An automated and high-throughput approach for enhanced precision of adenoviral titering

Paolo Bottega; Manlio Fusciello; Firas Hamdan; Jacopo Chiaro; Salvatore Russo; Federica D'Alessio; Mikaela Grönholm; Vincenzo Cerullo

doi:10.1016/j.omtm.2025.101410

An automated and high-throughput approach for enhanced precision of adenoviral titering

Mol Ther Methods Clin Dev. 2025 Jan 20;33(1):101410. doi: 10.1016/j.omtm.2025.101410. eCollection 2025 Mar 13.

Authors

Paolo Bottega^{1

2

3

4}, Manlio Fusciello^{1

2

3

4}, Firas Hamdan^{1

2

3

4}, Jacopo Chiaro^{1

2

3

4}, Salvatore Russo^{1

2

3

4}, Federica D'Alessio^{1

2

3

4}, Mikaela Grönholm^{1

2

3

4}, Vincenzo Cerullo^{1

2

3

4

5}

Affiliations

¹ Department of Pharmaceutical Biosciences, University of Helsinki, Faculty of Pharmacy ImmunoViroTherapy Lab, Drug Research Program, Viikinkaari 5E, 00790 Helsinki, Finland.
² Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Fabianinkatu 33, 00710 Helsinki, Finland.
³ Translational Immunology Program (TRIMM), Faculty of Medicine Helsinki University, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland.
⁴ Digital Precision Cancer Medicine Flagship (iCAN), University of Helsinki, 00014 Helsinki, Finland.
⁵ Department of Molecular Medicine and Medical Biotechnology and CEINGE, Naples University Federico II, 80131 Naples, Italy.

Abstract

Accurate quantification of viral vectors and vaccines is a crucial step required before any downstream use of virus preparations. The conventional immunocytochemistry-based method of adenovirus quantification has been widely used, but there are many areas for improvement toward accuracy and resource consumption savings to reduce viral miscalculation and wastage of vaccination materials. In this work, a one-step approach is implemented for optimized adenoviral quantification that uses a single antibody coupled with automated, high-throughput image acquisition and subsequent batch analysis. First, cells are infected with the adenovirus of interest and stained using the Hexon protein. Then, multichannel automated image acquisition via the Invitrogen EVOS M7000 Imaging System is performed. Last is an automated large batch analysis of acquired images via the EVOS Analysis Software, accomplished via precise training of the software allowing for virus infection quantification and additional parameters (counts, circularity, area and intensity of targets). It was found that the implemented approach yielded precise and accurate quantification of both oncolytic viral vaccines and gene therapy vectors in a time- and resource-effective manner when compared with conventional methodologies.

Keywords: adenovirus; hexon protein; highthroughput microscopy; immunocytochemistry; multiplicity of infection; oncolytic vaccines; vaccines; vaccines titering; vector quantification; viruses.