziquantel or oxamniquine having similar and immunizing effect.

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Abstract 4079Poster Board III-1014MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Parental characteristics, somatic fetal growth, and season of birth influence innate and adaptive cord blood cytokine responses

Immunologic responses at birth likely relate to subsequent risks for allergic diseases and wheezing in infancy; however, the influences of parental characteristics and prenatal factors on neonatal immune responses are incompletely understood. This study investigates potential correlations between urban parental, prenatal, and perinatal factors on innate and adaptive stimuli-induced cytokine responses. Five hundred sixty and 49 children of parents with and without allergic disease or asthma, respectively, were enrolled into a prospective birth cohort study (Urban Environment and Childhood Asthma). Cord blood mononuclear cells were incubated with innate and adaptive immune stimuli, and cytokine responses (ELISA) were compared with season of birth, parental characteristics, in utero stressors, and fetal growth. Many cytokine responses varied by season of birth, including 2-fold to 3-fold fluctuations with specific IFN-alpha and IFN-gamma responses. Birth weight was inversely associated with IFN-gamma responses to respiratory syncytial virus (R = -0.16), but positively associated with IL-8 responses to a variety of innate stimuli (R = 0.08-0.12). Respiratory syncytial virus-induced cytokine responses were 21% to 54% lower in children of mothers with asthma. Cytokine responses were generally lower in babies born to parents with allergy/asthma. Innate cytokine responses are associated with parental allergic or airway disease, somatic fetal growth, ethnicity, and season of birth. Collectively, these findings suggest that urban prenatal exposures and familial factors affect the development of the fetal immune system.

The Synthetic Plasmodium falciparum Circumsporozoite Peptide PfCS102 as a Malaria Vaccine Candidate: A Randomized Controlled Phase I Trial

BackgroundFully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults.Methodology and Principal FindingsThirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 µg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-γ production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-γ secreting CD8+ T cell responses. Responses were only marginally boosted after the 3rd vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 µg was less immunogenic in comparison to 30 and 100 µg that induced similar responses. AS02A formulations with 30 µg or 100 µg PfCS102 induced about 10-folds higher antibody and IFN-γ responses than Montanide formulations.Conclusions/SignificancePfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 µg formulated with AS02A appeared the most appropriate choice for such studies.Trial RegistrationSwissmedic.ch 2002 DR 1227

Interferon-gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown.Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed.Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals.Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection.

MVA.85A Boosting of BCG and an Attenuated, phoP Deficient M. tuberculosis Vaccine Both Show Protective Efficacy Against Tuberculosis in Rhesus Macaques

BackgroundContinuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection.Methods and FindingsHere, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNγ responses. Antigen 85A-specific IFNγ secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNγ levels post-infection as the strongest immunocorrelate for protection (spearman's rho: −0.60).ConclusionsBoth the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.

In Vivo and In Vitro Effects of Antituberculosis Treatment on Mycobacterial Interferon-γ T Cell Response

BackgroundIn recent years, the impact of antituberculous treatment on interferon (IFN)-γ response to Mycobacterium tuberculosis antigens has been widely investigated, but the results have been controversial. The objective of the present study was: i) to evaluate longitudinal changes of IFN-γ response to M. tuberculosis-specific antigens in TB patients during antituberculous treatment by using the QuantiFERON-TB Gold (QFT-G) assay; ii) to compare the differences in T-cell response after a short or prolonged period of stimulation with mycobacterial antigens; iii) to assess the CD4+ and CD8+ T cells with effector/memory and central/memory phenotype; iv) to investigate the direct in vitro effects of antituberculous drugs on the secretion of IFN-γ.Principal Findings38 TB patients was evaluated at baseline and at month 2 and 4 of treatment and at month 6 (treatment completion). 27 (71%) patients had a QFT-G reversion (positive to negative) at the end of therapy, while 11 (29%) TB patients remained QFT-G positive at the end of therapy. Among the 11 patients with persistent positive QFT-G results, six had a complete response to the treatment, while the remaining 5 patients did not have a resolution of the disease. All 27 patients who became QFT-G negative had a complete clinical and microbiological recovery of the TB disease. In these patients the release of IFN-γ is absent even after a prolonged 6-day incubation with both ESAT-6 and CFP-10 antigens and the percentage of effector/memory T-cells phenotype was markedly lower than subjects with persistent positive QFT-G results. The in vitro study showed that antituberculous drugs did not exert any inhibitory effect on IFN-γ production within the range of therapeutically achievable concentrations.ConclusionsThe present study suggests that the decrease in the M. tuberculosis-specific T cells responses following successful anti-TB therapy may have a clinical value as a supplemental tool for the monitoring of the efficacy of pharmacologic intervention for active TB. In addition, the antituberculous drugs do not have any direct down-regulatory effect on the specific IFN-γ response.

Screening of tuberculosis by interferon-γ assay before biologic therapy for rheumatoid arthritis

Infection with Mycobacterium tuberculosis (M. tuberculosis) is a critical complication in anti-TNF therapies. In 141 BCG vaccinated healthy individuals and 71 rheumatoid arthritis (RA) patients as screening before anti-TNF therapies, M. tuberculosis specific immune responses were evaluated by tuberculin skin test (TST) and enzyme-linked immunospot assay (ELISPOT), which detected antigen specific IFN-gamma secreting cells in peripheral blood mononuclear cells simulated with either purified protein derivative (PPD), early secretory antigen target 6 (ESAT-6) or culture filtrate protein 10 (CFP-10). Induration over 5 mm in TST was found in 87.9% of controls and 21.4% of RA patients. Erythema size in TST was significantly suppressed in RA patients, especially those receiving prednisolone (PSL), whereas the PPD specific IFN-gamma secretion was less attenuated. Significant responses to either ESAT-6 or CFP-10 in ELISPOT were detected in 14.1% of RA patients including those having positive TST, while the ELISPOT assay was negative in all healthy individuals and 73.3% of RA patients having positive TST. Of ELISPOT positive RA patients, mean dosage of PSL was 4.58 mg and 1.25 mg in TST negative and positive patients, respectively. Thus, ELISPOT is useful for screening of tuberculosis in RA patients, even in those receiving corticosteroids.