Cytosine-Phosphate-Guanine Oligodeoxynucleotides Containing GACGTT Motifs Enhance the Immune Responses Elicited by Keyhole Limpet Hemocyanin Antigen in Dairy Cattle

Abstract

Adjuvants are important components of vaccine formulations. Effective adjuvants line innate and adaptive immunity by signaling through pathogen recognition receptors. Synthetic cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) have been shown to have potentials as adjuvants for vaccines. However, the immunostimulatory effect of CpG is species-specific and depends on the sequence of CpG motifs. A CpG ODN (2135), containing 3 identical copies of GTCGTT motif, was previously reported to have the strongest effects on bovine peripheral blood mononuclear cells (PBMC). Based on the sequence of 2135, we replaced the GTCGTT motif with 11 other sequences containing CG and investigated their effects on bovine lymphocyte proliferation. Results showed that the CpG ODNs containing 3 copies of GACGTT motif had the highest lymphocyte stimulation index (7.91±1.18), which was significantly (P<0.05) higher than that of 2135 (4.25±0.56). The CpG ODNs containing 3 copies of GACGTT motif also significantly increased the mRNA expression of interferon (IFN)-α, interleukin (IL)-12, and IL-21 in bovine PBMC. When dairy cows were immunized with the keyhole limpet hemocyanin (KLH) antigen formulated with CpG ODNs containing 3 copies of GACGTT, production of KLH-specific antibodies in serum and in milk whey was significantly (P<0.05) enhanced. IFN-γ in whole blood stimulated by KLH was also significantly (P<0.05) increased in cows immunized with KLH plus CpG ODNs. Our results indicate that CpG ODNs containing 3 copies of the GACGTT motifs is a potential adjuvant for bovine vaccines.

Introduction

Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) have been shown to induce a spectrum of immunostimulatory responses. Accumulated lines of evidence indicate that Toll-like receptor 9 (TLR9) is involved in the recognition of CpG ODNs (Hemmi et al., 2000; Takeshita et al., 2001; Knuefermann et al., 2007). Interaction between CpG ODNs and TLR9 leads to the phosphorylation and activation of a number of transcription factors, including activator protein (AP)-1 and nuclear factor-κB, which ultimately regulates subsequent receptor expression and cytokine production (Schnare et al., 2001). CpG ODNs have been documented to activate B cells, macrophages, dendritic cells (DC), nature killer (NK) cells, and T-cells. CpG-treated human and murine B cells showed upregulated proliferation, resistance to apoptosis, cytokine production [interleukin (IL)-6 and IL-10 mainly], expression of costimulatory molecules, and antigen-presenting function (Hartmann and Krieg, 2000; Zhu and Marshall, 2001). Macrophages, DC, and other antigen-presenting cells have been indicated to have enhanced expression of class II major histocompatibility complex, costimulatory molecules (CD40, CD80, and CD86), and cytokines [IL-1β, IL-6, IL-12, interferon (IFN)-γ] (Jakob et al., 1998; Stacey et al., 2000). The lytic activity and IFN-γ production of NK cells were also found to be increased after CpG ODN stimulation (Lenert et al., 2003; Roda et al., 2005). Some studies demonstrated that CpG ODNs have direct stimulation of the cytotoxic response of T-cells (Bendigs et al., 1999; Chen et al., 2004; Graham et al., 2007).

The immune responses elicited by CpG ODNs lead to the consideration of using CpG as an adjuvant. Formulation of CpG ODNs in vaccines dramatically increased the production of antigen-specific antibodies and enhanced Th 1 type immunity as characterized by an increased production of IgG₂, secretion of IFN-γ, and lytic activity of cytotoxic T lymphocytes in mice (Klinman et al., 1999; Hunolstein et al., 2000; Chen et al., 2004). Other studies also demonstrated that CpG ODNs, when used as an adjuvant, are less toxic and induce less undesired side effects at the site of injection without compromising the production of IgG₂ in comparison with other adjuvants (Chu et al., 1997; Weiner et al., 1997; Weeratna et al., 2000).

The response of mammalian cells to CpG ODNs is species-specific, presumably due, as least in part, to the recognition by TLR9 in different species. It is in general agreement that the optimal motif for mice and birds is GACGTT, and the most stimulatory motif for other species, including human, bovine, ovine, and swine, is GTCGTT (Zhang et al., 2001, 2008). These claims remain controversial. The first immunostimulatory CpG motif for bovine species, AACGTT, was identified in 1998 (Brown et al., 1998). A sequence containing AACGTT motifs has been demonstrated to increase the secretion of IL-12, tumor necrosis factor (TNF)-α, and nitric oxide in bovine macrophages (Shoda et al., 2001). After testing a panel of 38 CpG ODNs, the sequence that effectively stimulated human cells (2135=TCGTCGTTTGTCGTTTTGTCGTT) was shown to be the optimal sequence for bovine cells in terms of lymphocyte proliferation response and IFN-γ secretion, a marker of Th1 type responses (Pontarollo et al., 2002). However, these CpG ODNs, with different motifs, were not compared based on the same sequence and copies of motifs, which might lead to the biased conclusion that GTCGTT is the optimal motif for bovine cells. In the present study, 11 different hexamer CpG motifs were used to replace GTCGTT in 2135 without changing the flanking sequences and their effects on proliferation of bovine peripheral blood mononuclear cells (PBMC) were analyzed. The CpG ODNs that had the highest stimulation index (SI) in the proliferation assay was further examined for its ability to upregulate the expression of selected cytokine genes by real-time PCR and for adjuvant activity in cattle vaccinated with a model antigen, keyhole limpet hemocyanin (KLH).

