Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786 as a fusion ('MAP fusion protein particle'). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Heterologous peptide display on chromatin nanofibers: A new strategy for peptide vaccines.

Chromatin organization starts from a "beads-on-a string" 10 nm fiber, a basic nucleosomal structure consisting of DNA and core histones. Given its regular nucleosome array on DNA backbone where N-terminal tails of each histone are exposed on the surface of chromatin fiber, we hypothesized that chromatin can be utilized as a heterologous peptide carrier to elicit a peptide-specific immune response. The plasmid DNA containing the Widom's clone 601 sequence and the recombinant chimeric histones containing the peptide derived from ras oncogene (G12V) were used to assemble the chromatin fiber in vitro. The immunogenicity of the assembled chromatin was tested in mice as a single vaccine component or formulated with adjuvants. G12V tagged-chromatin co-administered with adjuvants induced higher antibody responses against the G12V peptide than vaccination with adjuvant alone, while chimeric histones did not generate a significant antibody response. Interestingly, splenocytes from mice vaccinated with the G12V tagged-chromatin vaccine did not generate significant antigen-specific cytokine responses. Our studies suggest that chromatin can be utilized as an effective carrier of antigenic peptides for inducing specific antibody responses.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

Comparison of gene expression of immune mediators in lung and pulmonary lymph node granulomas from cattle experimentally infected with Mycobacterium bovis.

The cellular infiltrates and macrophage activation pathways may differ in granulomas found in the lungs and pulmonary lymph nodes of cattle infected with Mycobacterium bovis. The aim of this study was to compare the histopathology and gene expression profiles of cytokines and immune mediators for cattle which had these lesions in both sites. Ten Friesian-cross, 15-16 month old cattle were challenged intratracheally with 5 × 10(3)CFU of virulent M. bovis and killed and necropsied at 28 weeks after infection. Seven animals were found to have gross TB granulomas in both their lungs and pulmonary lymph nodes (PLN) and these lesions were fully encapsulated with central necrosis and mineralisation. Neutrophil infiltration was clearly involved in granuloma in lung whereas neutrophils were limited in lesions of PLN. Comparisons were made of immune mediators from these two sites from the same animals as well as those between lesioned PLN tissues and non-lesioned prescapular lymph nodes (PSLN). Gene expressions of the immune mediators were normalised using a housekeeping gene (U1), a monocyte/macrophage marker (CD14) and a common leucocyte marker (CD45). mRNA expression of IFN-γ, IL-17A, IRF5(1) and arginase 1 (Arg1) was significantly up-regulated in lung compared to that for PLN (p<0.05), while mRNA expression of IFN-γ, IL-12p40, TNF-α and iNOs for PLN was significantly higher than that for PSLN (p<0.05). In addition, IL-10 mRNA expression was significantly higher for lung compared to PLN when normalised for CD45 (p<0.05). The results suggested that the stronger proinflammatory immune response in the lesioned lung may be a consequence of enhanced expression of IRF5 promoting IFN-γ and IL-17 production. In contrast, Arg1 expression in the lungs could facilitate the infection through competing with iNOs for l-arginine, preventing generation of nitric oxide for clearance of M. bovis infection.

Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine.

Diverse cytokine profile from mesenteric lymph node cells of cull cows severely affected with Johne's disease.

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, is able to dampen or distort immune responses at the mucosal sites and coexist with a massive infiltration of immune cells in the gastrointestinal tract. Knowledge of the mechanism by which M. avium subsp. paratuberculosis subverts the immune response at the mucosal level in cattle is important for the development of improved disease control strategies, including new vaccines and diagnostic tests. In this study, 38 cull cows from herds infected with M. avium subsp. paratuberculosis were divided into four groups, based on M. avium subsp. paratuberculosis culture from gut tissues and histopathological lesion scores. Cytokine gene expression and secretion from M. avium subsp. paratuberculosis sonicate-stimulated peripheral blood mononuclear cell (PBMC) and mesenteric lymph node (MLN) cultures of the animals were compared. Antigen stimulation of MLN cells from the severely lesioned group resulted in significant upregulation of the mRNA expression of five cytokines, gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-13, IL-17A, and tumor necrosis factor alpha (TNF-α), which have a diverse range of functions, while there was no significant upregulation of these cytokines by the other groups. There were major differences between the responses of the PBMC and MLN cultures, with higher levels of secreted IFN-γ released from the MLN cultures and, conversely, higher levels of IL-10 released from the PBMC cultures. The upregulation of all five cytokines from cells at the site of infection in the severely lesioned animals suggested a dysregulated immune response, contributing to a failure to clear infection in this group of animals.

