Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

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A Zebrafish Model for Developing Vaccines against Tuberculosis

Maailmanlaajuisesti tuberkuloosi on yksi merkittävimmistä kuolemansyistä aiheuttaen vuosittain 1.5 miljoonan ihmisen kuoleman. Maailman terveysjärjestön mukaan, tuberkuloosia aiheuttava patogeeni, Mycobacterium tuberculosis, tartuttaa joka vuosi 10 miljoonaa ihmistä. Sairaus voi olla etenevä tai latentti. Latentissa infektiossa elimistön immuunipuolustus pystyy rajaamaan infektion granuloomarakenteiden sisään. Arviolta 1.7 miljardilla ihmisellä on tälläinen oireeton, latentti tuberkuloosi, ja siten 5-10% elinikäinen riski aktiiviselle sairaudelle. 1920-luvulla kehitetty, ainoa saatavilla oleva tuberkuloosirokote, Bacillus Calmette-Guérin, suojaa ainostaan imeväisikäisiä tuberkuloosin vakavampia tautimuotoja vastaan. Nykyisin ei ole saatavilla rokotetta, mikä antaisi tehokkaan suojan aikuisille tai estäisi latentin infektion uudelleenaktivaation. Viimeaikoina rokotetutkimus on keskittynyt turvallisempiin menetelmiin, kuten alayksikkörokotteisiin, jotka eivät sisällä elävää patogeenia. Huolimatta tuberkuloositutkimuksen pitkästä historiasta, tuberkuloosin taudinaiheuttamismekanismeja sekä mykobakteerien ja immuunipuolustuksen välistä vuorovaikutusta ei edelleenkään täysin tunneta. Yksi tuberkuloositutkimuksen haasteista on ollut käyttökelpoisten mallieläinten puute. Tässä tutkimuksessa hyödynnettiin seeprakalamallia (Danio Rerio), jonka Mycobacterium marinum-infektio muistuttaa monilta piirteiltään ihmisen tuberkuloosia. Tiedetään, että laboratorio- olosuhteissa, korkea infektioannos aiheuttaa etenevän sairauden ja matala- annoksinen infektio puolestaan latentin infektion. Tässä tutkimuksessa osoitettiin, että heikentämällä kalan immuunipuolustusta deksametasonilla, latentin infektiot uudelleenaktivoituvat. Tämän väitöskirjatutkimuksen päätavoitteena oli löytää uusia rokoteantigeeneja tuberkuloosia vastaan. Tavoitteen saavuttamiseksi valitsimme lupaavia M. marinumin geenejä sekä kirjallisuuden että lähetti-RNA-sevensointituloksen perusteella. In vivo- tutkimuksessa testattiin DNA-rokoteantigeeneinä yhteensä 22 eri geeniä. Näistä seitsemän on erityisesti reaktivaatiossa ilmentyvää ja 15 kirjallisuuden perusteella valittua geeniä. 16 eri antigeenin teho testattiin primaari-infektiota vastaan. Testatuista antigeeneistä neljä (cdh, PE5, PE31 ja RpfE) antoivat suojan neljän viikon kuluttua infektiosta bakteerimääritykseen perustuvassa tutkimuksessa. Näistä yksi (RpfE) paransi myös kalojen selviytymistä 12 viikon infektionjälkeisessä serannassa. Lisäksi testattiin 15 eri antigeenin mahdollinen suojavaikutus latentin infetion uudelleen aktivaatiota vastaan deksametasoni-hoitoon perustuvan koemallin avulla. Tutkimuksessa löydettiin kolme antigeenia, RpfB, MMAR_4207 ja MMAR_4110, jotka estivät uudelleenaktivaatiota. Tässä väitöskirjatutkimukssa osoitetaan, että seeprakalan M. marinum- infektiomalli sekä deksametasoniin perustuva latentin infektion uudelleenaktivaatiomalli ovat käyttökelpoisia testattaessa uusien DNA-rokotteiden tehoa. Johtuen M. tuberculosiksen ja M. marinumin genomien samankaltaisuudesta, löydettyjen lupaavien antigeenien M. tuberculosis-homologit ovat mahdollisilta kohdegeenejä