IL-10 secreting CD21⁺ B cells are present in jejunal Peyer's patches of sheep during fetal development.

We recently reported a novel interleukin-10 (IL-10)-secreting CD21(+) B cell population in jejunal Peyer's patches (JPP) of sheep with a regulatory function (Bregs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21(+) B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21(+) B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for Bregs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21(+) B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21(+) B cells in PPs prior to antigen exposure.

Human dendritic cells expressing hepatitis C virus core protein display transcriptional and functional changes consistent with maturation.

Hepatitis C virus (HCV) causes a chronic liver infection, which may result in cirrhosis and hepatocellular carcinoma. Impairment of the maturation process in dendritic cells (DCs) may be one of the mechanisms responsible for immune evasion of HCV. The core and NS3 proteins are among the most conserved HCV proteins and play a key role in viral clearance. To evaluate the effects of these proteins on DCs, monocyte-derived immature DCs (iDCs) were transfected with in vitro transcribed (IVT) HCV core or NS3 RNA and treated with maturation factors. Neither core nor NS3 had an inhibitory effect on DC maturation; however, transfection of iDCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of iDCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison with NS3 or GFP RNA treatments. Forty-two of these genes are involved in DC maturation. The effects of core on maturation of iDCs were confirmed with a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-γ secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.

Fetal immunization by a DNA vaccine delivered into the oral cavity.

Infectious diseases are the main cause of neonatal morbidity and mortality in humans. The World Health Organization estimated that in 1995 approximately 8 million infants died within the first year of life from infectious diseases, including 5 million during the first week of life. Some of the salient pathogens involved include herpes simplex virus, human immunodeficiency virus, hepatitis B virus, human cytomegalovirus, group B streptococcus, hemophilus and chlamydia. Infection with these pathogens usually occurs at the end of pregnancy, during birth or by breastfeeding. To reduce the risk of disease transmission, caesarian sections, prophylactic treatment with antibiotics or maternal antiviral therapy during the last trimester are used where available, together with improved neonatal care. None of these approaches, however, completely eliminates the risk of neonatal infection. Therefore, active or passive immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. Here, we demonstrate that a single immunization with a DNA vaccine delivered into the amniotic fluid in the oral cavity induces high serum antibody titers and a cell-mediated immune response, combined with induction of local immunity in the oral cavities of fetal lambs.

Natural and inducible regulatory B cells are widely distributed in ovine lymphoid tissues.

Regulatory B cells that produce IL-10 are now recognized as an important component of the immune system. We previously confirmed that IL-10 secreting CD21+ regulatory B cells (Breg cells) were present in ovine jejunal Peyer's patches (JPP) and this IL-10 production suppressed IL-12 and IFN-γ secretion. It is not known, however, whether ovine Breg cells are restricted to JPP or are present in other lymphoid tissues. Therefore, CD21+ B cells were purified from sheep JPP and from a variety of mucosal and systemic lymphoid tissues using magnetic cell sorting. Purified CD21+ B cells were stimulated with a TLR9-agonist, CpG oligodeoxynucleotide (CpG ODN), and the frequency of spontaneous and inducible (i) IL-10-secreting B cells was evaluated by ELISPOT. Spontaneous IL-10 secreting CD21+ B cells were present in mucosal (jejunal PP, parabronchial lymph nodes (LN), mesesnteric LN, and palatine tonsils) and systemic (spleen and blood) lymphoid tissues. Mucosal lymphoid tissues (parabronchial and mesenteric LNs and JPP) had the highest frequency of cells spontaneously secreting IL-10 while tonsils had the lowest. The frequency of B cells spontaneously secreting IL-10 was lowest in blood and spleen. There was large inter-animal variation in the frequency of CD21+ B cells spontaneously secreting IL-10 and no significant difference was detected following CpG ODN stimulation. When comparing within individual animals there was, however, a consistent increase in the frequency of CD21+ cells secreting IL-10 following CpG ODN stimulation versus stimulation with GpC control ODN. The presence of inducible (i)Breg cells in ovine mucosal tissues supports previous evidence from mice indicating that B cells have the capacity to modulate inflammatory responses. The presence of iBreg cells in ruminants may also provide a novel therapeutic target for both immunomodulatory drugs and vaccines designed to control antigen-specific mucosal inflammation.

