Research in Veterinary Science 86 (2009) 399–405

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Research in Veterinary Science

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Immunoadjuvant effects of bacterial genomic DNA and CpGoligodeoxynucleotides on avian influenza virus subtype H5N1inactivated oil emulsion vaccine in chicken

Yimeng Wang a,b,c,*, Chunqiao Shan b, Shuangxi Ming a, Yan Liu a,b, Yuchun Du c, Guotuo Jiang a,b

a School of Biological and Food Engineering, Dalian Polytechnic University, Dalian 116034, PR Chinab Dalian Sanyi Animal Medicine Co. Ltd., Dalian 116036, PR Chinac Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA

a r t i c l e i n f o

Article history:Accepted 16 September 2008

Keywords:CpG ODNBacterial genomic DNAAvian influenza virus subtype H5N1inactivated oil emulsion vaccineAdjuvantImmunization approachesImmune responses

0034-5288/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.rvsc.2008.09.006

* Corresponding author. Address: Department of Bof Arkansas, 705 West Putman Street, Apartment K 1Tel.: +1 479 575 6944; fax: +1 479 575 5555.

E-mail address: ammenwang@gmail.com (Y. Wang

a b s t r a c t

This study investigated the immunoadjuvant effects of three types of bacterial genomic DNA and CpG oli-gonucleotides (CpG ODN) on the avian influenza virus (AIV) subtype H5N1 inactivated oil emulsion vac-cine under two immunization strategies. The genomic DNA extracted from Escherichia coli O2,Staphylococcus aureus, Streptococcus faecalis FQ68, and synthetic CpG ODN were used as adjuvants, andtheir effects on the AIV oil emulsion vaccine were examined in chickens. The results indicated that whenadministered separately from the vaccine, adjuvants induced lower haemagglutination inhibition (HI)titres and serum IgG titres but resulted in higher concentrations of IFN-c and IL-10. In contrast, whencombined with the oil emulsion vaccine prior to inoculation, CpG ODN induced higher HI, IgG titresand IFN-c concentration but resulted in lower IL-10 concentration. These data suggest that, dependingon the immunization approaches, adjuvants may exert distinct immune effects in chickens receivingAIV H5N1 oil emulsion vaccine: the prior incorporation of CpG ODN into the vaccine may augment boththe humoral and Th1 type immune responses, while separate inoculation of adjuvants has not shown bet-ter adjuvanticity.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The highly pathogenic avian influenza A (H5N1) threatens theglobal economy and human welfare since it broke out in HongKong. Millions of birds have been killed, the related industrieshave been seriously affected. In addition to high pathogenicity,high variability is another feature of this virus. Research suggeststhat currently circulating strains of H5N1 viruses are becomingmore capable of causing disease in animals than earlier H5N1viruses (Ligon, 2005). An influenza pandemic may be imminent,and an effective poultry vaccine would be a valuable epidemo-logical tool.

Many kinds of bacterial genomic DNA and synthetic oligode-oxynucleotide containing unmethylated CpG motifs (CpG ODN),can be used to stimulate the vertebrate immune system andtrigger a cascade of immune responses, including stimulationof B cell proliferation, elevation of the antibody titres, activation

ll rights reserved.

iological Sciences, University, Fayetteville, AR 72701, USA.

).

of lymphocytes and degradation of the death rate of the chal-lenged chickens (Linghua et al., 2007a; Vleugels et al., 2002).CpG dinucleotides in vertebrate DNA are generally one-quarteras prevalent in bacterial DNA, and the cytosines in CpG dinucle-otides are highly methylated in vertebrate DNA, but not inmicroorganisms (Krieg et al., 1995). These differences make bac-terial CpG motifs function as a danger signal to the vertebrateimmune system, with triggering a cascade of immune responses.In our previous report, probiotics genomic DNA markedly re-duced the humoral responses when inoculated at the same sitebut separately from AIV oil emulsion vaccine (Yimeng et al.,2007). However, many other reports demonstrated that co-administration of CpG ODN with antigens could augment antigenspecific cell mediated and humoral immunity (Chu et al., 1997;Roman et al., 1997).

