Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis),responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cellmediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex andpoorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (falsepositiveresults) has been crucial to develop a more proper antigen. In the present study, we selected sixM. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by insilicoanalysis and evaluated them in experimental and natural infection; none of these antigens hadbeen previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animalswith both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected withMycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually inducedan IFN-g response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the mostvaluable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAPinfected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B amongTST and MTC specific-PCR positive animals, although this result needs to be proven in further studieswith a higher sample size. Our data confirm the feacibility to implement bioinformatic screening toolsand suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate theircontribution to bTB diagnosis.Fil: Eirin, Maria Emilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Macías, Analia Florencia. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Magnano, Gabriel Gustavo. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Morsella, Claudia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Mendez, Laura. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Bianco, María Verónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Severina, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Alito, Alicia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Pando, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Singh, Mahavir. No especifíca;Fil: Spallek, Ralph. No especifíca;Fil: Paolicchi, Fernando Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

A Human Lung Challenge Model to Evaluate the Safety and Immunogenicity of PPD and Live Bacillus Calmette-Guérin.

Rationale: A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed.Objectives: We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guérin (BCG) in a lung-oriented controlled human infection model.Methods: We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry.Measurements and Main Results: The frequency of adverse events was low (9.4%; n = 10), similar in the challenge versus control groups (P = 0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 104 cfu, respectively, based on changes in BAL cellular profiles (P = 0.02) and antibody responses (P = 0.01) at incremental doses before versus after challenge. At 104 versus 103 cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3; P < 0.001) and dysregulated proteins (64 vs. 0; P < 0.001). Immune responses were highly setting specific (in vitro vs. in vivo) and compartment specific (BAL vs. blood) and localized to the challenged lung segments.Conclusions: A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates

The identification of aptamers against serum biomarkers of human tuberculosis

>Magister Scientiae - MScTuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment

Antigen stimulation of peripheral blood mononuclear cells from Mycobacterium bovis infected cattle yields evidence for a novel gene expression program

<p>Abstract</p> <p>Background</p> <p>Bovine tuberculosis (BTB) caused by <it>Mycobacterium bovis </it>continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed <it>in vivo </it>in the absence of exogenous antigen stimulation. In the present study, we hypothesized that the PBMC from BTB-infected cattle would display a distinct gene expression program resulting from exposure to <it>M. bovis</it>. A functional genomics approach was used to examine the immune response of BTB-infected (<it>n </it>= 6) and healthy control (<it>n </it>= 6) cattle to stimulation with bovine tuberculin (purified protein derivative – PPD-b) <it>in vitro</it>. PBMC were harvested before, and at 3 h and 12 h post <it>in vitro </it>stimulation with bovine tuberculin. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes.</p> <p>Results</p> <p>250 gene spot features were significantly differentially expressed in BTB-infected animals at 3 h post-stimulation contrasting with only 88 gene spot features in the non-infected control animals (<it>P </it>≤ 0.05). At 12 h post-stimulation, 56 and 80 gene spot features were differentially expressed in both groups respectively. The results provided evidence of a proinflammatory gene expression profile in PBMC from BTB-infected animals in response to antigen stimulation. Furthermore, a common panel of eighteen genes, including transcription factors were significantly expressed in opposite directions in both groups. Real-time quantitative reverse transcription PCR (qRT-PCR) demonstrated that many innate immune genes, including components of the TLR pathway and cytokines were differentially expressed in BTB-infected (<it>n </it>= 8) versus control animals (<it>n </it>= 8) after stimulation with bovine tuberculin.</p> <p>Conclusion</p> <p>The PBMC from BTB-infected animals exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to <it>M. bovis </it>antigen stimulation, providing evidence of a novel gene expression program due to <it>M. bovis </it>exposure.</p

Identification of Antigens Specific to Non-Tuberculous Mycobacteria: The Mce Family of Proteins as a Target of T Cell Immune Responses

The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests

Detection and Characterization of Mycobacterial Infections Occurring in Phacochoerus africanus (Gmelin, 1788) (Common Warthog)

