Measuring CMI responses using the PrimeFlow RNA assay: A new method of evaluating BVDV vaccination response in cattle

Current methods for evaluating bovine viral diarrhea virus (BVDV) vaccination response typically rely on measurement of humoral responses as determined by virus neutralizing antibody titers (VNT) against BVDV. While VNT are correlated with increased protection, research has also shown that cell mediated immunity (CMI) is an important component of a protective response against BVDV. For example, improved protection against BVDV by modified-live viral (MLV) vaccines as compared to killed vaccines is thought to be due to better CMI induced by the MLV. The goal of this work was to evaluate the cell mediated response in vaccinated calves using a novel PrimeFlow RNA assay that incorporates cell surface marker staining with intracellular RNA expression of cytokines and viral RNA detection. Results from this study evaluating mRNA for IFN-γ and IL-2 at 24 h post-BVDV stimulation are similar to previous studies in which IFN-γ was detected in the CD4⁺ and CD8⁺ T cell population. However, a novel observation was the detection of IFN-γ mRNA in the NK cell population in vaccinated animals. The NK cell population contributed a significant portion of the IFN-γ produced. This study also demonstrated a decrease in the frequency and amount of BVDV in PBMCs, harvested from vaccinated calves and exposed to BVDV in vitro. Collectively data from this study highlights the association between an increase in IFN-γ and a decreased infection rate of isolated PBMC's, based on the frequency and amount of BVDV positive cells following in vitro exposure. This new method combines not only the ability to evaluate cellular responses, but also the ability to understand potential antiviral properties associated with cellular responses. This is the first assay to describe and simultaneously measure CMI responses and intracellular viral RNA quantity as a method to evaluate protective responses associated with vaccination.