Although there are many studies on the immunoadjuvant ef-fects of CpG ODN or bacterial genomic DNA on vaccines in manyanimals, their effects on AIV oil emulsion vaccine (subtypeH5N1) in chickens have been rarely studied. In addition,although having a long history, many conventional adjuvantshave been shown to cause the excessively severe immune re-sponse (Th2 biased immune response). In this study, with theaim of identifying a nucleotide adjuvant with the capability of

400 Y. Wang et al. / Research in Veterinary Science 86 (2009) 399–405

inducing gentler and Th1 immune responses, we examined theimmunoadjuvant effects of three bacterial genomic DNA andCpG ODN in chickens.

2. Materials and methods

2.1. Bacteria strains and animals

Escherichia coli and Staphylococcus aureus, both pathogenic forthe domestic fowl, were purchased from China Center of Veteri-nary Culture Collection. Streptococcus faecalis FQ68 was identifiedin our lab, and is a Gram-positive probiotics. Hanlanhe male chick-ens were purchased from Dalian Chicken Farm.

2.2. Reagents

AIV H5N1standard haemagglutinin, positive control serum andre-assortant AIV vaccine, inactivated oil emulsion (H5N1 subtype)were purchased from Harbin Weike Biotechnology DevelopmentCompany. Proteinase K, lysozyme, lysostaphin and RNase A werepurchased from Amresco, Inc., Sigma, Shanghai Hi-tech Group Co.Ltd., and TIANGEN Biotech (Beijing) Co. Ltd., respectively. ChickIFN-c and IL-10 ELISA Kits were purchased from RapidBio Lab,USA, and AIV H5 Antibody ELISA Kit was purchased fromDr. Ehrenstorfer in Germany.

2.3. Adjuvants preparation

The highly pure phosphorothioate CpG ODN (5’-TCGTCGTTTGACGTTTCTCGAC-3’) (B-class) was synthesized in theTaKaRa Biotech Co. Ltd., CpG ODN with no LTA/LPS-ligands wasresuspended in sterile phosphate-buffered saline (PBS) buffer at aconcentration of 1 mg/ml and stored at �20 �C.

Genomic DNA was extracted by phenol–chloroform method asfollows: 1, O2 genomic DNA extraction: E. coli O2 was cultured tosaturation state in LB nutrient medium. The strain was resus-pended in Tris–Hcl and digested by RNase A, 20% SDS and protein-ase K after centrifugation and separation from the nutrientmedium. Phenol/chloroform/isoamyl alcohol (25:24:1) was usedto extract the genomic DNA twice. After adding the isopropanol,the stripped genomic DNA pellet was collected with a glass hook,washed with 70% ethanol and dissolved in PBS; 2, S. aureus geno-mic DNA extraction: the lysostaphin was used to break cell walls,and the other steps were same to 1; 3, FQ68 genomic DNA extrac-tion: FQ68 strain was cultured in MRS nutrient medium and thelysozyme was used to break cell walls, and the other steps weresame to 1. The injection containing genomic DNA would be con-firmed on a 0.6% agarose gel containing ethidium-bromide, ana-lyzed by an UV spectrophotometer (TU-1800, Beijing PurkinjeGeneral Instrument Co. Ltd., China) for DNA concentration andchecked on SDS–PAGE.

