Kun Taek Park

Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease.

Control of Johnes disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P<0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RT-PCR revealed genes encoding IFN-γ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johnes disease.

Demonstration of allelic exchange in the slow-growing bacterium Mycobacterium avium subsp. paratuberculosis, and generation of mutants with deletions at the pknG, relA, and lsr2 loci

ABSTRACT Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johnes disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10−7 to 2.9 × 10−7. Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.

Deletion of relA abrogates the capacity of Mycobacterium avium paratuberculosis to establish an infection in calves

Previous comparative studies in goats revealed deletion of relA but not pknG abrogates the capacity of Mycobacterium avium subsp. paratuberculosis (Map) to establish a persistent infection. The immune response elicited by the mutant cleared infection. The objective of the present study was to extend the studies in calves and compare the proliferative response elicited by the relA deletion mutant (ΔrelA) and Map using flow cytometry and quantitative reverse transcription real-time PCR (qRT-PCR). Six 3-day-old calves were divided into two groups. Three were vaccinated with ΔrelA and 3 inoculated with wild type Map. The calves were challenged with Map 1 month later and necropsied 3 months post challenge. Three untreated calves were used as uninfected controls. Examination of tissues revealed the ΔrelA mutant was immune eliminated. Bacterial load of Map was significantly reduced in the calves vaccinated with ΔrelA and challenged with Map in comparison with calves inoculated and challenged with Map. A vigorous CD4 memory T cell response was detected at necropsy in PBMC from both infected groups. CD8 positive NK cells proliferated in the presence and absence of antigen stimulation in both treated groups but not in the uninfected group. IFN-γ, IL17, and IL22 gene expression were up-regulated with an associated increase in their transcription factors, Tbet and RORC, in both treated groups. TGF-β, IL-10, and FoxP3 were not up-regulated, indicating no activation of regulatory T cells. The findings show that the immune response to ΔrelA is clearly different than the response to Map. Understanding the immunological basis for this difference should facilitate development of a vaccine that elicits sterile immunity.

Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johnes disease in ruminants and possibly associated with human Crohns disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification.

Characterization and use of new monoclonal antibodies to CD11c, CD14, and CD163 to analyze the phenotypic complexity of ruminant monocyte subsets.

The sequencing of the bovine genome and development of mass spectrometry, in conjunction with flow cytometry (FC), have afforded an opportunity to complete the characterization of the specificity of monoclonal antibodies (mAbs), only partially characterized during previous international workshops focused on antibody development for livestock (1991, Leukocyte Antigens in Cattle, Sheep, and Goats; 1993, Leukocyte Antigens of Cattle and Sheep; 1996, Third Workshop on Ruminant Leukocyte Antigens). The objective of this study was to complete the characterization of twelve mAbs incompletely characterized during the workshops that reacted with molecules predominantly expressed on bovine monocytes and use them to provide further information on the phenotypic complexity of monocyte subsets in ruminants. Analysis revealed that the mAbs could be grouped into three clusters that recognize three different molecules: CD11c, CD14, and CD163. Following characterization, comparison of the patterns of expression of CD14 and CD163 with expression of CD16, CD172a, and CD209 revealed the mononuclear cell population is comprised of multiple subsets with differential expression of these molecules. Further analysis revealed the epitopes recognized by mAbs to CD14 and CD163 are conserved on orthologues in sheep and goats. In contrast to CD14 that is also expressed on sheep and goat granulocytes, CD163 is a definitive marker for their monocytes.

Development and characterization of a monoclonal antibody specific for bovine CD209.

Dendritic cells (DC) play a central role in tailoring the immune response to pathogens. Effector activity is mediated through pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPS). C-type lectin receptors (CLR) comprise a group of PRRs that recognize a broad range of pathogens. CD209 (DC-specific ICAM3-grabbing non-integrin, DC-SIGN) is a CLR expressed on DC that plays a critical role on DC function and pathogen recognition. It facilitates DC migration to peripheral tissues and local lymph nodes and mediates T cell activation by binding ICAM-2 (CD102) and ICAM-3 (CD50). The absence of monoclonal antibody (mAb) to bovine CD209 has limited the ability to characterize the phenotype and function of DC in cattle. To address this issue we developed and used a mAb to CD209 to characterize the phenotype of CD209 expressing cells in bovine blood using flow cytometry. Initial analysis has revealed the CD209 positive population in blood is comprised of multiple phenotypically defined subsets.

