Progress in the development and use of monoclonal antibodies to study the evolution and function of the immune systems in the extant lineages of ungulates.

Details on the origin and function of the immune system are beginning to emerge from genomic studies tracing the origin of B and T cells and the major histocompatibility complex. This is being accomplished through identification of DNA sequences of ancestral genes present in the genomes of lineages of vertebrates that have evolved from a common primordial ancestor. Information on the evolution of the composition and function of the immune system is being obtained through development of monoclonal antibodies (mAbs) specific for the MHC class I and II molecules and differentially expressed on leukocytes differentiation molecules (LDM). The mAbs have provided the tools needed to compare the similarities and differences in the phenotype and function of immune systems that have evolved during speciation. The majority of information currently available on evolution of the composition and function of the immune system is derived from study of the immune systems in humans and mice. As described in the present review, further information is beginning to emerge from comparative studies of the immune systems in the extant lineages of species present in the two orders of ungulates, Perissodactyla and Artiodactyla. Methods have been developed to facilitate comparative research across species on pathogens affecting animal and human health.

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

Advances in Understanding of the Immune Response to Mycobacterial Pathogens and Vaccines through Use of Cattle and Mycobacterium avium subsp. paratuberculosis as a Prototypic Mycobacterial Pathogen.

Lack of understanding of the immune response to mycobacterial pathogens has impeded progress in development of vaccines. Infection leads to development of an immune response that controls infection but is unable to eliminate the pathogen, resulting in a persistent infection. Although this puzzle remains to be solved, progress has been made using cattle as a model species to study the immune response to a prototypic mycobacterium, Mycobacterium a. paratuberculosis (Map). As chronicled in the review, incremental advances in characterizing the immune response to mycobacteria during the last 30 years with increases in information on the evolution of mycobacteria and relA, a gene regulating the stringent response, have brought us closer to an answer. We provide a brief overview of how mycobacterial pathogens were introduced into cattle during the transition of humankind to nomadic pastoralists who domesticated animals for food and farming. We summarize what is known about speciation of mycobacteria since the discovery of Mybacterium tuberculsis Mtb, M. bovis Mbv, and Map as zoonotic pathogens and discuss the challenges inherent in the development of vaccines to mycobacteria. We then describe how cattle were used to characterize the immune response to a prototypic mycobacterial pathogen and development of novel candidate vaccines.

Supplementing a Saccharomyces cerevisiae fermentation product modulates innate immune function and ameliorates bovine respiratory syncytial virus infection in neonatal calves.

The objectives of this study were to determine the effects of oral supplementation with Saccharomyces cerevisiae fermentation products (SCFP; SmartCare and NutriTek; Diamond V, Cedar Rapids, IA) on immune function and bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. Twenty-four Holstein × Angus, 1- to 2-d-old calves (38.46 ± 0.91 kg initial body weight [BW]) were assigned two treatment groups: control or SCFP treated, milk replacer with 1 g/d SCFP (SmartCare) and calf starter top-dressed with 5 g/d SCFP (NutriTek). The study consisted of one 31-d period. On days 19 to 21 of the supplementation period, calves were challenged via aerosol inoculation with BRSV strain 375. Calves were monitored twice daily for clinical signs, including rectal temperature, cough, nasal and ocular discharge, respiration effort, and lung auscultation. Calves were euthanized on day 10 postinfection (days 29 to 31 of the supplementation period) to evaluate gross lung pathology and pathogen load. Supplementation with SCFP did not affect BW (P = 0.762) or average daily gain (P = 0.750), percentages of circulating white blood cells (P < 0.05), phagocytic (P = 0.427 for neutrophils and P = 0.460 for monocytes) or respiratory burst (P = 0.119 for neutrophils and P = 0.414 for monocytes) activity by circulating leukocytes either before or following BRSV infection, or serum cortisol concentrations (P = 0.321) after BRSV infection. Calves receiving SCFP had reduced clinical disease scores compared with control calves (P = 0.030), reduced airway neutrophil recruitment (P < 0.002), reduced lung pathology (P = 0.031), and a reduced incidence of secondary bacterial infection. Calves receiving SCFP shed reduced virus compared with control calves (P = 0.049) and tended toward lower viral loads in the lungs (P = 0.051). Immune cells from the peripheral blood of SCFP-treated calves produced increased (P < 0.05) quantities of interleukin (IL)-6 and tumor necrosis factor-alpha in response to toll-like receptor stimulation, while cells from the bronchoalveolar lavage (BAL) of SCFP-treated calves secreted less (P < 0.05) proinflammatory cytokines in response to the same stimuli. Treatment with SCFP had no effect on virus-specific T cell responses in the blood but resulted in reduced (P = 0.045) virus-specific IL-17 secretion by T cells in the BAL. Supplementing with SCFP modulates both systemic and mucosal immune responses and may improve the outcome of an acute respiratory viral infection in preweaned dairy calves.

relA is Achilles' heel for mycobacterial pathogens as demonstrated with deletion mutants in Mycobacterium avium subsp. paratuberculosis and mycobacterium bovis bacillus Calmette-Guérin (BCG).