uusien tuberkuloosirokotteiden kehittämiselle.Tuberculosis (TB) is one of the most serious diseases worldwide, leading to the death of 1.5 million people annually. According to the World Health Organization, every year, 10 million people are infected by Mycobacterium tuberculosis, the causative agent of tuberculosis. Depending on the strength of the immune system, the infection develops into a granulomatous pulmonary disease, which can be progressive or is controlled by the immune system to maintain latency. An estimated 1.7 billion people have an asymptomatic latent TB, having a 5-10% risk of developing the active disease in their lifetime. The only licensed tuberculosis vaccine, the Bacillus Calmette Guérin, which was developed already in the 1920’s, protects only against severe forms of tuberculosis in infants. Currently, there is no vaccine available that effectively prevent tuberculosis in adults or the reactivation of a latent disease. The latest vaccine research focuses on safer immunization approaches, such as subunit vaccines, which do not contain live particles of the pathogen. Despite the long history of tuberculosis research, our understanding of the exact mechanisms of the diseases at the cellular level and the complex interactions between the bacilli and the immune system remain incomplete. One of the challenges in tuberculosis research has been a lack of appropriate animal models. A zebrafish (Danio rerio) model, based on an infection with Mycobacterium marinum leading to a disease, which resembles the main features of human tuberculosis, was utilized in this thesis. It is known that in an experimental set up, a high dose infection with M. marinum causes a progressive disease, whereas a low dose infection leads to the formation of a latent infection in adult zebrafish. In this study, it was shown that by suppressing the fish immune system with dexamethasone, the latent infection can be reactivated. The main goal of this thesis was to identify novel vaccine antigens to prevent tuberculosis. To this end, potential antigens were selected based on the literature and on a transcriptomic level analysis performed with in vitro reactivated M. marinum samples. In total, 22 different genes were tested as DNA vaccines. Seven of these were derived from reactivation-associated genes identified using the transcriptomic analysis and 15 genes were selected from the literature. 16 antigens were tested against the primary infection, and four (cdh, PE5, PE31 and RpfE) showed protection in a bacterial count analysis after 4 weeks of infection. Furthermore, the RpfE-derived antigen enhanced survival in a 12-weeks follow-up. Correspondingly, the preventive effects of 15 antigens against reactivation were tested with the dexamethasone-based reactivation method. With this model, three antigens, namely RpfB, MMAR_4207 and MMAR_4110, inhibited reactivation. This thesis showed that an adult zebrafish- M. marinum infection model, and the developed dexamethasone-induced reactivation model are feasible for testing the effectiveness of novel DNA vaccines. As the genomes of M. tuberculosis and M. marinum are highly similar, the M. tuberculosis homologues of the identified promising antigens are potential targets for developing novel vaccines and drugs against tuberculosis

A Zebrafish Model for Developing Vaccines against Tuberculosis

Maailmanlaajuisesti tuberkuloosi on yksi merkittävimmistä kuolemansyistä aiheuttaen vuosittain 1.5 miljoonan ihmisen kuoleman. Maailman terveysjärjestön mukaan, tuberkuloosia aiheuttava patogeeni, Mycobacterium tuberculosis, tartuttaa joka vuosi 10 miljoonaa ihmistä. Sairaus voi olla etenevä tai latentti. Latentissa infektiossa elimistön immuunipuolustus pystyy rajaamaan infektion granuloomarakenteiden sisään. Arviolta 1.7 miljardilla ihmisellä on tälläinen oireeton, latentti tuberkuloosi, ja siten 5-10% elinikäinen riski aktiiviselle sairaudelle. 1920-luvulla kehitetty, ainoa saatavilla oleva tuberkuloosirokote, Bacillus Calmette-Guérin, suojaa ainostaan imeväisikäisiä tuberkuloosin vakavampia tautimuotoja vastaan. Nykyisin ei ole saatavilla rokotetta, mikä antaisi tehokkaan suojan aikuisille tai estäisi latentin infektion uudelleenaktivaation. Viimeaikoina rokotetutkimus on keskittynyt turvallisempiin menetelmiin, kuten alayksikkörokotteisiin, jotka eivät sisällä elävää patogeenia. Huolimatta tuberkuloositutkimuksen pitkästä historiasta, tuberkuloosin taudinaiheuttamismekanismeja sekä mykobakteerien ja immuunipuolustuksen välistä vuorovaikutusta ei edelleenkään täysin tunneta. Yksi tuberkuloositutkimuksen haasteista on ollut käyttökelpoisten mallieläinten puute. Tässä tutkimuksessa hyödynnettiin seeprakalamallia (Danio Rerio), jonka Mycobacterium marinum-infektio muistuttaa monilta piirteiltään ihmisen tuberkuloosia. Tiedetään, että laboratorio- olosuhteissa, korkea infektioannos aiheuttaa etenevän sairauden ja matala- annoksinen infektio puolestaan latentin infektion. Tässä tutkimuksessa osoitettiin, että heikentämällä kalan immuunipuolustusta deksametasonilla, latentin infektiot uudelleenaktivoituvat. Tämän väitöskirjatutkimuksen päätavoitteena oli löytää uusia rokoteantigeeneja tuberkuloosia vastaan. Tavoitteen saavuttamiseksi valitsimme lupaavia M. marinumin geenejä sekä kirjallisuuden että lähetti-RNA-sevensointituloksen perusteella. In vivo- tutkimuksessa testattiin DNA-rokoteantigeeneinä yhteensä 22 eri geeniä. Näistä seitsemän on erityisesti reaktivaatiossa ilmentyvää ja 15 kirjallisuuden perusteella valittua geeniä. 16 eri antigeenin teho testattiin primaari-infektiota vastaan. Testatuista antigeeneistä neljä (cdh, PE5, PE31 ja RpfE) antoivat suojan neljän viikon kuluttua infektiosta bakteerimääritykseen perustuvassa tutkimuksessa. Näistä yksi (RpfE) paransi myös kalojen selviytymistä 12 viikon infektionjälkeisessä serannassa. Lisäksi testattiin 15 eri antigeenin mahdollinen suojavaikutus latentin infetion uudelleen aktivaatiota vastaan deksametasoni-hoitoon perustuvan koemallin avulla. Tutkimuksessa löydettiin kolme antigeenia, RpfB, MMAR_4207 ja MMAR_4110, jotka estivät uudelleenaktivaatiota. Tässä väitöskirjatutkimukssa osoitetaan, että seeprakalan M. marinum- infektiomalli sekä deksametasoniin perustuva latentin infektion uudelleenaktivaatiomalli ovat käyttökelpoisia testattaessa uusien DNA-rokotteiden tehoa. Johtuen M. tuberculosiksen ja M. marinumin genomien samankaltaisuudesta, löydettyjen lupaavien antigeenien M. tuberculosis-homologit ovat mahdollisilta kohdegeenejä uusien tuberkuloosirokotteiden kehittämiselle.Tuberculosis (TB) is one of the most serious diseases worldwide, leading to the death of 1.5 million people annually. According to the World Health Organization, every year, 10 million people are infected by Mycobacterium tuberculosis, the causative agent of tuberculosis. Depending on the strength of the immune system, the infection develops into a granulomatous pulmonary disease, which can be progressive or is controlled by the immune system to maintain latency. An estimated 1.7 billion people have an asymptomatic latent TB, having a 5-10% risk of developing the active disease in their lifetime. The only licensed tuberculosis vaccine, the Bacillus Calmette Guérin, which was developed already in the 1920’s, protects only against severe forms of tuberculosis in infants. Currently, there is no vaccine available that effectively prevent tuberculosis in adults or the reactivation of a latent disease. The latest vaccine research focuses on safer immunization approaches, such as subunit vaccines, which do not contain live particles of the pathogen. Despite the long history of tuberculosis research, our understanding of the exact mechanisms of the diseases at the cellular level and the complex interactions between the bacilli and the immune system remain incomplete. One of the challenges in tuberculosis research has been a lack of appropriate animal models. A zebrafish (Danio rerio) model, based on an infection with Mycobacterium marinum leading to a disease, which resembles the main features of human tuberculosis, was utilized in this thesis. It is known that in an experimental set up, a high dose infection with M. marinum causes a progressive disease, whereas a low dose infection leads to the formation of a latent infection in adult zebrafish. In this study, it was shown that by suppressing the fish immune system with dexamethasone, the latent infection can be reactivated. The main goal of this thesis was to identify novel vaccine antigens to prevent tuberculosis. To this end, potential antigens were selected based on the literature and on a transcriptomic level analysis performed with in vitro reactivated M. marinum samples. In total, 22 different genes were tested as DNA vaccines. Seven of these were derived from reactivation-associated genes identified using the transcriptomic analysis and 15 genes were selected from the literature. 16 antigens were tested against the primary infection, and four (cdh, PE5, PE31 and RpfE) showed protection in a bacterial count analysis after 4 weeks of infection. Furthermore, the RpfE-derived antigen enhanced survival in a 12-weeks follow-up. Correspondingly, the preventive effects of 15 antigens against reactivation were tested with the dexamethasone-based reactivation method. With this model, three antigens, namely RpfB, MMAR_4207 and MMAR_4110, inhibited reactivation. This thesis showed that an adult zebrafish- M. marinum infection model, and the developed dexamethasone-induced reactivation model are feasible for testing the effectiveness of novel DNA vaccines. As the genomes of M. tuberculosis and M. marinum are highly similar, the M. tuberculosis homologues of the identified promising antigens are potential targets for developing novel vaccines and drugs against tuberculosis

