Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response

Abstract

Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against fish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine ex vivo and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor α (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches.

Keywords: DNA vaccine; VHSV glycoprotein G; erythrocytes; immune response; proteome; rainbow trout; red blood cells; transcriptome.

Figure 1

General workflow of experimental steps from sample collection to data analysis.

Figure 2

Monitoring transfection of rainbow trout red blood cells (RBCs). Fluorescent micrographs of RBCs transfected with (A) pmTFP1 and (B) pmTFP1GVHSV at six days post-transfection at 14°C, monitored by teal fluorescent protein (TFP). Fluorescent images taken with the IN Cell 6000 imaging system, augmentation 60x. (C) RBC transfection with 2, 4, and 8 μg of plasmid pmTFP1GVHSV was confirmed by GVHSV gene RT-qPCR, at one day (white bars), three days (gray bars) and six days (black bars) post-transfection. The eukaryotic 18S rRNA gene was used as an endogenous control. Data are displayed as mean ± SD (n = 3). Two-way ANOVA with Tukey's multiple comparisons test was performed between plasmid concentrations (black lines and asterisks) and times post-transfection (gray lines and asterisks). (D) Time course of transfected RBCs (black bars) and transfected RTS11 (gray bars) with 4 μg of pmTFP1GVHSV at one, three and six days post-transfection monitored by GVHSV RT-qPCR. The eukaryotic 18S rRNA gene was used as an endogenous control. Data are displayed as mean ± SD (n = 3 for RBCs and n = 2 for RTS11). Two-way ANOVA with Sidak's multiple comparisons test was performed between cell types at the different times post-transfection. ^*, ^**, ^***, and ^****, represent the P values < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. Overlay flow cytometry histogram of representative GVHSV immunostaining of (E) RBCs and (F) RTS11 cells transfected with 4 μg of pmTFP1 or pmTFP1GVHSV at three days post-transfection (gray histogram, pmTFP1-transfected; green histogram, pmTFP1GVHSV-transfected). Representative immunofluorescence of GVHSV intracellular immunostaining of (G) RBCs and (H) RTS11 cells transfected with 4 μg of pmTFP1 or pmTFP1GVHSV at three days post-transfection (protein [APC, red] and nuclei [DAPI, blue]). Fluorescence images were taken with 60 × magnification.

Figure 3

Functional pathway analysis of differentially expressed genes (DEGs) in the FACS single-cell sorted GVHSV-expressing RBC transcriptome profile. DEGs in pmTFP1GVHSV with FDR Q value < 0.05 and fold change P value < 0.05. Overrepresented GO terms were identified by the Cytoscape ClueGO app with Reactome and GO Immune Process terms. The Reactome Pathways multilevel pie chart shows (A) upregulated and (B) downregulated overrepresented terms in pmTFP1GVHSV-expressing RBCs. The GO Immune Process multilevel pie chart shows (C) upregulated overrepresented terms in pmTFP1GVHSV-expressing RBCs. Asterisks denote GO-term significance (^**p < 0.01).

Figure 4

Functional pathway analysis of differentially expressed proteins (DEPs) in the GVHSV-transfected RBC proteome profile. DEPs in pmTFP1GVHSV with (−1.5 < log₂ fold change < 1.5) and an FDR P-value < 0.001. Overrepresented GO terms were identified by the Cytoscape ClueGO app, with Reactome Pathways, Reactome Reactions and GO Immune Process terms. The Reactome Pathways multilevel pie chart shows (A) upregulated and (B) downregulated overrepresented terms in pmTFP1GVHSV-transfected RBCs. The Reactome Reactions multilevel pie chart shows (C) upregulated and (D) downregulated overrepresented terms in pmTFP1GVHSV-transfected RBCs. The GO Immune Process multilevel pie chart shows (E) upregulated overrepresented terms in pmTFP1GVHSV-transfected RBCs. Asterisks denote GO-term significance (^**p < 0.01).

Figure 5

Gene expression evaluation to validate functional pathway analysis of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in GVHSV-transfected RBCs transcriptome and proteome profiles, respectively. RBCs were transfected with pmTFP1 and pmTFP1GVHSV plasmids for 6 days at 14°C. Afterwards, gene expression was evaluated by RT-qPCR. Data are displayed as mean ± SD (n = 5). The EF1α gene was used as an endogenous control. The Wilcoxon test was performed between pmTFP1GVHSV- and pmTFP1-transfected RBCs (control, red line). ^*and ^**represent P-values < 0.05 and < 0.01, respectively.

Figure 6

Immune proteins expression in GVHSV-transfected RBCs. (A) Immune protein expression measured by flow cytometry and calculated by the formula MRFI (mean relative fluorescence intensity) = fluorescence in pmTFP1GVHSV- transfected RBCs MRFI (mean relative fluorescence intensity) = fluorescence in pmTFP1GVHSV- transfected RBCs /fluorescence in pmTFP1-transfected RBCs. (B) Representative immunofluorescence of pmTFP1- and pmTFP1GVHSV-transfected RBCs (protein [APC, red] and nuclei [DAPI, blue]). Fluorescence images were taken with 60 × magnification.

Figure 7

Protection conferred by conditioned medium (CM) from GVHSV-transfected RBCs against VHSV infection in RTG-2 cells. RTG-2 cells pretreated with CM from RBCs transfected with pmTFP1 (black bars) and pmTFP1GVHSV (gray bars) plasmids (diluted 1/5 and 1/125 in MEM 10% FBS), at three (A) and six (B) days post-transfection. Cells were then infected with VHSV (MOI 1 × 10⁻²) for 24h at 14°C. The positive control is non-pretreated RTG-2 cells infected with VHSV. VHSV infectivity was evaluated by means of focus forming units (FFU)/mL. FFUs were stained with 2C9 antibody against the N protein of VHSV. Data are displayed as mean ± SD (n = 3). The Mann Whitney test was performed between treatments at each dilution. (C) ifn1 and mx gene expression in RTG-2 cells quantified by RT-qPCR after treatment with CM from pmTFP1- (black bars) and pmTFP1GVHSV- (gray bars) transfected RBCs (diluted 1/5 in MEM 10% FBS) at three and six days post-transfection. The eukaryotic 18S rRNA gene was used as an endogenous control. Data are displayed as mean ± SD (n = 3). The Mann Whitney test was performed between conditions. ^*, ^**, and ^****, represent P-values < 0.05, < 0.01, and < 0.0001, respectively.

Figure 8

M1 and M2 macrophage markers in RTS11 cells co-cultured with GVHSV-transfected RBCs. Rainbow trout RBCs transfected with pmTFP1 or pmTFP1GVHSV for three days at 14°C. Afterwards, transfected RBCs were co-cultured with the RTS-11 cell line at 14°C for three days. Then, the il12β, inos, and il10 gene expression profiles were quantified by RT-qPCR. Gene expression was normalized against eukaryotic 18S rRNA and compared to control cells (RTS11 co-cultured with pmTFP1-transfected RBCs, red line) (fold-change). Data represent the mean ± SD (n = 6). A Wilcoxon test was performed between both conditions.