Materials and Methods

In vitro studies

CpG oligodeoxynucleotides

The ODNs were synthesized (Mission Biotech) based on the sequence of ODN 2135 (TC-GTCGTT-T-GTCGTT-TT-GTCGTT), which was previously demonstrated to induce strong immunostimulatory effects on bovine PBMC (Pontarollo et al., 2002). Totally 11 different motifs were randomly selected to replace GTCGTT. All CpG ODNs contained 3 copies of the active motif on the nuclease-resistant phosphorothioate backbone (Table 1). A sequence of ODN containing 3 copies of GTGCTT was used as the control CpG (Ctrl ODN). All ODNs were resolved in sterile nonpyrogenic water at a concentration of 250 μM and stored at −70°C.

Table 1.

List of Cytokine-Phosphate-Quanine Oligodeoxynucleotides Synthesized on a Phosphorothioate Backbone Used in the Lymphocyte Proliferation Assay

CpG ODNs	Sequence (5′–3′)
Ctrl ODN	TC-GTGCTT-T-GTGCTT-TT-GTGCTT
GTCGTT	TC-GTCGTT-T-GTCGTT-TT-GTCGTT
ATCGAT	TC-ATCGAT-T-ATCGAT-TT-ATCGAT
GTCGAT	TC-GTCGAT-T-GTCGAT-TT-GTCGAT
ATCGGT	TC-ATCGGT-T-ATCGGT-TT-ATCGGT
GTCGGT	TC-GTCGGT-T-GTCGGT-TT-GTCGGT
GCCGTT	TC-GCCGTT-T-GCCGTT-TT-GCCGTT
ACCGTT	TC-ACCGTT-T-ACCGTT-TT-ACCGTT
ATCGTT	TC-ATCGTT-T-ATCGTT-TT-ATCGTT
AGCGTT	TC-AGCGTT-T-AGCGTT-TT-AGCGTT
GGCGTT	TC-GGCGTT-T-GGCGTT-TT-GGCGTT
GACGTT	TC-GACGTT-T-GACGTT-TT-GACGTT
AACGTT	TC-AACGTT-T-AACGTT-TT-AACGTT

CpG, cytokine-phosphate-quanine; ODN, oligodeoxynucleotides.

Isolation of PBMC

Bovine PBMC were isolated according to the method previously described (Mehrzad et al., 2008) with modifications. Briefly, Heparin-anticoagulated blood was mixed with an equal volume of Ca²⁺-free Hank's balanced salt solution (HBSS; Gibco/BRL), carefully layered onto Ficoll-Paque Plus (GE Healthcare), and centrifuged at 1500 g for 40 minutes at 4°C. The buffy coat was washed with HBSS and suspended in RPMI (Roswell Park Memorial Institute) 1640 (Gibco/BRL) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco/BRL), 1% antibiotic–antimycotic solution (Sigma-Aldrich Co.), and 1% l-glutamine at the concentration of 10⁷ cells/mL.

Lymphocyte proliferation assay

Lymphocyte proliferation assay was carried out as described (Mehrzad and Zhao, 2008) with modifications. Freshly isolated PBMC were suspended in RPMI 1640 supplemented with 10% heat inactivated FBS), 1% antibiotic–antimycotic solution, 1% l-glutamine, and 0.05 M 2-mercaptoethanol (Sigma) and added to 96-well plates at 10⁵ cells/well with the presence of various CpG ODNs (3 μM) or Con A (2 μg/mL; Sigma) in the final volume of 100 μL. Each treatment was repeated in triplicates and the plates were incubated at 37°C, 5% CO₂, for 54 hours. Thereafter, the cells were pulsed with [methyl-³H] thymidine (0.4 μCi/well) and incubated for additional 18 hours before being harvested. The radioactivity of each sample was measured using a liquid scintillation counter. The SI was calculated by dividing the mean counts per minute (CPM) of each treatment over that of the medium control.

In vitro stimulation, RNA extraction, and reverse transcription

Bovine PBMC were added to 24-well plates (3×10⁶ cells/well) with or without selected CpG ODNs (3 μM) and incubated at 37°C, 5% CO₂, for 6 hours. PBMC were centrifuged and total RNA was extracted using Trizol (Invitrogen) according to the manufacturer's instructions. The reverse transcription (RT) reaction was started by adding 2 μg of total RNA and 0.5 μg of oligo(dT_12-18) to 12 μL of water, then heating at 70°C for 10 minutes. After the samples were cooled on ice, 10 mM dithiothreitol, 0.5 mM of each deoxynucleotide triphosphate (dNTP), 5× first strand buffer, and 200 U Superscript II RNase H⁻ Reverse Transcriptase (Gibco/BRL) were added. The mixture was stabilized at 25°C for 10 minutes and subsequently incubated at 42°C for 50 minutes for the RT reaction to proceed. Thereafter, the temperature was raised to 70°C for 15 minutes to inactivate the reverse transcriptase. Synthesized cDNA was examined for its quality and stored at −20°C until being used for real-time PCR.

Quantitative real-time PCR

Amplification by LightCycler real-time PCR was carried out as described (Lee et al., 2006). Primers for specific bovine genes are listed in Table 2. The reaction conditions for each gene were optimized using a QuantiTect SYBR Green PCR kit (Qiagen) in a LightCycler system (Roche) and applied to the following protocol. The cDNA was analyzed in 20 μL PCR mixture containing a final concentration of 0.5 μM primer, 1 μL of cDNA, and 2× QuantiTect SYBR green PCR master mix. The PCR master mix contained HotStartaq DNA polymerase, SYBR green PCR buffer, and dNTP mix including dUTP, SYBR green I, ROX (passive reference dye), and MgCl₂ (2.5 mM). The PCR mixture was added into a cold PCR capillary, centrifuged, and placed in the LightCycler system. The LightCycler was programmed in 3 steps: (1) denaturation at 95°C for 15 minutes; (2) amplification for 50 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for a duration depending on the product length (5 seconds per 100 bp); and (3) melting curve analysis at 95°C for 5 seconds, 65°C for 15 seconds and 95°C for 40 seconds, followed by cooling at 40°C. In each reaction, the cycle number at which the fluorescence rose appreciably above background fluorescence was determined as the crossing point (CP).

