An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region- positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Natural Infection of Dairy Cows with Bovine Leukemia Virus Affects Immunoglobulin Levels in Saliva and Serum but Not Milk.

Bovine leukemia virus (BLV) is a retroviral infection that disrupts the immune function of infected animals. It is widespread among U.S. dairy cattle. In this pilot study, the average total IgA and IgM concentrations in milk, saliva, and serum samples from BLV ELISA-positive (ELISA+) dairy cows were compared against samples from BLV ELISA-negative (ELISA-) cows using the Kruskal-Wallis test (with ties). The results from ELISA+ cows were also stratified by lymphocyte count (LC) and proviral load (PVL). In milk and saliva from ELISA+ cows, the average total IgA and IgM concentrations were decreased compared to ELISA- cows, although this was only statistically significant for saliva IgM in cows with low PVL (p = 0.0424). Numerically, the average total IgA concentrations were 33.6% lower in milk and 23.7% lower in saliva, and the average total IgM concentrations were 42.4% lower in milk and 15.5% lower in saliva. No significant differences were observed in the total serum IgA concentrations, regardless of PVL and LC. The total serum IgM from ELISA+ cows was significantly decreased (p = 0.0223), with the largest decreases occurring in the highest PVL and LC subgroups. This pilot study is a first step in investigating the impact of BLV on mucosal immunity and will require further exploration in each of the various stages of disease progression.

Bovine leukemia virus detection and dynamics following experimental inoculation.

Bovine leukemia virus (BLV) infects more than 40% of the United States cattle population and impacts animal health and production. Control programs aiming to reduce disease prevalence and incidence depend on the ability to detect the BLV provirus, anti-BLV antibodies, and differences in blood lymphocyte counts following infection. These disease parameters also can be indicative of long-term disease progression. The objectives of this study were to determine the timing and to describe early fluctuations of BLV-detection by qPCR, ELISA, and lymphocyte counts. Fifteen Holstein steers were experimentally inoculated with 100 µL of a blood saline inoculum. Three steers served as in-pen negative controls and were housed with the experimentally infected steers to observe the potential for contract transmission. Five additional negative controls were housed separately. Steers were followed for 147 days post-inoculation (DPI). Infections were detected in experimentally infected steers by qPCR and ELISA an average of 24- and 36 DPI, respectively. Significant differences in lymphocyte counts between experimentally infected and control steers were observed from 30 to 45 DPI. Furthermore, a wide variation in peak proviral load and establishment was observed between experimentally infected steers. The results of this study can be used to inform control programs focused on the detection and removal of infectious cattle.

Mycobacterium avium Subspecies paratuberculosis Drives an Innate Th17-Like T Cell Response Regardless of the Presence of Antigen-Presenting Cells.

The gastrointestinal disease of ruminants is clinically known as Johne's disease (JD) and is caused by Mycobacterium avium subspecies paratuberculosis (MAP). An accumulative effect by insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines have made the control of JD difficult. Currently lacking in the model systems of JD are undefined correlates of protection and the sources of inflammation due to JD. As an alternative to commonly studied immune responses, such as the Th1/Th2 paradigm, a non-classical Th17 immune response to MAP has been suggested. Indeed MAP antigens induce mRNAs encoding the Th17-associated cytokines IL-17A, IL-17F, IL-22, IL-23, IL-27, and IFNγ in CD3+ T cell cultures as determined by RT-qPCR. Although not as robust as when cultured with monocyte-derived macrophages (MDMs), MAP is able to stimulate the upregulation of these cytokines from sorted CD3+ T cells in the absence of antigen-presenting cells (APCs). CD4+ and CD8+ T cells are the main contributors of IL-17A and IL-22 in the absence of APCs. However, MAP-stimulated MDMs are the main contributor of IL-23. In vivo, JD+ cows have more circulating IL-23 than JD- cows, suggesting that this proinflammatory cytokine may be important in the etiology of JD. Our data in this study continue to suggest that Th17-like cells and associated cytokines may indeed play an important role in the immune responses to MAP infection and the development or control of JD.

Inflammatory Th17 responses to infection with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle and their potential role in development of Johne's disease.

