Sources of variability in Luminex bead-based cytokine assays: Evidence from twelve years of multi-site proficiency testing.

Bead array assays, such as those sold by Luminex, BD Biosciences, Sartorius, Abcam and other companies, are a well-established platform for multiplexed quantification of cytokines and other biomarkers in both clinical and discovery research environments. In 2011, the National Institute of Allergy and Infectious Diseases (NIAID)-funded External Quality Assurance Program Oversight Laboratory (EQAPOL) established a proficiency assessment program to monitor participating laboratories performing multiplex cytokine measurements using Luminex bead array technology. During every assessment cycle, each site was sent an assay kit, a protocol, and blinded samples of human sera spiked with recombinant cytokines. Site results were then evaluated for performance relative to peer laboratories. After over a decade of biannual assessments, the cumulative dataset contained over 15,500 bead array observations collected at more than forty laboratories in twelve countries. These data were evaluated alongside post-assessment survey results to empirically test factors that may contribute to variability and accuracy in Luminex bead-based cytokine assays. Bead material, individual technical ability, analyte, analyte concentration, and assay kit vendor were identified as significant contributors to assay performance. In contrast, the bead reader instrument model and the use of automated plate washers were found not to contribute to variability or accuracy, and sample results were found to be highly-consistent between assay kit-manufacturing lots and over time. In addition to these statistical analyses, subjective evaluations identified technical ability, instrument failure, protocol adherence, and data transcription errors as the most common causes of poor performance in the proficiency program. The findings from the EQAPOL multiplex program were then used to develop recommended best practices for bead array monitoring of human cytokines. These included collecting samples to assay as a single batch, centralizing analysis, participating in a quality assurance program, and testing samples using paramagnetic-bead kits from a single manufacturer using a standardized protocol.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA "breathing" on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening.

Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. Many currently known MC activators are inadequate for in vivo applications, however, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by using high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistically evaluated using quality control rules and the B-score calculation, and compounds with B-scores of >3.0 were chosen as mast cell activators (hits). These hits were re-evaluated with secondary and tertiary HTS assays, followed by further statistical analysis. From these hits, we selected 15 compounds that caused degranulation in murine and human MCs, with potential for flexible chemical modification for further study. Among these 15 compounds, ST101036, ST029248, and ST026567 exhibited higher degranulation potency than other hit compounds in both human and mouse MCs. In addition, the 15 compounds identified promote de novo synthesis of cytokines and induce the release of eicosanoids from human and mouse MCs. HTS enabled us to identify small-molecule MC activators with unique properties that may be useful as vaccine adjuvants.

Impact of early life exposure to ionizing radiation on influenza vaccine response in an elderly Japanese cohort.

The objective of this study was to evaluate effects of whole body radiation exposure early in life on influenza vaccination immune responses much later in life. A total of 292 volunteers recruited from the cohort members of ongoing Adult Health Study (AHS) of Japanese atomic bomb (A-bomb) survivors completed this observational study spanning two influenza seasons (2011-2012 and 2012-2013). Peripheral blood samples were collected prior to and three weeks after vaccination. Serum hemagglutination inhibition (HAI) antibody titers were measured as well as concentrations of 25 cytokines and chemokines in culture supernatant from peripheral blood mononuclear cells, with and without in vitro stimulation with influenza vaccine. We found that influenza vaccination modestly enhanced serum HAI titers in this unique cohort of elderly subjects, with seroprotection ranging from 18 to 48% for specific antigen/season combinations. Twelve percent of subjects were seroprotected against all three vaccine antigens post-vaccination. Males were generally more likely to be seroprotected for one or more antigens post-vaccination, with no differences in vaccine responses based on age at vaccination or radiation exposure in early life. These results show that early life exposure to ionizing radiation does not prevent responses of elderly A-bomb survivors to seasonal influenza vaccine.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Development and implementation of a proficiency testing program for Luminex bead-based cytokine assays.

Luminex bead array assays are widely used for rapid biomarker quantification due to the ability to measure up to 100 unique analytes in a single well of a 96-well plate. There has been, however, no comprehensive analysis of variables impacting assay performance, nor development of a standardized proficiency testing program for laboratories performing these assays. To meet this need, the NIH/NIAID and the Cancer Immunotherapy Consortium of the Cancer Research Institute collaborated to develop and implement a Luminex assay proficiency testing program as part of the NIH/NIAID-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke University. The program currently monitors 25 domestic and international sites with two external proficiency panels per year. Each panel includes a de-identified commercial Luminex assay kit with standards to quantify human IFNγ, TNFα, IL-6, IL-10 and IL-2, and a series of recombinant cytokine-spiked human serum samples. All aspects of panel development, testing and shipping are performed under GCLP by EQAPOL support teams. Following development testing, a comprehensive site proficiency scoring system comprised of timeliness, protocol adherence, accuracy and precision was implemented. The overall mean proficiency score across three rounds of testing has remained stable (EP3: 76%, EP4: 75%, EP5: 77%); however, a more detailed analysis of site reported results indicates a significant improvement of intra- (within) and inter- (between) site variation, suggesting that training and remediation for poor performing sites may be having a positive impact on proficiency. Through continued proficiency testing, identification of variables affecting Luminex assay outcomes will strengthen efforts to bring standardization to the field.

Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen.

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

Molecular measurement of T cell receptor excision circles.

This chapter provides protocols necessary for quantifying human, mouse, and nonhuman primate signal joint T cell receptor excision circles (sjTRECs) produced during T cell receptor alpha (TCRA) gene rearrangement. These non-replicated episomal circles of DNA are generated by the recombination process used to produce antigen-specific T cell receptors. The number of sjTRECs per mg of thymus tissue or per 100,000 lysed cells has been shown to be a molecular marker of thymopoiesis and naïve T cells. This technology is beneficial to investigators interested in quantitating the level of naïve T cell production occurring under various circumstances in a variety of systems, and complements traditional phenotypic analyses of thymopoiesis. This chapter specifically describes procedures required for rapid detection and quantitation of sjTRECs in thymus tissue or isolated cells using real-time quantitative polymerase chain reaction (PCR). The sjTREC assay system comprises species-specific forward and reverse primers for amplification of a unique site on the T cell receptor δ (TCRD) sjTREC, a fluorescently labeled (FAM/ZEN/IABkFQ) species-specific real-time probe, and a species-specific sjTREC DNA plasmid standard for quantitation.

Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication.

Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IkappaBalpha required to activate NF-kappaB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IkappaBalpha depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IkappaBalpha depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-kappaB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.

Thymic involution and immune reconstitution.

Chronic thymus involution associated with aging results in less efficient T-cell development and decreased emigration of naïve T cells to the periphery. Thymic decline in the aged is linked to increased morbidity and mortality in a wide range of clinical settings. Negative consequences of these effects on global health make it of paramount importance to understand the mechanisms driving thymic involution and homeostatic processes across the lifespan. There is growing evidence that thymus tissue is plastic and that the involution process might be therapeutically halted or reversed. We present here progress on the exploitation of thymosuppressive and thymostimulatory pathways using factors such as keratinocyte growth factor, interleukin 7 or sex steroid ablation for therapeutic thymus restoration and peripheral immune reconstitution in adults.