2.4. The production of AIV H5N1 oil emulsion vaccine incorporatingbacterial genomic DNA or CpG ODN

Commercial AIV oil emulsion vaccine was used to emulsify theadjuvants to ensure the simultaneous release of adjuvants andantigen in vivo. The commercial AIV oil emulsion vaccine wasmixed with adjuvant at the ratio of 5:1 in a high-speed homoge-nizer (YQ-3, Jintan, Jiangsu, China). After mixing at 12,000 rpmfor 7 min, the oil emulsion was assessed as follows: the mixed oilemulsion vaccine did not disperse on the surface of water andthere was no delamination after 24 h at 4 �C. The control oil emul-sion vaccine was mixed with the same amount of sterile PBS andproduced in the same way.

2.5. Animals and immunization

In order to compare the adjuvanticity in humoral response orcytokine secretion induced by free CpG ODN, pathogenic or probi-otics genomic DNA, the first set of tests was carried out as follows.Thirty-day-old chickens were randomly allocated into five groupsof 12 chickens each. These chickens were injected with the oilemulsion vaccine followed by 50lg nucleotide adjuvant into thebreast muscle. The adjuvants of A, B, C, D groups were CpG ODN,E. coli O2 genomic DNA, S. aureus genomic DNA and FQ68 genomicDNA, respectively. The control group (E) was injected with the oilemulsion vaccine followed by sterile PBS. The amount of commer-cial vaccine was administrated according to manufacturer’sinstruction (0.5 ml). The second set of tests was designed to exam-ine the immunoadjuvant effects of bacterial genomic DNA and CpGODN when they were incorporated into the vaccine. Thirty-day-oldchickens were divided into five groups, and each group with 10chickens. Groups A, B, C, and D were injected with oil emulsionvaccine in a combination of 50 lg nucleotide adjuvants of CpGODN, E. coli O2 genomic DNA, S. aureus genomic DNA and FQ68genomic DNA, respectively. The control group (E) was injectedwith the vaccine incorporating the same amount of sterile PBS.At day 21 post inoculation, five groups were immunized again withthe same regime in the second set. The amount of antigen inocu-lated in these five groups was same. All chickens did not receiveany vaccines previously. The vaccination procedures were per-formed according to the policies of local Animal Care Committee.

2.6. Sample collection

Serum was collected weekly to monitor normal hematologicalstatus by the HI and IgG titres. The serum samples were storedat �20 �C until assayed by ELISA and HI tests. All serum sampleswere used for HI tests, and three randomly selected serum samplesin every group were used for each ELISA test.

2.7. The AIV H5 specific antibody titres assay

HI titre in serum was assayed by Haemagglutination and hae-magglutination inhibition tests, according to the Chinese NationalStandard of diagnostic techniques for highly pathogenic avianinfluenza, and the Office International des Epizooties (OIE) stan-dards (OIE, 2005).

The antibody of serum IgG was measured with a commercialELISA kit (Dr. Ehrenstorfer, Germany), which was based on com-petitive binding. The serum samples (not diluted) to be testedand the labeled monoclonal antibody were competitive to bondthe standard antigen. After incubation, the antigen was detectedspectrophotometrically at 450/630 nm. The serum IgG titres wereinversely proportional to OD values (the lower the OD value, thehigher the IgG titre).

2.8. IFN-c and IL-10 assay

IFN-c and IL-10 concentrations were assessed by commercialELISA kits (RapidBio, USA), which is standard sandwich ELISA.

2.9. Statistical analysis

Data were analyzed using the statistical software program Sy-stat 13 (SPSS 13). Distribution of data was determined usingdescriptive statistics. Data which were not normally distributedwere transformed by ranking. Differences were investigated usingone-way analysis of variance (ANOVA). Means of the rank werecompared using LSD’s multiple comparison test. A p-value of<0.05 was considered significant.