Chronic intestinal inflammation typically associated with late stage Johne's disease (JD) in cattle occurs despite a lack of significant expression of the typical proinflammatory cytokines IFNγ and TNFα derived from Th1- like T cells. In contrast, these cytokines appear to be relatively abundant during early infections with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of JD in cattle. The roles of non-classical immune responses, such as those associated with Th17 cells, in response to MAP infection and development of clinical JD are less clear. In this review, we examine literature suggesting that Mycobacterial infections, including Mycobacterium tuberculosis, Mycobacterium bovis, and MAP, are all associated with expression of Th17 promoting cytokines (IL-23, IL-22, IL-17a). We discuss the possibility that Th17 associated cytokines, particularly IL-23, may act as contributing factors in development and maintenance of inflammation characteristic of clinical JD. An as yet relatively unexplored source of chronic inflammation due to over expression of IL-1α and IL-1ß is also presented. We further discuss the fact that, as with the typical Th1-like cytokines IFNγ and TNFα , IL-17a is not significantly expressed in CD4+ T cells from cows with clinical JD, possibly due to T cell exhaustion. Finally, we present the notion that the Th17 driving cytokine IL-23 expressed by infected macrophages and associated epithelial cells may contribute to chronic inflammation during later stages of JD.

Mycobacterium avium sp. paratuberculosis (MAP) induces IL-17a production in bovine peripheral blood mononuclear cells (PBMCs) and enhances IL-23R expression in-vivo and in-vitro.

Johne's disease (JD) is a chronic inflammatory gastrointestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of JD is difficult largely due to insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines. Correlates of protection are lacking in model systems of JD and the sources of inflammation due to JD are not well characterized. Commonly studied immune responses, such as the Th1/Th2 paradigm, do not adequately explain host responses to MAP. A potential role for non-classical immune responses to MAP, such as that mediated by Th17 cells, has been suggested. Indeed, MAP antigens induce mRNAs encoding the cytokines IL-23 and IL-17a in bovine peripheral blood mononuclear cells (PBMCs). IL-23 and IL-17a production have both been associated with Th17-like immune responses. Th17 cells are also defined by surface expression of the IL-23 receptor (IL-23R). To determine the relative prevalence of potential Th17 cells in PBMCs from MAP test positive and MAP test negative cows, PBMCs were isolated and analyzed by immunostaining and flow cytometry. Fresh PBMCs from MAP test positive cows (n = 12) contained a significantly higher proportion of IL-23R positive cells in populations of CD4+, CD8+, and Yδ + T cells than in cells from MAP test negative cows (n = 12; p < 0.05). Treatment with MAP antigens increased the percentage of all T cell subsets with surface expression of IL-23R when compared to untreated (n = 12; p < 0.05) cells. ELISA results for IL-17a secretion revealed a higher concentration of IL-17a secreted from PBMCs treated with MAP antigen (n = 20) than from PBMCs not treated with MAP antigens (n = 20) (p < 0.001), regardless of the JD test status of source cows. Also, we observed a moderate negative correlation between JD diagnostic scores for JD + cows and plasma IL-17a concentration (n = 42; r = -0.437; p-value < 0.004). Plasma with low and mid JD- scores (n = 31; n = 9; 0.1 ≤ X < 0.3) had significantly more IL-17a when compared to plasma with high JD- scores (n = 10; 0.3 ≤ X < 0.46; p-values < 0.05). Similarly, plasma with low JD + score values (0.55 ≤ X < 1.0; n = 9) had significantly more IL-17a when compared to plasma with high JD + score values (X ≥ 2.0; n = 21; p < 0.05). Overall, plasma from JD + cows (0.55 < X ≤ 2.86; n = 41) had significantly less IL-17a than plasma from JD- cows (0 < X ≤ 0.46; n = 70). Our data suggests that Th17-like cells may indeed play a role in early immune responses to MAP infection and development or control of JD.

T and B cell activation profiles from cows with and without Johne's disease in response to in vitro stimulation with Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) is a chronic wasting disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). JD is particularly problematic on US dairy farms: estimates show that over 50% of farms are MAP-contaminated and as many as 91% of dairy herds could be infected. Although estimates vary widely, JD may cost the dairy industry between $200 million and $1.5 billion every year. One major obstacle to JD management is that JD is difficult to detect in many animals, in part due to the variable immunity against MAP demonstrated by JD+ cattle. To characterize the diversity of immune responses against MAP, peripheral blood mononuclear cells from 154 JD test negative and 96 JD test positive cows from the same dairy herds were stimulated with MAP in vitro. The activation of CD4+, CD8+ and γδ T cells and surface IgM+ B cells was measured using flow cytometry. CD4+CD45R0+ T cells, γδ+MHCII+ and γδ+MHCII- T cells and SIgM+ B cells from JD test positive cows all exhibited increased proportions expressing CD25 after MAP stimulation, while CD8+ T cells did not demonstrate increased CD25 expression in response to MAP.

