Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene

Fowl pox virus (FPV) has been modified to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to co-express a novel host cytokine from such recombinants and characterize its immune response. In this study, previously constructed H5-recombinant Fowl po...

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Main Author:	Nor Majid, Nadzreeq
Format:	Thesis
Language:	English
Published:	2016
Subjects:	Gene expression Cytokines Poultry - Virus diseases
Online Access:	http://psasir.upm.edu.my/id/eprint/83071/1/FBSB%202016%2048%20ir.pdf
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spelling	my-upm-ir.830712022-01-11T02:15:14Z Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene 2016-11 Nor Majid, Nadzreeq Fowl pox virus (FPV) has been modified to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to co-express a novel host cytokine from such recombinants and characterize its immune response. In this study, previously constructed H5-recombinant Fowl pox viruses co-expressing host IL-15 (rFPV) was inoculated into specific-pathogen-free (SPF) chickens. T-lymphocyte populations and selected cytokine expression namely IL-15 and IL-18 of the vaccinated chickens were evaluated to add the relatively limited knowledge of chicken IL-15 cytokine gene compared to that of mammalian. It is hypothesized that vaccination with H5-recombinant Fowl pox viruses co-expressing IL-15 gene is able to show a higher population percentage of CD4+ and CD8+ T lymphocytes, and high IL-15 and IL-18 expressions, compared to H5-recombinant vaccines alone, in chickens. Prior to in vivo characterization, recombinant viruses were propagated in Chicken Embryonic Fibroblast (CEF) primary cell line. Stability of H5 gene from influenza strain A/Chicken/Malaysia/5858/2004 (1695 kb), and IL-15 (695 kb) gene integrations was confirmed by using Polymerase Chain Reaction (PCR) with specific primers after three passages. Propagations and plaque assays were done until desired titres of recombinant viruses were obtained. Parental (FP9 wild-type) and recombinant virus vaccines (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. The immunogenicity of the recombinant viruses was analyzed based on evaluation of T-lymphocytes cell population via flow cytometry, from Peripheral Blood Mononuclear Cell (PBMC) of 14 and 28-days-old vaccinated chickens. Chickens inoculated with rFPV/H5/IL-15 had a higher increased in CD4+ T cells population relative to rFPV/H5 in both time points. However, the result showed that rFPV/H5/IL-15 was not significant (P>0.05) in inducing CD8+ T cells. In general, the percentage of CD4+ and CD8+ lymphocytes cell population in chickens immunized with rFPV/H5/IL-15 were statistically higher compared to chickens immunized with rFPV/H5 and FP9 wild-type virus (P<0.05). Specific gene expressions of SPF chickens inoculated with rFPV were analyzed by quantitative real-time PCR (qRT-PCR), following extraction of spleen from 14-day-old SPF chickens at days 2, 4 and 6 post-infection. Two target genes chosen were IL-15 and IL-18 genes. The rFPV/H5/IL-15 group showed an increased level of IL-15 and IL-18 genes expression up to 2 and 3.5 folds, respectively, within 6 days post-vaccination, compared with other inoculated groups. rFPV/H5 group showed an increased level of IL-15 gene expression at day 2 and maintained at day 4 until day 6, while the IL-18 expression was decreasing within 6 days. Overall, the FP9 wild type group showed a low cytokine expression level as compared to the recombinant virus groups. While histopathology results showed successful vaccination of rFPV into chicken cells, weekly weighing suggested that inoculation with rFPV might not influence any weight changes. In summary, this study showed modulation immunogenicity of FP9 Wild Type, rFPV/H5, and rFPV/H5/IL-15, with rFPV/H5/IL-15 being the best vaccine candidate compared to others. Gene expression Cytokines Poultry - Virus diseases 2016-11 Thesis http://psasir.upm.edu.my/id/eprint/83071/ http://psasir.upm.edu.my/id/eprint/83071/1/FBSB%202016%2048%20ir.pdf text en public masters Universiti Putra Malaysia Gene expression Cytokines Poultry - Virus diseases Abdul Razak, Mariatulqabtiah
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Abdul Razak, Mariatulqabtiah
topic	Gene expression Cytokines Poultry - Virus diseases
