Evaluation of Adjuvant Effect of Cytosine-Guanosine-Oligodeoxynucleotide in Meat-Type Chickens Coadministered In Ovo with Herpesvirus of Turkey Vaccine

Abstract

Herpesvirus of turkey (HVT) increases activation of T cells in 1-day-old chickens when administered in ovo. This study evaluated whether adding cytosine-guanosine oligodeoxynucleotides (CpG ODNs) to the HVT vaccine could enhance the adjuvant effect of HVT. We used a CpG ODN dose of 10 μg per egg. The experimental groups were (1) diluent-only control (sham), (2) HVT, (3) HVT+CpG ODN, (4) HVT+non-CpG ODN, (5) CpG ODN, and (6) non-CpG ODN control. Cellular response evaluation included measuring the frequencies of macrophages (KUL01⁺MHC-II⁺), gamma delta T cells (γδTCR⁺MHC-II⁺), CD4⁺, and CD8⁺ T cell subsets, including double-positive (DP) cells. In addition, CD4⁺ and CD8⁺ T cell activation was evaluated by measuring the cellular expression of major histocompatibility complex class II (MHC-II), CD44 or CD28 costimulatory molecules. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT. The findings showed that (1) HVT vaccination induced significantly higher frequencies of γδ⁺MHC-II⁺ and CD4⁺CD28⁺ T cells when compared with sham chickens. Frequencies of DP and CD4⁺CD28⁺ T cells in HVT-administered birds were significantly higher than those observed in the non-CpG ODN group. (2) Groups receiving HVT+CpG ODN or CpG ODN alone were found to have significantly increased frequencies of activated CD4⁺ and CD8⁺ T cells, when compared with HVT. Our results show that CpG ODN administration in ovo with or without HVT significantly increased frequencies of activated CD4⁺ and CD8⁺ T cells.

Introduction

The Herpesvirus of turkey (HVT) (Witter et al., 1970) is the most commonly used vaccine licensed for commercial use in poultry against Marek's disease (MD) (reviewed in Romanutti et al., 2020). The HVT, formerly known as serotype 3 but currently recognized as Meleagrid alphaherpesvirus 1, along with two additional serologically similar viruses, Gallid alphaherpesvirus 2 (formerly known as serotype 1 or Marek's disease virus-1 [MDV-1]) and Gallid alphaherpesvirus 3 (formerly known as serotype 2 or MDV-2), encompasses the genus Mardivirus (ICTVdB-Management, 2006).

In this study, we refer to these three species as MDV-1, MDV-2, and HVT. The HVT vaccine is typically given either alone to meat-type (broiler) chickens or in combination with MDV-1 and MDV-2 vaccines to egg-type/layer and broiler breeder chickens (Dunn and Gimeno, 2013). Meat-type chickens are commonly vaccinated with HVT through the in ovo route at 18 days of embryonation (ED) (Abdul-Cader et al., 2018b). Chicks vaccinated with HVT through the in ovo route are known to be better protected against challenge with oncogenic MDV-1 than those vaccinated at hatch (Sharma and Graham, 1982).

There are also recombinant monovalent or polyvalent HVT-based vaccines (rHVTs) carrying heterologous viral antigen genes commercially available (reviewed in Romanutti et al., 2020). Our previous research has shown that, in addition to conferring protection against MD, in ovo vaccination with HVT can accelerate the development of immunocompetence in specific pathogen-free avian supplies (SPAFAS) layer (egg-type) chickens (Gimeno et al., 2015) and commercial meat-type chickens (broilers and broiler breeders) (Boone et al., 2023; Boone et al., 2020).

Toll-like receptors (TLRs) constitute a major class of pattern or pathogen recognition receptors, which play a key role in the initiation of host innate immune responses (Kulkarni et al., 2011). Avian TLR21, a functional orthologue of mammalian TLR9, can recognize viral and bacterial DNA containing unmethylated cytosine-guanosine (CpG) motifs and aid in the induction of immune responses (Brownlie et al., 2009). The TLR21 agonist, synthetic CpG oligodeoxynucleotides (CpG ODN), has been successfully used as an immunostimulatory as well as vaccine adjuvant in chickens (Abdul-Cader et al., 2016; St. Paul et al., 2013).

Others and we have previously shown that CpG ODN administered in ovo either alone (Abdul-Cader et al., 2018a; Gunawardana et al., 2019; Thapa et al., 2015) or in combination with monovalent (Bavananthasivam et al., 2018b) and recombinant (Gaghan et al., 2023) HVT vaccines can provide immunostimulatory effects and aid in boosting vaccine-induced responses in chickens.

