Automated, label-free TCID50 assay to determine the infectious titer of virus-based therapeutics

Hochdorfer, Daniel; Businger, Ramona; Hotter, Dominik; Seifried, Carina; Solzin, Johannes

doi:10.1016/j.jviromet.2021.114318

Cited by 14 publications

(11 citation statements)

References 16 publications

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“…To determine the quantity of infectious virus present in our samples, TCID 50 assay was performed as described previously ( Hochdorfer et al, 2022 ). Briefly, BHK-21 (C13) cells were plated in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP

Gautam

Xin

Garcia

et al. 2022

Front. Bioeng. Biotechnol.

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Purification of viruses, especially for therapeutic purposes, is a tedious and challenging task. The challenges arise due to the size and surface complexity of the virus particles. VSV-GP is a promising oncolytic virus, which has been approved for phase I clinical trials by the Food and Drug Administration (FDA) of United States and Paul Ehrlich Institute (PEI) of Germany. The virus particles of VSV-GP are larger in size than vectors commonly used for gene therapy (e.g., adenovirus, adeno-associated virus, etc.). The current established proprietary clinical-grade manufacturing process for the purification of VSV-GP encompasses several chromatographic and non-chromatographic steps. In this study, we describe a new single-step purification process for the purification of VSV-GP virus, using cation exchange convective flow column with relatively higher yields. The purified virus was characterized for its quality attributes using TCID50 assay (for viral infectivity), host cell protein contaminant ELISA, SDS-PAGE, size exclusion chromatography (SEC), and cryo-electron microscopy. Furthermore, the purified viral therapeutic material was tested in vivo for its efficacy and safety. All these characterization methods demonstrated a therapeutic virus preparation of high purity and yield, which can be readily used for various studies.

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Section: Methodsmentioning

confidence: 99%

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP

Gautam

Xin

Garcia

et al. 2022

Front. Bioeng. Biotechnol.

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“…Despite significant advancements in automation of the assay-readout [8], traditional endpoint titration methods still suffer from limited precision and/or low sample throughput. To overcome this analytical bottleneck, we sought ways to replace the existing readout methods, typically based on detection of cell lysis, with an assay setup benefiting from the detection of more subtle and earlier virus-induced cellular changes.…”

Section: Cell Rounding Occurs Early After Infection and Correlates Wi...mentioning

confidence: 99%

High Throughput Determination of Infectious Virus Titers by Kinetic Measurement of infection-Induced Changes in Cell Morphology

Hotter,

Kunzelmann,

Kiefer

et al. 2024

Preprint

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Infectivity assays are the key analytical technology for development of manufacturing processes for virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 hours post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV &lt; 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for VSV-GP and NDV, but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.

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“…Although there has been extensive development in molecular and cellular biology methods and imaging techniques applied to antiviral testing, the assays developed are often limited by the availability of reagents and equipment that can be used in biosafety containment facilities [ 147 ]. Therefore, future trends are focused on the development of accurate, simplified, and fast cell-based assays for sensitive and quick high-throughput screening (HTS) of antivirals [ 182 , 183 , 184 , 185 , 186 , 187 ]. An additional complication encountered when testing natural products as antiviral agents is the tedious nature of identifying bioactive components, leaving the mechanism of action of the crude extract or the metabolites unknown [ 151 ].…”

Section: Antiviral Activitymentioning

confidence: 99%

Current Landscape of Methods to Evaluate Antimicrobial Activity of Natural Extracts

et al. 2023

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Natural extracts have been and continue to be used to treat a wide range of medical conditions, from infectious diseases to cancer, based on their convenience and therapeutic potential. Natural products derived from microbes, plants, and animals offer a broad variety of molecules and chemical compounds. Natural products are not only one of the most important sources for innovative drug development for animal and human health, but they are also an inspiration for synthetic biology and chemistry scientists towards the discovery of new bioactive compounds and pharmaceuticals. This is particularly relevant in the current context, where antimicrobial resistance has risen as a global health problem. Thus, efforts are being directed toward studying natural compounds’ chemical composition and bioactive potential to generate drugs with better efficacy and lower toxicity than existing molecules. Currently, a wide range of methodologies are used to analyze the in vitro activity of natural extracts to determine their suitability as antimicrobial agents. Despite traditional technologies being the most employed, technological advances have contributed to the implementation of methods able to circumvent issues related to analysis capacity, time, sensitivity, and reproducibility. This review produces an updated analysis of the conventional and current methods to evaluate the antimicrobial activity of natural compounds.

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Automated, label-free TCID50 assay to determine the infectious titer of virus-based therapeutics

Cited by 14 publications

References 16 publications

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP

High Throughput Determination of Infectious Virus Titers by Kinetic Measurement of infection-Induced Changes in Cell Morphology

Current Landscape of Methods to Evaluate Antimicrobial Activity of Natural Extracts

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