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7Skanwasher 300, Skatron Instruments AS, Lier, Norway. 8Titretek ELISA, plate reader, supplied by Labsystems, Brussels,. Belgium. 9SAS, Version 6.12; SAS ...
Research Note Stimulatory Effect of CpG Sequences on Humoral Response in Chickens B. Vleugels,1 C. Ververken, and B. M. Goddeeris Physiology and Immunology of Domestic Animals, Department of Animal Science, Faculty of Agricultural and Applied Biological Sciences, K. U. Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium ABSTRACT Oligonucleotides containing the dinucleotide CpG have an immunostimulatory effect in mammals. The CpG motif is interpreted as a signal of prokaryote invasion by the innate immune system and consequently activates defense mechanisms. The goal of this study was to investigate whether the immunostimulatory actions of CpG oligonucleotides take place in birds as well. To this end, birds were immunized with BSA and the serum antibody response was followed. A significantly higher BSA-specific response was observed in the CpG-treated group. Moreover, immuno-

stimulatory DNA resulted in more persistent responses to immunization. After only one immunization, titers as high as in booster responses were observed in the CpGtreated birds. The effects were shown to depend on the presence of an unmethylated CpG core sequence in the DNA, because the reversal of CpG to GpC even caused a decrease in antibody response. These findings demonstrate that CpG oligonucleotides could be a valuable adjuvant for poultry vaccines.

(Key words: chicken, oligonucleotide, adjuvant, antibody, enzyme linked immunosorbent assay) 2002 Poultry Science 81:1317–1321

Studies in mammals have shown a positive effect of bacterial or viral DNA on immune responses (Ballas et al., 1996; Cowdery et al., 1996; Sparwasser et al., 1998; Hartmann and Krieg, 1999). These stimulatory actions are attributed to CpG dinucleotides in a particular base context, called CpG motifs, present within these prokaryotic nucleic acids (Krieg et al., 1995; Manzel and McFarlane, 1999). It has been hypothesized that stimulatory CpG motifs trigger a response by the immune system because of their bacterial characteristics, which prompt the antigen-presenting cells (APC) of the innate immune system into action (Krieg, 1999). CpG occurs 5 to 20 times less frequently in vertebrate genomes than in bacterial DNA, in which the dinucleotide occurs at its expected frequency (Krieg et al., 1995). Because of this difference in frequency and the methylation of the CpG in vertebrate genomes (Jones et al., 1999), the detection of unmethylated CpG sequences is experienced by the immune system as an indicator of prokaryote presence. Actions to eliminate this potential threat are undertaken when CpG oligonucleotides are recognized by the Toll-Like Receptor 9 (Hemmi et al., 2000). The pathways involved are similar to the pattern recognition of other bacterial cell components but cause a different temporal

response (Sparwasser et al., 1997; Hartmann and Krieg, 1999). Several groups have proposed a consensus base sequence for immunostimulatory DNA sequences (ISS). In mice, the core of ISS consists of an unmethylated, covalent CpG bond, preceded at its 5′ side by two purines, preferentially GpA, and followed at the 3′ end by two pyrimidines, either TpC or TpT (Krieg et al., 1995; Sato et al., 1996; Hassett et al., 1999; McCluskie et al., 2000). Almost all activity can be abolished by methylation or removal of the CpG dinucleotide (Krieg et al., 1995; Krieg, 1999; Leitner et al., 2000). Changes in the purines and pyrimidines surrounding the CpG dinucleotide have an impact on the response because they determine the effects of the innate immune system on the antigen-dependent response. They are also responsible for differences in activity between species. Moreover, sequences consisting of CpG repeats or a CpG dinucleotide preceded by a C and followed by a G, have been described as inhibitory (Krieg et al., 1998). Several studies demonstrate a rise in cytokine and antibody concentration in serum of CpG-treated mice (Davis et al., 1998). Similar effects were also found in humans and primates (Davis et al., 2000). Changes in the surrounding sequences can be used to optimize and tailor the oligo (Sato et al., 1996; Krieg, 1999) for use in vaccines or studies.

2002 Poultry Science Association, Inc. Received for publication January 15, 2002. Accepted for publication May 3, 2002. 1 To whom correspondence should be [email protected].

Abbreviation Key: ABTS = 2,2′-azino di-(3′ethylbenzathiazoline-6sulfonate); dpi = days postimmunization; IFA = incomplete Freund’s adjuvant; ISS = immunostimulatory DNA sequences; oc = control oligo; ov = treatment oligo; TMB = 3,3′,5,5′ tetramethylbenzidine.