For example, CpG ODN was shown to reduce MDV-1 replication in vitro (Bavananthasivam et al., 2018b), whereas in ovo CpG ODN administration, either alone (Abdul-Cader et al., 2018a; Gunawardana et al., 2019) or along with a recombinant HVT-laryngotracheitis (rHVT-LT) vaccine (Gaghan et al., 2023), augmented the cellular frequencies of monocytes/macrophages, gamma delta (γδ) T cells, as well as conventional CD8⁺ and CD4⁺ T cells, including their activation in the spleen and lung of 1-day-old chicks.

The objective of this study was to evaluate whether addition of 10 μg per egg of CpG ODN to the HVT vaccine in ovo would enhance the adjuvant effect of HVT. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT. In particular, we have evaluated the cellular frequencies of macrophages (KUL01⁺MHC-II⁺), γδ T cells (γδTCR⁺MHC-II⁺), and CD4⁺/CD8⁺ T cell subsets, including double-positive (DP) T cells. In addition, T cell activation was also measured, as determined by the CD4⁺ and CD8⁺ T cell expression of major histocompatibility complex class II (MHC-II), CD44, or CD28 costimulatory molecules.

Materials and Methods

Chicken and embryonated chicken eggs

Commercially available meat-type chickens (broiler breeders) (Aviagen, Inc., Pageland, SC) were used.

Experimental design

Two experiments were performed. Experiment 1 was performed to assess the effect of in ovo vaccination with HVT, with and without CpG ODN, on humoral immune responses that evaluated IgG responses subsequent to inoculation with keyhole limpet hemocyanin (KLH). Experiment 2 was performed to assess the effect of in ovo vaccination with HVT, with and without CpG ODN, on various splenocyte phenotypes of the innate immune system (monocytes/macrophages, γδ T cells) and those of T cell-mediated immunity.

Both experiments used commercial broiler breeder embryonated chicken eggs. All eggs were incubated and hatched at the College of Veterinary Medicine (North Carolina State University). The two experiments were approved by the institutional animal care and use committee (IACUC). Before in ovo vaccination, eggs were arbitrarily grouped into the different treatment groups. In experiment 1, chickens were wing banded at hatch and then were housed in Animal Biosafety Level 2 floor pens with ad libitum access to water and food. In both experiments, hatchability was evaluated in all groups.

In experiment 1, 131 embryonated eggs were vaccinated with HVT at 18 ED [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated], or not given the vaccine, vaccine diluent, or adjuvant. At hatch, 15 chickens in each group were inoculated through the subcutaneous route with 1 mg of KLH. An additional second dose of 1 mg of KLH was given at 15 days of age and blood was collected a week later for detection of anti-KLH antibodies. An additional 15 chickens that did not receive the vaccine, vaccine diluent, or adjuvant were used as controls in this experiment.

In experiment 2, 563 embryonated eggs were vaccinated with HVT at 18 ED [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated]. At hatch, 70 chickens per group were humanely euthanized with carbon dioxide (CO₂) and splenocytes were collected for flow cytometry.

Each flow sample consisted of spleens collected from 10 chicks and the 10 spleens were pooled per sample, and there was a total of 7 samples per group. Also at hatch, five chickens, per group, were humanely euthanized with CO₂ and spleens and lungs were collected for DNA extraction to monitor vaccine replication.

Vaccine and adjuvant preparation and vaccination procedures

A commercial cell-associated HVT vaccine, strain FC-126, was used. The dose of the vaccine given was 3,040 plaque forming units. A synthetic analogue of the ligand for TLR21, CpG ODN 2007, Class B (5′-TCG TCG TTG TCG TTT TGT CGT T-3′), was used as an adjuvant, as well as non-CpG ODN (5′-TGC TGC TTG TGC TTT TGT GCT T-3′) (InvivoGen, San Diego, CA). Class B CpG ODN consisted of a complete phosphorothioate backbone. The CpG ODN and non-CpG ODN were prepared following the manufacture's recommendation.

A dose of 10 μg of CpG ODN and 10 μg of non-CpG ODN was added to HVT or given alone. We determined this dose from a previous study where we defined which dose could be administered with an rHVT vaccine without affecting titers of the vaccine virus and we also evaluated effect on hatchability (Gaghan et al., 2023). Vaccination with the commercial HVT vaccine alone or with supplementation with CpG ODN and non-CpG ODN, or CpG ODN and non-CpG ODN alone was conducted at 18 ED by the intra-amniotic route, manually, as described (Sharma and Graham, 1982).

KLH enzyme-linked immunosorbent assay

Following the manufacturer's recommendations, a commercial enzyme-linked immunosorbent assay kit, chicken anti-KLH IgG (Alpha Diagnostic International, Inc., San Antonio, TX), was used to assess anti-KLH antibody activity levels (U/mL).