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows.

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.

IFN-gamma enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis.

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism.

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.

Effect of recombinant cytokines on leucocytes and physiological changes in bovine mammary glands during early involution.

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rbolL-2) (2 x 10(5) units, n = 6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 microg, n = 4) and recombinant bovine interleukin-1beta (rbolL-1beta) (10 microg, n = 10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rbolL-2 and rboGM-CSF were produced in a yeast expression system, while rbolL-1beta was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rbolL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rbolL-1beta induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rbolL-1beta compared with control PBS quarters. In addition, infusion of glands with rbolL-1beta lowered the citrate:lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rbolL-1beta at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rbolL-1beta, also increased the rate of mammary gland involution during the early dry period.

Ability of T cell subsets and their soluble mediators to modulate the replication of Mycobacterium bovis in bovine macrophages.

Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with Bacillus Calmette-Guerin (BCG) were obtained and expanded in vitro by incubation with purified protein derivative. The ability of these cells to modulate the replication of virulent Mycobacterium bovis in autologous-infected macrophages was compared to cells from non-vaccinated controls. Cells from non-vaccinated animals were shown to confer a significant degree of mycobacteriostatic activity to autologous-infected macrophages. This activity was not inhibited by including a neutralizing antibody versus interferon-gamma (IFN-gamma), and was dependent on direct contact between PBMCs and infected macrophages. Addition of autologous PBMCs from BCG-vaccinated cattle was shown to significantly enhance macrophage resistance to M. bovis, and this increased macrophage resistance was partly abrogated by including a neutralizing antibody to IFN-gamma. Addition of T cells from non-vaccinated animals to infected macrophages was associated with a modest increase in macrophage release of TNF-alpha and nitric oxide, whereas PBMCs from vaccinated animals increased very significantly the release of these factors. Neutralization of nitric oxide (NO), by inclusion of monomethyl-L-arginine, significantly diminished the ability of PBMCs from vaccinated animals to enhance macrophage resistance to M. bovis, but had no impact on the ability of T cells from naive animals to modulate macrophage function. The ability of naive cells to increase macrophage anti-M. bovis activity was largely mediated by CD4+ T cells, whereas both CD4+ T cells and CD8+ T cells conferred macrophage resistance to M. bovis in vaccinated animals. These data highlight the role of IFN-gamma and NO in the immune resistance of cattle to M. bovis.

Mucosal immunity in the brushtail possum (Trichosurus vulpecula): detection of antibody in serum and at female reproductive sites after intranasal immunization.

Vaccination strategies for the brushtail possum, which rely upon stimulation of mucosal immunity, are being developed for biocontrol purposes. As little is known about how to stimulate possum immune responses via a mucosal site, groups of possums were immunized intranasally with keyhole limpet haemocyanin (KLH) alone or in combination with known or novel mucosal adjuvants. Antigen-specific antibody titres in female reproductive secretions were measured by ELISA and compared with antibody titres in the serum. Antigen-induced lymphocyte proliferative responses were measured as an indicator of cell-mediated responses. Intranasal immunization with KLH alone stimulated a weak serum antibody response that was significantly increased when KLH was given with cholera toxin subunit B (CTB), recombinant possum tumour necrosis factor alpha (TNF alpha) or live Mycobacterium bovis bacillus Calmette Guerin (BCG). Antibody titres in secretions from ovarian follicles and the uterus were very low in animals administered KLH alone. Significantly higher antibody titres to KLH were present in the reproductive secretions of possums immunized with KLH plus CTB, BCG or heat-killed Mycobacterium vaccae. Antibody titres were lower in mucosal secretions than in the serum, but there was a significant correlation between the two. In addition, coadministration of live BCG with KLH produced a strong antigen-specific cell-mediated response to KLH. This study has shown that an immune response to a protein antigen can be stimulated in possums by intranasal immunization and that antigen-specific antibodies can be detected in secretions from the female reproductive tract.