Use of Benzonase^® Nuclease to Prevent Clumping of Stored Peripheral Blood Mononuclear Cells (PBMCs) During Vaccine Evaluation

Ex vivo cytokine and memory T cell responses to the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 in vaccinated volunteers

Journal of Immunology (2008) 180, 1451

Maria Cecilia Huaman^1,2, Laura B. Martin¹, Elissa Malkin¹, David L. Narum¹, Louis H. Miller¹, Siddhartha Mahanty^1,3, and Carole A. Long^1,2

¹ Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD USA

² Laboratory of Malaria and Vector Research, and 3 Laboratory of Parasitic Diseases, NIAID, NIH, Rockville, MD USA

Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials

Journal of Immunological Methods (2007) 322, 57

Marta Bull¹, Deborah Lee², Jason Stucky², Ya-Lin Chiu³, Abbe Rubin³, Helen Horton^2,4, M. Juliana McElrath^2,4,5

¹ Children’s Hospital and Regional Medical Center, Seattle, WA USA ² Program in Infectious Diseases, and ³ Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, WA USA

⁴ Department of Medicine and 5 Laboratory Medicine, University of Washington School of Medicine, Seattle, WA USA

Peripheral blood mononuclear cells (PBMCs) isolated from whole blood have important applications in vaccine research – for example, during quantification of vaccine induced T cell responses. T cell response assay methods initially required freshly isolated cells for optimal signal detection, posing a serious practical limitation for sample handling during large clinical trials. Frozen PBMCs (particularly PBMCs prepared from stored blood) tend to clump together upon thawing, preventing further analysis. In 2001, Smith et al demonstrated that inclusion of Benzonase^® Nuclease in PBMC thawing buffer prevented cell clumping, allowing implementation of PBMC cryopreservation. This method has since been widely adopted in vaccine evaluation studies, including the two profiled here. In the first study (Huaman 2008), Benzonase Nuclease-treated PBMCs were analyzed by ELISPOT and Luminex^® xMAP^® multiplex assays to determine cytokine levels following stimulation with a 42-amino acid fragment of Plasmodium falciparum merozoite surface protein-1 (MSP1). This allowed researchers to assess potential suitability of the MSP142 fragment as an anti-malarial vaccine. In the second study (Bull 2007), researchers determined optimal blood processing techniques for use in HIV-1 vaccine evaluation. Defining best practices is critical to prepare for future multicenter, large-scale phase IIB and III trials. Benzonase Nuclease was routinely included in PBMC thawing buffer prior to analysis with ELISPOT assays or intracellular cytokine staining.

Taken together, these studies demonstrate an important role of Benzonase Nuclease in developing vaccines against some of the world’s most serious infectious diseases.

Additional Reference

1. Smith JG, Liu X, Kaufhold RM, Clair J, Caulfield MJ. 2001. Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus. Clin. Diagn. Lab. Immunol.. 8(5):871-879. https://doi.org/10.1128/cdli.8.5.871-879.2001