DNA extraction and quantitative polymerase chain reaction

The DNA was extracted from the spleens and lungs by using the Gentra Puregene Tissue kit (QIAGEN, Inc., Germantown, MD) following the manufacturer's recommendations. Real-time polymerase chain reaction (PCR) assay was performed as described previously (Gimeno et al., 2016). Gene-specific primers for a 62-bp fragment that lies between open reading frames HVT072 and HVT073 of the HVT genome (F: 5′-CGGGCCTTACGTTTCACCT-3′ and R: 5′-GCGCCGAAAAGCTAGAAAAG-3′), and chicken glyceraldehyde-3-phosphate dehydrogenase (F: 5′-GGAGTCAACGGATTTGGCC-3′ and R: 5′-TTTGCCAGAGAGGACGGC-3′), were used (Gimeno et al., 2016).

The PCR amplifications were done by using a QuantStudio 3 (Life Technology Holdings, Singapore) in a 20 μL PCR containing 50 ng of DNA and 0.2 μM of each primer. SYBR^® green-based master mix (PowerUp™ SYBR™ Green Master Mix for quantitative polymerase chain reaction (qPCR) workflow, ThermoFisher Scientific Baltics UAB, Vilnius, Lithuania) was used. All reactions were run in triplicates. The relative quantification of the load of HVT DNA was evaluated by the comparative Ct method as reported (Gimeno et al., 2008).

Flow cytometry

Spleens were collected from chicks at hatch and single-cell suspensions were prepared for immunophenotyping as previously described (Laursen et al., 2018). In brief, a total of 10⁶ cells were resuspended in 100 μL fluorescence-activated cell sorting buffer (phosphate-buffered saline with 1% bovine serum albumin) and were plated per well on 96-well round-bottomed plates. Primary antibodies were added to each well (0.5–1 μg/10⁶ cells) and stained for 30 min on ice with fluorescent mouse monoclonal antibodies directed to bind various chicken markers. Table 1 lists all the antibodies and their fluorochrome format used in each of the four panels.

Table 1.

Staining Panels for Flow Cytometry Analysis of 1-Day-Old Meat-Type Chicken Splenocytes

Panel ^a	Antigen	Clone	Fluorochrome
1		—	L/D near IR^b
	Macrophage	KUL01	FITC
	MHC-II	CIa	R-PE
2		—	L/D near IR
	CD3	CT-3	PB™
	CD4	CT-4	R-PE/CY7
	CD8 $^{α}$	CT-8	APC
	MHC-II	CIa	R-PE
3		—	L/D near IR
	CD3	CT-3	PB
	CD4	CT-4	R-PE/CY7
	CD8 $^{α}$	CT-8	APC
	CD44	AV-6	FITC
	CD28	AV-7	R-PE
4	—	—	L/D near IR
	CD45	LT40	APC
	CD3	CT-3	PB
	TCR-γ/δ	TCR 1	R-PE
	MHC-II	CIa	FITC

Four panels were used in this study: panel 1 used antibodies directed against chicken macrophages (KULO1⁺) with surface expression of MHC-II; panel 2 used antibodies directed against chicken CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell subsets with surface expression of MHC-II; panel 3 used antibodies directed against chicken CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell subsets with surface expression of CD44 and CD28; and panel 4 used antibodies directed against chicken TCR gamma delta (γδ) cells with surface expression of MHC-II.

In all panels, exclusion of dead cells was performed by staining the cells with the L/D fixable near-IR dead cell stain (Invitrogen, Carlsbad, CA).

APC, allophycocyanin; CY7, cyanine 7; FITC, fluorescein isothiocyanate; IR, infrared; L/D, Live/Dead™; MHC-II, major histocompatibility complex class II; PB, Pacific Blue; PE, phycoerythrin; TCR, T cell receptor.

All the monoclonal antibodies were purchased from Southern Biotech (Birmingham, AL). In all the panels, cell viability dye, Live/Dead™ near infrared (Invitrogen, Carlsbad, CA), was used to exclude dead cells. Stained cells along with the compensation and staining controls were washed and fixed with 4% paraformaldehyde before data acquisition using an Attune NxT Flow Cytometer (ThermoFisher Scientific). Data analysis was carried out using the FlowJo software (Tree Starr, Ashland, OR). The gating strategy applied to the staining panels included gating mononuclear single-cell splenocytes using forward and side scatter parameters of width (W) and height (H) to exclude doublet cells followed by gating on the live cell population (shown in Figs. 2A –4A).

FIG. 1.

Effect of in ovo vaccination with HVT supplemented with CpG ODN at 18 ED on accelerating the maturation of antibody immune responses in meat-type chickens. A conventional HVT vaccine was given at 3,040 plaque forming units [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated]. Antibody responses were measured 7 days after the second exposure of KLH. Anti-KLH IgG antibody levels (U/mL) were assessed by ELISA. Mean data from 15 chickens are presented. All groups are compared with the HVT treatment group; the error bars represent standard deviation. CpG ODN, cytosine-guanosine oligodeoxynucleotides; ED, days of embryonation; ELISA, enzyme-linked immunosorbent assay; HVT, herpesvirus of turkey; KLH, keyhole limpet hemocyanin.