INTRODUCTION

addressed:

Bart.

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Lipford et al. (1997) demonstrated a tailor-made oligo, which specifically induced interleukin-12 but not tumor necrosis factor-α. This reduction is mainly desired to prevent the occurrence of septic shock. We hope to ascertain whether CpG-containing oligodeoxynucleotides cause similar effects in the avian species as in mammals, i.e., activation of the innate immune system, resulting in a better response by the adaptive immune system.

oligonucleotide, in which the CpG sequences were reversed (oc), was injected into the fourth group. This arrangement resulted in four treatment groups: a sham-injected group (c), a group receiving 20 µg of BSA (b), a group injected with 20 µg of BSA and 20 µg of oligo ov (ov), and a group that was treated with 20 µg of BSA and 20 µg of oligo oc (oc). The primary immunization was given at 14 d of age, and the secondary immunization was at 35 d of age.

Sampling MATERIALS AND METHODS Experimental Birds Broilers of a commercial line (Hybro G2) were hatched and kept in open floor pens at a maximum initial density of 15 birds per m2. Wood shavings were used as litter. The birds had access to food and water ad libitum. Ambient temperature and ventilation were regulated in keeping with standard breeding practices. A series of commercial pelleted feed mixes for broiler rearing (Krix3) were used as prescribed by the manufacturer.

Oligonucleotides Oligos [control oligo (oc): 5′-GCTAGAGCTTAGGCT-3′ with GpC and treatment oligo (ov): 5′-GCTAGACGTTAGCGT-3′ with CpG] were prepared by Biosource International.4 All other reagents were purchased from Sigma5 unless otherwise stated. At 14 d of age, groups of 10 chicks each were randomly grouped and a treatment was assigned to each of the groups. All birds were injected subcutaneously with 100 µL.

Experimental Design The first experiment was designed to establish the minimally required dose of CpG DNA to be stimulatory. Different amounts of CpG (0, 1, 5, 10, 15, and 20 µg/bird) were injected in chickens in combination with 20 µg of BSA in PBS and incomplete Freund’s adjuvant (IFA) (50:50). Injections without IFA were administered to establish the quality of CpG as adjuvant. The immune response was monitored and compared to sham-injected animals. In the second experiment, a control group received an injection of 100 µL PBS:IFA (50:50). All other birds were injected with 20 µg of BSA dissolved in PBS:IFA (50:50). In the treatment group, an additional 20 µg of the synthetic oligo (ov) were mixed into the BSA solution. To check the specificity of the CpG sequence in the oligo, a twin

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Chicks were supplied by Avibel, Halle-Zoersel, Belgium. Feed was obtained from Hendrix, Merksem, Belgium. 4 Biosource, Nivelles, Belgium. 5 Sigma Chemical Co., St. Louis, MO. 6 KPL, Kirkegaard and Perry Laboratories, Gaithersburg, MD. 7 Skanwasher 300, Skatron Instruments AS, Lier, Norway. 8 Titretek ELISA, plate reader, supplied by Labsystems, Brussels, Belgium. 9 SAS, Version 6.12; SAS Institute Inc., Cary, NC. 3

Blood samples were collected from the wing vein before injection and at 4, 7, 14, and 21 d postimmunization (dpi). In the second experiment, a second injection was also given on 21 dpi (35-d-old chickens), and blood was sampled 4, 7, and 14 d thereafter. Blood was allowed to clot at room temperature and was subsequently centrifuged. The serum was pipetted into a new tube and stored frozen until assayed by ELISA.

ELISA Analysis of Blood Samples All assays were essentially as described by Mast et al. (1997), but minor modifications were made. It was established that a 1/10,000 dilution of a sheep anti-chicken polyclonal antibody5 yielded excellent results for the measurement of total Ig. Blocking with 0.25% casein reduced background in the IgM assay. In all other ELISA, 5% glycine was used as a blocking agent. To reduce background coloring in the ELISA, serum incubation and antibody binding steps were performed at 4 C instead of at room temperature. In the assays for IgG and IgM, the antibodies against chicken IgG and IgM were diluted 1/1,000. For IgG, 3,3′,5,5′ tetramethylbenzidine (TMB)6 was chosen as a substrate for peroxidase instead of 2,2′-azino di-(3′ethylbenzathiazoline-6-sulfonate) (ABTS). ABTS7 was used as a substrate in all other ELISA. Between steps samples were washed in a plate washer.7 Absorption, at 405 nm for ABTS and at 450 for TMB, was measured using a plate reader.8

Data Analyses The optical density of a 1/200 dilution of serum was used to follow the response in time for total Ig, IgM, IgG, and IgA. Sera were also titrated for their total antigenspecific Ig content.

