Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receiving antiretroviral therapy.

Immunogenicity, manufacturing feasibility, and safety of a novel, autologous dendritic cell (DC)-based immunotherapy (AGS-004) was evaluated in ten human immunodeficiency virus type 1 (HIV-1)-infected adults successfully treated with antiretroviral therapy (ART). Personalized AGS-004 was produced from autologous monocyte-derived DCs electroporated with RNA encoding CD40L and HIV antigens (Gag, Vpr, Rev, and Nef) derived from each subjects' pre-ART plasma. Patients received monthly injections of AGS-004 in combination with ART. AGS-004 was produced within a mean of 6 weeks and yielded 4-12 doses/subject Full or partial HIV-specific proliferative immune responses occurred in 7 of 9 evaluable subjects. Responses were specific for the AGS-004 presented HIV antigens and preferentially targeted CD8(+) T cells. Mild adverse events included flu-like symptoms, fatigue, and injection site reactions. No evidence of autoimmunity, changes in viral load, or significant changes in absolute CD4(+) and CD8(+) T cell counts were observed. This pilot study supports the further clinical investigation of AGS-004.

Optimization of a highly standardized carboxyfluorescein succinimidyl ester flow cytometry panel and gating strategy design using discriminative information measure evaluation.

The design of a panel to identify target cell subsets in flow cytometry can be difficult when specific markers unique to each cell subset do not exist, and a combination of parameters must be used to identify target cells of interest and exclude irrelevant events. Thus, the ability to objectively measure the contribution of a parameter or group of parameters toward target cell identification independent of any gating strategy could be very helpful for both panel design and gating strategy design. In this article, we propose a discriminative information measure evaluation (DIME) based on statistical mixture modeling; DIME is a numerical measure of the contribution of different parameters towards discriminating a target cell subset from all the others derived from the fitted posterior distribution of a Gaussian mixture model. Informally, DIME measures the "usefulness" of each parameter for identifying a target cell subset. We show how DIME provides an objective basis for inclusion or exclusion of specific parameters in a panel, and how ranked sets of such parameters can be used to optimize gating strategies. An illustrative example of the application of DIME to streamline the gating strategy for a highly standardized carboxyfluorescein succinimidyl ester (CFSE) assay is described.

Tn-MUC1 DC Vaccination of Rhesus Macaques and a Phase I/II Trial in Patients with Nonmetastatic Castrate-Resistant Prostate Cancer.

MUC1 is a glycoprotein expressed on the apical surface of ductal epithelial cells. Malignant transformation results in loss of polarization and overexpression of hypoglycosylated MUC1 carrying truncated carbohydrates known as T or Tn tumor antigens. Tumor MUC1 bearing Tn carbohydrates (Tn-MUC1) represent a potential target for immunotherapy. We evaluated the Tn-MUC1 glycopeptide in a human phase I/II clinical trial for safety that followed a preclinical study of different glycosylation forms of MUC1 in rhesus macaques, whose MUC1 is highly homologous to human MUC1. Either unglycosylated rhesus macaque MUC1 peptide (rmMUC1) or Tn-rmMUC1 glycopeptide was mixed with an adjuvant or loaded on autologous dendritic cells (DC), and responses were compared. Unglycosylated rmMUC1 peptide induced negligible humoral or cellular responses compared with the Tn-rmMUC1 glycopeptide. Tn-rmMUC1 loaded on DCs induced the highest anti-rmMUC1 T-cell responses and no clinical toxicity. In the phase I/II clinical study, 17 patients with nonmetastatic castrate-resistant prostate cancer (nmCRPC) were tested with a Tn-MUC1 glycopeptide-DC vaccine. Patients were treated with multiple intradermal and intranodal doses of autologous DCs, which were loaded with the Tn-MUC1 glycopeptide (and KLH as a positive control for immune reactivity). PSA doubling time (PSADT) improved significantly in 11 of 16 evaluable patients (P = 0.037). Immune response analyses detected significant Tn-MUC1-specific CD4+ and/or CD8+ T-cell intracellular cytokine responses in 5 out of 7 patients evaluated. In conclusion, vaccination with Tn-MUC1-loaded DCs in nmCRPC patients appears to be safe, able to induce significant T-cell responses, and have biological activity as measured by the increase in PSADT following vaccination. Cancer Immunol Res; 4(10); 881-92. ©2016 AACR.

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.