FIG. 2.

Effect of in ovo vaccination with HVT supplemented with CpG ODN at 18 ED on frequencies of macrophages and $γ δ T$ cells with expression of MHC-II in 1-day-old meat-type chickens. A conventional HVT vaccine was given at 3,040 plaque forming units [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated]. Splenic mononuclear single-cell suspensions were prepared and stained with fluorochrome-conjugated antibodies. (A) The strategy used to gate KUL01⁺ and γδ T cells expressing MHC-II. (B) The frequency of the KUL01⁺MHC-II⁺ cell population in the chicken groups. (C) The frequency of the TCRγδ MHC-II⁺ cell population in the chicken groups. A total of 10 spleens were pooled per sample, and there were 7 samples per group. All groups are compared with the HVT treatment group; **p ≤ 0.01. The error bars represent standard deviation. MHC-II, major histocompatibility complex class II; TCR, T cell receptor.

FIG. 3.

Effect of in ovo vaccination with HVT supplemented with CpG ODN at 18 ED on frequencies of CD4⁺ and CD8⁺ T cell subsets, DP T cells (CD3⁺CD4⁺CD8⁺), and CD4⁺ and CD8⁺ T cells with expression of MHC-II in 1-day-old meat-type chickens. A conventional HVT vaccine was given at 3,040 plaque forming units [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated]. Splenic mononuclear single-cell suspensions were prepared and stained with fluorochrome-conjugated antibodies. (A) The strategy used to gate CD4⁺, CD8⁺, CD3⁺CD4⁺CD8⁺, CD4⁺MHC-II⁺, and CD8⁺MHC-II⁺ T cells. (B) The frequency of the CD4⁺ T cell subset population in the chicken groups. (C) The frequency of the CD8⁺ T cell subset population in the chicken groups. (D) The frequency of the DP T cell population in the chicken groups. (E) The frequency of the CD4⁺ T cell subset population with MHC-II expression in the chicken groups. (F) The frequency of the CD8⁺ T cell subset population with MHC-II expression in the chicken groups. A total of 10 spleens were pooled per sample, and there were 7 samples per group. All groups are compared with the HVT treatment group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The error bars represent standard deviation. DP, double positive.

FIG. 4.

Effect of in ovo vaccination with HVT supplemented with CpG ODN at 18 ED on frequencies of CD4⁺ and CD8⁺ T cell subsets with expression of the activation markers, CD28, and CD44, in 1-day-old meat-type chickens. A conventional HVT vaccine was given at 3,040 plaque forming units [HVT], HVT supplemented with 10 μg CpG ODN [HVT+CpG ODN], HVT supplemented with 10 μg non-CpG ODN [HVT+non-CpG ODN], or 10 μg CpG ODN [CpG ODN], or 10 μg non-CpG ODN [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated]. Splenic mononuclear single-cell suspensions were prepared and stained with fluorochrome-conjugated antibodies. (A) The strategy used to gate CD4⁺ and CD8⁺ T cells with expression of CD28 and CD44. (B) The frequency of the CD4⁺ T cell subset population with CD28 expression in the chicken groups. (C) The frequency of the CD4⁺ T cell subset population with CD44 expression in the chicken groups. (D) The frequency of the CD8⁺ T cell subset population with CD28 expression in the chicken groups. (E) The frequency of the CD8⁺ T cell subset population with CD44 expression in the chicken groups. A total of 10 spleens were pooled per sample, and there were 7 samples per group. All groups are compared with the HVT treatment group; *p ≤ 0.05, **p ≤ 0.01. The error bars represent standard deviation.

Analysis of all samples was performed on the basic “live cell” gate, and the cells expressing mannose receptor C1-like B (monocyte/macrophage lineage, clone KUL01) and MHC-II were gated and referred to hereafter as “macrophages” in panel 1 (Kulkarni et al., 2022). For the γδ T cell analysis (panel 4), the live CD45⁺population followed by CD3⁺ population was used as the backbone gate, cells positive for the T cell receptor (TCR) γδ marker and MHC-II were gated and analyzed. To determine the frequencies of T cell subsets and the T cell subsets with MHC-II expression (panel 2), live CD3⁺ population was used as the backbone gate.

To evaluate T cell activation (panel 3), CD44 and CD28 were used as the target cell activation markers (Baaten et al., 2010; Linterman et al., 2014) such that the live CD44⁺/CD28⁺ cell populations served as the primary population (Cortes-Cerisuelo et al., 2017) from which the CD3⁺ cells (T cells) were gated as the secondary population, and further analyzed for the populations expressing CD4/CD8 markers. Results are presented as percentage instead of absolute numbers since each sample included spleens from ten 1-day old chickens.

