Effect of CpG-Oligonucleotide in Enhancing Recombinant Herpes Virus of Turkey-Laryngotracheitis Vaccine-Induced Immune Responses in One-Day-Old Broiler Chickens

Abstract

Infectious laryngotracheitis (ILT) is an economically important disease of chickens. While the recombinant vaccines can reduce clinical disease severity, the associated drawbacks are poor immunogenicity and delayed onset of immunity. Here, we used CpG-oligonucleotides (ODN) as an in ovo adjuvant in boosting recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccine-induced responses in one-day-old broiler chickens. Two CpG-ODN doses (5 and 10 μg/egg) with no adverse effect on the vaccine-virus replication or chick hatchability were selected for immune-response evaluation. Results showed that while CpG-ODN adjuvantation induced an increased transcription of splenic IFNγ and IL-1β, and lung IFNγ genes, the IL-1β gene expression in the lung was significantly downregulated compared to the control. Additionally, the transcription of toll-like receptor (TLR)21 in the spleen and lung and inducible nitric oxide synthase (iNOS) in the spleen of all vaccinated groups was significantly reduced. Furthermore, splenic cellular immunophenotyping showed that the CpG-ODN-10μg adjuvanted vaccination induced a significantly higher number of macrophages, TCRγδ+, and CD4+ T cells as well as a higher frequency of activated T cells (CD4+CD44+) when compared to the control. Collectively, the findings suggested that CpG-ODN can boost rHVT-LT-induced immune responses in day-old chicks, which may help in anti-ILT defense during their later stages of life.

Keywords: CpG-ODN; adjuvant; chickens; immune response; infectious laryngotracheitis.

Figure 1

Effect of CpG-ODN on rHVT-LT vaccine-virus replication in vitro. Monolayer cultures of chicken embryo fibroblasts (CEFs) were infected with one dose of vaccine and with three serial 10-fold dilutions. Vaccine ± CpG-ODN doses (5, 10, and 25 µg) were titrated in triplicate and the growth of the virus was determined through assessing the cytopathic effect on CEFs using an inverted microscope. Titration was evaluated through counting the number of plaque-forming units (PFUs). Different letters above the standard-error-of-mean bars indicate significant difference (p < 0.05) between the groups.

Figure 2

Effect of CpG-ODN adjuvantation on the immune-gene expression in the spleen. Recombinant HVT-LT (rHVT-LT) vaccine was adjuvanted with either 5 μg or 10 μg of CpG-ODN and administered in ovo on the day 18 of embryonation (ED18) via the amniotic route. Spleens from the day-old broiler chicks were collected in RNAlater for RNA extraction and cDNA synthesis. Real-time PCR to quantify the expression of IFNγ, IL-12, IFNβ, IL-1β, iNOS (inducible nitric oxide synthase), and TLR21 genes was performed along with the housekeeping gene (β-actin). The expression levels are shown as relative to β-actin. Different letters above the standard-error-of-mean bars indicate significant difference (p < 0.05) between the groups.

Figure 3

Effect of CpG-ODN adjuvantation on the immune-gene expression in the lung. Recombinant HVT-LT (rHVT-LT) vaccine was adjuvanted with either 5 μg or 10 μg of CpG-ODN and administered in ovo on the day 18 of embryonation (ED18) via the amniotic route. Lung tissues from the day-old broiler chicks were collected in RNAlater for RNA extraction and cDNA synthesis. Real-time PCR to quantify the expression of IFNγ, IL-12, IFNβ, IL-1β, iNOS (inducible nitric oxide synthase), and TLR21 genes was performed along with the housekeeping gene (β-actin). The expression levels are shown as relative to β-actin. Different letters above the standard-error-of-mean bars indicate significant difference (p < 0.05) between the groups.

Figure 4

Effect of CpG-ODN on macrophage responses in one-day-old chicks. Single-cell splenocyte suspensions were prepared to obtain mononuclear cells and stained with anti-chicken monoclonal antibodies against KUL-01 (macrophage/monocyte lineage marker) and TCRγδ receptor. (A) Basic gating strategy showing representative analysis plots to exclude doublets from the populations and gating on live cells. (B) Live cells were gated to obtain macrophage populations and representative plot showing KUL-01 hi and KUL-01 Lo cells along with the bar chart depicting frequencies found across the treatment groups. (C) Live cells were gated to obtain TCRγδ+ populations and representative plot showing γδT cells along with the bar chart depicting frequencies found across the treatment groups. Different letters above the standard-error-of-mean bars within each of the data set graphs indicate significant statistical difference (p < 0.05) between the groups.

Figure 5

Effect of CpG-ODN on T-cell responses in one-day-old chicks. Single-cell splenocyte suspensions were prepared to obtain mononuclear cells and stained with anti-chicken monoclonal antibodies against CD3, CD4, CD8, and CD44 markers. The basic gating strategy included excluding doublets from the populations and gating on live cells. (A) Representative plot showing CD3+ gated population and a quadrant plot gated on CD3+ cells showing T cell subsets, CD4+ (Q3), CD8+ (Q1), and double-positive (DP) (Q2) cells along with the bar chart depicting their cellular frequencies. (B) Representative plots showing cells expressing CD44 gated on live cells to obtain CD3+ cells followed by quadrant gating showing T-cell subsets, CD44+CD4+ (Q3), CD44+CD8+ (Q1), and CD44+ DP (Q2) cells along with the bar chart depicting their cellular frequencies. Different letters above the standard-error-of-mean bars within each of the data set graphs indicate significant statistical difference (p < 0.05) between the groups.