Strategies for loading dendritic cells with hepatitis C NS5a antigen and inducing protective immunity.

Dendritic cell (DC)-based vaccination strategies are promising for the treatment of cancers and infectious diseases including hepatitis C virus (HCV). As the induction of T cell-mediated immune responses by DC vaccination is highly dependent on efficient antigen loading of the DCs, the purpose of this study was to identify an optimal nonviral DC loading strategy for HCV NS5a. Furthermore, the efficacy of immunization with the NS5a-loaded DCs in comparison to plasmid encoding NS5a and NS5a protein was evaluated. Transfection of DCs with mRNA was most efficient with close to 100% of DCs expressing NS5a, whereas approximately 10% of protein-pulsed DCs and <1% of plasmid-transfected DCs expressed NS5a, suggesting remarkably different loading efficiencies. Vaccination of mice with NS5a mRNA-transfected DCs or NS5a protein-pulsed DCs resulted in significantly stronger CD4(+) and CD8(+) T-cell responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5, in comparison to vaccination with NS5a DNA-transfected DCs, plasmid encoding NS5 or rNS5a protein formulated with alum. Furthermore, vaccination with NS5a mRNA-transfected DCs was superior to vaccination with rNS5a-pulsed DCs. These data have important clinical implications, with mRNA-transfected DCs providing a safe and effective vaccination strategy against hepatitis C and possibly other pathogens.

Animal genomics for animal health report: critical needs, problems to be solved, potential solutions, and a roadmap for moving forward.

The first International Symposium on Animal Genomics for Animal Health, held at the World Organisation for Animal Health (OIE) Headquarter, 23-25 October, 2007, Paris, France, assembled more than 250 participants representing research organizations from 26 countries. The symposium included a roundtable discussion on critical needs, challenges and opportunities, and a forward look at the potential applications of animal genomics in animal health research. The aim of the roundtable discussion was to foster a dialogue between scientists working at the cutting edge of animal genomics research and animal health scientists. In an effort to broaden the perspective of the roundtable discussion, the organizers set out four priority areas to advance the use of genome-enabled technologies in animal health research. Contributions were obtained through open discussions and a questionnaire distributed at the start of the symposium. This symposium report provides detailed summaries ofthe outcome ofthe roundtable discussion for each of the four priority areas. For each priority, the problems needing to be solved, according to the views of the participants, are identified, including potential solutions, recommendations, and lastly, concrete steps that could be taken to address these problems. This report serves as a roadmap to steer research priorities in animal genomics research.

Innate immunity and new adjuvants.

Vaccination remains the most cost-effective biomedical approach to the control of infectious diseases in livestock. Vaccines based on killed pathogens or subunit antigens are safer but are often ineffective and require coadministration with adjuvants to achieve efficacy. Unfortunately, most conventional adjuvants are poorly defined, complex substances that fail to meet the stringent criteria for safety and efficacy desired in new generation vaccines. A new generation of adjuvants that work by activating innate immunity presents exciting opportunities to develop safer, more potent vaccines. In this review the authors highlight the role of innate immunity in protection against infectious disease and provide some examples of promising new adjuvants that activate innate immunity. They do not review the conventional adjuvants present in many vaccines since they have been reviewed extensively previously.

Vaccination in conservation medicine.

Unprecedented human population growth and anthropogenic environmental changes have resulted in increased numbers of people living in closer contact with more animals (wild, domestic, and peridomestic) than at any other time in history. Intimate linkage of human and animal health is not a new phenomenon. However, the global scope of contemporary zoonoses has no historical precedent. Indeed, most human infectious diseases classed as emerging are zoonotic, and many of these have spilled over from natural wildlife reservoirs into humans either directly or via domestic or peridomestic animals. Conservation medicine has recently emerged as a meaningful discipline to address the intersection of animal, human, and ecosystem health. Interest in the development of novel vaccines for wildlife encounters important challenges that may prevent progress beyond the conceptual phase. Although notable examples of successful wildlife immunisation programmes exist, depending upon key considerations, vaccination may or may not prove to be effective in the field. When implemented, wildlife vaccination requires a combination of novel zoonosis pathogen management strategies and public education to balance conservation, economic, and public health issues.