Data analysis and statistics

The statistical analyses for all experiments were performed using GraphPad Prism version 10 (GraphPad Software, LLC, Inc.). Data results of the HVT group were compared with all other treatment groups (HVT+CpG ODN, HVT+non-CpG ODN, CpG ODN, and non-CpG ODN) and the sham-inoculated group. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT.

Depending on the data distribution, the nonparametric test (Kruskal–Wallis), or the parametric test, one-way analysis of variance (ANOVA), and the Dunnett's multiple comparison post-test, was performed to identify the differences between groups. Data are presented in the figures as mean ± standard deviation and the results were considered statistically significant at p < 0.05.

Results

Hatchability and HVT vaccine virus replication

In ovo vaccination with HVT alone or combined with CpG ODN and non-CpG ODN on hatchability was assessed and there was no statistical significance found among the groups. The rate of hatchability for the HVT, HVT+CpG ODN, HVT+non-CpG ODN, CpG ODN, non-CpG ODN, and sham-inoculated groups was 91.5%, 85.0%, 90.0%, 85.5%, 84.4%, and 89.6%, respectively (data not shown).

Supplementing 10 μg of CpG ODN and 10 μg of non-CpG ODN to the HVT vaccine did not affect the in vivo virus replication as measured by qPCR in the spleen and lung samples from 1-day-old chickens (Table 2).

Table 2.

Replication of Herpesvirus of Turkey in the Spleen and Lung of Meat-Type Chickens at 1-Day of Age After In Ovo Inoculation at 18 Days of Embryonation

Treatments ^a	Spleen: frequency of positive chickens ^b	Spleen: ΔΔCt ± SD ^c HVT positive samples ^b	Lung: frequency of positive chickens ^b	Lung: ΔΔCt ± SD ^c HVT positive samples ^b	Spleen or lung: frequency of positive chickens ^b
HVT	4/5¹	737.5 ± 119.4¹	5/5¹	786.6 ± 191.7¹	5/5¹
HVT+CpG ODN	3/5¹	811.7 ± 60.7¹	4/5¹	835.1 ± 204.6¹	4/5¹
HVT+non-CpG ODN	3/5¹	718.9 ± 89.2¹	5/5¹	667.3 ± 44.3¹	5/5¹

A conventional HVT vaccine was given at 3,040 PFUs alone or supplemented with 10 μg of CpG ODN (HVT+CpG ODN) or 10 μg of non-CpG ODN (HVT+non-CpG ODN).

Different superscript numbers indicate that differences among groups were statistically significant in the ANOVA test (p < 0.05).

Results are presented as the average ΔΔCt of each group plus or minus the SD.

ANOVA, analysis of variance; CpG ODN, cytosine-guanosine oligodeoxynucleotides; HVT, herpesvirus of turkey; PFUs, plaque forming units; SD, standard deviation.

Antibody response

There were no significant differences in the anti-KLH IgG antibody levels (U/mL) when the HVT treatment group was compared with all other treatments and the sham-inoculated chickens at 21 days of age (Fig. 1).

Cellular responses

To quantitate the effect of different treatments on the splenic innate and the adaptive immune cell responses, the frequencies of macrophages and γδ T cells expressing MHC-II (Fig. 2), as well as different T cell subsets, CD4⁺, CD8⁺, and CD4⁺CD8⁺ (DP) T cells (Fig. 3), were determined. In addition, the activation of CD4⁺ and CD8⁺ T cells, as determined by their surface expression of MHC-II (Fig. 3), CD28, or CD44 molecules (Fig. 4), was also evaluated.

It is also of note here that to elucidate CpG ODN adjuvant-mediated effects, the statistical comparison of the results described below was focused on comparing the changes observed in the HVT-only treatment group with the rest of the groups, including the sham control. The numbers denoted in the parentheses below present the group mean frequency of cell populations.

Macrophages and TCR γδ cells

No significant differences were seen in the frequencies of KUL01⁺MHC-II⁺ cells among HVT and the other treatment groups, as well as the sham-inoculated chickens (Fig. 2B). However, a numerical increase in the frequency of these cells in the groups receiving HVT+CpG ODN (15.2%) or CpG ODN only (16.7%) was observed when compared with the HVT-only (10.2%) treatment. The frequencies of γδ T cells with MHC-II expression (γδMHC-II⁺) were significantly increased in the HVT (17.4%) treatment group when compared with the sham-inoculated chickens (12.3%) (Fig. 2C). No significance was observed in the frequency of γδ T cells in the HVT group when compared with the other treatment groups and sham-inoculated chickens (data not shown).

