Biology subjects

Thomas, K. N.

Publications and source records attributed to Thomas, K. N..

4 recordsLinked to original sources

bioRxiv2021

VaxArray Immunoassay for the Multiplexed Quantification of Poliovirus D-Antigen

Next generation poliovirus vaccines are critical to reaching global poliovirus eradication goals. Recent efforts have focused on creating inactivated vaccines using attenuated Sabin strains that maintain patient safety benefits and immunogenicity of conventional inactivated vaccines while increasing manufacturing safety and lowering production costs, and on developing novel oral vaccines using modified Sabin strains that provide critical mucosal immunity but are further attenuated to minimize risk of reversion to neurovirulence. In addition, there is a push to improve the analytical tools for poliovirus vaccine characterization. Conventional and Sabin inactivated poliovirus vaccines typically rely on standard plate-based ELISA as in vitro D-antigen potency assays in combination with WHO international standards as calibrants. While widely utilized, the current D-antigen ELISA assays have a long time to result (up to 72 hours), can suffer from lab-to-lab inconsistency due to non-standardized protocols and reagents, and are inherently singleplex. For D-antigen quantitation, we have developed the VaxArray Polio Assay Kit, a multiplexed, microarray-based immunoassay that uses poliovirus-specific human monoclonal antibodies currently under consideration as standardized reagents for characterizing inactivated Sabin and Salk vaccines. The VaxArray assay can simultaneously quantify all 3 poliovirus serotypes with a time to result of less than 3 hours. Here we demonstrate that the assay has limits of quantification suitable for both bioprocess samples and final vaccines, excellent reproducibility and precision, and improved accuracy over an analogous plate-based ELISA. The assay is suitable for adjuvanted combination vaccines, as common vaccine additives and crude matrices do not interfere with quantification, and is intended as a high throughput, standardized quantitation tool to aid inactivated poliovirus vaccine manufacturers in streamlining vaccine development and manufacturing, aiding the global polio eradication effort.

immunology↗

bioRxiv2021

Diversity and evolution of amphibian pupil shapes

Pupil constriction has important functional consequences for animal vision, yet the evolutionary mechanisms underlying diverse pupil sizes and shapes, often among animals that occupy optically similar environments, are poorly understood. We aimed to quantify the diversity and evolution of pupil shapes among amphibians and test for potential correlations to ecology based on functional hypotheses. Using photographs, we surveyed pupil shape and the orientation of the constricted pupil across adults of 1293 amphibian species, 72 families, and 3 orders, and additionally for larval life stages for all families of frogs and salamanders with a biphasic ontogeny. Pupil shape is exceptionally diverse in amphibians with evolutionary transitions throughout the amphibian tree of life. For amphibians with a biphasic life history, we found that pupils change in many species that occupy distinct habitats before and after metamorphosis. Finally, we found that non-elongated (round or diamond) constricted pupils were correlated with species inhabiting consistently dim light environments (burrowing and aquatic species) and that elongated pupils (vertical and horizontal) were more common in species with larger absolute eye sizes. We propose that amphibians provide a valuable group within which to explore the anatomical, physiological, optical, and ecological mechanisms underlying the evolution of pupil shape.

evolutionary biology↗

bioRxiv2021

Multiplexed VaxArray Immunoassay for Rapid Antigen Quantification in Measles and Rubella Vaccine Manufacturing

Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery. MR vaccine characterization currently relies on the 50% cell culture infectious dose (CCID50) assay, an endpoint assay with low reproducibility that requires 10-14 days to complete. For streamlining bioprocess analysis and improving measurement precision relative to CCID50, we developed the VaxArray Measles and Rubella assay kit, which is based on a multiplexed microarray immunoassay with a 5-hour time to result. Here we demonstrate vaccine-relevant sensitivity ranging from 345 - 800 IFU/mL up to 100,000 IFU/mL and specificity that allows simultaneous analysis in bivalent vaccine samples. The assay is sensitive to antigen stability and has minimal interference from common vaccine additives. The assay exhibits high reproducibility and repeatability, with 15% CV, much lower than the typical 0.3 log10 error (~65%) observed for the CCID50 assay. The intact protein concentration measured by VaxArray is reasonably correlated to, but not equivalent to, CCID50 infectivity measurements for harvest samples. However, the measured protein concentration exhibits equivalency to CCID50 for more purified samples, including concentrated virus pools and monovalent bulks, making the assay a useful new tool for same-day analysis of vaccine samples for bioprocess development, optimization, and monitoring.

immunology↗

bioRxiv2021

Genomic and Spectral Visual Adaptation in Southern Leopard Frogs during the Ontogenetic Transition from Aquatic to Terrestrial Light Environments

Many animals have complex life cycles where larval and adult forms have distinct ecologies and habitats that impose different demands on their sensory systems. While the adaptive decoupling hypothesis predicts reduced genetic correlations between life stages, how sensory systems adapt across life stages at the molecular level is not well understood. Frogs are a compelling system to study this question in because most species rely on vision as both aquatic tadpoles and terrestrial adults, but these habitats present vastly different light environments. Here we used whole eye transcriptome sequencing to investigate differential expression between aquatic tadpoles and terrestrial juveniles of the southern leopard frog (Lithobates sphenocephalus). Because visual physiology changes with light levels, we also tested how constant light or dark exposure affected gene expression. We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles, versus 5% for light/dark exposure. Analyses targeting a curated set of visual genes revealed significant differential expression between life stages in genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Light/dark exposure had a significant effect on a smaller set of visual genes. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Overall, we identified extensive expression-level differences in the eyes of tadpole and juvenile frogs related to observed morphological and physiological changes through metamorphosis, and corresponding adaptive shifts to optimize vision in aquatic versus terrestrial environments.

genomics↗