CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D☆
Introduction
Bovine herpes virus-1 (BHV-1) is an important pathogen in cattle, which causes a variety of clinical manifestations including rhinotracheitis, conjunctivitis, vulvovaginitis and abortions. Additionally, secondary opportunistic bacterial infections can lead to enhanced morbidity and mortality [1]. Previously, we have shown that the major glycoproteins of BHV-1, gB, gC and gD, induce protection in cattle when administered as a subunit vaccine formulated with an oil-based adjuvant [2]. However, as oil-based adjuvants also result in significant losses due to meat trimming, there is a need within the livestock industry to move towards adjuvants that induce minimal injection site reactions of short duration. Oil-based adjuvants also induce primarily Th2-type immune responses [3], [4], which often are not protective and sometimes may be detrimental [5], [6].
Recently, the potential of using bacterial DNA as an adjuvant has been demonstrated in mice and primates [7], [8]. In bacterial DNA, CpG dinucleotides are unmethylated and present at an expected frequency (1/16 bases) whereas they are under-represented (1/50 to 1/60 bases) and selectively methylated in vertebrate genomes [9]. Because of these differences, it has been suggested that vertebrates recognize bacterial DNA as foreign due to their evolutionary adoption of a non-self pattern recognition mechanism [10]. Immunostimulatory DNA sequences have been further studied in the form of synthetic oligodeoxynucleotides (ODN). In vitro, ODN containing CpG motifs can activate murine macrophages as demonstrated by their secretion of IL-12, TNF-α, IFN-α and IFN-β [11], [12]. CpG ODNs are also mitogenic for B cells [13], can stimulate dendritic cells [14], and indirectly induce NK cells to secrete IFN-γ and enhance their lytic activity [15], [16].
In mice, CpG ODN is a potent adjuvant when administered with an antigen. Furthermore, when combined with other adjuvants such as alum or Freund’s incomplete adjuvant (FIA), CpG ODN contributes to a synergistic immunostimulatory effect, which can enhance immune responses to a level comparable to that induced by Freund’s complete adjuvant (FCA) [8], [17], [18]. In contrast to oil-based adjuvants, CpG ODN promotes the generation of Th1-biased immune responses [19], [20] and elicits antigen-specific cellular and humoral immune responses that are far superior to those induced by alum [18], which is still the only adjuvant licensed for human use in many countries. In mice there is remarkably little injection site reaction or other toxicity associated with CpG ODN when employed as an adjuvant [8]. In addition, CpG DNA has strong immunomodulatory and immunostimulatory activity when applied as a therapy in murine models of allergy and cancer [21].
The potential of CpG ODN as an adjuvant has also been examined in large outbred animals. In Aotus monkeys, the antigenicity of a candidate malaria peptide vaccine was greatly enhanced by addition of CpG ODN to the formulation [22]. Orangutans are naturally infected with hepatitis B virus (HBV), but do not respond well to Energix-B® (SmithKline-Beecham, Rixensart, Belgium), a commercial HBV vaccine. The addition of CpG ODN to the Energix-B® formulation increased the level of seroprotection conferred by the vaccine from 8 to 100% after two immunizations [7].
Previously, we identified a CpG motif that elicits in vitro proliferative responses of bovine lymphocytes [23]. Since in vitro proliferation induced by an ODN is indicative of in vivo adjuvant potential, the most stimulatory ODN, which contains three copies of this motif, was chosen to adjuvant a truncated, secreted form of glycoprotein D (tgD) [24] in a BHV-1 challenge trial. Calves were immunized with tgD formulated with alum, CpG ODN or a combination of both. As reported for mice, CpG ODN alone or in combination with alum generated stronger and more balanced immune responses than alum, yet caused mild and transient tissue reaction. Furthermore, enhanced protection was observed, demonstrating that even in large animals, CpG DNA has potential as an adjuvant.
Section snippets
Cells, viruses and glycoproteins
Bovine viral diarrhea virus-free Madin Darby bovine kidney (MDBK) cells were cultured in minimal essential medium (MEM; Gibco-BRL, Grand Island, NY), supplemented with 5% fetal bovine serum (FBS; Gibco-BRL). The 108 strain of BHV-1 was propagated in these cells. Virus recovered from nasal fluids was quantified by plaque titration on MDBK cells in microtiter plates with an antibody overlay as previously described [25]. A truncated secreted version of BHV-1 gD (tgD) was constructed by terminating
Humoral immune responses induced by tgD
In order to assess the adjuvant activity of a CpG containing ODN in cattle, three groups of calves were vaccinated with BHV-1 tgD formulated with a combination of alum and different doses of ODN 2135. Two additional groups received either tgD in alum or tgD with the highest dose of ODN 2135. After one immunization there was no significant increase in total IgG, but after the second immunization all vaccinated groups, except the group immunized with tgD in alum, had significantly higher titers
Discussion
This is the first report demonstrating the potential of CpG DNA to enhance immune responses against an economically important pathogen in a veterinary species, as to date only rodent or primate models have been used to demonstrate the efficacy of this class of immunostimulant. These data demonstrate that ODN 2135 has an adjuvant effect when formulated at 10 mg per dose with BHV-1 tgD and that the addition of ODN 2135 to a tgD-alum formulation enhances the antibody titers in a dose-dependent
Acknowledgements
The authors are grateful to D. Willson for clinical assessments, and to B. Evans, B. Caroll, J. Mamer, C. Olson and T. Bruneau for care and handling of the animals. We thank T. King and L. Latimer for excellent technical assistance. Financial support was provided by grants from the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research, QIAGEN-GmbH, Germany, and Canadian Adaptation and Rural Development. L.A. Babiuk is recipient of a Canada research
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Published as VIDO’s Journal Series Number 304.
- 1
Present address: European Patent Office, P.B. 5818 Patentlaan 2, 2280 HV Rijswijk, The Netherlands.
- 2
Present address: Intervet International, Wim de Körverstraat 35, 5830 AA Boxmeer, The Netherlands.