Thesis (PhD)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex and the cause of bovine tuberculosis (bTB), has an extensive host range that includes livestock and wildlife. While warthogs are considered spill-over hosts for bTB, they could potentially become reservoir hosts, if conditions are favourable, i.e. increased population densities. With limited knowledge on the infection status of warthogs in South Africa and their epidemiological significance for other species, it is imperative to have readily available diagnostic tests for warthogs. Therefore, this study aimed to (i) establish reference cohorts of M. bovis-infected and uninfected warthogs; (ii) utilize these for the development and evaluation of diagnostic tools that can distinguish between infected and uninfected individuals; (iii) determine the seroprevalence of bTB in warthogs using the newly developed diagnostic tools; and (iv) characterize the genetic diversity of M. bovis isolates from warthogs. Three serological assays, i.e. the indirect PPD ELISA, the TB ELISA-VK® and the DPP® VetTB Assay, could distinguish between M. bovis-infected and uninfected warthogs with high sensitivity (75-88%) and specificity (79-89%). The overall seroprevalence from four M. bovis-endemic locations was high, i.e. 38%. Furthermore, three tests measuring the cellmediated immune responses of warthogs were developed. A cytokine release assay measuring interferon gamma induced protein 10 was able to distinguish between M. bovisinfected and uninfected warthog with a sensitivity of 68% and a specificity of 84%. The comparative intradermal tuberculin test classified 100% of culture-negative warthogs as test negative and 81% of culture-positive warthogs as test positive. Lastly, CXCL9, 10, 11, IFNG and TNFA gene expression were significantly increased in whole blood from M. bovis-infected warthogs in response to antigen stimulation, with CXCL10 showing the greatest mean fold increase. High genetic diversity of M. bovis isolates from warthogs was confirmed through spoligotyping and whole genome sequencing. Two distinct clades of M. bovis were identified by WGS, even though they shared the same spoligotype patterns This study has demonstrated that warthogs develop measurable and specific immune responses to M. bovis infection, which can be used to identify infected individuals ante-mortem. Furthermore, these tests will facilitate epidemiological studies of bTB in warthogs. With a high culture prevalence in warthogs from bTB endemic areas such as uMhkuze Nature Reserve and the Greater Kruger National Park, and high seroprevalence, warthogs seem to be highly susceptible to M. bovis infection. This suggests that warthogs may be an ideal sentinel species and strengthens the case that, under certain circumstances, they could be maintenance hosts. The genetic diversity of M. bovis isolates and the identification of two distinct clades challenges the current hypothesis that a single dominant strain circulates within a specific geographical location. Warthogs as a species should receive greater attention as potential disease maintenance hosts or as sentinels for bTB disease surveillance.AFRIKAANSE OPSOMMING: Mycobacterium bovis, 'n lid van die Mycobacterium tuberculosis kompleks en die oorsaak van bees-tuberkulose (bTB), het 'n uitgebreide gashere stelsel wat strek vanaf vee tot-en-met wild. Terwyl vlakvarke as oorspoel gashere vir bTB beskou word, kan hulle potensieël instandhoudingsgashere wees, indien toestande gunstig is, d.w.s. wanneer daar ‘n hoë bevolkingsdigtheid is. Met ‘n beperkte kennis, ten opsigte van vlakvarke se infeksiestatus, in Suid-Afrika asook hul epidemiologiese betekenis vir ander spesies, is dit noodsaaklik om algemeen beskikbare diagnostiese toetse vir vlakvarke te hê wat besmette individue kan identifiseer. Daarom het hierdie studie daarop gemik om (i) verwysingskohorte van M. bovis-besmette en onbesmette vlakvarke te bevestig; (ii) dié te gebruik vir die ontwikkeling en evaluering van diagnostiese instrumente wat tussen besmette en onbesmette individue kan onderskei; (iii) die seroprevalensie van bTB in vlakvarkke te bepaal, deur gebruik te maak van die nuut ontwikkelde diagnostiese instrumente; en (iv) die genetiese diversiteit van M. bovis-isolate van vlakvarke te karakteriseer. Drie serologiese toetse, naamlik die indirekte PPD ELISA, die TB ELISA-VK® en die DPP® VetTB-assay, kan onderskei tussen M. bovis-besmette en onbesmette vlakvarke met hoë sensitiwiteit (75-88%) en spesifisiteit (79-89%). Die algehele seroprevalensie van vier M. bovis-endemiese gebiede was hoog, d.w.s. 38%. Nog drie toetse was ontwikkel wat die selbemiddelde immuunreaksie van vlakvarke gemeet het. 'n Sitokien-vrystellingstoets wat interferon-gamma-geïnduseerde proteïen 10 meet was in staat om te onderskei tussen M. bovis-besmette en onbesmette vlakvarke met 'n sensitiwiteit van 68% en 'n spesifisiteit van 84%. Die vergelykende intradermale tuberkulintoets het 100% van kultuur-negatiewe vlakvarke as toets-negatief geklassifiseer en 81% van kultuur-positiewe vlakvarke as toets positief geklassifiseer. Laastens, was die geen-uitdrukking van CXCL9, 10, 11, IFNG en TNFA beduidend hoër in die bloed van M. bovis-besmette vlakvarke, in reaksie op antigeenstimulasie, met CXCL10 wat die grootste gemiddelde vouverhoging toon. Hoë genetiese diversiteit van M. bovis-isolate, vanuit vlakvarke, is bevestig deur middel van spoligotipering en heelgenoom volgorde bepaling. Twee verskillende stamme van M. bovis is deur heelgenoom volgorde bepaling geïdentifiseer, alhoewel hulle dieselfde spoeligotipepatrone gedeel het. Hierdie studie het getoon dat vlakvarke ‘n meetbare en spesifieke immuunreaksie op M. bovis-infeksie ontwikkel, wat gebruik kan word om besmette individue ante-mortem (voor die dood) te identifiseer. Verder sal hierdie toetse epidemiologiese studies van bTB in vlakvarke fasiliteer. Met 'n hoë kultuurvoorkoms in vlakvarke afkomstig vanuit bTB-endemiese gebiede soos uMhkuze Natuur Reservaat en die Grooter Kruger Nationale Park, as ook die hoë seroprevalensie, lyk vlakvarke hoogs vatbaar vir M. bovis-infeksie. Dit dui daarop dat vlakvarke 'n ideale brandwag spesie kan wees en versterk die aanname dat hulle onder sekere omstandighede instandhoudingsgashere kan wees. Die genetiese diversiteit van M. bovis-isolate en die identifisering van twee duidelike stamme daag die huidige hipotese uit, wat voorstel dat 'n enkele dominante stam versprei word binne 'n bepaalde geografiese gebied. Vlakvarke as 'n spesie moet groter aandag geniet as ‘n moontlike instandhoudingsgasheer of as brandwag vir bTB