Table 2.

Sequences of Primers for Bovine Cytokines and Toll-Like Receptor 9 Used in Real-Time PCR

Gene	Primer	Sequence (5′–3′)	Product length	Accession no.
β-actin	F38	CCT TTT ACA ACG AGC TGC GTG TG	391	AH00130
	R428	ACG TAG CAGAGC TTC TCC TTG ATG
IFN-α	F368	ACT TCC ACA GAC TCA CTC TC	124	Z46508
	R491	CTC CTG AAA CTC TCC TGC AA
IFN-γ	F296	TTC AGA GCC AAA TTG TCT CC	185	M29867
	R480	CTG GAT CTG CAG ATC ATC CA
IL-6	F209	TCA TTA AGC GCA TGG TCG ACA AA	105	NM173923
	R313	TCA GCT TAT TTT CTG CCA GTG TCT
IL-12	F623	TTA TTG AGG TCG TGG TAG AAG CTG	115	U11815
	R737	GGT CTC AGT TGC AGG TTC TTG G
IL-21	F62	CAG TGG CCC ATA AGT CAA GC	137	AB073021
	R198	TAC ATC TTC TGG AGC TGG CA
TNF-α	F2377	TCT TCT CAA GCC TCA AGT AAC AAG C	418	AF011926
	R2794	CCA TGA GGG CAT TGG CAT AC
TLR9	F259	TGG CTG TTC CTG AAG TCT GT	169	NM183081
	R353	AAG TGG TGG ATG CGG TTG GA

IFN, interferon; IL, interleukin; TLR9, Toll-like receptor 9.

Relative quantification

The expression of each gene was analyzed using a relative quantification method. A slope was determined from the exponential phase, under the optimized real-time PCR amplification conditions, of each target gene or the reference gene (bovine β-actin). The amplification efficiency (E) was calculated based on the slope, where E=10^[−1/slope]. The expression of selected genes was calibrated to that of β-actin, for each treatment and converted to the relative gene expression ratio, where \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes {10} {9} {7} {6} \begin{document} \begin{align*} \hbox{Relative gene expression ratio} = \frac{(E_{\rm target})^{(\hbox{Control CP}_{\rm target} - \hbox{treatment CP}_{\rm target})}}{({\rm E}_{\rm ref})^{(\hbox{Control CP}_{\rm reference} - {\rm treatment\ CP}_{\rm reference})}} \end{align*} \end{document}

In vivo studies

Animals

Sixteen 2–3-year-old, pregnant, and clinically healthy Holstein cows in their late stage of lactation were selected based on milk somatic cell counts (<500,000 cells/mL) and the absence of bacteria in aseptically collected milk samples. All cows were housed in a free-stall enclosed barn (located at Pingtung, Taiwan) and milked twice daily. The use and care of all animals in this study were approved by the Animal Care and Use Committee of National Pingtung University of Science and Technology.

Immunization protocol

Vaccines were prepared by emulsifying Freund's incomplete adjuvant (FICA; Sigma) with an equal volume of KLH (Sigma) with or without CpG ODNs in nonpyrogenic water (Table 3). The total volume for each dose of immunization was 2 mL. Animals were randomly assigned into 4 groups (n=4 each): (1) FICA (the control); (2) KLH+FICA; (3) KLH+FICA+CpG; (4) KLH+FICA+cCpG. Cows received the primary immunization (D0) subcutaneously at the right side of the neck and were boosted with the same formulation 2 weeks later (D14) at the left side of the neck. Serum samples were prepared from blood collected on D0, D7, D14, D21, D28, D35, D70, and D140 for analysis of KLH-specific antibody. Heparin-anticoagulated whole blood was collected from the tail vein on D0, D14, and D28 for PBMC isolation and IFN-γ assay. Milk samples (20 mL) were collected on D0, D7, D14, D21, D28, and D35 before the cows moved to the dry period. Milk samples were poured into 40 mL round-bottom centrifuge tubes and centrifuged at 44,000 g for 30 minutes at 4°C. After centrifugation, the fat layer was removed and the skimmed milk was carefully decanted into new centrifuge tubes followed by centrifugation at 44,000 g for 30 minutes at 4°C. The clear whey was collected and stored in aliquots at −20°C.

Table 3.

Composition of Vaccine Formulations

Vaccine group	FICA	KLH	CpG ^a	cCpG ^b	Total volume
(1) FICA	1 mL	—	—	—	2 mL
(2) FICA+KLH	1 mL	2 mg	—	—	2 mL
(3) FICA+KLH+CpG	1 mL	2 mg	0.5 mg	—	2 mL
(4) FICA+KLH+cCpG	1 mL	2 mg	—	0.5 mg	2 mL

CpG ODN=TC-GACGTT-T-GACGTT-TT-GACGTT.

cCpG ODN=TC-GAGCTT-T-GAGCTT-TT-GAGCTT.

cCpG, control CpG; FICA, Freund's incomplete adjuvant; KLH, keyhole limpet hemocyanin.

