Indoor Biotechnologies
  • Charlottesville, United States
Recent publications
Antibodies are widely used in medicinal and scientific research due to their ability to bind to a specific antigen. Most often, antibodies are composed of heavy and light chain domains. Under physiological conditions, light chains are produced in excess, as compared to the heavy chain. It is now known that light chains are not silent partners of the heavy chain and can modulate the immune response independently. In this work, the first crystal structure of a light chain dimer originating from mice is described. It represents the light chain dimer of 6A8, a monoclonal antibody specific to the allergen Der f 1. Building on the unexpected occurrence of this kind of dimer, we have demonstrated that this light chain is stable in solution alone. Moreover, enzyme-linked immunosorbent assays (ELISA) have revealed that, when the light chain is not partnered to its corresponding heavy chain, it interacts non-specifically with a wide range of proteins. Computational studies were used to provide insight on the role of the 6A8 heavy chain domain in the specific binding to Der f 1. Overall, this work demonstrates and supports the ongoing notion that light chains can function by themselves and are not silent partners of heavy chains.
Background: T cell responses to natural SARS-CoV-2 infection may be more robust and longer lived than antibody responses, thus preventing re-infection. Accurate assessment of vaccine-induced T cell responses is critical for understanding the magnitude and longevity of vaccine-induced immunity across patient cohorts. Aims: To establish a simple, accurate and rapid whole blood test to determine natural and vaccine-induced SARS-CoV-2 immunity via a cytokine release assay. Methods: Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with PCR-confirmed previous infection (n=29), suspected infection (n=30) or with no history of exposure (n=69); and in donors pre- and post-vaccination (n=32). Cytokines were measured by enzyme immunoassay and multiplex array. Results: Cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) were highly elevated in PCR-confirmed or suspected SARS-CoV-2 infected donors at 20->2000pg/ml and 20-1000pg/ml, respectively, compared to history negative controls (<20-90pg/ml). Receiver operating curves showed IL-2 as the superior biomarker with AUC of 0.99 compared to IFN-γ (0.94). Following vaccination, 100% of PCR-confirmed donors and 94% of unexposed individuals demonstrated a positive IL-2 response. Mean IL-2 levels increased ~18-fold from 12pg/ml pre-vaccination to 202pg/ml and 216pg/ml after the 1 and 2 vaccine doses, respectively. No other cytokines were suitable biomarkers for distinguishing SARS-CoV-2 infection or vaccination responses. Conclusion: This rapid, whole blood-based T cell test can be utilised to make accurate and comparable assessments of vaccine-induced T cell immunity across multiple population cohorts, and aid decision making on public health policies and vaccine efficacy.
Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30–37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3.
Outbreak caused by coronavirus disease 2019 (COVID-19) started in Wuhan, Hubei Province, China and quickly spread around the world. Current evidence is contradictory on the association of asthma with COVID-19 and associated severe outcomes. Type 2 inflammation may reduce the risk for severe COVID-19. Whether asthma diagnosis may be a risk factor for severe COVID-19 especially for those with severe disease or non-allergic phenotypes deserve further attention and clarification. In addition, COVID-19 does not appear to provoke asthma exacerbations and asthma therapeutics should be continued for patients with exposure to COVID-19. Changes in the intensity of pollinization, earlier start and extension of pollinating season, and increase in production and allergenicity of pollen are known direct effects that air pollution has on physical, chemical, and biological properties of the pollen grains. They are influenced and triggered by meteorological variables that could partially explain the effect on COVID-19. SARS-CoV-2 is capable to persist in the environment and can be transported by bioaerosols which can further influence the transmission rate and seasonality of coronavirus. The Covid-19 pandemic has changed the behavior of adults and children globally. A general trend during the pandemic has been human isolation indoors due to school lockdowns, loss of job or implementation of virtual work at home. A consequence of this behavior change would presumably be changes in indoor allergen exposures and reduction of inhaled outdoor allergens. Therefore, lockdowns during the pandemic might have improved some specific allergies, while worsening others, depending on the housing conditions.