T cell response

No significant differences were observed in the frequencies of CD4⁺ T cells among HVT and the other treatment groups, as well as the sham-inoculated chickens (Fig. 3B). However, a numerical increase in the frequency of CD4⁺ T cells in the groups receiving HVT+CpG ODN (12.8%) or CpG ODN only (12.1%) was observed when compared with HVT-only (10.7%) treatment. The frequencies of the CD8⁺ T cells in the CpG ODN (47.3%) and non-CpG ODN (48.8%) groups were significantly increased when compared with the HVT (44.0%) group (Fig. 3C).

A significant increase in the frequency of the DP cells in the HVT (23.0%) group was observed when compared with non-CpG ODN (19.5%) group (Fig. 3D). The frequencies of CD4⁺MHC-II⁺ cells were significantly higher in both the HVT+CpG ODN (10.9%) and CpG ODN (9.9%) groups when compared with the HVT (6.5%) group (Fig. 3E). Frequencies of CD8⁺MHC-II⁺ cells were also significantly higher in both the HVT+CpG ODN (21.7%) and CpG ODN (22.2%) groups when compared with the HVT (16.5%) group (Fig. 3F).

The HVT (44.7%) group induced a significantly higher frequency of CD4⁺CD28⁺ cells in comparison with the non-CpG group (34.1%) and sham-inoculated chickens (24.7%) (Fig. 4B). Although not statistically significant, a numerical increase in the frequency of CD4⁺CD28⁺ cells in the groups receiving HVT+CpG ODN (49.3%) was observed when compared with HVT-only (44.7%) or CpG ODN-only (44.3%) treatments. No significant differences were seen in the frequencies of CD4⁺CD44⁺ cells among HVT and the other treatment groups, as well as the sham-inoculated chickens (Fig. 4C). No significant differences were seen in the frequencies of CD8⁺CD28⁺ (Fig. 4D) and CD8⁺CD44⁺ (Fig. 4E) cells among HVT and the other treatment groups, as well as the sham-inoculated chickens.

In addition to the figures presented here, a summary highlighting the significant (p < 0.05) changes in the splenic cellular frequencies in chickens receiving different treatments compared with the group receiving HVT vaccine alone is given in Table 3.

Table 3.

Summary of All Study Results in Meat-Type Chickens When All Groups Are Compared with Herpesvirus of Turkey (Evaluation of Adjuvant Effect)

Treatments ^a	KLH ^b	KUL01⁺ MHC-II⁺ cells ^b	$γ ∕ δ$ MHC-II⁺ cells ^b	CD3⁺ CD4⁺ MHC-II⁺ cells ^b,c	CD3⁺ CD8⁺ MHC-II⁺ cells ^b,c	CD3⁺ CD4⁺ cells ^b	CD3⁺ CD8⁺ cells ^b	CD3⁺ CD4⁺ CD8⁺ cells ^b	CD3⁺ CD4⁺ CD28⁺ cells ^b	CD3⁺ CD8⁺ CD28⁺ cells ^b	CD3⁺ CD4⁺ CD44⁺ cells ^b	CD3⁺ CD8⁺ CD44⁺ cells ^c
Sham	=	=	−	=	=	=	=	=	−	=	=	=
HVT+CpG ODN	=	=	=	+	+	=	=	=	=	=	=	=
HVT+non-CpG ODN	=	=	=	=	=	=	=	=	=	=	=	=
CpG ODN	=	=	=	+	+	=	+	=	=	=	=	=
Non-CpG ODN	=	=	=	=	=	=	+	−	−	=	=	=

An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT.

A conventional HVT vaccine was given at 3,040 PFUs supplemented with 10 μg of CpG ODN (HVT+CpG ODN) or 10 μg of non-CpG ODN (HVT+non-CpG ODN), or 10 μg CpG ODN alone [CpG ODN], or 10 μg non-CpG ODN alone [non-CpG ODN], or 0.1 mL of vaccine diluent only [sham inoculated], and compared with HVT given at 3,040 PFU; all treatments were given at 18 days of embryonation.

All data are compared with HVT, + indicates a statistically significant difference or increased frequency, = indicates no statistically significant difference, and − indicates a statistically significant decrease or reduced frequency.

There were no significant differences among the HVT+CpG ODN and CpG ODN groups for these cell subsets.

CD, cluster of differentiation; KLH, keyhole limpet hemocyanin.

Discussion

In previous studies, we have demonstrated that in ovo vaccination with HVT at 18 ED can hasten the development of immunocompetence in 1-day-old egg-type SPAFAS chickens (Gimeno et al., 2015) and commercial meat-type chickens (Boone et al., 2023; Boone et al., 2020). In ovo vaccination with HVT had positive effects on certain aspects of innate and cell-mediated immunity, specifically T cell activation (Boone et al., 2023). Addition of a TLR3 agonist, polyinosinic-polycytidylic acid, with HVT could enhance the frequency of activated monocytes/macrophages (Boone et al., 2023).