Detection of KLH-specific antibodies in serum and milk

The antibody titer was measured by ELISA as described (Lee et al., 2005) with modifications. Briefly, 96-well flat-bottom plates were coated with KLH (1 μg/mL) and incubated overnight at 4°C. Then, the coating solution was removed followed by blocking the plates with 2% gelatin (Sigma) in PBS at 37°C for 1 hour. After washing the plates twice with PBS-T (containing 0.05% Tween 20), samples diluted with PBS-T (1:10,000 and 1:100 for serum and whey samples, respectively) were added into the wells. The plates were incubated at 4°C overnight. Thereafter, the plates were washed 3 times, and incubated with rabbit anti-bovine IgG₁, IgG₂, or IgM (Bethyl Laboratories, Inc.), diluted in PBS containing 0.2% Tween 20 for 1 hour at 37°C. After washing, alkaline-phosphatase-labeled sheep anti-rabbit IgG (1:2000) (Kirkegaard and Perry Laboratories, Inc.) was added to the wells and incubated for another 1 hour at 37°C. Substrate was added and the plates were read at OD₄₀₅ nm.

Interferon-γ

A bovine IFN-γ ELISA kit (BioSource International) was used to determine the production of IFN-γ in whole blood in response to KLH. Briefly, 100 μL of KLH in RPMI 1640 (150 μg/mL) was added to 500 μL whole blood from each cow. The mixture was incubated at 37°C, 5% CO₂, for 24 hours and centrifuged at 1700 g, 4°C, for 20 minutes. The supernatant was collected for analyzing the level of IFN-γ according to the manufacture's instructions.

Statistical methods

All data were analyzed by the statistical software, SAS (Version 9.0). For lymphocyte proliferation assay and the relative gene expression ratio, differences between the treatments were analyzed by analysis of variance (ANOVA) and compared by Duncan's multiple comparison. For the antibody responses and IFN-γ secretion, data were analyzed by ANOVA and Duncan's multiple comparison was used to test the significance between treatment groups throughout the study and at each time point. A P value of <0.05 was considered significant.

Results

In vitro studies

Lymphocyte proliferation in response to different CpG motifs

CpG ODNs containing 3 copies of GTCGTT, AGCGTT, GGCGTT, and GACGTT motif significantly (P<0.05) increased bovine lymphocyte proliferation in comparison with the Ctrl ODN (Fig. 1). GACGTT motif had the highest SI (7.91±1.18) and was significantly (P<0.05) higher than that of GTCGTT (SI=4.25±0.56).

FIG. 1.

Stimulation of bovine lymphocyte proliferation by CpG oligodeoxynucleotides (ODNs) (3 μM) containing different motifs as listed in Table 1. The stimulation index (SI) was calculated by dividing the mean counts per minute (CPM) of each treatment by that of the medium control. Data are represented as the mean SI±SEM of 8 cows. *The mean SI is significantly different from that of the control ODN (Ctrl ODN) at P<0.05.

Upregulation of cytokines and TLR9 induced by GTCGTT and GACGTT CpG ODNs in bovine PBMC

As shown in Fig. 2, AGCGTT and GGCGTT CpG ODN did not upregulate mRNA expression of these genes. GTCGTT CpG ODN only significantly (P<0.05) increased the expression of IL-12 in comparison with the control. On the other hand, GACGTT CpG ODN significantly (P<0.05) upregulated the expression of IFN-α, IL-12, and IL-21. Neither IFN-γ nor IL-6 mRNA expression was significantly increased by the CpG ODNs. The PBMC stimulated by GACGTT CpG ODN had a significantly (P<0.05) increased gene expression of TNF-α and TLR9. However, the increase was relatively small (relative gene expression ratio was <2).

FIG. 2.

The expression of cytokine and Toll-like receptor 9 (TLR9) genes in bovine peripheral blood mononuclear cells (PBMC) stimulated by CpG ODNs containing 3 copies of GTCGTT, AGCGTT, GGCGTT, or GACGTT motif. Freshly isolated bovine PBMC were incubated with each CpG ODN (3 μM) for 6 hours. The expression of the target gene was calibrated by that of the reference gene, bovine β-actin, and converted to the relative gene expression ratio. Data are presented as the mean±SEM. of 5 cows and only means >2 are valid for statistical analysis. Treatments with different superscript letters in the same group are significantly (P<0.05) different. The group of bars without superscript letters indicates that the treatments were not significantly different.

In vivo studies

Antibody responses against KLH antigen in serum

The CpG ODN containing 3 copies of GACGTT motif was formulated with KLH antigen to examine its effect on antibody responses and antigen-induced IFN-γ secretion in dairy cattle. The responses of KLH-specific antibodies, including total IgG, IgG₁, IgG₂, and IgM, in serum were illustrated in Fig. 3. In cows immunized with KLH-antigen, all the subclasses of KLH-specific antibody peaked on D21 or D28, which were 1 or 2 weeks after the boost (D14). Formulation of CpG ODN containing 3 copies of GACGTT motif with KLH antigen significantly (P<0.05) increased the production of KLH-specific total IgG, IgG₁, IgG₂, and IgM in serum in comparison with other formulations and the control. Moreover, the antibody response (for all subclasses) in cows immunized with KLH+FICA+CpG was more persistent, which remained significantly (P<0.05) higher than other treatments and the control until the end of the study (D140), except IgG₁. The antibody response of cows immunized with KLH+FICA+cCpG was not significantly different from that of cows immunized with KLH+FICA. In addition, cows immunized with KLH+FICA or KLH+FICA+cCpG did not have an increased production of KLH-specific IgG₂ in serum.

FIG. 3.

The response of keyhole limpet hemocyanin (KLH)-specific (A) total IgG, (B) IgG₁, (C) IgG₂, and (D) IgM in serum as determined by ELISA. Cows were immunized twice on D0 and D14 with (1) Freund's incomplete adjuvant (FICA), (2) KLH+FICA, (3) KLH+FICA+CpG, or (4) KLH+FICA+control CpG (cCpG). Data are expressed as the mean±SEM of the OD values read at 405 nm from 4 animals at each time point. Treatments with different superscript letters in the legend box are significantly (P<0.05) different throughout the study. *The mean OD value is significantly different from that of the adjuvant control (FICA) at P<0.05.

