Protective Efficacy of BCG Vaccination in Calves Vaccinated at Different Ages.

, the causative agent of bovine tuberculosis (bTB), is a globally prevalent pathogen with significant animal welfare, economic and public health impacts. In the UK, the control of bTB relies on detection via tuberculin skin tests with ancillary interferon gamma (IFN-γ) release assays, followed by culling infected animals. Vaccination with Bacille Calmette-Guérin (BCG) could be an important element of bTB control, and a number of studies have demonstrated its protective efficacy, particularly when young calves are vaccinated.

CD205 antigen targeting combined with dendritic cell recruitment factors and antigen-linked CD40L activation primes and expands significant antigen-specific antibody and CD4(+) T cell responses following DNA vaccination of outbred animals.

Dendritic cell antigen targeting primes robust immune responses in mouse models. Optimizing this immunization strategy in the actual hosts that require protection will advance development of efficacious contemporary vaccines. In a proof-of-concept study, we tested the immunogenicity of a single, low dose of a novel multi-component DNA construct expressing a CD205-targeted antigen fused to a CD40L minimal functional domain for linked DC activation.

The calf model of immunity for development of a vaccine against tuberculosis.

Tuberculosis (TB) remains a major threat to public health. The identification of safe TB vaccine candidates beyond Mycobacterium bovis BCG, is an exciting prospect for control of human TB and necessary in the context of the human immunodeficiency virus (HIV) pandemic. Selection of vaccine candidates for human trials which are ultimately targeted for use in children less than 5 years of age or in newborns will require an animal model that closely approximates immune function and disease.

Isolation and purification of afferent lymph dendritic cells that drain the skin of cattle.

Dendritic cells (DCs) are central to the induction of immune responses and are a pivotal control point that determines the outcome of infectious challenge. Cannulation of afferent lymphatic vessels allows the isolation of large numbers of lymph DCs. First, lymph nodes that are draining the skin are surgically removed (takes approximately 1 h).

Characterisation of bovine inducible nitric oxide synthase.

Inducible nitric oxide (iNOS) is an enzyme that catalyzes the production of the reactive nitrogen intermediate nitric oxide (NO). NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of cell death (apoptosis). NO plays a major role in the killing of intracellular pathogens as part of the innate immune response.

DNA vaccine construct incorporating intercellular trafficking and intracellular targeting motifs effectively primes and induces memory B- and T-cell responses in outbred animals.

We developed a vaccine construct in which a BVP22 domain and an invariant-chain major histocompatibility complex class II-targeting motif capable of enhancing dendritic cell antigen uptake and presentation were fused to a sequence encoding a B- and T-cell antigen from the Anaplasma marginale major surface protein 1a and tested whether this construct would prime and expand immune responses in outbred calves. A single inoculation with this construct effectively primed the immune responses, as demonstrated by a significant enhancement of CD4(+) T-cell proliferation compared to that in calves identically inoculated but inoculated with a DNA construct lacking the targeting domains and compared to that in calves inoculated with an empty vector. These proliferative responses were mirrored by priming and expansion of gamma interferon-positive CD4(+) T cells and immunoglobulin G responses against the linked B-cell epitope.

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease.

Rapid and long-term disappearance of CD4+ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale.

In humans and ruminants infected with Anaplasma, the major surface protein 2 (MSP2) is immunodominant. Numerous CD4(+) T cell epitopes in the hypervariable and conserved regions of MSP2 contribute to this immunodominance. Antigenic variation in MSP2 occurs throughout acute and persistent infection, and sequentially emerging variants are thought to be controlled by variant-specific Ab.

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle.

Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists.

Toll-like receptors (TLR) have been described as partially sharing signalling pathways but showing unique ligand specificity and tissue distribution. Here, the response of bovine macrophages (Mphi) and dendritic cells (DC), both derived from monocytes, was compared by exposing them to the TLR-specific ligands lipopolysaccharide, poly(I:C)-double-stranded RNA, and CpG-DNA, as well as inactivated Gram-negative and Gram-positive bacteria, shown to bind to TLR. The production of NO, superoxide anion, interleukin-10 (IL-10), IL-12 and tumour necrosis factor (TNF) was determined.

Maturation of bovine dendritic cells by lipopeptides.

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation.

Differences in cytokine synthesis by the sub-populations of dendritic cells from afferent lymph.

Two phenotypically distinct subpopulations of dendritic cells (SIRPalpha+ CC81Ag- DC and SIRPalpha- CC81Ag+ DC) have previously been identified in bovine afferent lymph which show functional differences when assayed in vitro. The purpose of this study was to investigate whether differences in cytokine production between the two subpopulations might occur which could influence the bias of the immune response they stimulate. Qualitative and quantitative polymerase chain reactions were used to detect specific mRNA transcripts and flow cytometry and enzyme-linked immunosorbent assays were used to detect protein production.

CpG ODN 2006 and IL-12 are comparable for priming Th1 lymphocyte and IgG responses in cattle immunized with a rickettsial outer membrane protein in alum.