Dairy Cows Naturally Infected with Bovine Leukemia Virus Exhibit Abnormal B- and T-Cell Phenotypes after Primary and Secondary Exposures to Keyhole Limpet Hemocyanin.

Bovine leukemia virus (BLV) is a retrovirus that is highly prevalent in US dairy herds: over 83% are BLV infected and the within-herd infection rate can be almost 50% on average. While BLV is known to cause lymphosarcomas, only 5% or fewer infected cattle will develop lymphoma; this low prevalence of cancer has historically not been a concern to dairy producers. However, more recent research has found that BLV+ cows without lymphoma produce less milk and have shorter lifespans than uninfected herdmates. It has been hypothesized that BLV infection interferes with normal immune function in infected cattle, and this could lead to reduced dairy production. To assess how naturally infected BLV+ cows responded to a primary and secondary immune challenge, 10 BLV+ and 10 BLV- cows were injected subcutaneously with keyhole limpet hemocyanin (KLH) and dimethyldioctadecylammonium bromide. B- and T-cell responses were characterized over the following 28 days. A total of 56 days after primary KLH exposure, cows were re-injected with KLH and B- and T-cell responses were characterized again over the following 28 days. BLV+ cows produced less KLH-specific IgM after primary immune stimulation; demonstrated fewer CD45R0+ B cells, altered proportions of CD5+ B cells, altered expression of CD5 on CD5+ B cells, and reduced MHCII surface expression on B cells ex vivo; exhibited reduced B-cell activation in vitro; and displayed an increase in BLV proviral load after KLH exposure. In addition, BLV+ cows had a reduced CD45R0+γδ+ T-cell population in the periphery and demonstrated a greater prevalence of IL4-producing T cells in vitro. All together, our results demonstrate that both B- and T-cell immunities are disrupted in BLV+ cows and that antigen-specific deficiencies can be detected in BLV+ cows even after a primary immune exposure.

Bovine Leukemia Virus and Mycobacterium avium subsp. paratuberculosis Are Not Associated with Shiga Toxin-Producing Escherichia coli Shedding in Cattle.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis in cattle, and Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. Both diseases are chronic in nature and can lead to the disruption of normal immunological or physiological processes. Cattle are the major reservoir of Shiga toxin-producing Escherichia coli (STEC), a cause of foodborne illness in humans. We tested the hypothesis that cattle infected with BLV or MAP are more likely to shed STEC. We conducted a cross-sectional study during the summers of 2011 and 2012 in 11 Michigan cattle herds. A fecal sample from each animal was collected for STEC culture, and multiplex PCR for stx1, stx2, and eaeA was used to screen suspect colonies for STEC confirmation. Antibody detection enzyme-linked immunosorbent assays for BLV and MAP were used to screen serum from each animal. Flow cytometry was used to quantify the percentage of lymphocytes, monocytes, and neutrophils in a subsample (n =497) of blood samples. Of the animals sampled, 34.9% were BLV positive, 2.7% were MAP positive, and 16% were shedding STEC. Cattle in the dairy herds had a higher frequency of BLV and MAP than did those in beef herds, but more cattle in beef herds were shedding STEC. Neither BLV nor MAP was associated with STEC shedding (P values of 0.6838 and 0.3341, respectively). We also observed no association between STEC status and the percentage of neutrophils (P value of 0.3565), lymphocytes (P value of 0.8422), or the lymphocyte-to-monocyte ratio (P value of 0.1800). Although controlling both BLV and MAP is important for overall herd health and productivity, we found no evidence that controlling BLV and MAP has an impact on STEC shedding in cattle.

MicroRNAs Encoded by Bovine Leukemia Virus (BLV) Are Associated with Reduced Expression of B Cell Transcriptional Regulators in Dairy Cattle Naturally Infected with BLV.