Antibody responses against KLH antigen in milk

The response of KLH-specific antibodies, including total IgG, IgG₁, IgG₂, and IgM, in milk was illustrated in Fig. 4. Formulation of CpG ODN containing 3 copies of GACGTT motif with KLH antigen significantly (P<0.05) increased the production of KLH-specific total IgG, IgG₁, and IgM in milk in comparison with other formulations and the control. Cows vaccinated with KLH+FICA or KLH+FICA+cCpG only significantly (P<0.05) increased the production of KLH-specific IgG₁ in milk. None of the formulations increased the production of KLH-specific IgG₂ in milk.

FIG. 4.

The response of KLH-specific (A) total IgG, (B) IgG₁, (C) IgG₂, and (D) IgM in whey as determined by ELISA. Cows were immunized twice on D0 and D14 with (1) FICA, (2) KLH+FICA, (3) KLH+FICA+CpG, or (4) KLH+FICA+cCpG. Data are expressed as the mean±SEM of the OD values read at 405 nm from 4 animals at each time point. Treatments with different superscript letters in the legend box are significantly (P<0.05) different throughout the study. *The mean OD value is significantly different from that of the adjuvant control (FICA) at P<0.05.

Production of IFN-γ in whole blood incubated with KLH

Antigen-specific secretion of IFN-γ in whole blood was measured on D0, D14, and D28 using a commercially available ELISA kit. As shown in Fig. 5, the secretion of IFN-γ in whole blood after KLH stimulation was significantly (P<0.05) increased in cows immunized with KLH+FICA+CpG on D14 in comparison with other formulations and the control. On D28, the secretion of IFN-γ in whole blood after KLH stimulation in cows immunized with KLH+FICA+CpG was still significantly (P<0.05) higher than those in cows received FICA and KLH+FICA+cCpG, but not KLH+FICA.

FIG. 5.

The expression of interferon (IFN)-γ mRNA in whole blood stimulated by KLH (25 μg/mL). Cows were immunized twice on D0 and D14 with (1) FICA, (2) KLH+FICA, (3) KLH+FICA+CpG, or (4) KLH+FICA+cCpG, and whole blood samples were taken on D0, D14, and D28. Data are presented as the mean±SEM of the OD values read at 405 nm from 4 animals. *The mean OD value is significantly (P<0.05) different from that of cows immunized with the adjuvant control (FICA).

Discussion

Eleven different motifs were used to replace GTCGTT without changing the flanking region in 2135 (TCGTCGTTTGTCGTTTTGTCGTT) and their stimulatory effects on bovine lymphocyte proliferation were examined based on the same general structure. Results showed that GACGTT had the highest SI (7.91±1.18), which was higher (P<0.05) than that of 2135 (4.25±0.56). CpG ODNs containing GACGTT were highly stimulatory for mice (Krieg et al., 1995) and rabbits (Rankin et al., 2001), and show only limited effects in humans (Hartmann and Krieg, 2000). Our results indicated that when all the CpG ODNs were synthesized with exactly the same structure, the one with 3 copies of GACGTT motif had the highest SI in the bovine lymphocyte proliferation assay. This surprising result warrants further studies.

Our results on cytokine and TLR9 gene expression by bovine PBMC also supported the notion that CpG ODN containing 3 copies of GACGTT motifs was more stimulatory than other CpG ODNs. GACGTT demonstrated stimulatory effects similar to those of 2135 (Fig. 2). In addition, GACGTT, but not 2135, significantly increased IFN-α mRNA expression even though the relative gene expression ratio was only slightly over 2. It is known that PBMC respond differently to various classes of CpG ODNs. The structure of CpG ODNs used in our study belongs to the K (or referred as B) type, which only induces minimum expression of IFN-γ and IFN-α in human and bovine PBMC (Verthelyi et al., 2001; Mena et al., 2003). The expression of IL-12 was significantly upregulated in PBMC stimulated by GACGTT and GTCGTT CpG ODNs. A stronger induction of IFN-α and IL-12 may be beneficial to enhancing Th1 type immune responses when the CpG ODN is formulated to a vaccine as an adjuvant.

To the best of our knowledge, this is the first investigation on the expression of IL-21 in response to CpG ODN stimulation. IL-21 is a crucial cytokine for the function of B cells, the cytotoxicity of lymphocytes, and the development and differentiation of NK cells (Parrish-Novak et al., 2000; EBERT, 2009; Konforte et al., 2009). CpG ODNs containing GACGTT, but not GTCGTT, motifs significantly increased the expression of IL-21. For IL-6, the induction of mRNA in CpG ODN-stimulated bovine PBMC was not significant in our assay. The expression of IL-6 in bovine PBMC was upregulated after stimulation with 10 μM CpG ODNs (Zhang et al., 2001). The concentration applied in our study was only 3 μM, which might have compromised the stimulatory effect. In addition, large variation among individuals in response to CpG ODNs was reported in humans, cows, and pigs (Kamstrup et al., 2001; Rankin et al., 2001; Leifer et al., 2003). The cellular receptor for CpG ODNs is TLR9 (Bauer et al., 2001). The expression of TNF-α and TLR9 was slightly increased in PBMC stimulated by the GACGTT, but not GTCGTT, CpG ODN. However, the relative gene expression ratio was relatively small (<2). In general, bovine PBMC treated by the GACGTT motif had more significantly upregulated cytokine genes than those treated by the GTCGTT motif. A report in humans indicated that genetic polymorphisms of TLR9 influenced the immune response to CpG ODNs (Kikuchi et al., 2005). Whether this is the case for bovine species requires further investigations.