Immunostimulatory oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) stimulate IL-12-dependent Th1 dominated cytokine and enhanced IgG responses when co-delivered with antigen to mice. However, the CpG ODN sequences that are optimal for each mammalian species may differ. Previously, we demonstrated that a CpG ODN containing the GTCGTT motif was optimal for stimulating bovine B cell proliferation, and induced IL-6, IL-12 and IFN-gamma production by peripheral blood mononuclear cells (PBMC).

Peripheral blood-derived bovine dendritic cells promote IgG1-restricted B cell responses in vitro.

Regulation of humoral responses involves multiple cell types including the requirements for cognate interactions between T and B cells to drive CD40-dependent responses to T-dependent antigens. A third cell type has also been shown to play an essential role, the dendritic cell (DC). We demonstrate that bovine peripheral blood-derived (PB)-DC are similar in function to features described for human interstitial DC including the production of signature type 2 cytokines [interleukin (IL)-13, IL-10].

A comparison of the anti-inflammatory and immuno-stimulatory activities of orf virus and ovine interleukin-10.

Orf virus causes pustular skin lesions (orf) in sheep, goats and humans. The virus encodes an interleukin-10 (orfvIL-10) that is identical in amino acid composition to ovine IL-10 (ovIL-10) over the C terminal two-thirds of the polypeptide, but not in the N terminal third. The immuno-suppressive and immuno-stimulatory activities of orfvIL-10 and ovIL-10 were compared.

DNA-encoded fetal liver tyrosine kinase 3 ligand and granulocyte macrophage-colony-stimulating factor increase dendritic cell recruitment to the inoculation site and enhance antigen-specific CD4+ T cell responses induced by DNA vaccination of outbred animals.

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals.

Cytokine responses of bovine dendritic cells and T cells following exposure to live or inactivated bovine respiratory syncytial virus.

We compared the effects of live or inactivated bovine respiratory syncytial virus (BRSV) on cytokine production by bovine monocyte-derived dendritic cells (MoDC). We also investigated the response of resting memory CD4(+) T cells to MoDC exposed to both viral preparations. Although BRSV did not appear to replicate in MoDC or to affect expression of major histocompatibility complex (MHC) class I, MHC class II, or CD80/86, a higher percentage of cells exposed to live virus appeared to undergo apoptosis/necrosis.

CD26 is expressed on a restricted subpopulation of dendritic cells in vivo.

Two major sub-populations of dendritic cells (DC) are present in afferent lymph draining the skin of cattle distinguished by expression of signal regulator protein alpha (SIRPalpha). The SIRPalpha(-) population expresses the uncharacterized bovine WC10 antigen (Ag). Initial N-terminal sequencing of the WC10 protein purified by affinity chromatography showed significant homology with human CD26.

Expression of caveolin by bovine lymphocytes and antigen-presenting cells.

Caveolin is a generic term for a family of proteins that include caveolin-1, -2 and -3. Although the distribution of these proteins varies between cells, caveolin-1 and -2 are commonly found coating membrane invaginations known as caveolae. Studies on human and murine cells suggest that caveolin/caveolae can be found in neutrophils, macrophages and mast cells, in which they are involved in the uptake of pathogens, but not in lymphocyte cell lines.

Caveolae and caveolin in immune cells: distribution and functions.

Caveolae are small, cholesterol-rich, hydrophobic membrane domains, characterized by the presence of the protein caveolin and involved in several cellular processes, including clathrin-independent endocytosis, the regulation and transport of cellular cholesterol, and signal transduction. Recently, caveolae have been identified as providing a novel route by which several pathogens are internalized by antigen-presenting cells and as centers for signal transduction. Here, we review the distribution and role of caveolae and caveolin in mammalian immune cells.

NK-like CD8(+) cells in immunologically naïve neonatal calves that respond to dendritic cells infected with Mycobacterium bovis BCG.

Pre-exposure to environmental mycobacteria and induction of an inappropriately biased immune response may be major factors affecting the efficacy of BCG; vaccination of neonates that have not been exposed to environmental mycobacteria may induce more effective immunity. Responses of neonatal calves to mycobacterial antigens using dendritic cells (DC) as antigen-presenting cells were investigated. In nonvaccinated, immunologically naive calves as young as 1 day old, a population of CD8(+) cells proliferated and produced IFN-gamma in response to BCG-infected DC.

Antigen-presenting cells from calves persistently infected with bovine viral diarrhoea virus, a member of the Flaviviridae, are not compromised in their ability to present viral antigen.

The aim of this study was to assess whether the infection of antigen-presenting cells (APC) in vivo, evident in calves persistently infected (PI) with bovine viral diarrhoea virus (BVDV), compromised their ability to stimulate virus-specific T cell responses. Major histocompatibility complex (MHC) molecule-identical cattle were identified from the inbred family at the Institute for Animal Health. One was PI and immunotolerant to BVDV.