Vaccine 21 (2002) 127–137 CpG-containing oligodeoxynucleotides, in combination with conventional adjuvants, enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein X.P. Ioannou a , S.M. Gomis a , B. Karvonen a , R. Hecker b , L.A. Babiuk a , S. van Drunen Littel-van den Hurk a,∗ a Veterinary Infectious Disease Organization, 120 Veterinary Road, Saskatoon, Sask., Canada S7N 5E3 b QIAGEN-GmbH, Max-Volmer-Street 4, 40724 Hilden, Germany Received 21 March 2002; received in revised form 16 July 2002; accepted 24 July 2002 Abstract Vaccine adjuvants must have the capacity to increase protective immune responses with minimal side effects. Conventional adjuvants not only cause undesirable tissue site reactions, but often induce T-helper type 2 (Th2)-biased responses which may be undesirable in certain disease scenarios. Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) are novel adjuvants known to promote Th1-type immune responses. In this study, we compared various mineral oil, metabolizable oil and non-oil adjuvants alone and in combination with CpG ODN for their ability to augment immune responses to a truncated secreted form of bovine herpesvirus (BHV) glycoprotein D (tgD). All adjuvants tested induced Th2-biased immune responses characterized by a predominance of serum IgG1 as well as interleukin-4 (IL-4) production by in vitro stimulated splenocytes. The inclusion of CpG ODN in these formulations not only increased immune responses, but more importantly enhanced serum IgG2a levels and production of interferon- (IFN-) by splenocytes, indicating a more balanced or Th1-type response. The use of a mineral oil-based adjuvant at reduced doses in combination with CpG ODN attenuated the tissue damage while not compromising the magnitude of the immune response in both mice and sheep. In addition, reduced amounts of mineral oil combined with CpG ODN induced a more balanced Th1/Th2 immune response than the mineral oil used alone. Our results clearly demonstrate that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing the amount of mineral oil and hence undesirable side effects of vaccine adjuvants. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Adjuvant; CpG ODN; Immunization; Vaccination 1. Introduction Presently, all conventional inactivated and subunit vac- cines for humans and animals are mixed with adjuvants to increase their ability to induce immune responses. Alu- minum compounds such as aluminum hydroxide [Al(OH) 3 ] and aluminum phosphate (AlPO 4 ) have been used widely in both human and veterinary vaccines since 1930 [1]. In- deed, these compounds are the only adjuvants licensed for use in humans. These adjuvants skew the immune response towards a T-helper type 2 (Th2) response, which is char- acterized by the secretion of Th2-type cytokines such as interleukin-4 (IL-4) and IL-5 and the generation of IgG1 and IgE, but weak or absent cytotoxic T lymphocyte (CTL) re- ∗ Corresponding author. Tel.: +1-306-966-7487; fax: +1-306-966-7478. E-mail address: vandenhurk@sask.usask.ca (S. van Drunen Littel-van den Hurk). sponses [2–5]. T-helper type 2-dominant immune responses are associated with certain immunopathological compli- cations such as allergy, asthma, and autoimmune disease [6,7]. Redirecting a Th2-dominated immune response to a Th1-biased response may prevent or even reverse such com- plications [7,8]. In addition, Th1-type immune responses are essential for the control of intracellular infections [9]. In a murine Leishmania major infection model resistance or susceptibility to the disease is linked to the development of a Th1 or Th2 response, respectively [10,11]. A similar situation is reported in mice infected with Schistosoma mansoni where susceptibility to disease is likewise asso- ciated with a Th2-type immune response [12]. The ability to modulate immunoglobulin isotype subclass distribution is therefore an important property for adjuvants. Synthetic oligodeoxynucleotides containing unmethylated CpG dinu- cleotides (CpG ODN) are novel adjuvants known to selec- tively induce Th1-dominated immune responses [13]. CpG 0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0264-410X(02)00378-X