However, addition of other commonly used MD vaccines (ie, CVI-988 or SB-1) did not have any effect on the adjuvant ability of HVT (Boone, article under preparation). In this study, considering the TLR21-mediated immunostimulatory and/or adjuvant effects of CpG ODN in chickens (Abdul-Cader et al., 2018a; Abdul-Cader et al., 2017; Bavananthasivam et al., 2018a; Bavananthasivam et al., 2018b; De Silva Senapathi et al., 2018; Gaghan et al., 2023), the HVT vaccine was given in ovo and supplemented with CpG ODN to evaluate cellular responses in chicks on the day of hatch.

To determine adjuvant-mediated effects, the statistical comparison was focused on comparing the results obtained in the HVT-only treatment group with those of the rest of the groups, including the sham control. An adjuvant effect was considered when HVT+CpG ODN, but not HVT+non CpG ODN, or CpG ODN, or non-CpG ODN, induced significantly increased effects on any of the immune parameters examined when compared with HVT. The results showed that both the HVT+CpG ODN and CpG ODN-only treatments could augment CD4⁺ and CD8⁺ T cell activation, thus, indicating the immunostimulatory effect of CpG ODN.

These changes noted with CD4⁺ and CD8⁺ T cell activation were expected since both T cell subsets, CD4⁺ and CD8α ⁺, express the receptor for CpG (Brisbin et al., 2012), thus, direct interactions between the cells and CpG ODN are possible. However, because CpG ODN with or without HVT had the same effect, it did not fit our definition of adjuvant for this study.

T cell-mediated responses play a critical role in the vaccine-induced protective immunity against avian viral diseases (reviewed in Hao et al., 2021). Our previous study has shown that in ovo HVT vaccination can enhance T cell responses, which included increased splenic frequencies of CD4⁺ and CD8⁺ T cells as well as their activation status, as determined by the T cell upregulation of MHC-II, CD44 or CD28 molecules (Boone et al., 2023; Boone et al., 2020; Gimeno et al., 2015; Gimeno et al., 2012).

MHC-II upregulation on T cells indicates activation of these cells (Holling et al., 2004; Holling et al., 2002), and although CD28 is a costimulatory receptor expressed on the activated T cells (Koskela et al., 1998), CD44 is an adhesion receptor involved in the activated T cell migration between lymphoid tissues and the site of infection (Baaten et al., 2010). In this study, the HVT vaccine given alone was found to significantly increase the frequencies of CD4⁺CD28⁺ cells when compared with the sham-inoculated chickens, as has been reported in a previous study (Boone et al., 2023), and this effect was also evident in the HVT+CpG ODN group.

We also found that the treatments with HVT+CpG ODN vaccine or CpG ODN only led to significantly increased frequencies of CD4⁺ and CD8⁺ T cells expressing MHC-II when compared with the HVT-only treatment group, suggesting treatment-mediated T cell activation. Furthermore, although not statistically significant, a numerical increase in the frequencies of CD4⁺ T cells was observed in the group receiving HVT+CpG ODN when compared with HVT-only treatment. These observations were in support of previous studies that have demonstrated the ability of CpG ODN in enhancing T cell, specifically the CD4⁺ T cell, responses when given in ovo (Abdul-Cader et al., 2018a; De Silva Senapathi et al., 2018; Gaghan et al., 2023; Gunawardana et al., 2019).

Although the treatment with both HVT+CpG ODN and the CpG ODN only produced similar cellular changes, when compared with the HVT-only group, the finding is still noteworthy. This is because the CpG adjuvantation of HVT can not only provide TLR-mediated general immunostimulatory effects but also can enhance vaccine antigen-specific T cell activation, which plays a key role in MD protection. Ample research shows that CpG ODN has been used in combination with other vaccine antigens to enhance protective immune responses (Bavananthasivam et al., 2018b; Mallick et al., 2011; Zhang et al., 2008).

The observation that the administration of HVT+CpG ODN or CpG ODN-only yielded similar effects also raises a possibility of both viral (HVT) DNA and the adjuvant CpG DNA competing for binding to TLR21 that may have caused diminished adjuvant effects (Tarkowski et al., 2010; Zähringer et al., 2008), which needs further investigation. The CD8⁺ T cell analysis in this study showed that the CpG ODN treatment induced a significant increase in the frequency of this cell type when compared with the HVT-only group; however, any inference from this observation remains inconclusive since a similar significant increase in the CD8⁺ T cell population was also seen in the group receiving non-CpG ODN.

A higher frequency of CD8⁺ T cells induced by the CpG ODN treatment may be due to the activation of other DNA sensing mechanisms such as the cytosolic cyclic GMP–AMP synthase system, which is present in chickens and shown to induce broad antiviral activities (Li et al., 2020).