Review: Capripoxvirus Diseases: Current Status and Opportunities for Control.

Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine.

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 naïve 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.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004.

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.

Evidence for the existence of regulatory and effector B cell populations in Peyer's patches of sheep.

IL-10 secreting CD21(+) B cells exist in sheep Peyer's patches (PP). It's not known however, whether all PP B cells are regulatory or whether an effector population also exists in this tissue. To further characterize the subpopulations of B cells in PP's, highly purified B cells were negatively sorted from jejunal PP and fractionated according to co-expression of CD72(+)CD21(+)or CD72(+)CD21(-) molecules and then stimulated with the TLR9-agonist, CpG ODN. IL-10, IL-12, IFN-γ, and IgM production were then assayed. We observed that only highly purified CD72(+)CD21(+) B cells spontaneously secreted high levels of IL-10, but they did not produce any IL-12, IFN-γ or IgM suggesting that this cell population contains regulatory B cells. In contrast, CD72(+)CD21(-) B cells did not secrete IL-10, but secreted IL-12, IFN-γ, and IgM, suggesting they include effector cells. In addition, B cells expressing surface IgA, IgM and IgG1 all secreted similar levels of IL-10. We further confirmed that only B cells produce IL-10, while other cells in the PP including DCs and T cells do not. Our investigations may provide evidence for the existence of two sub-populations in sheep PP; IL-10 secreting regulatory (CD72(+)CD21(+)) cells, and IL-12/IFN-γ/IgM-secreting effector (CD72(+)CD21(-)) cells.

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis.

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-gamma (IFN-gamma) responses induced by BCG, the CFP vaccines induced strong antibody responses but weak IFN-gamma 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-gamma 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.

Bovine alveolar macrophages: phenotypic and functional properties of subpopulations obtained by Percoll density gradient centrifugation.

Bronchoalveolar cells retrieved from conventionally raised, healthy calves were separated into four fractions on a discontinuous Percoll density gradient. The alveolar macrophage (AM) subpopulations and nonseparated AM were assayed for such phenotypic markers as la-antigen, ectoenzymes, and immune receptors, as well as for functional activity in antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, superoxide anion generation, and their influence on lectin-induced lymphocyte proliferation. The low-density fraction was composed of large cells with low Ia-antigen expression, low ADCC activity, and high ecto-enzyme and C3b-receptor activity. In contrast the high-density fraction contained mainly small, monocytelike cells, with high Ia expression and low-level expression of most other markers and functions. Two fractions of intermediary density overlapped in most of the characteristics, but could be distinguished on the basis of ADCC activity, interleukin-1 generation, and the level of leucine amino peptidase activity.

Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse model.

The nasal mucosa is an important arm of the mucosal system since it is often the first point of contact for inhaled antigens. The ineffectiveness of the simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies in appropriate delivery systems and adjuvants. We have evaluated biphasic lipid vesicles as a novel intranasal (i.n.) delivery system (designated as vaccine targeting adjuvant, VTA) containing bacterial antigens and CpG oligodeoxynucleotides (ODNs). Results show that administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in greater induction of IgA levels in serum (P< 0.05) and mucosal antibody responses such as IgA in nasal secretions and lung (P< 0.01) after immunization with a combined subcutaneous (s.c.)/i.n. as compared to s.c./s.c. approach. Based on antibody responses, VTA formulations were found to be suitable as delivery systems for antigens and CpG ODNs by the intranasal route, resulting in a Th2-type of immune response, characterized by IgG1 and IL-4 production at the systemic level.

Novel vaccines from biotechnology.