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3. Results

3.1. Antibody immune responses to AIV oil emulsion vaccine whenadministrated separately from adjuvant

3.1.1. HI titres in five groupsThe HI tests were used to assess the humoral immune response

induced by CpG ODN and three bacterial genomic DNA adminis-trated separately to the vaccine. Groups A (vaccine followed byCpG ODN) and B (vaccine followed by E. coli O2 genomic DNA) al-ways induced significantly lower HI titres than the control group(vaccine followed by PBS) from days 14 to 49 post-inoculation(p 6 0.003 at these six time points). Compared with the controlgroup, groups C (vaccine followed by S. aureus genomic DNA)and D (vaccine followed by FQ68 genomic DNA) induced signifi-cantly lower (p 6 0.026) HI titres in several test points. The HI ti-trea of group A were lower than those of groups B, C and D –group B had the lowest HI titre, while group D had the highest(Fig. 1A).

3.1.2. Titres of serum IgG in five groupsIn order to strengthen the credibility of HI tests results, serum

IgG titre was checked by ELISA. Three samples were randomly se-lected for ELISA antibody test at three time points (14, 28 and 42days post-inoculation). Groups A (vaccine followed by CpG ODN)and B (vaccine followed by E. coli O2 genomic DNA) resulted inOD significantly higher at 14 days post-inoculation (p = 0.022 and

A

B

Fig. 1. Antibodies to AIV vaccine when administrated separately from adjuvant.

0.019 in groups A and B, respectively) than those for the controlgroup (Fig. 1B).

3.2. Serum IFN-c and IL-10 concentrations

In order to monitor the cytokine secretion at the formulation ofadjuvant separation from the vaccine, the concentrations of serumIFN-c and IL-10 were detected by ELISA tests. There were similarIFN-c and IL-10 concentrations in all groups (p > 0.05) (Fig. 2).

3.3. Antibody immune responses to AIV oil vaccine whencoadministrated with adjuvant

3.3.1. HI titres in five groupsIn the second set of experiments, we first combined the vaccine

with the respective adjuvant, and then injected the combinationinto chickens. In addition, a second vaccination was used to boostthe adjuvant effects. The HI tests were used to trace the humoralresponse during the whole immunization process (Fig. 3A). Com-pared with the control group, group A (vaccine incorporatingCpG ODN) induced significantly higher HI titres at days 21, 28,49, 70 after the first inoculation (p = 0.041, 0.015, 0.023 and0.003, respectively). When combined with the vaccine, FQ68 geno-mic DNA induced similar HI titres to the control group at all timepoints (p > 0.05). However, compared with the control group, bothgroups B (vaccine incorporating O2 genomic DNA) and C (vaccineincorporating S. aureus genomic DNA) resulted in lower HI titres

(A) HI titres to AIV vaccine. (B) Serum IgG titres to AIV vaccine (OD values).

A

B

Fig. 2. Concentrations of serum IFN-c and IL-10 when vaccine was administrated separately from adjuvant. (A) Concentration of serum IFN-c (pg/ml). (B) Concentration ofserum IL-10 (pg/ml).

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from days 14 to 42 after the first inoculation and similar HI titres atdays 49, 56 and 70 after the first inoculation (p > 0.05).

3.3.2. Titres of serum IgG in five groupsThe humoral immune response resulted from HI tests was

examined by ELISA test (Fig. 3B). At days 14, 42 after the first inoc-ulation, the OD values of group A (vaccine incorporating CpG ODN)were lower than those of the control group (p > 0.05), and at 28days after the first inoculation, there was significant difference be-tween these two groups (p = 0.001). However, the differences be-tween each of three bacterial genomic adjuvant groups and thecontrol group were not significant at these time points (p > 0.05).

3.4. Serum IFN-c and IL-10 concentrations

The cytokine concentration was monitored in the second set(Fig. 4). The IFN-c concentration of every group increased withtime (from 14 to 42 days after the first immunization), and was sig-nificantly higher (42 days after the first immunization) (p = 0.005)in group A (vaccine incorporating CpG ODN) than that of the con-trol group. Three bacterial genomic adjuvant groups induced thesimilar or slightly higher IFN-c concentration than the controlgroup (Fig. 4A). In contrast, the IL-10 concentration of every groupdecreased with time over the days the experiment ran. Groups Aand D (vaccine incorporating FQ68 genomic DNA) induced signifi-cantly lower (14 days after the first immunization) (p = 0.002 and0.006, respectively) IL-10 concentrations than the control group.The amount of differences between groups B or C (vaccine incorpo-rating O2 or S. aureus genomic DNA) and the control group differedat three time points (Fig. 4B).

