MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

Histidine hydrogen bonding in MHC at pH 5 and pH 7 modeled by molecular docking and molecular dynamics simulations

Hydrogen bonds play important roles in maintaining the structure of proteins and in the formation of most biomolecular protein-ligand complexes. All amino acids can act as hydrogen bond donors and acceptors. Among amino acids, Histidine is unique, as it can exist in neutral or positively charged forms within the physiological pH range of 5.0 to 7.0. Histidine can thus interact with other aromatic residues as well as forming hydrogen bonds with polar and charged residues. The ability of His to exchange a proton lies at the heart of many important functional biomolecular interactions, including immunological ones. By using molecular docking and molecular dynamics simulation, we examine the influence of His protonation/deprotonation on peptide binding affinity to MHC class II proteins from locus HLA-DP. Peptide-MHC interaction underlies the adaptive cellular immune response, upon which the next generation of commercially-important vaccines will depend. Consistent with experiment, we find that peptides containing protonated His residues bind better to HLA-DP proteins than those with unprotonated His. Enhanced binding at pH 5.0 is due, in part, to additional hydrogen bonds formed between peptide His+ and DP proteins. In acidic endosomes, protein His79β is predominantly protonated. As a result, the peptide binding cleft narrows in the vicinity of His79β, which stabilizes the peptide - HLA-DP protein complex. © 2014 Bentham Science Publishers.

Two years survival rate of Class II ART restorations in primary molars using two ways to avoid saliva contamination

AIM To compare the survival rates of Class II Atraumatic Restorative Treatment (ART) restorations placed in primary molars using cotton rolls or rubber dam as isolation methods. METHODS A total of 232 children, 6-7 years old, both genders, were selected having one primary molar with proximal dentine lesion. The children were randomly assigned into two groups: control group with Class II ART restoration made using cotton rolls and experimental group using rubber dam. The restorations were evaluated by eight calibrated evaluators (Kappa > 0.8) after 6, 12, 18 and 24 months. RESULTS A total of 48 (20.7%) children were considered dropout, after 24 months. The cumulative survival rate after 6, 12, 18 and 24 months was 61.4%, 39.0%, 29.1% and 18.0%, respectively for the control group, and 64.1%, 55.1%, 40.1% and 32.1%, respectively for the rubber dam group. The log rank test for censored data showed no statistical significant difference between the groups (P = 0.07). The univariate Cox Regression showed no statistical significant difference after adjusting for independent variables (P > 0.05). CONCLUSION Both groups had similar survival rates, and after 2 years, the use of rubber dam does not increase the success of Class II ART restorations significantly.

Studies of MHC class I antigen presentation & the origins of the immunopeptidome

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer.

Studies of MHC class I antigen presentation & the origins of the immunopeptidome

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer.

Association of immunoproteasomes with the endoplasmic reticulum

Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three c-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the c-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immuno¯uorescence experiments with anticalreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. c-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the ®rst direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Non-inherited maternal HLA alleles are associated with rheumatoid arthritis

Background. Rheumatoid arthritis (RA) is strongly associated with a series of HLA-DRB1 alleles that encode a conserved sequence of amino acids (70Q/R K/R R A A74) in the DRβ1 chain, known as the shared epitope (SE). However 30% of patients are negative for DRB1*04 and 15% are SE-negative. Exposure to these alleles as non-inherited maternal antigens (NIMA) might explain this discrepancy. We undertook a family study to investigate the role of NIMA in RA. Methods. One hundred families, including the RA proband and both parents, were recruited. HLA-DRB1 genotyping was performed using an allele-specific polymerase chain reaction by standard methods. The frequencies of NIMA and non-inherited paternal antigens (NIPA) were compared using contingency tables and a two-tailed P test. We then reviewed four previously published studies of NIMA in RA and conducted an analysis of the combined data Results. We identified 36 families in which the proband was DRB1*04-negative and 13 in which the proband lacked the SE. There was an excess of DRB1*04 and SE NIMA (P=0.05) compared with NIPA. Combined analysis with previous studies showed that 53/231 mothers (23%) versus 25/205 fathers (12%) had a non-inherited DRB1*04 (P=0.003) and 30/99 mothers versus 18/101 fathers had a non-inherited SE allele (P=0.03). Conclusion. A role for HLA NIMA in RA is suggested by these results.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3+, CD4+, CD8+ and [gamma][delta] T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for [gamma][delta] T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Immuno-fluorescence staining patterns of leukocyte subsets in the skin of taurine and indicine cattle

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein–Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein–Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25+ cells labelled regularly shaped cells that showed a wide range of intensities of staining.