Due to the in vitro effects of CpG ODN containing 3 copies of GACGTT motif, it was formulated with the KLH antigen to examine its effect on responses of KLH-specific antibodies and antigen-induced IFN-γ secretion in dairy cattle. The production of KLH-specific total IgG, IgG₁, IgG₂, and IgM in serum was elevated (P<0.05) in cows immunized with KLH+FICA+CpG in comparison with other vaccine formulations and the adjuvant control. It is noteworthy that an increased (P<0.05) production of IgG₂ in serum was only observed in cows immunized with KLH+FICA+CpG, but not KLH+FICA and KLH+FICA+cCpG, in comparison with the control. Several previous studies have demonstrated that CpG ODNs containing 3 copies of GTCGTT motifs (assigned as 2006, 2007, and 2135) enhanced both humoral and cell-mediated immune responses of vaccines in bovine species (Ioannou et al., 2002; Rankin et al., 2002; Zhang et al., 2003; Wedlock et al., 2005a, 2005b; Mapletoft et al., 2006). Among all the subclasses of antibody, IgG₂ has been the isotype that is analyzed most frequently as an indication of Th1 immune responses. CpG ODNs, when used as the adjuvant, is a strong inducer of Th1 immune responses, as characterized by increased production of IgG₂ and IFN-γ expression in immune cells. Our results showed that CpG ODNs with GACGTT motifs not only enhanced the production of antigen-specific IgG₁ and IgM, but also IgG₂ which was not increased by other formulations. Both IgM and IgG₂ are crucial opsonins for bovine phagocytes. Further, the responses of KLH-specific antibodies in serum were significantly much more persistent in cows immunized with KLH+FICA+CpG and remained high until 140 days after the primary immunization. Similar results were also reported in a recent study (Kovacs-Nolan et al., 2009), where CpG ODN alone, or in conjunction with other types of adjuvant, significantly induced long-lasting humoral responses in cows immunized with a hen egg lysozyme antigen.

The levels of KLH-specific total IgG, IgG₁, and IgM, in milk were also elevated (P<0.05) in cows immunized with KLH+FICA+CpG. The increase of total IgG in milk was almost completely due to the increased level of IgG₁ because they had very similar kinetic profiles. Immunization with KLH+FICA or KLH+FICA+cCpG only increased the level of KLH-specific IgG₁ in milk, but not other subclasses. None of the vaccine formulations raised the level of KLH-specific IgG₂ in milk. The concentration of antibodies in normal milk drops dramatically, as much as ∼100-fold lower, in comparison with those in colostrum and serum. Therefore, it was not surprising to see diminished antigen-specific antibody responses in milk. In bovine milk, the ratio of IgG₂/IgG₁ is much lower than that in serum. IgG₂ has a higher affinity to FcRn, the receptor responsible for transportation of IgG across blood/milk barrier, and is recycled from milk back to blood at a faster rate (Medesan et al., 1998; Cianga et al., 1999). Therefore, the concentration of IgG₂ is very limited in milk, which might not be able to reflect the increase of KLH-specific IgG₂ in serum. In milk, the transient nature of IgM production was more obvious. The level of IgM dropped to that close to the basal level on D28.

The addition of CpG ODN with GACGTT motifs directed the immune responses to type Th1 as implied by the data of IFN-γ secretion in whole blood. This is in agreement with several other studies that used CpG ODN to enhance the efficacy of vaccines in bovine species (Rankin et al., 2002; Zhang et al., 2003; Wedlock et al., 2005b; Mapletoft et al., 2006). CpG ODNs did not increase the mRNA expression of IFN-γ in the in vitro assay. However, they significantly increased the expression of IL-12, which plays an important role in the development of Th1 immune cells, especially during the early stage of immunization.

Our results indicate that the CpG ODN containing 3 copies of GACGTT motif is a strong inducer of bovine lymphocyte proliferation, upregulated cytokine gene expression in bovine PBMC, and enhanced production of opsonic antibodies and secretion of IFN-γ. Although these preliminary data are still inconclusive, application of this CpG ODN as an adjuvant requires further investigations and may have positive effects on vaccines for dairy cows.

Footnotes

Acknowledgments

This study was supported by Grant NSC 95-2313-B-020-006 from the National Science Council of Taiwan and grants from the Dairy Cattle Genetics Research and Development Council of the Canadian Dairy Network and the Natural Sciences and Engineering Research Council of Canada.

Author Disclosure Statement

No competing financial interests exist.

References

BAUER

, KIRSCHNING

C.J.

, HACKER

, REDECKE

, HAUSMANN

, AKIRA

, WAGNER

, LIPFORD

G.B.

2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. U. S. A., 98:9237–9242.

BENDIGS

, SALZER

, LIPFORD

G.B.

, WAGNER

, HEEG

1999. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells. Eur. J. Immunol., 29:1209–1218.

BROWN

W.C.

, ESTES

D.M.

, CHANTLER

S.E.

, KEGERREIS

K.A.

, SUAREZ

C.E.

1998. DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for bovine B cells. Infect. Immun., 66:5423–5432.

CHEN

Y.F.

, LIN

C.W.

, TSAO

Y.P.

, CHEN

S.L.

2004. Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumor growth in C57BL/6 mice. J. Virol., 78:1333–1343.

CHU

R.S.

, TARGONI

O.S.

, KRIEG

A.M.

, LEHMANN

P.V.

, HARDING

C.V.

1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med., 186:1623–1631.

CIANGA

, MEDESAN

, RICHARDSON

J.A.

, GHETIE

, WARD

E.S.

1999. Identification and function of neonatal Fc receptor in mammary gland of lactating mice. Eur. J. Immunol., 29:2515–2513.

EBERT

E.C.

2009. Interluekin-21 up-regulates perforin-mediated cytotoxic activity of human intra-epithelial lymphocyte. Immunology, 127:206–215.

GRAHAM

S.P.

, SAYA

, AWINO

, NGUGI

, HYANJUI

J.K.

, HECKER

, TARACHA

E.L.N.