Statistical Analyses Data were analyzed using the PROC MIXED method of SAS software,9 which is well suited for dealing with repeated measurement data and missing values. The covariance structure was determined using the Schwartz Bayesian goodness-of-fit criterion and the Akaike information criterion. The closer to 0 these criteria are, the better the model fits. Simple covariance was chosen. Some values are combined in Table 1. Once the covariance structure was established, P-values were obtained using least squares means and estimate state-

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STIMULATORY EFFECT OF CpG SEQUENCES TABLE 1. Comparison of covariance structure using the Schwartz Bayesian goodness-of-fit criterion (SBC) and the Akaike’s information criterion (AIC)1 SBC/AIC for dependent variable Covariance structure Simple Autoregressive

Ig

IgG

IgM

−221/−82 −345/−213

−345/−97 −1,203/−1,403

−251/45 −450/−897

1

SBC and AIC, the closer the value is to zero the better the covariance structure model fits.

ments in PROC MIXED. The Tukey-Kramer adjustments were used to make significant comparisons possible between the mean values.

RESULTS To determine a suitable stimulatory dose, various concentrations of CpG oligos were combined with an immunization against BSA. BSA-specific serum antibodies were determined. A dose of 20 µg CpG oligonucleotide gave an improvement (P < 0.05) compared to injection with only a traditional adjuvant (data not shown). To establish the possible use of CpG as adjuvant, a mixture of BSA and CpG was also added without IFA. This addition resulted in a small response, whereas injection of protein alone did not (data not shown). In the second experiment, a greater rise in BSA-specific total Ig, IgM, and IgG was observed after treatment with at least 20 µg of CpG containing oligonucleotides. This effect could even be boosted by a second injection (Figure 1). The effects were significant (P < 0.05) for total Ig on 14 and 21 dpi for the primary immunization and on 4 and 21 dpi for the booster immunization. Analysis of the BSAspecific IgG response yielded the same results. For IgM, difference (P < 0.05) was only observed on 14 dpi after the first immunization. For IgA no difference was observed in either case (data not shown). Removal of the CpG from the oligo resulted in decreased antibody response. Moreover, the BSA-specific serum antibodies were present longer when CpG oligos were added to the vaccine. Furthermore, we found that CpG-containing oligonucleotides can enhance the antibody response but cause a delay in time of appearance. This shift in time kinetics was observed with all groups that received an oligonucleotide injection (P < 0.05). Compared to sham-injected birds, all treatment groups showed a distinct response in time, demonstrated by a treatment × dpi interaction (P < 0.001). Comparison of the three treatment groups among themselves revealed that CpG and GpC delayed the responses (P < 0.05); total Ig and IgG responses were delayed for about 3 to 4 d. For IgM, however, this effect was not significant. Defining total Ig serum titers revealed the same results as the analysis of the fixed 1/200 dilutions. Depending on the sample time, antibody concentrations that were 4 to 32 times greater were observed in the circulation of CpGtreated birds (Figure 2). This finding could already be inferred from the analysis of the 1/200 dilutions, which had shown that, at the time of the booster injection, the antibody concentration had not yet dropped to baseline level in the

CpG-treated group. At 14 dpi, for the primary and secondary immunization, a four-to-five-times greater concentration was observed in the CpG-treated group compared to the BSA:IFA group. The oligonucleotide in which the CpG was reversed lowered the titers of anti-BSA antibodies in comparison to the BSA:IFA group.