Bovine leukemia virus (BLV) is estimated to infect over 83% of dairy herds and over 40% of all dairy cows in the United States. While, BLV only causes leukemia in a small proportion of animals, research indicates that BLV+ cattle exhibit reduced milk production and longevity that is distinct from lymphoma development. It is hypothesized that BLV negatively affects production by interfering with cattle immunity and increasing the risk of secondary infections. In particular, BLV+ cows demonstrate reduced circulating levels of both antigen-specific and total IgM. This study investigated possible mechanisms by which BLV could interfere with the production of IgM in naturally infected cattle. Specifically, total plasma IgM and the expression of genes IGJ, BLIMP1, BCL6, and PAX5 in circulating IgM+ B cells were measured in 15 naturally infected BLV+ and 15 BLV- cows. In addition, BLV proviral load (PVL) (a relative measurement of BLV provirus integrated into host DNA) and the relative expression of BLV TAX and 5 BLV microRNAs (miRNAs) were characterized and correlated to the expression of selected endogenous genes. BLV+ cows exhibited lower total plasma IgM and lower expression of IGJ, BLIMP1, and BCL6. While, BLV TAX and BLV miRNAs failed to correlate with IGJ expression, both BLV TAX and BLV miRNAs exhibited negative associations with BLIMP1 and BCL6 gene expression. The results suggest a possible transcriptional pathway by which BLV interferes with IgM production in naturally infected cattle.

Reduced humoral immunity and atypical cell-mediated immunity in response to vaccination in cows naturally infected with bovine leukemia virus.

Bovine leukemia virus (BLV) is a retrovirus that is widely distributed across US dairy herds: over 83% of herds are BLV-infected and within-herd infection rates can approach 50%. BLV infection reduces both animal longevity and milk production and can interfere with normal immune health. With such a high prevalence of BLV infection in dairy herds, it is essential to understand the circumstances by which BLV negatively affects the immune system of infected cattle. To address this question, BLV- and BLV+ adult, lactating Holstein dairy cows were vaccinated with Bovi-Shield GOLD® FP® 5 L5 HB and their immune response to vaccination was measured over the course of 28days. On day 0 prior to vaccination and days 7, 14 and 28 post-vaccination, fresh PBMCs were characterized for T and B cell ratios in the periphery. Plasma was collected to measure titers of IgM, IgG1 and IgG2 produced against bovine herpesvirus 1 (BHV1), Leptospira hardjo and L. pomona, as well as to characterize neutralizing antibody titers produced against BHV1 and bovine viral diarrhea virus types 1 and 2. On day 18 post-vaccination, PBMCs were cultured in the presence of BHV1 and flow cytometry was used to determine IFNγ production by CD4+, CD8+ and γδ T cells and to investigate CD25 and MHCII expression on B cells. BLV+ cows produced significantly lower titers of IgM against BHV1, L. hardjo and L. pomona and produced lower titers of IgG2 against BHV1. γδ T cells from BLV+ cows displayed a hyper reactive response to stimulation in vitro, although no differences were observed in CD4+ or CD8+ T cell activation. Finally, B cells from BLV+ cows exhibited higher CD25 expression and reduced MHCII expression in response to stimulation in vitro. All together, data from this study support the hypothesis that BLV+ cows fail to respond to vaccination as strongly as BLV- cows and, consequently, may have reduced protective immunity when compared to healthy BLV- cows.

Regulatory T cells and immune profiling in johne's disease lesions.

Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease of ruminants. Hallmark symptoms of clinical Johne's disease include diarrhea, progressive weight loss, and premature death; symptoms due largely to chronic inflammation in the small intestine. MAP colonizes resident macrophages within the ileum of the small intestine, subsequently establishing a persistent infection in the host. It has been proposed that regulatory T cells may play a role in the progression of Johne's disease, either through promotion of tolerance to MAP or via a loss in homeostasis that subsequently allows widespread inflammation. In this report, we evaluated the presence of Tregs, as well as other immune parameters, in the ileum and draining lymph nodes of MAP associated lesions. A lesion classification scheme was developed to categorize severity of MAP-induced lesions within infected tissues and subsequently regulatory T cell presence and overall immune activity were assessed corresponding to lesions of varying severity, in comparison to tissues from healthy control animals. Our results revealed a relationship between animal health and overall lesion severity within the infected tissues, as well as a relationship between bacterial burden and severity of pathology. Regulatory T cell abundance was shown to decrease with increasing lesion severity. Within the ileum, the expression of many Th1, Th2, and Treg-associated genes increased in mild lesions and decreased in severe lesions, whereas in the lymph nodes the expression of these genes tended to increase with increasing lesion severity. Based on our results, we conclude that a local loss of T cell (including Treg) activity occurs within severe ileal lesions associated with MAP, resulting in a loss of homeostasis that ultimately leads to the progression of clinical Johne's disease.

Enhanced production of human influenza virus in PBS-12SF cells with a reduced interferon response.

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>10(7) PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNα/ß receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down ∼90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNα stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model.

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFß. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

Bovine leukemia virus: a major silent threat to proper immune responses in cattle.