Studies with Mycobacterium avium subsp. paratuberculosis (Map) in cattle revealed deletion of relA, a global regulator gene, abrogated ability of the mutant to establish a persistent infection, attributed to development of an immune response that cleared infection. Analysis of the recall response demonstrated presence of CD8 cytotoxic T cells that kill intracellular bacteria. Replication of the primary response demonstrated the CTL response could be elicited with the ΔMap/relA mutant or the target of the immune response, a 35 kD membrane protein. Follow up comparative studies with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and a BCG relA (ΔBCG/relA) deletion mutant revealed deletion of relA enhanced the CTL response compared to BCG. Analysis of the cytokine profile of cells proliferating in response to stimulation with BCG or BCG/relA showed increased expression of IFN-γ, TNF-α, and IL-17 by cells stimulated with ΔBCG/relA in comparison with BCG. The proliferative and CTL responses were markedly reduced in response to stimulation with heat killed BCG or ΔBCG/relA. Intracellular bacterial killing was mediated through the perforin, granzyme B (GnzB), and the granulysin pathway. The data indicate relA is the Achilles' heel for pathogenic mycobacteria and deletion may be key to improving efficacy of attenuated vaccines for mycobacterial pathogens.

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 by MAP2121c. 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 peptide-based vaccine for Mycobacterium avium subspecies paratuberculosis.

Recent efforts to develop a live attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease (JD), revealed relA is important in Map virulence. Deletion of the relA gene impairs the ability of Map to establish a persistent infection. Analysis of the basis for this observation revealed infection with a relA deletion mutant (ΔrelA) elicits development of cytotoxic CD8 T cells (CTL) with the ability to kill intracellular bacteria. Further analysis of the recall response elicited by ΔrelA vaccination showed a 35 kDa membrane peptide (MMP) is one of the targets of the immune response, suggesting it might be possible to develop a peptide-based vaccine based on MMP. To explore this possibility, ex vivo vaccination studies were conducted with MMP alone and incorporated into a nanoparticle (NP) vector comprised of poly (D, L-lactide-co-glycolide) and monophosphoryl lipid A (PLGA/MPLA). As reported, ex vivo vaccination studies showed CD8 CTL were elicited with classic and monocyte derived dendritic cells (cDC and MoDC) pulsed with MMP alone and incorporated into a PGLA/MPLA vector. Incorporation of MMP into a NP vector enhanced the ability of CD8 CTL to kill intracellular bacteria. The findings indicate incorporation of MMP into a PGLA/MPLA nanoparticle vector is one of the possible ways to develop a MMP based vaccine for Johne's disease.

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/ΔrelA, 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.

Evaluation of antigen specific interleukin-1ß as a biomarker to detect cattle infected with Mycobacterium bovis.

Bovine tuberculosis (bTB) is a major world-wide health problem that has been difficult to control, due to the lack of an effective vaccine and limited ability of the tuberculin skin test (TST) and the ancillary whole blood interferon-gamma (IFN-γ) release assay (IGRA) to detect all infected animals. A 6 h cytokine flow cytometric IFN-γ (CFC) assay was developed in effort to overcome these limitations and expand methods for studying the mechanisms of bTB immunopathogenesis. The present study was conducted to evaluate IL-1ß as a biomarker to use in conjunction with the IFN-γ CFC assay to improve the diagnostic accuracy for bTB. Three animal groups with predefined Mbv infection status were used for analysis of IL-1ß in plasma from whole blood cultures stimulated with ESAT-6/CFP-10 for 20-24 h. Parallel stimulations were performed for enumeration of IFN-γ producing T cells. Data analysis showed that Mbv infected animals have a higher frequency of IFN-γ producing CD4+ T cells and plasma IL-1ß than animals exposed to non-tuberculous mycobacteria (NTM) or uninfected control animals, with a significant correlation between the two readouts, thus allowing differentiation between the three animal groups. IL-1ß has the potential to serve as an additional biomarker for detecting cattle infected with Mbv.