Targeting antigen to diverse APCs inactivates memory CD8+ T cells without eliciting tissue-destructive effector function

Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases. Copyright © 2010 by The American Association of Immunologists, Inc.

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Identifying Genetic Risk Factors in Canine Autoimmune Disorders

Autoimmune diseases are more common in dogs than in humans and are already threatening the future of some highly predisposed dog breeds. Susceptibility to autoimmune diseases is controlled by environmental and genetic factors, especially the major histocompatibility complex (MHC) gene region. Dogs show a similar physiology, disease presentation and clinical response as humans, making them an excellent disease model for autoimmune diseases common to both species. The genetic background of canine autoimmune disorders is largely unknown, but recent annotation of the dog genome and subsequent development of new genomic tools offer a unique opportunity to map novel autoimmune genes in various breeds. Many autoimmune disorders show breed-specific enrichment, supporting a strong genetic background. Furthermore, the presence of hundreds of breeds as genetic isolates facilitates gene mapping in complex autoimmune disorders. Identification of novel predisposing genes establishes breeds as models and may reveal novel candidate genes for the corresponding human disorders. Genetic studies will eventually shed light on common biological functions and interactions between genes and the environment. This study aimed to identify genetic risk factors in various autoimmune disorders, including systemic lupus erythematosus (SLE)-related diseases, comprising immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis arteritis (SMRA) as well as Addison s disease (AD) in Nova Scotia Duck Tolling Retrievers (NSDTRs) and chronic superficial keratitis (CSK) in German Shepherd dogs (GSDs). We used two different approaches to identify genetic risk factors. Firstly, a candidate gene approach was applied to test the potential association of MHC class II, also known as a dog leukocyte antigen (DLA) in canine species. Secondly, a genome-wide association study (GWAS) was performed to identify novel risk loci for SLE-related disease and AD in NSDTRs. We identified DLA risk haplotypes for an IMRD subphenotype of SLE-related disease, AD and CSK, but not in SMRA, and show that the MHC class II gene region is a major genetic risk factor in canine autoimmune diseases. An elevated risk was found for IMRD in dogs that carried the DLA-DRB1*00601/DQA1*005011/DQB1*02001 haplotype (OR = 2.0, 99% CI = 1.03-3.95, p = 0.01) and for ANA-positive IMRD dogs (OR = 2.3, 99% CI = 1.07-5.04, p-value 0.007). We also found that DLA-DRB1*01502/DQA*00601/DQB1*02301 haplotype was significantly associated with AD in NSDTRs (OR = 2.1, CI = 1.0-4.4, P = 0.044) and the DLA-DRB1*01501/DQA1*00601/DQB1*00301 haplotype with the CSK in GSDs (OR=2.67, CI=1.17-6.44, p= 0.02). In addition, we found that homozygosity for the risk haplotype increases the risk for each disease phenotype and that an overall homozygosity for the DLA region predisposes to CSK and AD. Our results have enabled the development of genetic tests to improve breeding practices by avoiding the production of puppies homozygous for risk haplotypes. We also performed the first successful GWAS for a complex disease in dogs. With less than 100 cases and 100 controls, we identified five risk loci for SLE-related disease and AD and found strong candidate genes involved in a novel T-cell activation pathway. We show that an inbred dog population has fewer risk factors, but each of them has a stronger genetic risk. Ongoing studies aim to identify the causative mutations and bring new knowledge to help diagnostics, treatment and understanding of the aetiology of SLE-related diseases.

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

Interferon-gamma (Ifn gamma), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifn gamma to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifn gamma, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/ nitric oxide adduct (DETA/NO), and Nos2(-/-) mice identified Nitric oxide (NO) induced by Ifn gamma as a key regulator of aggregation of APECs. Further studies with Nos2(-/-) APECs revealed that some Ifn. responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifn gamma and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifn gamma contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifn gamma and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or ``group behavior'' of APECs are discussed in the context of host resistance to infectious organisms.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.