Bovine leukemia virus (BLV) infection is widespread in the US dairy industry and the majority of producers do not actively try to manage or reduce BLV incidence within their herds. However, BLV is estimated to cost the dairy industry hundreds of millions of dollars annually and this is likely a conservative estimate. BLV is not thought to cause animal distress or serious pathology unless infection progresses to leukemia or lymphoma. However, a wealth of research supports the notion that BLV infection causes widespread abnormal immune function. BLV infection can impact cells of both the innate and adaptive immune system and alter proper functioning of uninfected cells. Despite strong evidence of abnormal immune signaling and functioning, little research has investigated the large-scale effects of BLV infection on host immunity and resistance to other infectious diseases. This review focuses on mechanisms of immune suppression associated with BLV infection, specifically aberrant signaling, proliferation and apoptosis, and the implications of switching from BLV latency to activation. In addition, this review will highlight underdeveloped areas of research relating to BLV infection and how it causes immune suppression.

A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis.

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model.

Vaccination remains a major tool for prevention and progression of Johne's disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne's disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne's Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA-APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne's disease vaccine candidate screening and evaluation.

Regulatory T Cell Activity and Signs of T Cell Unresponsiveness in Bovine Paratuberculosis.

Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a wasting disease of ruminants displaying a long subclinical stage of infection followed by clinical disease characterized by severe diarrhea, wasting, and premature death. Immunologically, subclinical disease is characterized by a Th1 response effective at controlling intracellular infections such as that caused by MAP. In late subclinical disease, the Th1 response subsides and a non-protective Th2 response becomes prominent. One hypothesis for this shift in immune paradigm is that a population of MAP-reactive regulatory T cells (Tregs) develops during subclinical infection, limiting Th1-type responses to MAP antigens. To investigate this, we sought to accomplish the following: (1) determine if CD4(+)CD25(-) T cells exposed to MAP-infected macrophages develop a Treg phenotype, (2) develop a method to expand the relative abundance of Tregs in bovine peripheral blood lymphocyte populations, and (3) identify functional activities of expanded Tregs when combined with autologous peripheral blood mononuclear cells (PBMCs) and live MAP. We found that CD4(+)CD25(-) T cells exposed to MAP-infected macrophages from cows with Johne's disease do not show signs of a Treg phenotype and appear unresponsive to MAP antigens. A method for Treg expansion was successfully developed; however, based on results obtained in the subsequent functional studies it appears that these Tregs are not MAP-specific. Overall, it seems that T cell unresponsiveness, rather than Treg activity, is driving the Th1-to-Th2 immune shift observed during Johne's disease. Further, we have successfully developed a method to enrich non-specific bovine Tregs that exert suppressive effects against Th1 cytokine production.

Infection of Primary Bovine Macrophages with Mycobacterium avium Subspecies paratuberculosis Suppresses Host Cell Apoptosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is able to survive intracellularly in macrophages by preventing normal phagosome maturation processes utilized to destroy bacteria. Infected macrophages often undergo apoptotic cell death to efficiently present bacterial antigens to the host adaptive immune system in a process known as efferocytosis. Recent studies with Mycobacterium tuberculosis (MTB) showed that macrophages infected with MTB are less likely to undergo apoptosis than control, uninfected cells. It is proposed that regulation of macrophage apoptosis is an important immune evasion tactic for MTB. Based on the similarity of MAP and MTB, we hypothesized that MAP-infected macrophages would be resistant to apoptosis compared to uninfected cells within the same culture and to cells from uninfected cultures. Our results demonstrate that, indeed, populations of MAP-infected macrophages contain fewer apoptotic cells than similar populations of control cells, and that MAP infection reduces the sensitivity of infected macrophages to induction of apoptosis by H(2)O(2). We further demonstrate that MAP-infected cells contain reduced caspase activity for caspases 3/7, 8, and 9. Reduced caspase activity in MAP-infected macrophages is also maintained after H(2)O(2) induction. This reduction in caspase activity is accompanied by a pronounced reduction in transcription of caspase genes encoding caspases 3, 7, and 8, but not for caspase 9, when compared to control, uninfected cells. Furthermore, MAP infection drastically effects the expression of several host cell proteins important for regulation of apoptosis. Studies using mutant MAP strains demonstrate the importance of bacterial specific factors in the control of host macrophage apoptosis. Together these data demonstrate that MAP specific factors may prevent caspase activity and caspase gene transcription as well as apoptosis signaling protein expression, resulting in decreased spontaneous host cell apoptosis and decreased sensitivity to apoptosis inducing agents.