Vaccination continues to be the main approach to protecting animals from infectious diseases. Until recently, all licensed vaccines were developed using conventional technologies. However, the introduction of modern molecular biological tools and genomics, combined with a better understanding of not only which antigens are critical in inducing protection, but an appreciation of host defences that must be stimulated, has opened a new opportunity to develop safer and more effective vaccines. The authors describe the current and future trends in vaccine development and stress that in addition to identifying and producing the protective antigens, it is critical to formulate and deliver these vaccines appropriately to maximise the potential of modern advances in pathogenesis and vaccinology.

Rotavirus particles function as immunological carriers for the delivery of peptides from infectious agents and endogenous proteins.

A major problem in the development of useful animal subunit vaccines has been the generation of immune responses to weakly immunogenic molecules. For this purpose a new and effective delivery system has been devised. This system is based upon the inner capsid of bovine rotavirus. Under the appropriate conditions, the inner capsid protein, designated BP6, can be made to self-assemble in vitro and form spherical particles. These particles possess an inherent capacity to target to cells of the immune system. Exploitation of these properties has led to the development of technology to couple antigens to the VP6 particles such that the sphere acts as a novel immunological carrier. This is based on a "binding peptide" derived from another rotavirus peptide, VP4, as well as on more traditional techniques of chemical coupling. We have coupled peptides or proteins to this carrier via the binding peptide and have shown that every epitope tested to date gave excellent immune responses. Furthermore, using this carrier, immunity has been developed without the use of adjuvants. This has far-reaching implications for animal and human immunization.

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.

Sequence and expression of a bovine herpesvirus-1 gene homologous to the glycoprotein K-encoding gene of herpes simplex virus-1.

In the bovine herpes virus-1 (BHV-1) genome, a gene equivalent to the glycoprotein k (gK)-encoding gene of other herpesviruses was identified and sequenced. The primary translation product is predicted to comprise 338 amino acids (aa) and to exhibit a molecular mass of 37.5 kDa. It possesses characteristics typical for membrane glycoproteins including a potential cleavable signal sequence, three transmembrane domains and two potential N-linked glycosylation sites. Comparison to the gK proteins of the other herpesviruses revealed aa sequence homologies of 46, 44, 53, 43, and 46% with the gK counterparts of herpes simplex viruses-1 and 2 (HSV-1 and 2), equine herpesvirus-1 (EHV-1), Marek's disease virus (MDV) and varicella zoster virus (VZV), respectively. A 30-kDa primary translation product was identified following in vitro translation of in vitro transcribed mRNA. When canine microsomal membranes were added to the translation reaction, a 38-kDa glycosylated protein was detected. Treatment with endoglycosidase F or H (endo or H) removed the glycosyl groups and reduced the apparent molecular mass of the 38-kDa glycoprotein.

Effect of recombinant bovine interleukin-1 beta in normal calves and in calves infected with bovine herpesvirus type 1.

Bovine herpesvirus-1 (BHV-1) is an important pathogen of respiratory infections in cattle. Its continuing importance lies in its ability to predispose infected hosts to bacterial infections. In this present study, we determined whether the immunoregulatory effects induced by interleukin-1 (IL-1) could stimulate appropriate host defense mechanisms to influence the course of BHV-1 infection in cattle. We first evaluated the effect of different doses (10-1000 ng/kg) of IL-1 in normal cattle. A single administration of IL-1 was able to induce a dose-dependent increase in polymorphonuclear (PMN) cells as well as monocytes in peripheral blood. The number of CD3+ lymphocytes and gamma/delta T cells in peripheral circulation decreased transiently in a dose-dependent manner. In the disease model, the effect of IL-1 administration (300 ng/kg) 24 h before, at the time of, and 24 h after the BHV-1 challenge was assessed. As a single therapeutic modality, IL-1 did not significantly reduce the establishment or progression of BHV-1-induced disease. Nevertheless, our results demonstrated that the significant modulation of diverse immune parameters did not exacerbate disease. Thus, the use of IL-1 as an adjunct therapy or as a vaccine adjuvant in cattle can be safely considered in situations where BHV-1 infection is likely to occur.