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

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

Evaluation of two mutants of \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e as candidates for a live attenuated vaccine for Johne’s disease

Control of Johne’s 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 \u3c 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 RTPCR revealed genes encoding IFN-ƴ, 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 Johne’s disease

Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen-presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded byMAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL

A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria

Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/relA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/relA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb

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

[This corrects the article DOI: 10.1371/journal.pone.0165247.]

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

Aims: We evaluated the potential of a nanoparticle (NP) delivery system to improve methods of delivery of candidate peptide-based vaccines for Paratuberculosis in cattle. Methods and Results: Peptides derived from Mycobacterium avium subsp. paratuberculosis (Map), and the pro-inflammatory monophosphoryl lipid A (MPLA) were incorporated in polymeric NPs based on poly (D,L-lactide-co- glycolide) (PLGA). The PLGA/MPLA NPs carriers were incubated with macrophages to examine their effects on survival and function. PLGA/MPLA NPs, with and without Map antigens, are efficiently phagocytized by macrophages with no evidence of toxicity. PLGA/MPLA NP formulations did not alter the level of expression of MHC I or II molecules. Expression of TNFa and IL12p40 was increased in Map-loaded NPs. T-cell proliferation studies using a model peptide from Anaplasma marginale demonstrated that a CD4 T- cell recall response could be elicited with macrophages pulsed with the peptide encapsulated in the PLGA/MPLA NP. Conclusions: These findings indicate PLGA/MPLA NPs can be used as a vehicle for delivery and testing of candidate peptide-based vaccines. Significance and Impact of the Study: These results will assist on more in depth studies on PLGA NP delivery systems that may lead to the development of a peptide-based vaccine for cattle

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