4. Discussion

The immunoadjuvant effects of CpG ODN and bacterial genomicDNA in mice and other animal models have been well reported.However, the effects in chicken, especially on AIV H5N1 oil emul-sion vaccine remain unclear. This study investigated the immuno-adjuvant effects of CpG ODN and three bacterial genomic DNAs onAIV H5N1 oil emulsion vaccine in chickens. We firstly comparedthe different adjuvanticity of gram-negative pathogenic, gram-po-sitive pathogenic, gram-positive probiotics genomic DNA and CpGODN on AIV oil emulsion vaccine. We then compared the differentimmune effects induced by different approaches to administratenucleotide adjuvant and vaccine in vivo. Our results indicated thatCpG ODN possessed the immune potential when incorporated intovaccine. In addition, we preliminarily demonstrated that combina-tion of CpG ODN and antigen induced a Th1/Th2 mixed responsewith a tendency towards Th1 type response in chicken.

Antibody titres, isotypes or a combination of these factors playimportant roles against bacterial infection, viral challenge (Linghuaet al., 2007a; Ricardo and Patricia, 2001). In China, HI tests aremostly used to monitor antibody levels induced by AIV oil emul-sion vaccine. Therefore, the HI titre was considered as the key fac-tor in the appraisal of the vaccine adjuvant in this study. Theinjection of these nucleotide adjuvants alone (without antigen)dramatically reduced HI titres, while the co-administration of theantigen and CpG ODN excellently increased HI titres. Undoubtedly,the latter method of delivering adjuvant contributes to improvethe immune effects induced by AIV oil emulsion vaccine.

In addition to humoral immunity, cellular immunity also playsimportant roles in the viral challenge or tumor extension. The

A

B

Fig. 3. Antibodies to AIV vaccine when co-administrated with adjuvant. (A) HI titres to AIV vaccine. (B) Serum IgG titres to AIV vaccine (OD values).

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treatment of a variety of chicken diseases, like Marek disease, de-pends on cellular immunity. IL-10 is produced primarily by T cellsand monocytes (Coutelier et al., 1988; Fuchs et al., 1996) and IFN-cis also mainly secreted by activated T cells, so their productions re-flect the cell mediated immune levels. When CpG ODN or bacterialgenomic DNA were administrated separately to the vaccine, theconcentrations of IFN-c and IL-10 were only slightly increased atthe early phase of post immunization, which demonstrated thatthese nucleotide adjuvants alone were of little benefit for the cellmediated immune response. When combined with the vaccine,CpG ODN augmented the secretion of IFN-c, suggesting that thecellular immunity (Th1 type response) had been boosted.

It is well known that CpG ODN administration parenterallyinduces strong Th1 components like IL-2, IL-12, IFN-c, IgG2a(Cowdery et al., 1996; Halpern et al., 1996; Klinman et al., 1996;Linghua et al., 2007b). It is also known that IFN-c decreases theIgG1/IgG2a ratio, whereas IL-10 increases the IgG1/IgG2a ratio onmice models (Crawley et al., 2003). Th1 cytokine IFN-c is an impor-tant B cell switch factor for the induction of antigen-specific IgG2a-secreting B cells (Coutelier et al., 1988; Coutelier et al., 1991). Inthis study, higher concentration of IFN-c induced by CpG ODN inthe second set of tests demonstrated the type of immune responseswitched to Th1-biased response. Because IL-10 have been shownto inhibit the transcription mRNA of Th1 cytokines, down-regulatemajor histocompatibility complex class II expression on monocytesand inhibit monocyte production of proinflammatory cytokines,including IL-1, IL-2, IL-6, IL-8, tumor necrosis factor-a, and IFN-c

