Plasma Proteome Responses in Salmonid Fish Following Immunization

Abstract

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout ( Oncorhynchus mykiss , Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.

Exploration of the Nurse Shark (Ginglymostoma cirratum) Plasma Immunoproteome Using High-Resolution LC-MS/MS

“…Plasma samples from the five, repeatedly sampled nurse sharks were prepared for proteomic analysis at the University of Aberdeen Proteomics facility, as detailed in 14 . Briefly, 1 μl of plasma was diluted with 99 µl 50 mM ammonium bicarbonate and proteins were reduced in 2 mM dithiothreitol for 25 min at 60°C, S-alkylated in 4 mM iodoacetamide for 30 min at 25°C in the dark, digested with porcine trypsin (Promega) overnight at 37°C, then freeze-dried.…”

“…To establish the extent of variation in protein abundances between the control and HSA-immunized sharks across the time course, principal component analyses (PCA) were performed in R using the prcomp function, and visualized using ggplot2 v3.2.1 ( 41 ). As there was only a single control shark, statistical analyses at the level of individual proteins were restricted to using the four immunized sharks, assuming protein levels at day 0 as an internal baseline control for each animal ( 14 ). We filtered the data to retain proteins with LFQ values present in at least 8 out of 12 timepoints in every immunized shark across the timecourse (n=260 proteins), ensuring the analyses were based on consistently identified peptides.…”

“…For instance, LC-MS/MS has been used to characterize human plasma proteomes ( 7 , 8 ), and to assist in the identification of biomarkers for diseases such as cancer (e.g., 9 , 10 ). Such tools have also been applied to characterize proteomes in non-mammalian species (e.g., 11 – 14 ), permitting the identification and quantification of many proteins simultaneously and circumventing the need for specific mAbs (reviewed by 15 ). However, such methods require a comprehensive sequence database to match the enzymatically digested peptides detected during LC-MS/MS back to their original proteins ( 16 – 19 ).…”

“…We also generated a high-quality in silico proteome for this species via the assembly of a novel transcriptome built with PacBio and Illumina data. Our previous immunization study on rainbow trout ( Oncorhynchus mykiss ), also using LC-MS/MS proteomics ( 14 ), identified abundance changes in 278 plasma proteins, including both classical immune proteins (e.g., complement components) and proteins not usually associated with immune responses in mammals (e.g., apolipoproteins). A separate study by Morro and colleagues ( 27 ) identified a greater number of plasma proteins (1822) in rainbow trout using LC-MS/MS label-free proteomics in combination with enrichment of low abundance proteins.…”

Histopathological analysis of zebrafish after introduction of non-biodegradable polyelectrolyte microcapsules into the circulatory system

“…Various blood parameters are usually considered reliable and comprehensive markers of the physiological state of any vertebrate organism ( Tahir et al, 2018 ; Bakke et al, 2020 ; Huang et al., 2019 ). Most current approaches require blood extraction to analyze its parameters; however, continuous real-time signal acquisition directly from an animal bloodstream has long been a desirable goal in physiological research ( Wang et al, 2017 ; Gray et al, 2018 ).…”