A chemical genetics approach to identify targets essential for the viability of mycobacteria

Master'sJOINT M.SC. IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVER

Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease

<p>Abstract</p> <p>Background</p> <p>Bovine tuberculosis is an enduring disease of cattle that has significant repercussions for human health. The advent of high-throughput functional genomics technologies has facilitated large-scale analyses of the immune response to this disease that may ultimately lead to novel diagnostics and therapeutic targets. Analysis of mRNA abundance in peripheral blood mononuclear cells (PBMC) from six <it>Mycobacterium bovis </it>infected cattle and six non-infected controls was performed. A targeted immunospecific bovine cDNA microarray with duplicated spot features representing 1,391 genes was used to test the hypothesis that a distinct gene expression profile may exist in <it>M. bovis </it>infected animals <it>in vivo</it>.</p> <p>Results</p> <p>In total, 378 gene features were differentially expressed at the <it>P </it>≤ 0.05 level in bovine tuberculosis (BTB)-infected and control animals, of which 244 were expressed at lower levels (65%) in the infected group. Lower relative expression of key innate immune genes, including the Toll-like receptor 2 (<it>TLR2</it>) and <it>TLR4 </it>genes, lack of differential expression of indicator adaptive immune gene transcripts (<it>IFNG, IL2, IL4</it>), and lower <it>BOLA </it>major histocompatibility complex – class I (<it>BOLA</it>) and class II (<it>BOLA-DRA</it>) gene expression was consistent with innate immune gene repression in the BTB-infected animals. Supervised hierarchical cluster analysis and class prediction validation identified a panel of 15 genes predictive of disease status and selected gene transcripts were validated (<it>n </it>= 8 per group) by real time quantitative reverse transcription PCR.</p> <p>Conclusion</p> <p>These results suggest that large-scale expression profiling can identify gene signatures of disease in peripheral blood that can be used to classify animals on the basis of <it>in vivo </it>infection, in the absence of exogenous antigenic stimulation.</p