Introduction (contexte de la recherche) Les produits non standardisés parfois utilisés en clinique peuvent nuire à l’innocuité et l’efficacité de l’immunothérapie. Identité, qualité et pureté des médicaments sont des facteurs clés d’innocuité, d’efficacité et d’acceptabilité. Une non-standardisation peut entraîner l’inefficacité par manque de pureté, faible puissance et toxicité par une puissance inconstante et la présence de contaminants. La poudre de graine d’Arachis hypogaea L. (arachide) allégée en graisses (PDAH) est une immunothérapie orale approuvée par les agences d’évaluation américaine et européenne, pour réduire les réactions allergiques, dont l’anaphylaxie, lors d’exposition accidentelle à l’arachide chez les enfants de 4 à 17 ans. Objectif Évaluation du matériel source de farine d’arachide (partiellement dégraissée −12 % de matières grasses, farine d’arachide légèrement grillée de la société Golden Peanut and Tree Nuts). Méthodes La puissance relative des allergènes immunodominants a été évaluée par une norme de référence qualifiée par ELISA ; les aflatoxines ont été quantifiées par chromatographie liquide à ultra-haute performance (UHPLC). D’autres caractéristiques ont aussi été évaluées. La proportion de lots rejetés lors du test du principe actif entre 2018 et 2021 est rapportée. Résultats La proportion de lots rejetés a été de 57 % (n/N = 8/14) en 2018, 37 % (n/N = 3/8) en 2019, 57 % (n/N = 4/7) en 2020 et 60 % (n/N = 3/5) en 2021. De plus, les lots rejetés différaient de la norme de plusieurs ordres de grandeur. Les raisons du rejet ont été la qualité (les critères d’acceptation de la puissance pour la sélection n’ont pas été satisfaits pour ≥ 1 composant allergénique) ou l’innocuité (niveau d’aflatoxines proche du seuil d’acceptabilité). Conclusions La majorité de la farine d’arachide provenant d’une source unique n’a pas satisfait aux critères du test du principe actif et de développement du produit PTAH. Assurer la standardisation entre lots, l’identité, la qualité et la pureté du médicament lors des phases de développement du produit, est une condition clé de l’optimisation de l’innocuité et de l’efficacité de l’immunothérapie.
Quantitative risk assessment (QRA) for allergens exists in many different forms with different requirements placed on the risk assessor depending on the question that needs to be answered. An electronic workshop held in October 2020 and comprising representatives from a wide range of food allergy and allergen stakeholder groups identified that a summary of current best in class guidance, identified gaps, potential improvements & harmonization of allergen QRA arising largely from cross contact would be very beneficial. The current manuscript provides an introduction to allergen QRA and an overview of inputs potentially needed for different QRA methods, when deemed feasible and necessary. It also introduces the European branch of the International Life Sciences Institute (ILSI Europe) Expert Group (EG), created to attempt to achieve consensus on the methodologies needed for allergen QRAs by food business operators, and their implementation. Areas of focus include proactive assessments for food production under normal conditions, both in the upstream supply chain and in food production facilities, and reactive assessments as part of an allergen incident response. As a follow-up report to the October 2020 electronic workshop, the current manuscript provides an overview of allergen QRA and insights into the guidance being developed. This manuscript will itself be followed by more detailed guidance for allergen QRA published open access as an ILSI Europe report.
The study aimed to document the method standardization and assessment of micronutrient and inflammatory markers in women from indigenous tribal communities of Jharkhand using a low volume, high throughput assay. This cross-sectional study was done among women of the reproductive age group from Sauria Paharia and Santhal tribal households in selected villages. Capillary blood samples were collected from the women during a household survey to estimate ferritin, soluble Transferrin Receptor(sTfR), Retinol Binding Protein (RBP4), and inflammatory biomarkers, C-Reactive Protein (CRP) and α-1 Acid Glycoprotein (AGP) using a multiplex assay. Vitamin D and hemoglobin were estimated using an LC-MS technique and cyanmethemoglobin method respectively. A multiplex Luminex based method was developed and standardized. The assay was used to estimate biomarkers in samples from 413 women (178 and 235 from Sauria Paharia and Santhal tribes, respectively). Over 51% of women had raised CRP or AGP levels. Iron status was significantly better in Sauria Paharia compared to Santhal women. Anemia prevalence was 72% among Santhal women. The proportion of women with iron deficiency increased after adjusting for inflammation. The overall prevalence of Vitamin A deficiency and insufficiency was 25% and 34%, respectively with similar prevalence in both tribes. All Santhal women had sufficient Vitamin D levels, while 25% and 20% of Sauria Paharia women had insufficient and deficient Vitamin D levels, respectively. Our low volume, high throughput multiplex assays may provide a feasible approach for assessing nutritional biomarkers in nutritionally vulnerable hard-to reach communities.
Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) technology offers the unique potential for unequivocally deleting allergen genes at the source. Compared to prior gene editing approaches, CRISPR boasts substantial improvements in editing efficiency, throughput, and precision. CRISPR has demonstrated success in several clinical applications such as sickle cell disease and β-thalassemia, and preliminary knockout studies of allergenic proteins using CRISPR editing show promise. Given the advantages of CRISPR, as well as specific DNA targets in the allergen genes, CRISPR gene editing is a viable approach for tackling allergy, which may lead to significant disease improvement. This review will highlight recent applications of CRISPR editing of allergens, particularly cat allergen Fel d 1, and will discuss the advantages and limitations of this approach compared to existing treatment options.
Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.
We developed a SARS-CoV-2 spike subunit vaccine formulation containing dual TLR ligand liposome adjuvant. The vaccine-induced robust systemic neutralizing antibodies and completely protected mice from a lethal challenge. Two immunizations protected against lung injury and cleared the virus from lungs upon challenge. The adjuvanted vaccine also elicited systemic and local anti-Spike IgA which can be an important feature for a COVID-19 vaccine.
Background Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen-specific T cell reactivity in a cohort of CR allergic children with asthma. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild-to-moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. Results Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL-4 production and a Th2 polarized profile in 54.9% of participants, but IFNγ production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. Conclusions Our results demonstrate that in children with mild-to-moderate asthma, CR-specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes.
Background The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5‐specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods A Phl p 5‐specific ELISA system was assessed with respect to accuracy, precision, inter‐assay (within laboratory) and inter‐laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter‐assay variation (max. GCV 24%) and especially inter‐laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions Based on the collaborative study results, the assessed Phl p 5‐specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.
Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub‐Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE‐binding glycans recognized up to 2008 were considered to be “classical” cross‐reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE‐binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose‐alpha‐1,3‐galactose (alpha‐gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha‐gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross‐reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub‐Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.
Background: Little is known about environmental food allergen exposure on school surfaces. Objective: Compare the distribution of major food allergens in floor dust and table wipe samples from elementary schools, and dust samples from students' homes. Methods: In this sub-study of the School Inner-City Asthma Study-II, 103 table wipe samples and 98 floor dust samples from cafeterias and classrooms in 18 elementary schools were analyzed for milk, peanut, cashew, hazelnut and egg by multiplex array. Home kitchen floor and bed dust samples from 90 students were also analyzed. Results: Food allergens were detectable in schools, but at significantly lower levels than in homes (p<0.001). In schools, milk and peanut were detected in all table wipe samples; milk and egg were detected in all floor dust samples. Cafeteria table wipe samples contained significantly higher levels of milk, peanut, hazelnut, and egg, compared with classrooms. Cafeteria floor dust samples contained higher levels milk than classrooms. Peanut-restrictive policies did not consistently reduce environmental peanut exposure in schools. Peanut allergen was lower in dust from homes of students with peanut allergy (n=5) compared to without (n=85) (p<0.001). Reassuringly, peanut allergen in the schools of peanut-allergic students was not significantly different than in their homes. Conclusion: Food allergens were readily detectable on tables and floors in elementary schools, but at levels lower than in students' homes. For peanut-allergic students, the levels of detectable peanut in their schools were not higher than their homes. The low levels of detectable food allergens in school environments are unlikely to result in severe allergic reactions.
Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small‐molecule ligands. Ligand‐binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds. Pollen and plant food allergens of the family 10 of pathogenesis‐related proteins bind a variety of small molecules such as glycosylated flavonoid derivatives, flavonoids, cytokinins, and steroids in vitro. However, their natural ligand binding was reported to be highly specific. Insect and mammalian lipocalins transport odorants, pheromones, catecholamines, and fatty acids with a similar level of specificity, while the food allergen β‐lactoglobulin from cow's milk is notably more promiscuous. Non‐specific lipid transfer proteins from pollen and plant foods bind a wide variety of lipids, from phospholipids to fatty acids, as well as sterols and prostaglandin B2, aided by the high plasticity and flexibility displayed by their lipid‐binding cavities. Ligands increase the stability of allergens to thermal and/or proteolytic degradation. They can also act as immunomodulatory agents that favor a Th2 polarization. In summary, ligand‐binding allergens expose the immune system to a variety of biologically active compounds whose impact on the sensitization process has not been well studied thus far.
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15 members
Lisa D Vailes
  • Purified Allergens
Stephanie Filep
  • Immunoassay Group
Sayeh Agah
  • Allergen Manufacturing, R & D
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Charlottesville, United States
Head of institution
Martin D. Chapman