Safety of CpG oligodeoxynucleotides in veterinary species.

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.

Safety and efficacy of CpG-containing oligodeoxynucleotides as immunological adjuvants in rabbits.

The efficacy of oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) as an adjuvant for rabbits was assessed alone and in combination with aluminum hydroxide (CpG/alum). The CpG/alum combination elicited a greater immune response to several antigens compared to Freund's adjuvant. A non-CpG/alum combination did not have the same effects as CpG/alum suggesting that the adjuvanticity was related to the CpG motifs. In addition, we formulated one of the antigens with combinations of CpG ODN and 30 or 10% Emulsigen (Em) [CpG/Em (30%) and CpG/Em (10%)]. Both CpG/Em (30%) and CpG/Em (10%) were more effective than Em, and equivalent to CpG/alum. The CpG/Em (10%) combination caused minimal tissue damage. Our results demonstrate that the addition of CpG ODN to aluminum hydroxide or to 10% Em significantly improves the efficiency of these adjuvants, without enhancing tissue reaction.

Biological activity of immunostimulatory CpG DNA motifs in domestic animals.

Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in domestic animals.

CpG-containing oligodeoxynucleotides, in combination with conventional adjuvants, enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein.

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-gamma (IFN-gamma) 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.

The immunogenicity and protective efficacy of bovine herpesvirus 1 glycoprotein D plus Emulsigen are increased by formulation with CpG oligodeoxynucleotides.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved.

Oligodinucleotides containing CpG motifs stimulate vertebrate immune cells in vitro, have proven efficacy in murine disease models and are currently being tested in human clinical trials as therapies for cancer, allergy, and infectious disease. As there are no known immunostimulatory motifs for veterinary species, the potential of CpG DNA as a veterinary pharmaceutical has not been investigated. Here, optimal CpG motifs for seven veterinary and three laboratory species are described. The preferential recognition of a GTCGTT motif was strongly conserved across two vertebrate phyla, although a GACGTT motif was optimal for inbred strains of mice and rabbits. In a subsequent adjuvanticity trial, the in vitro screening methodology was validated in sheep, representing the first demonstration of CpG DNA efficacy in a veterinary species. These results should provide candidate immunostimulant and therapeutic drugs for veterinary use and enable the testing of CpG DNA in large animal models of human disease.