Interleukin-15 activated bovine natural killer cells express CD69 and produce interferon-γ

Interleukin (IL)-15 is an essential cytokine in natural killer (NK) cell development and survival. In humans, IL-15 shows overlapping properties with IL-2 due to partly shared receptors and signal transduction and both cytokines synergize equally well with IL-12 in the induction of interferon (IFN)-γ production from NK cells. Bovine NK cells however, have been reported to produce less IFN-γ after in vitro IL-12 stimulation when exposed to human IL-15 in comparison to bovine IL-2. We therefore wanted to determine if homologous IL-15 is needed for adequate stimulation of bovine NK cells. Biologically active recombinant bovine IL-15 (rbIL-15) produced in mammalian cells by the use of a modified expression vector stimulated NK cells to a dose-dependent IFN-γ production in the presence of IL-12. In contrast to earlier findings, we also detected potent IFN-γ production from bovine NK cells stimulated by human IL-15 and IL-12. Finally, we describe a monoclonal antibody recognizing bovine CD69 and show the expression of this early activation marker on bovine NK cells ex vivo and following rbIL-15 stimulation.

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

Characterization and expression of monoclonal antibody-defined molecules on resting and activated bovine αβ, γδ T and NK cells.

Monoclonal antibodies (mAbs) specific for leukocyte differentiation molecules (LDMs) were developed during the past few decades to expand reagents for research in ruminants, pigs, and horses. The specificity of some of the mAb-defined molecules was determined through participation in international workshops. Other molecules identified with mAbs during this time, and more recently with mAbs developed after the workshops, have remained partially characterized. Efforts are now underway to characterize the specificity of these mAbs. As reported here, flow cytometry (FC) was used to screen two sets of hybridomas to determine how many of the hybridomas produce mAbs that detect molecules with up-regulated expression on activated lymphocytes or NK cells. Thirty four hybridomas were identified. Comparison of the patterns of reactivity of the mAbs showed some of the mAbs formed clusters that recognize 5 different molecules. FC showed one cluster recognized CD25. Use of mass spectrometry showed 4 clusters recognized orthologues of CD26, CD50, gp96 and signaling lymphocytic activation molecule family member 9 (SLAMF9). Verification and documentation that CD26, CD50, and SLAMF9 were only up-regulated on activated cells was obtained with PBMC from calves vaccinated with a Mycobacterium avium paratuberculosis mutant, Map-relA. CD26 and CD50 were up-regulated on NK cells, CD4 and CD8 T cells and γδ T cells. SLAMF9 was only up-regulated on CD4, CD8, and γδ T cells. gp96 was detected on granulocytes, monocytes and activated NK cells. Detection was attributable to the binding of gp96 to its receptor CD91.

Depletion of CD4 T lymphocytes at the time of infection with M. avium subsp. paratuberculosis does not accelerate disease progression

A calf model was used to determine if the depletion of CD4 T cells prior to inoculation of Mycobacterium avium subsp. paratuberculosis (Map) would delay development of an immune response to Map and accelerate disease progression. Ileal cannulas were surgically implanted in 5 bull calves at 2 months of age. Two calves were depleted of CD4 T cells by intravenous injection of anti-bovine CD4 antibody administered 24h prior to inoculation with Map. The two CD4-depleted calves and one non-depleted calf were inoculated via ileal cannula with 1 × 10(8)cfu live Map every 3 days for a total of 4 inoculations. Two additional calves served as non-depleted and uninfected controls. Injection with the anti-CD4 mAb reduced the frequency of CD4 T cells from a pre-depletion average of 15% to less than 1% in PBMC at 24h. However, a consistent proliferative response dominated by CD4 T cells, developed in both treated and untreated calves over the course of the 6-month study period. Recovery of Map from serial biopsies obtained from the CD4-depleted and non-depleted calves after Map infection did not differ. In addition, CD4 depletion did not increase the level of Map shed in the feces over the non-depleted animal.