DISCUSSION CpG oligonucleotides have been shown to be potent immunostimulatory substances and are under investigation to become widely used tools in immunomodulation, vaccines, and allergy therapy (Pisetsky, 1996). To our knowledge, this study is the first to assess their use in the avian species. After comparing several studies, we chose to use a mouse sequence, described by Klinman et al. (1996). It contains two CpG motifs, one of which was shown to be very efficient in B-cell stimulation. However, studies have shown that the optimal CpG motif for B-cell stimulation differs between humans and mice. The best stimulatory sequence in humans is GTCGTT, whereas the optimal mouse sequence is GACGTT (Krieg, 1999; Hartmann et al., 2000). The extent to which the mouse sequence is the most effective one for use in chickens was not investigated and remains a subject for future experiments. Chickens were immunized at 14 d of age, because those 1 wk or younger are not yet fully competent for B-cell responses (Mast and Goddeeris, 1999). A suitable dose of oligonucleotides was established to be 20 µg. Most likely, when less of the CpG oligo is administrated, clearance and degradation by nucleases are too high, resulting in lower responses. When responses of chicks treated with CpG were compared to those injected only with BSA:IFA, a greater BSA-specific antibody response was observed. Upon primary immunization, total Ig, IgG, and IgM were demonstrated to be generally higher in CpG-treated birds than in the BSA controls and were statistically significantly higher at specific time points. On 14 and 21 dpi, total Ig and IgG responses of the CpG-treated group were significantly higher than the other groups. After a booster injection, a significantly higher total Ig and IgG response was observed in the CpG treatment group on 4 and 21 dpi. No effect on IgM was detectable upon secondary immunization, although others have shown a CpG-related increase in IgM (Klinman et al., 1996; Davis et al., 1998). The rise of serum IgM background levels with age (Figure 1C) has also been observed in other species, for instance in the pig (Bianchi et al., 1992, 1999). IgA antibodies, which are important for mucosal immunity, were not affected by any

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FIGURE 2. Antibody titers in the differently immunized groups. Each group was injected with 20 µg of BSA in a 50:50 mixture with incomplete Freund’s adjuvant (IFA; IFA:CpG; IFA:GpC). Secondary immunization with the same antigen was performed at 21 d postimmunization (dpi) under identical conditions. Titers were calculated for the three treatment groups. This figure shows a distinct difference in concentration; the end values of the IFA- and IFA:GpC-treated groups were already reached after the first injection in the IFA:CpG group.

FIGURE 1. Antibody responses in the differently immunized groups. Total Ig (A), IgG (B), and IgM (C) BSA-specific responses to a subcutaneous injection of 20 µg of BSA in combination with different adjuvants were measured in an ELISA on 1/200 serum dilutions. Arrows indicate injections. The experiment consisted of a control group (䊉, no BSA added), a BSA-immunized group [䊊, BSA:IFA (50:50)], a CpG-treated group [䉮, BSA:IFA (50:50) + 20 µg CpG] and a GpC control group (▼, BSA:IFA (50:50) + 20 µg GpC). Each point in the graph is the average of 10 birds. Standard errors were calculated per treatment and at each point in time. *Statistically significant difference between CpG-treated group and the others as calculated by PROC MIXED. IFA = incomplete Freund’s adjuvant.

of the treatments. The use of CpG DNA as a mucosal adjuvant is still under investigation but the majority of data suggest a strong and positive effect of CpG (Horner et al., 1998). Analysis of the BSA-specific total Ig titers revealed the same results as the analysis of the fixed 1/200 dilution, in which at the time of the booster injection, the antibody concentration had not yet declined to baseline level in the CpG-treated group. At 14 dpi, for the primary and the secondary immunizations, antibody concentrations that were four to five times higher were observed in the CpGtreated group compared to BSA controls. The longer persistence of antibodies in circulation and the higher titer might even make a secondary immunization redundant. This result could be very important economically and practically, as each vaccination implies extra labor and costs. A reduction in the antibody titer was observed when the GpC oligonucleotides were used. Possible explanations can be searched for in the field of antisense technology. Oligonucleotides can be used as antisense molecules to impair translation of mRNA (Agrawal, 1999). Antisense basepairing of the GpC oligo could impair translation in this way. In the eukaryotic genome, CpG islands are generally closely associated with housekeeping genes and the promoter regions of certain genes (Kundu and Rao, 1999). This preferred localization of strong basepairs, (G or C) in and around the promoter regions of genes could also draw the oligonucleotides to preferentially bind to the promoter regions and inhibit transcription. Certain oligonucleotide sequences have been shown to be immunoinhibitory (Krieg et al.,1998). However, up to now, all nucleotides described with suppressive characteristics contained CpG.

STIMULATORY EFFECT OF CpG SEQUENCES

In conclusion, CpG oligonucleotides can significantly enhance antigen-specific responses in chicken. After one injection, levels and duration comparable to a normal booster immunization were observed within the CpG-treated group. The improved response and the absence of immunization-induced lesions (Weeratna et al., 2000) make CpG containing nucleotides very promising adjuvants in chicken vaccines. In view of a growing resistance to the use of antibiotics, this is an extra advantage in the development of new vaccination strategies.

ACKNOWLEDGMENTS The authors thank C. Borgers, I. Geerts, and A. Respen for their skilled technical assistance.

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