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Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines

View Article: PubMed Central - PubMed

ABSTRACT

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

Electronic supplementary material: The online version of this article (doi:10.1186/s13567-017-0423-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Specific binding of anti-chCD205 and internalization of antigen delivery complex. A The recombinant parts of chicken CD205 receptor used for monoclonal antibody preparation and the monoclonal antibody characterization. The CD205B and CD205F proteins were purified from inclusion bodies on DEAE Sepharose and separated on Tris-Tricine polyacrylamide gel stained by Coomassie blue (left). Monoclonal mouse anti-CD205 antibodies were analyzed using Western blots. Results of two representative detections of monoclonal anti-CD205 specific for CD205B or CD205F are shown. B CD205 expression on leukocytes. Splenocytes and peripheral leukocytes were stained with anti-chicken CD205 antibody and antibodies recognizing each of the indicated antigens. Histograms represent the CD205 fluorescence for cells gated for positive expression of the markers indicated at the left. The results of one representative of three experiments are shown. C Median fluorescence intensity (MFI) of CD205 stained cells and standard deviation. D Internalization of streptavidin-CD205-FITC complex by chicken primary splenocytes one (left) and two (right) hours of incubation in different temperatures. Dotted line plot non-labelled cells (biot—control IgG); Black line plot—FITC labelled cells incubated in 4 °C; Gray line plot—FITC labelled cells incubated in 40 °C.
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Fig2: Specific binding of anti-chCD205 and internalization of antigen delivery complex. A The recombinant parts of chicken CD205 receptor used for monoclonal antibody preparation and the monoclonal antibody characterization. The CD205B and CD205F proteins were purified from inclusion bodies on DEAE Sepharose and separated on Tris-Tricine polyacrylamide gel stained by Coomassie blue (left). Monoclonal mouse anti-CD205 antibodies were analyzed using Western blots. Results of two representative detections of monoclonal anti-CD205 specific for CD205B or CD205F are shown. B CD205 expression on leukocytes. Splenocytes and peripheral leukocytes were stained with anti-chicken CD205 antibody and antibodies recognizing each of the indicated antigens. Histograms represent the CD205 fluorescence for cells gated for positive expression of the markers indicated at the left. The results of one representative of three experiments are shown. C Median fluorescence intensity (MFI) of CD205 stained cells and standard deviation. D Internalization of streptavidin-CD205-FITC complex by chicken primary splenocytes one (left) and two (right) hours of incubation in different temperatures. Dotted line plot non-labelled cells (biot—control IgG); Black line plot—FITC labelled cells incubated in 4 °C; Gray line plot—FITC labelled cells incubated in 40 °C.

Mentions: The specificity of newly prepared anti-chCD205 monoclonal antibodies was tested by ELISA against recombinant antigens CD205B and CD205F (data not shown). The specificity of the positive clones was verified on Western blot, where the recombinant proteins CD205B and CD205F were detected using different clones of monoclonal antibodies (Figure 2A). For the purpose of antigen delivery, it was necessary to select antibodies recognizing natural surface receptor CD205 by flow cytometry on chicken splenocytes and blood cells. Flow cytometry data were gated to exclude cell debris according to the cell granularity and size (SSC/FSC parameters). CD205 was expressed at very low levels on T cell subsets of leukocytes (CD4+, TCRγδ+, and CD8a+) as well as splenocytes. As shown in Figures 2B and C, higher expression levels of CD205 were seen on KUL01+ CD11c+, MHCII+ cells and Bu1+ cells.Figure 2


Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines
Specific binding of anti-chCD205 and internalization of antigen delivery complex. A The recombinant parts of chicken CD205 receptor used for monoclonal antibody preparation and the monoclonal antibody characterization. The CD205B and CD205F proteins were purified from inclusion bodies on DEAE Sepharose and separated on Tris-Tricine polyacrylamide gel stained by Coomassie blue (left). Monoclonal mouse anti-CD205 antibodies were analyzed using Western blots. Results of two representative detections of monoclonal anti-CD205 specific for CD205B or CD205F are shown. B CD205 expression on leukocytes. Splenocytes and peripheral leukocytes were stained with anti-chicken CD205 antibody and antibodies recognizing each of the indicated antigens. Histograms represent the CD205 fluorescence for cells gated for positive expression of the markers indicated at the left. The results of one representative of three experiments are shown. C Median fluorescence intensity (MFI) of CD205 stained cells and standard deviation. D Internalization of streptavidin-CD205-FITC complex by chicken primary splenocytes one (left) and two (right) hours of incubation in different temperatures. Dotted line plot non-labelled cells (biot—control IgG); Black line plot—FITC labelled cells incubated in 4 °C; Gray line plot—FITC labelled cells incubated in 40 °C.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5382389&req=5

Fig2: Specific binding of anti-chCD205 and internalization of antigen delivery complex. A The recombinant parts of chicken CD205 receptor used for monoclonal antibody preparation and the monoclonal antibody characterization. The CD205B and CD205F proteins were purified from inclusion bodies on DEAE Sepharose and separated on Tris-Tricine polyacrylamide gel stained by Coomassie blue (left). Monoclonal mouse anti-CD205 antibodies were analyzed using Western blots. Results of two representative detections of monoclonal anti-CD205 specific for CD205B or CD205F are shown. B CD205 expression on leukocytes. Splenocytes and peripheral leukocytes were stained with anti-chicken CD205 antibody and antibodies recognizing each of the indicated antigens. Histograms represent the CD205 fluorescence for cells gated for positive expression of the markers indicated at the left. The results of one representative of three experiments are shown. C Median fluorescence intensity (MFI) of CD205 stained cells and standard deviation. D Internalization of streptavidin-CD205-FITC complex by chicken primary splenocytes one (left) and two (right) hours of incubation in different temperatures. Dotted line plot non-labelled cells (biot—control IgG); Black line plot—FITC labelled cells incubated in 4 °C; Gray line plot—FITC labelled cells incubated in 40 °C.
Mentions: The specificity of newly prepared anti-chCD205 monoclonal antibodies was tested by ELISA against recombinant antigens CD205B and CD205F (data not shown). The specificity of the positive clones was verified on Western blot, where the recombinant proteins CD205B and CD205F were detected using different clones of monoclonal antibodies (Figure 2A). For the purpose of antigen delivery, it was necessary to select antibodies recognizing natural surface receptor CD205 by flow cytometry on chicken splenocytes and blood cells. Flow cytometry data were gated to exclude cell debris according to the cell granularity and size (SSC/FSC parameters). CD205 was expressed at very low levels on T cell subsets of leukocytes (CD4+, TCRγδ+, and CD8a+) as well as splenocytes. As shown in Figures 2B and C, higher expression levels of CD205 were seen on KUL01+ CD11c+, MHCII+ cells and Bu1+ cells.Figure 2

View Article: PubMed Central - PubMed

ABSTRACT

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

Electronic supplementary material: The online version of this article (doi:10.1186/s13567-017-0423-8) contains supplementary material, which is available to authorized users.

No MeSH data available.