Reno Pontarollo

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.

TLR9−/− and TLR9+/+ mice display similar immune responses to a DNA vaccine

Plasmid DNA continues to attract interest as a potential vaccine‐delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll‐like receptor 9‐deficient (TLR9−/−) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen‐specific antibody and at stimulating antigen‐specific interferon‐γ (IFN‐γ)‐secreting cells in TLR9−/− mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D☆

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

Recent advances in the use of DNA vaccines for the treatment of diseases of farmed animals

DNA-based vaccination constitutes one of the most recent approaches to vaccine development. This technology is in principle one of the most simple and yet versatile methods of inducing both humoral and cellular immune responses, as well as protection against a variety of infectious agents. However, although immune responses have been induced in a number of larger species, most information on the efficacy of DNA immunization has been generated in mice. In this review the information available to date about the use of DNA vaccines in farmed animals, including cattle, pigs and poultry, is presented. The areas that need specific attention in the future to bring this technology to the market are discussed, including the issues concerning delivery, safety, compatibility of plasmids in multivalent vaccines and the potential of using immune stimulants as part of a DNA vaccine.

Topical delivery of plasmid DNA using biphasic lipid vesicles (Biphasix)

The development of non‐invasive methods for the delivery of vaccines through the skin will greatly improve the safety and the administration of human and veterinary vaccines. In this study we examined the efficiency of topical delivery of plasmids by assessing the localization of gene expression using luciferase as a reporter gene and induction of immune responses using a plasmid encoding for the bovine herpesvirus type‐1 glycoprotein D (pgD). Topical administration of plasmids in a lipid‐based delivery system (biphasic lipid vesicles – Biphasix) resulted in gene expression in the lymph node, whereas with intradermal injection, antigen expression was found in the skin. Following administration of plasmid with the gene gun, antigen expression was observed in both the skin as well as in the draining lymph nodes. Transcutaneous immunization with pgD formulated in biphasic lipid vesicles elicited gD‐specific antibody responses and a Th2‐type cellular response. In contrast, immunization by the intradermal route resulted in the stimulation of a Th1‐type response. These findings have implications for both vaccine design and tailoring of specific immune responses.

Priming by DNA immunization augments T-cell responses induced by modified live bovine herpesvirus vaccine

DNA vaccines have several advantages over conventional vaccines. One of the most important characteristics is the presentation of antigen via both MHC class I and class II receptors. Although this generally results in strong T-cell responses, antibody production and protection achieved by DNA immunization are unfortunately not always adequate. In contrast, modified live virus (MLV) vaccines usually induce adequate antibody and moderate cellular responses, whereas killed vaccines tend to elicit weak immune responses in general. A DNA prime-MLV boost regimen should result in enhanced cellular immunity and possibly improved antibody production. To test this hypothesis, plasmids encoding bovine herpesvirus-1 (BHV-1) glycoproteins B and D were delivered by gene gun to the genital mucosa of cattle prior to immunization with modified live BHV-1 vaccine. The immune responses induced were compared to those of an MLV-vaccinated group and a negative control group. Although significantly enhanced T-cell responses were induced by priming with the DNA vaccine, there was no increase in antibody titres. Similar levels of protection were induced by the MLV vaccine alone and the DNA prime and MLV boost regimen, which suggests that there is no correlation between the induction of T-cell responses and protection from BHV-1 challenge.

BoLA class I allele diversity and polymorphism in a herd of cattle

Major histocompatibility complex class I genes are among the most polymorphic genes characterized. The high level of polymorphism is essential for generating host immune responses. In humans, three distinct genomic loci encode human leukocyte antigen (HLA) class I genes, allowing individuals to express up to six different HLA class I molecules. In cattle, the number of distinct genomic loci are currently at least six, and the number of different bovine leukocyte antigens (BoLA) class I molecules that are expressed in individual animals are variable. The extent of allele variation within the cattle population is unknown. In this study, the number and variety of BoLA class I sequences expressed by 36 individuals were determined from full-length BoLA class I cDNA clones. Twenty distinct BoLA class I alleles were identified, with only four being previously reported. The number of expressed BoLA class I alleles in individual animals ranged between one and four, with none of the animals having an identical complement of BoLA class I molecules. Variation existed in the number of BoLA class I alleles expressed as well as the composition of expressed alleles, however, several BoLA class I alleles were found in multiple individual animals. Polymorphic amino acid sites were analyzed for positive and negative selection using the ADAPTSITE program. In the antigen recognition sites (ARS), there were eight positions that were predicted to be under positive selection and three positions that were predicted to be under negative selection from 62 positions. In contrast, for non-antigen recognition sites (non-ARS), there were three positions that were predicted to be under positive selection and 20 that were predicted to be under negative selection from 278, indicating that positive selection of amino acids occurs at a greater frequency within the antigen recognition sites.

Effects of intradermally administered plasmid deoxyribonucleic acid on ovine popliteal lymph node morphology.

In the last decade it has become apparent that bacterial deoxyribonucleic acid (DNA) is recognized as a “danger signal” by the mammalian immune system. To investigate this interaction, sheep were injected intradermally two centimeters distal to the lateral prominence of the fibular head with 400 μg of purified plasmid DNA. Over a 28‐day period ultrasound measurements indicated a progressive increase in size of both plasmid and saline (controls) treated popliteal lymph nodes and at Day 30 macroscopic and histological measurements of the lymph nodes were determined. Compared with the contralateral control lymph nodes, plasmid exposed lymph nodes were heavier (2.8 ± 0.1g vs. 2.0 ± 0.6 g) and displayed prominent histological changes in the cortex and medulla. Average medullary cord thickness (114.2 ± 25.2 μm) and the average distance across medullary sinuses (64.4 ± 2.5 μm) were significantly greater after plasmid exposure relative to contralateral controls (62.7 ± 14.9 μm and 36.5 ± 1.0 μm, respectively). Total number of germinal centers (71.4 ± 17.7) and the total area of germinal centers (4.0 ± 1.3 mm2) within the cortex of popliteal lymph nodes exposed to plasmid were also significantly greater than the controls (40.4 ± 11.4 and 1.6 ± 0.5 mm2, respectively). Our results demonstrate that a single exposure to plasmid DNA has long term effects on regional lymph node weight and morphology. Anat Rec 262:186–192, 2001.