, NENE

2007. Immunostimulatory CpG oliodeoxynucleotides enhance the induction of bovine CD4+ cytotoxic T-lymphocyte responses against the polymorphic immunodominant molecule of the protozoan parasite Theileria parva. Vet. Immunol. Immunopathol., 115:383–389.

HARTMANN

, KRIEG

A.M.

2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol., 164:944–952.

10.

HEMMI

, TAKEUCHI

, KAWAI

, KAISHO

, SATO

, SANJO

, MATSUMOTO

, HOSHINO

, WAGNER

, TAKEDA

, SHIZO

2000. A Toll-like receptor recognizes bacterial DNA. Nature, 408:740–745.

11.

HUNOLSTEIN

C.V.

, TELONI

, MARIOTTI

, RECCHIA

, OREFICI

, NISINI

2000. Synthetic oligodeoxynucleotide containing CpG motif induces an anti-polysaccharide type 1-like immue response after immunization of mice with Haemophilus influenzae type b conjugate vaccine. Int. Immunol., 12:295–303.

12.

IOANNOU

X.P.

, GRIEBEL

, HECKER

, BABIUK

L.A.

, VAN DRUNEN LITTLE-VAN DEN HURK

2002. The immunogenicity and protective efficacy of bovine herpesvirus 1 glycoprotein D plus emulsigen are increased by formulation with CpG oligodeoxynucleotides. J. Virol., 76:9002–9010.

13.

JAKOB

, WALKER

P.S.

, KRIEG

A.M.

, UDEY

M.C.

, VOGEL

J.C.

1998. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role of dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J. Immunol., 161:3042–3049.

14.

KAMSTRUP

, VERTHELYI

, KLINMAN

D.M.

2001. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. Vet. Microbiol., 78:353–362.

15.

KIKUCHI

, LIAN

Z.-X.

, KIMURA

, SELMI

, YANG

G.X.

, GORDON

S.C.

, INVERNIZZI

, PODDA

, COPPEL

R.L.

, ANSARI

A.A.

, IKEHARA

, MIYAKAWA

, GERSHWIN

M.E.

2005. Genetic polymorphisms of toll-like receptor 9 influence the immune response to CpG and contribute to hyper-IgM in primary biliary cirrhosis. J. Autoimmun., 24:347–352.

16.

KLINMAN

D.M.

, BARNHART

K.M.

, CONOVER

1999. CpG motifs as immune adjuvants. Vaccine, 17:19–25.

17.

KNUEFERMANN

, BAUMGARTEN

, KOCH

, SCHWEDERSKI

, VELTEN

, EHRENTRAUT

, MERSMANN

, MEYER

, HOEFT

, ZACHAROWSKI

, GROHé

2007. CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo. Respir. Res., 8:72.

18.

KONFORTE

, SIMARD

, PAIGE

C.J.

2009. IL-21: an executor of B cell fate. J. Immunol., 182:1781–1787.

19.

KOVACS-NOLAN

, MAPLETOFT

J.W.

, LATIMER

, BABIUK

L.A.

, VAN DRUNEN LITTEL-VAN DEN HURK

2009. CpG oligonucleotide, host defense peptide and polyphosphazene act synergistically, inducing long-lasting, balanced immune responses in cattle. Vaccine, 27:2048–2054.

20.

KRIEG

A.M.

, YI

A.K.

, MATSON

, WALDSCHMIDT

T.J.

, BISHOP

G.A.

, TEASDALE

, KORETZKY

G.A.

, KLINMAN

D.M.

1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature, 374:546–549.

21.

LEE

J.-W.

, BANNERMAN

D.D.

, PAAPE

M.J.

, HUANG

M.-K.

, ZHAO

2006. Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR. Vet. Res., 37:219–229.

22.

LEE

J.-W.

, O'BRIEN

C.N.

, GUIDRY

A.J.

, PAAPE

M.J.

, SHAFER-WEAVER

K.A.

, ZHAO

2005. The effect of a trivalent vaccine against Staphylococcus aureus mastitis on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis. Can. J. Vet. Res., 69:11–18.

23.

LEIFER

C.A.

, VERTHELYI

, KLINMAN

D.M.

2003. Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides. J. Immunol., 26:313–319.

24.

LENERT

, RASMUSSEN

, ASHMAN

R.F.

, BALLAS

Z.K.

2003. Structural characterization of the inhibitory DNA motif for the type A (D)-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells. DNA Cell Biol., 22:621–631.

25.

MAPLETOFT

J.W.

, OUMOUNA

, TOWNSEND

H.G.

, GOMIS

, BABIUK

L.A.

, VAN DRUNEN LITTEL-VAN DEN HURK

2006. Formulation with CpG oligodeoxynucleotides increases cellular immunity and protection induced by vaccination of calves with formalin-inactivated bovine respiratory syncytial virus. Virology, 353:316–323.

26.

MEDESAN

, CIANGA

, MUMMERT

, STANESCU

, GHETIE

, WARD

E.S.

1998. Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site. Eur. J. Immunol., 28:2092–2100.

27.

MEHRZAD

, JANSSEN

, DUCHATEAU

, BURVENICH

2008. Increase in Escherichia coli inoculum dose accelerates CD8 cells trafficking in the premiparous bovine mammary gland. J. Dairy Sci., 91:193–201.

28.

MEHRZAD

, ZHAO

2008. T lymphocyte proliferative capacity and CD4+/CD8+ ratio in primiparous and pluriparous lactating cows. J. Dairy Res., 75:457–465.

29.

MENA

, NICHANI

A.K.

, POPOWYCH

, IOANNOU

X.P.

, GODSON

D.L.

, MUTWIRI

G.K.

, HECKER

, BABIUK

L.A.