spellingShingle	Gene expression Cytokines Poultry - Virus diseases Nor Majid, Nadzreeq Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
description	Fowl pox virus (FPV) has been modified to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to co-express a novel host cytokine from such recombinants and characterize its immune response. In this study, previously constructed H5-recombinant Fowl pox viruses co-expressing host IL-15 (rFPV) was inoculated into specific-pathogen-free (SPF) chickens. T-lymphocyte populations and selected cytokine expression namely IL-15 and IL-18 of the vaccinated chickens were evaluated to add the relatively limited knowledge of chicken IL-15 cytokine gene compared to that of mammalian. It is hypothesized that vaccination with H5-recombinant Fowl pox viruses co-expressing IL-15 gene is able to show a higher population percentage of CD4+ and CD8+ T lymphocytes, and high IL-15 and IL-18 expressions, compared to H5-recombinant vaccines alone, in chickens. Prior to in vivo characterization, recombinant viruses were propagated in Chicken Embryonic Fibroblast (CEF) primary cell line. Stability of H5 gene from influenza strain A/Chicken/Malaysia/5858/2004 (1695 kb), and IL-15 (695 kb) gene integrations was confirmed by using Polymerase Chain Reaction (PCR) with specific primers after three passages. Propagations and plaque assays were done until desired titres of recombinant viruses were obtained. Parental (FP9 wild-type) and recombinant virus vaccines (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. The immunogenicity of the recombinant viruses was analyzed based on evaluation of T-lymphocytes cell population via flow cytometry, from Peripheral Blood Mononuclear Cell (PBMC) of 14 and 28-days-old vaccinated chickens. Chickens inoculated with rFPV/H5/IL-15 had a higher increased in CD4+ T cells population relative to rFPV/H5 in both time points. However, the result showed that rFPV/H5/IL-15 was not significant (P>0.05) in inducing CD8+ T cells. In general, the percentage of CD4+ and CD8+ lymphocytes cell population in chickens immunized with rFPV/H5/IL-15 were statistically higher compared to chickens immunized with rFPV/H5 and FP9 wild-type virus (P<0.05). Specific gene expressions of SPF chickens inoculated with rFPV were analyzed by quantitative real-time PCR (qRT-PCR), following extraction of spleen from 14-day-old SPF chickens at days 2, 4 and 6 post-infection. Two target genes chosen were IL-15 and IL-18 genes. The rFPV/H5/IL-15 group showed an increased level of IL-15 and IL-18 genes expression up to 2 and 3.5 folds, respectively, within 6 days post-vaccination, compared with other inoculated groups. rFPV/H5 group showed an increased level of IL-15 gene expression at day 2 and maintained at day 4 until day 6, while the IL-18 expression was decreasing within 6 days. Overall, the FP9 wild type group showed a low cytokine expression level as compared to the recombinant virus groups. While histopathology results showed successful vaccination of rFPV into chicken cells, weekly weighing suggested that inoculation with rFPV might not influence any weight changes. In summary, this study showed modulation immunogenicity of FP9 Wild Type, rFPV/H5, and rFPV/H5/IL-15, with rFPV/H5/IL-15 being the best vaccine candidate compared to others.
format	Thesis
qualification_level	Master's degree
author	Nor Majid, Nadzreeq
author_facet	Nor Majid, Nadzreeq
author_sort	Nor Majid, Nadzreeq
title	Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
title_short	Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
title_full	Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
title_fullStr	Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
title_full_unstemmed	Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene
title_sort	characterization of t-lymphocyte populations and selected cytokine expressions of chickens vaccinated with h5-recombinant fowl pox viruses co-expressing il-15 gene
granting_institution	Universiti Putra Malaysia
publishDate	2016
url	http://psasir.upm.edu.my/id/eprint/83071/1/FBSB%202016%2048%20ir.pdf
_version_	1747813342610718720

Characterization of T-lymphocyte populations and selected cytokine expressions of chickens vaccinated with H5-recombinant fowl pox viruses co-expressing IL-15 gene

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