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Polynucleotide immunization has captured the imagination of numerous researchers and commercial companies around the world as a novel approach for inducing immunity in animals. Clearly, the ‘proof-of-principle’ has been demonstrated both in rodents and various animal species. However, to date, no commercial veterinary vaccine has been developed, or to our knowledge, is in the licensing phase. The present review summarizes the types of pathogens and host species for which polynucleotide immunization has been tried. We have tried to identify possible barriers to commercialization of this technology and areas that need attention if this promising technology is ever to become a reality in the commercial arena.

Recent advances in our understanding of virulence factors of viruses and the proteins or glycoproteins involved in inducing neutralizing antibodies or cell mediated immunity are forming the foundation for the development of a new generation of viral vaccines. Using bovine herpesvirus as an example, we have identified glycoproteins gB, gC, and gD as important targets for inducing neutralizing antibody responses, with gD being able to induce the highest neutralizing and cellular responses. For subunit vaccine development, the glycoproteins were produced in both prokaryotic and eukaryotic expression systems. Glycoproteins produced in eukaryotic systems were very effective in stimulating a broad range of immune responses in cattle. These glycoproteins were then formulated into effective vaccines that prevented both virus shedding and clinical disease. Herpesviruses also served as an excellent model for the identification and deletion of specific genes which lead to attenuation. In herpesviruses, two major classes of genes can be deleted. Class I includes glycoprotein genes that are nonessential for virus replication in vitro, and Class II includes genes involved in nucleic acid metabolism. these gene deleted regions can then be replaced with genes coding for protective antigens of other pathogens to develop multivalent vaccines in a single vector. Similar approaches are being used for other viruses including vaccinia virus and adenovirus. Finally, we introduced plasmids coding for protective antigens, gB, gC, and gD, into animals and developed immunity to these antigens. This approach has the potential to revolutionize vaccination regimes of the future.

Human and murine immature DCs (iDCs) are highly efficient in antigen capture and processing, while as mature cells they present antigen and are potent initiators of cell-mediated immune responses. Consequently, iDCs are logical targets for vaccine antigens. Originally discovered for their antimicrobial activity, and thought of as strictly part of the innate immune system, studies with defensins such as human β (beta)-defensin 2 (hBD2) and murine β-defensin 2 (mBD2) have shown that they can function as chemo-attractant for iDCs and, in vaccination strategies, can enhance antigen-specific adaptive immune responses. Most studies to date have been conducted in mice. In contrast, little is known about defensins in cattle. To expand our understanding of the role of defensins in modulating immune responses in cattle, DCs were generated from bovine monocytes and the immature state of these bovine DCs was characterized phenotypically and through functional assays. By day 3 (DC3), bovine monocyte-derived DCs stained positively for DC-specific receptors CD1, CD80/86, CD205, DC-Lamp and MMR. When compared to conventional 6-day DC cultures or DCs cultured for 10 days with and without maturation factors, these DC3 were functionally at their most immature stage. Fourteen of the 16 known bovine β-defensins were synthesized and the synthetic peptides were screened for their ability to attract bovine iDCs. Bovine DC3 were consistently attracted to BNBD3, an analog of BNBD3 (aBNBD3), BNBD9 and bovine EBD in vitro and to aBNBD3 in vivo. These results are the first to describe chemotactic ability of synthetic bovine β-defensins for immature bovine monocyte-derived DCs.

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty naive horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.

Culture filtrate protein (CFP) vaccines have been shown to be effective in small animal models for protecting against tuberculosis while immunisation with these types of vaccines in cattle has been less successful. A study was conducted in cattle to evaluate the ability of selected adjuvants and immunomodulators to stimulate protective immune responses to tuberculosis in animals vaccinated with Mycobacterium bovis CFP. Seven groups of cattle (n = 5) were vaccinated with M. bovis CFP formulated with either Emulsigen™ or Polygen™ adjuvant alone or in combination with a specific oligodeoxynucleotides (ODN), polyinosinic acid: polycytidylic acid (poly I:C) or poly I:C and recombinant granulocyte-macrophage colony stimulating factor. Two additional groups were vaccinated subcutaneously with BCG or non-vaccinated. In contrast to the strong interferon-γ (IFN-γ) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-γ responses. The addition of CpG ODN to CFP significantly enhanced cell-mediated responses and elevated antibody responses to mycobacterial antigens. Of the CFP vaccinated groups, the strongest IFN-γ responses to CFP vaccines were measured in animals vaccinated with CFP/Emulsigen + CpG or CFP/Polygen + CpG. The animals in these two groups, together with those in the BCG and non-vaccinated groups were challenged intratracheally with virulent M. bovis at 13 weeks after the first vaccination and protection was assessed, by examination for presence of tuberculous lesions in the lungs and lymph nodes, 13 weeks later at postmortem. While BCG gave the best overall protection against tuberculosis, significant protection was also seen in animals vaccinated with CFP/Emulsigen + CpG. These results establish an important role for CpG ODN in stimulating protective Th1 responses to tuberculosis in cattle and indicate that a sub-unit protein vaccine can protect these animals against tuberculosis.

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.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.