J. Frank T. Griffin

Animal models of protective immunity in tuberculosis to evaluate candidate vaccines

While the etiology of tuberculosis is well understood, the nature of the protective immune response to the causative mycobacteria has remained a mystery. There is an urgent need to define protective immunity critically, and to develop alternative animal models to evaluate the efficacy of new-generation vaccines against tuberculosis in a cost-effective way.

A strategic approach to vaccine development: animal models, monitoring vaccine efficacy, formulation and delivery

One of the most pressing issues in animal and human medicine currently is the need to develop new and more effective vaccines, against an ever increasing range of infectious diseases. Traditional vaccines have involved the use of killed microorganisms, live attenuated cultures or antigenic extracts. In spite of extensive research, there have been very few newly developed vaccines for humans or animals, over the past 20 years. Recent developments in our understanding of the pathways of immunity required to produce protection against different infections, allow immunological principles to be incorporated into the design new and better vaccines. This paper outlines the key variables that need to be taken into consideration when developing a new vaccine. Three critical areas will be considered in the review: (1) use of an appropriate experimental animal model of infection; (2) targeting appropriate immune markers for protection; (3) design of optimal vaccine formulation and delivery systems.

CLONING AND SEQUENCING OF EXPRESSED DRB GENES OF THE RED DEER (CERVUS ELAPHUS) MHC

The expressed major histocompatibility complex (Mhc) class II DRB genes of 50 unrelated deer were examined by reverse transcription polymerase chain reaction, cloning, and sequencing of DRB exon 2. Deer, like other mammals, have at least one highly polymorphic Mhc class II DRB gene. Thirty-four different sequences were identified. Most of the variation in amino acid composition occurred at positions that have been shown to form the peptide binding site (PBS). Eighteen deer-specific substitutions were found, 11 of these occurred in the PBS. Significantly higher rates of replacement substitutions than silent substitutions were found in the deer sequences, indicating strong positive selection pressure for diversity in DRB sequences. Between one and four DRB sequences were found per deer. Inheritance of these sequences in pedigrees showed Mendelian segregation with up to two expressed DRB genes per haplotype. Sheep are the only other ruminant in which the presence of more than one expressed DRB gene has been demonstrated. Phylogenetic trees were constructed in an attempt to assign the deer DRB sequences to specific loci, but no clear segregation of the DRB sequences for different loci was found. It would seem likely that sequence exchange between the loci has occurred. As has been shown in other species, the α-helix and β-sheet regions of exon 2 appeared to have different evolutionary histories.

Vaccination against tuberculosis: Is BCG more sinned against than sinner?

While extensive experimental studies of tuberculosis (Tb) have provided the foundation data for the discovery of cell‐mediated immunity, there remains much to be disclosed about the critical pathways of immunity involved in this infectious process and the factors necessary to produce protective immunity. Studies on the actiology and pathology of this disease have failed to elucidate the mechanisms of protective immunity. Although Tb research has been neglected for the past 30 years, the re‐emergence of Tb worldwide as a significant zoonotic disease has re‐focused research in this area. Scientific solutions for the control of Tb in man or domestic animals have not been found using empirical methods. Composite studies involving animal models of experimental infection will be necessary to critically evaluate vaccine efficacy and elucidate the basic immunological mechanisms involved in both disease and immunity. Available data which suggest that disease‐related hypersensitivity and immunity are dissociable highlight the prospect that immunity to infection may be induced without compromising the continued need for ongoing systems of immunodiagnosis to exclude disease. In populations with a high prevalence of disease it is likely that a combination of immunodiagnosis, chemotherapy and immunoprophylaxis will be required to eradicate the disease.

Veterinary Tuberculosis Vaccine Development

Tuberculosis caused by Mycobacterium bovis in domestic livestock and wildlife is a significant problem in many countries worldwide. Wildlife reservoirs of tuberculosis confound programs for tuberculosis eradication from domestic livestock. Successful vaccination against tuberculosis in domestic animals or wildlife could contribute to tuberculosis eradication. Bacille Calmette-Guérin (BCG) has been used as the prototype vaccine for domestic livestock and wildlife. The majority of studies have been carried out with BCG-vaccinated animals challenged experimentally with M. bovis. Although protection against disease has been evident in all these studies, protection against infection has rarely occurred. Results obtained with BCG vaccination of cattle, deer, ferrets, opossums, and rabbits are presented here and highlight the need for appropriate animal models for vaccination and control of the variables that influence the efficacy of BCG vaccine. Refinement of the existing animal models is essential for the advancement of tuberculosis vaccine research of relevance to animals and humans.

Innate immune markers that distinguish red deer (Cervus elaphus) selected for resistant or susceptible genotypes for Johne’s disease

While many factors contribute to resistance and susceptibility to infectious disease, a major component is the genotype of the host and the way in which it is expressed. Johne’s disease is a chronic inflammatory bowel disease affecting ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). We have previously identified red deer breeds (Cervus elaphus) that are resistant; have a low rate of MAP infection and do not progress to develop Johne’s disease. In contrast, susceptible breeds have a high rate of MAP infection as seen by seroconversion and progress to develop clinical Johne’s disease. The aim of this study was to determine if immunological differences exist between animals of resistant or susceptible breeds. Macrophage cultures were derived from the monocytes of deer genotypically defined as resistant or susceptible to the development of Johne’s disease. Following in vitro infection of the cells with MAP, the expression of candidate genes was assessed by quantitative PCR as well as infection rate and cell death rate. The results indicate that macrophages from susceptible animals show a significantly higher upregulation of inflammatory genes (iNOS, IL-1α, TNF-α and IL-23p19) than the macrophages from resistant animals. Cells from resistant animals had a higher rate of apoptosis at 24 hours post infection (hpi) compared to macrophages from susceptible animals. The excessive expression of inflammatory mRNA transcripts in susceptible animals could cause inefficient clearing of the mycobacterial organism and the establishment of disease. Controlled upregulation of inflammatory pathways coupled with programmed cell death in the macrophages of resistant animals may predispose the host to a protective immune response against this mycobacterial pathogen.

Global gene expression profiling of monocyte-derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johnes disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype. Cells were infected with MAP in vitro and gene expression analysed by RNA-Seq. Transcriptome analysis revealed a more disrupted gene expression profile in macrophages from susceptible animals compared with cells from resistant animals in terms of the number of genes up- or downregulated. Highly upregulated genes were related to chemotaxis (CXCL10, CSF3, and CCL8) and type 1 interferon signalling (RSAD2, IFIT1, IFIT2, ISG12, ISG15, USP18, and HERC6). Upregulation of these genes was observed to be greater in macrophages from susceptible animals compared to cells from resistant animals in response to in vitro MAP infection. These data support the use of transcriptomic approaches to enable the identification of markers associated particularly with susceptibility to MAP infection.

Production and Characterization of Monoclonal Antibodies against a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis

ABSTRACT The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johnes disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.

Isolation of High-Affinity Single-Chain Antibodies against Mycobacterium avium subsp. paratuberculosis Surface Proteins from Sheep with Johne's Disease

ABSTRACT Johnes disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools—necessary to understand disease processes and to identify subclinical infection—are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johnes disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 106-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 103M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer ( Cervus elaphus ) with bovine tuberculosis

BackgroundBovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15).ResultsThe presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection.ConclusionsThe results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer.