Abstract
Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.
Highlights
The safety and potency of poultry vaccines has traditionally been assessed through in vivo vaccination and challenge tests[1]
Primary chicken bone marrow-derived dendritic cells (DCs), which have been characterized in previous s tudies[14,15], were stimulated with a commercially available inactivated poultry vaccine against infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) in a mineral oil adjuvant in a water-in-oil formulation, and analyzed by LC–MS/MS to evaluate changes in the proteome In addition, chBDMCs were stimulated with inactivated IBV antigens only to discriminate between the effects of a single inactivated antigen and a vaccine
LC–MS/MS led to the identification of 2,828 proteins (Supplementary Table S1), 991 of which were identified multiple times, because several UniProtKB accession codes were present for these individual protein sequences, and these were manually removed based on identical expression levels in all samples
Summary
The safety and potency of poultry vaccines has traditionally been assessed through in vivo vaccination and challenge tests[1]. There is a global intent to replace in vivo vaccine tests for in vitro alternatives[2] This has already led to the development of an enzyme-linked immunosorbent assay (ELISA) as an in vitro antigen quantification method to assess inactivated vaccines for Newcastle disease virus (NDV) for potency[3]. One approach to discover biomarkers of immune responsiveness to inactivated poultry vaccines is the investigation of in vitro vaccine-stimulated dendritic cells by proteomic analysis using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Both infectious bronchitis virus (IBV) and NDV cause respiratory tract infections that can disseminate to other tissues, leading to reduced egg production and mortality in chickens[12,13]. This study may generate new hypotheses about the mechanisms by which an inactivated viral poultry vaccine activates chicken DCs, as well as new insights in the cellular processes involved in chBMDCs maturation after stimulation with LPS
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