Macrophages and γδ T cells play an important role in avian innate immune responses against many pathogens, including viruses (Elshafiee et al., 2022; Laursen et al., 2018). We have previously shown the ability of HVT to induce significantly higher frequencies of γδ T cells and activated monocytes/macrophages, compared with sham-inoculated chickens (Boone et al., 2023). Enhancement of macrophage and γδ T cell responses in chickens has been reported after the in ovo administration of CpG DNA (Abdul-Cader et al., 2018a; Abdul-Cader et al., 2017; De Silva Senapathi et al., 2018; Gaghan et al., 2023; Gunawardana et al., 2019).

In this study, HVT-only administration induced significantly higher γδ T cells expressing MHC-II when compared with the sham-inoculated chickens; this effect was also evident in the HVT+CpG ODN or CpG ODN-only groups, indicating a treatment-related immunostimulatory effect on these cells. An increase in the frequency of macrophages (KUL01⁺MHC-II) was also observed in the HVT+CpG ODN and CpG ODN groups when compared with the sham control, HVT, HVT+non-CpG, and non-CpG-only groups, suggesting an augmenting effect of CpG ODN or the adjuvanted HVT vaccine on this cell type.

In support of this observation, our recent study has shown that in ovo adjuvantation of CpG ODN with rHVT-LT can increase the splenic frequencies of macrophages in chickens at hatch (Gaghan et al., 2023). Collectively, the analysis of macrophage and γδ T cell populations suggests that CpG ODN can augment innate immune cell responses and that its adjuvantation with HVT may help in the induction of a vaccine-specific host defense. The ability of CpG ODN to augment innate immune cell responses is most likely due to the fact that chicken macrophages and γδ T cells express TLR21 (Alkie et al., 2019).

Another important effect of CpG ODN molecules, specifically those of Class B type, is the activation of B cells (Krug et al., 2001). Chicken B cells can express TLR21 (St. Paul et al., 2012) and that the in ovo inoculation with 50 μg of class B CpG ODN in SPAFAS chickens can induce increased proliferation of B cells in the lungs and spleen of 1-day-old chicks (Abdul-Cader et al., 2018a). However, this study that used 10 μg of Class B CpG ODN found no significant differences in the anti-KLH serum antibody responses between the treatment groups in chickens at 21 days of age.

A previous study also reported that an inoculation of 10 μg of Class B CpG ODN subcutaneously in SPAFAS chickens at 7 and 21 days posthatch resulted in no significant changes in the serum antibody response (St. Paul et al., 2014). Along similar lines, our previous study investigating the in ovo effect of HVT in meat-type chickens also found no significant vaccine-induced effect on the anti-KLH responses (Boone et al., 2023; Boone et al., 2020). The lack of effect on the humoral responses may be related to the dose of CpG ODN (Abdul-Cader et al., 2018a) or the type of the bird (Koenen et al., 2002) used in the studies, and thus warrants further evaluation of effects of CpG ODN on IgM⁺ B cells in chickens.

In our previous study, we found that doses of CpG ODN higher than 10 μg adversely affected the HVT vaccine titers (Gaghan et al., 2023), thus this study chose to use 10 μg dose of the adjuvant, which emphasizes the importance of selecting an appropriate adjuvant dose when combining it with a vaccine. The dose used here did not affect the frequency of chickens positive for replication of the virus or the viral genome load. Based on a previous publication (Boone et al., 2023), the frequency of chickens and viral genome load for the vaccine formulations with and without CpG ODN are within the expected range at day of age in the spleen and lung.

In summary, our results demonstrated that in ovo HVT, when given alone, enhanced innate cellular and T cell activation responses. CpG administration in ovo with or without HVT significantly increased frequencies of activated CD4⁺ and CD8⁺ T cells, when compared with the group that received the HVT vaccine only. Thus, even though CpG ODN was a strong immunomodulant, it did not fit the criteria of adjuvancy to the HVT vaccine in this study. Further studies evaluating cytokine responses in parallel with percentage of immune cells subsets as well as and function of lymphoid cells are warranted.

Footnotes

Acknowledgments

Authors thank personnel at Laboratory Animal Resources for their help in the husbandry of the chickens, Aviagen, Inc. for kindly supplying the embryonated chicken eggs, and Janet Dow and the University of North Carolina at Chapel Hill Flow Cytometry Core Facility for assistance in acquiring the flow data.

Authors' Contributions

A.C.B. contributed to conceptualization, methodology, data curation, formal analysis, investigation, and writing—original draft. R.R.K. was involved in conceptualization, methodology, data curation, investigation, supervision, and writing—review and editing. A.L.C., C.G., J.M. carried out methodology, data curation, investigation, and writing—review and editing.

T.V. took charge of methodology and writing—review and editing. J.E. carried out methodology and writing—review and editing. I.M.G. was in charge of conceptualization, methodology, data curation, investigation, resources, supervision, and writing—review and editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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