Versatile tool for competence management - an e-portfolio management system for higher education in applied sciences

In the line of outcome based education and transferability of credits, we study a Higher Education case and propose an e-Portfolio solution as a versatile tool for assessment tasks. The solution tackles the problems firstly, of accreditation of prior achievements from both institutional and experiential learning. Secondly, the developed tool carries the process of learning outcome definitions management (derived from the real employment world), and the learner self-assessment and self-reflection as well as the guidance and support for these. The e-Portfolio management system, ePofo, supports the identification, assessment, recognition and accreditation of prior learning achievements and learning outcomes. Additionally, it is a tool to present the sectoral qualification requirements to the students, to derive learning needs and define the learning outcomes, thereby structuring the teaching. It also provides support for the learner‟s own management of achievements and competences, and finally for presenting the profile to potential employers.peerReviewe

Integrating fish models in tuberculosis vaccine development

Tuberculosis is a chronic infection by Mycobacterium tuberculosis that results in over 1.5 million deaths worldwide each year. Currently, there is only one vaccine against tuberculosis, the Bacillus Calmette-Guérin (BCG) vaccine. Despite widespread vaccination programmes, over 10 million new M. tuberculosis infections are diagnosed yearly, with almost half a million cases caused by antibiotic-resistant strains. Novel vaccination strategies concentrate mainly on replacing BCG or boosting its efficacy and depend on animal models that accurately recapitulate the human disease. However, efforts to produce new vaccines against an M. tuberculosis infection have encountered several challenges, including the complexity of M. tuberculosis pathogenesis and limited knowledge of the protective immune responses. The preclinical evaluation of novel tuberculosis vaccine candidates is also hampered by the lack of an appropriate animal model that could accurately predict the protective effect of vaccines in humans. Here, we review the role of zebrafish (Danio rerio) and other fish models in the development of novel vaccines against tuberculosis and discuss how these models complement the more traditional mammalian models of tuberculosis.publishedVersionPeer reviewe

Immunization of adult zebrafish for the preclinical screening of DNA-based vaccines

Abstract The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases

Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish

Abstract Roughly one third of the human population carries a latent Mycobacterium tuberculosis infection, with a 5–10% lifetime risk of reactivation to active tuberculosis and further spreading the disease. The mechanisms leading to the reactivation of a latent Mycobacterium tuberculosis infection are insufficiently understood. Here, we used a natural fish pathogen, Mycobacterium marinum, to model the reactivation of a mycobacterial infection in the adult zebrafish (Danio rerio). A low-dose intraperitoneal injection (∼40 colony-forming units) led to a latent infection, with mycobacteria found in well-organized granulomas surrounded by a thick layer of fibrous tissue. A latent infection could be reactivated by oral dexamethasone treatment, which led to disruption of the granuloma structures and dissemination of bacteria. This was associated with the depletion of lymphocytes, especially CD4⁺ T cells. Using this model, we verified that ethambutol is effective against an active disease but not a latent infection. In addition, we screened 15 mycobacterial antigens as postexposure DNA vaccines, of which RpfB and MMAR_4207 reduced bacterial burdens upon reactivation, as did the Ag85-ESAT-6 combination. In conclusion, the adult zebrafish-M. marinum infection model provides a feasible tool for examining the mechanisms of reactivation in mycobacterial infections, and for screening vaccine and drug candidates