, GRIEBEL

2003. Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by class A and class B CpG-oligodeoxynucleotide. Oligonucleotides, 13:245–259.

30.

PARRISH-NOVAK

, DILLON

S.R.

, NELSON

, HAMMOND

, SPRECHER

, GROSS

J.A.

, JOHNSTON

, MADDEN

, WEST

, SCHRADER

, BURKHEAD

, HEIPEL

, BRANDT

, KUIJPER

J.L.

, KRAMER

, CONKLIN

, PRESNELL

S.R.

, BERRY

, SHIOTA

, BORT

, HAMBLY

, MUDRI

, CLEGG

, MOORE

, GRANT

F.J.

, LOFTON-DAY

, GILBERT

, RAYOND

, CHING

, YAO

, SMITH

, WEBSTER

, WHITMORE

, MAURER

, KAUSHANSKY

, HOLLY

R.D.

, FOSTER

2000. Interleukin-21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature, 408:57–63.

31.

PONTAROLLO

R.A.

, RANKIN

, BABIUK

L.A.

, GODSON

D.L.

, GRIEBEL

P.J.

, HECKER

, KRIEG

A.M.

, VAN DRUNEN LITTLE-VAN DEN HURK

2002. Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs. Vet. Immunol. Immunopathol., 84:43–59.

32.

RANKIN

, PONTAROLLO

, GOMIS

, KARVONEN

, WILLSON

, LOEHR

B.I.

, GODSON

D.L.

, BABIUK

L.A.

, HECKER

, VAN DRUNEN LITTLE-VAN DEN HURK

2002. CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D. Vaccine, 20:3014–3022.

33.

RANKIN

, PONTAROLLO

, IOANNOU

, KRIEG

A.M.

, HECKER

, BABIUK

L.A.

, VAN DRUNEN LITTLE-CAN DEN HURK

2001. CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense Nucleic Acid Drug Dev., 11:333–340.

34.

RODA

J.M.

, PARIHAR

, CARSON

W.E.

3rd.

2005. CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J. Immunol., 175:1619–1627.

35.

SCHNARE

, BARTON

G.M.

, HOLT

A.C.

, TAKEDA

, AKIRA

, MEDZHITOV

2001. Toll-like receptors control activation of adaptive immune responses. Nat. Immunol., 2:947–950.

36.

SHODA

L.K.M.

, KEGERREIS

K.A.

, SUAREZ

C.E.

, MWANGI

, KNOWLES

D.P.

, BROWN

W.C.

2001. Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-α, and NO production by bovine macrophages. J. Leukoc. Biol., 70:103–112.

37.

STACEY

K.J.

, SESTER

D.P.

, SWEET

M.J.

, HUME

D.A.

2000. Macrophage activation by immunostimulatory DNA. Curr. Top. Microbiol. Immunol., 247:41–58.

38.

TAKESHITA

, LEIFER

C.A.

, GURSEL

, ISHII

K.J.

, TAKESHITA

, GURSEL

, KLINMAN

D.M.

2001. Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol., 167:3555–3558.

39.

VERTHELYI

, ISHII

K.J.

, GURSEL

, TAKESHITA

, KLINMAN

D.M.

2001. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol., 166:2372–2377.

40.

WEDLOCK

D.N.

, DENIS

, SKINNER

M.A.

, KOACH

, DE LISLE

G.W.

, VORDERMEIER

H.M.

, HEWINSON

R.G.

, VAN DRUNEN LITTLE-CAN DEN HURK

, BABIUK

L.A.

, HECKER

, BUDDLE

B.M.

2005a. Vaccination of cattle with CpG oligodeoxynucleotdie-formulated mycobacterial protein vaccine and Mycobacterium bovis BCG induced levels of protection against bovine tuberculosis superior to those induced by vaccination with BCG alone. Infect. Immun., 73:3540–3546.

41.

WEDLOCK

D.N.

, SKINNER

M.A.

, DE LISLE

G.W.

, VORDERMEIER

H.M.

, HEWINSON

R.G.

, HECKER

, VAN DRUNEN LITTLE-VAN DEN HURK

, BABIUK

L.A.

, BUDDLE

B.M.

2005b. Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis. Vet. Immunol. Immunopathol., 106:53–63.

42.

WEERATNA

R.D.

, MCCLUSKIE

M.J.

, XU

, DAVIS

H.L.

2000. CpG DNA induces stronger immune responses with less toxicity than other adjuvants. Vaccine, 18:1755–1762.

43.

WEINER

G.J.

, LIU

H.-M.

, WOOLDRIDGE

J.E.

, DAHLE

C.E.

, KRIEG

A.M.

1997. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization. Proc. Natl. Acad. Sci. U. S. A., 94:10833–10837.

44.

ZHANG

, PALMER

G.H.

, ABBOTT

J.R.

, HOWARD

C.J.

, HOPE

J.C.

, BROWN

W.C.

2003. CpG ODN 2006 and IL-12 are comparable for priming Th1 lymphocyte and IgG responses in cattle immunized with a reckettsial outer membrane protein in alum. Vaccine, 21:3307–3318.

45.

ZHANG

, SHODA

L.K.

, BRAYTON

K.A.

, ESTES

D.M.

, PALMER

G.H.

, BROWN

W.C.

2001. Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif. J. Interferon Cytokine Res., 21:871–881.

46.

ZHANG

, TIAN

, ZHOU

2008. In vivo administration effects of various oligodeoxynucleotides containing synthetic immunostimulatory motifs in the immune response to pseudorabies attenuated virus vaccine in newborn piglets. Vaccine, 26:224–233.

47.

ZHU

F.G.

, MARSHALL

J.S.

2001. CpG-containing oligodeoxynucleotides induce TNF-alpha and IL-6 production but not degranulation from murine bone marrow-derived mast cells. J. Leukoc. Biol., 69:253–262.