(de Waal Malefyt et al., 1991; Fuchs et al., 1996), the decreasedproduction of IL-10 indicated that CpG ODN switched the immuneresponse to Th1-biased in chicken. This result was consistent withother reports in many animal models (Chu et al., 2000; Coutelieret al., 1991; Cowdery et al., 1996; Crawley et al., 2003; de WaalMalefyt et al., 1991; Deml et al., 1999; Guo et al., 2007; Klinmanet al., 1996; Zhang et al., 2006).

There were significantly different immune effects induced bythe aforementioned two ways of delivering CpG ODN and oilemulsion vaccine. A potential explanation is: When CpG ODNwas incorporated into oil emulsion vaccine before the immuniza-tion, the antigen would be released together with CpG ODNin vivo. In this scenario, CpG ODN could not only induce thematuration of APCs, which capture the antigen and take up thesignal of the antigen to B cells, but also trigger the differentia-tion of Th0 to Th1. Thus both the humoral and Th1 responsescould be increased, which was observed in the second set oftests. When CpG ODN was administrated separately to oil emul-sion vaccine, the antigen would be released in about two weekspost-inoculation, causing that CpG ODN took effect before therelease of antigen in vivo. In this case, the activated APCs couldnot capture the antigen and consequently take up the signal ofthe antigen to B cells. On the other hand, CpG ODN might stim-ulate or activate T cells, leading to higher concentrations of IL-10and IFN-c. This might be a feasible explanation for the results ofthe first set of the tests and our previous publication (Yimenget al., 2007).

A

B

Fig. 4. Concentrations of serum IFN-c and IL-10 when vaccine was co-administrated with adjuvant. (A) Concentration of serum IFN-c (pg/ml). (B) Concentration of serum IL-10 (pg/ml).

404 Y. Wang et al. / Research in Veterinary Science 86 (2009) 399–405

A series of studies have shown that CpG ODN and bacterialgenomic DNA is mainly recognized by the toll-like receptor (TLR)nine in mice and humans (Hemmi et al., 2000; Tsujimoto et al.,2006). However, chicken genome lacks the TLR9 gene, so CpG mo-tifs triggered signaling in chicken must be mediated by otherreceptor(s), but the detailed mechanism is unclear now.

This study compared the adjuvanticity induced by CpG ODNand three types of bacterial genomic DNA, indicating that the for-mer was better. And the comparison of immune effects induced bythree types of bacterial genomic DNA demonstrated that genomicDNA from different strains showed varied immune adjuvanticity.This may be related to the immunosuppressive CpG motifs (CpG–N) frequency in natural bacterial genomic DNA (Yamada et al.,2002); or the length of bacterial genomic DNA or its complex spa-tial structure affects its immune activity (Cho et al., 2006).

Disappointingly, we have not detected and removed the LPS/LTA in bacterial genomic DNA extractive, since they affect the hosthealth and adjuvanticity of bacterial genomic DNA. However, therewas no pathological or toxic symptoms in chickens immunizedwith bacterial genomic DNA.

Taken together, we demonstrated that CpG ODN could mark-edly reduce the humoral immune response and slightly enhancethe cytokine secretion when administrated separately to the vac-cine, characterized by decreased HI titres and increased IFN-c, IL-10 concentrations. However, CpG ODN could enhance the humoraland Th1 immunity when incorporated into oil emulsion vaccine,characterized by increased HI titres, IFN-c concentration and de-creased IL-10 concentration. The significant effects of CpG ODN

suggest that CpG ODN can be used as an effective adjuvant againstthe virus challenge.

Acknowledgement

This work was supported by the Provincial Institutions Cooper-ation Fund of Liaoning Province (No. 200402011).

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