Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio).

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines.

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

Tuberculosis (TB) remains a major global health challenge and the development of a better vaccine takes center stage in fighting the disease. For this purpose, animal models that are capable of replicating the course of the disease and are suitable for the early-stage screening of vaccine candidates are needed. A Mycobacterium marinum infection in adult zebrafish resembles human TB. Here, we present a pre-clinical screen for a DNA-based tuberculosis vaccine in the adult zebrafish using an M. marinum infection model. We tested 15 antigens representing different types of mycobacterial proteins, including the Resuscitation Promoting factors (Rpf), PE/PPE protein family members, other membrane proteins and metabolic enzymes. The antigens were expressed as GFP fusion proteins, facilitating the validation of their expression in vivo. The efficiency of the antigens was tested against a low-dose intraperitoneal M. marinum infection (≈ 40 colony forming units), which mimics a primary M. tuberculosis infection. While none of the antigens was able to completely prevent a mycobacterial infection, four of them, namely RpfE, PE5_1, PE31 and cdh, led to significantly reduced bacterial burdens at four weeks post infection. Immunization with RpfE also improved the survival of the fish against a high-dose intraperitoneal injection with M. marinum (≈ 10.000 colony forming units), resembling the disseminated form of the disease. This study shows that the M. marinum infection model in adult zebrafish is suitable for the pre-clinical screening of tuberculosis vaccines and presents RpfE as a potential antigen candidate for further studies.

DNA vaccination boosts Bacillus Calmette-Guérin protection against mycobacterial infection in zebrafish.

Despite the widespread use of the current Bacillus Calmette-Guérin (BCG) vaccine, tuberculosis is still a major cause of morbidity and mortality worldwide. Vaccination with BCG does not prevent a Mycobacterium tuberculosis infection, nor does it inhibit the reactivation of latent tuberculosis. Here, we show that adult zebrafish are modestly and variably protected from a mycobacterial infection by BCG vaccination. An intraperitoneal (i.p.) BCG vaccination was associated with enhanced survival upon a high-dose (20,000 bacteria) Mycobacterium marinum infection. In addition, BCG-vaccinated fish were more able to restrict a low-dose (30 bacteria) intraperitoneal infection with M. marinum, as indicated by lower bacterial loads at six weeks post infection (wpi). However, the vaccination could not completely prevent an infection. A qRT-PCR analysis comparing BCG-vaccinated and unvaccinated fish upon a mycobacterial infection indicated that the induction of Tumor necrosis factor (TNF) was more modest in vaccinated fish. The partial protection gained by BCG could be boosted by a DNA vaccine combining Ag85B, ESAT6 and a resuscitation-related gene RpfE, suggesting that this combination of antigens could be useful for a future BCG booster vaccine. We conclude that zebrafish is a useful early-phase preclinical model for studying subunit vaccines designed for boosting the effects of BCG.

Animal models in tuberculosis research - where is the beef?

INTRODUCTION: Tuberculosis (TB) is a major global health problem, and new drugs and vaccines are urgently needed. As clinical trials in humans require tremendous resources, preclinical drug and vaccine development largely relies on valid animal models that recapitulate the pathology of human disease and the immune responses of the host as closely as possible. AREAS COVERED: This review describes the animal models used in TB research, the most widely used being mice, guinea pigs and nonhuman primates. In addition, rabbits and cattle provide models with a disease pathology resembling that of humans. Invertebrate models, including the fruit fly and the Dictyostelium amoeba, have also been used to study mycobacterial infections. Recently, the zebrafish has emerged as a promising model for studying mycobacterial infections. The zebrafish model also facilitates the large-scale screening of drug and vaccine candidates. EXPERT OPINION: Animal models are needed for TB research and provide valuable information on the mechanisms of the disease and on ways of preventing it. However, the data obtained in animal studies need to be carefully interpreted and evaluated before making assumptions concerning humans. With an increasing understanding of disease mechanisms, animal models can be further improved to best serve research goals.

Adequate Th2-type response associates with restricted bacterial growth in latent mycobacterial infection of zebrafish.

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.

An adult zebrafish model for preclinical tuberculosis vaccine development.

Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the current live vaccine, Bacillus Calmette-Guérin. Tuberculosis vaccine research has been hampered by a scarcity of animal models which replicate human disease and are suitable for large-scale studies. We have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes an infection resembling human tuberculosis in adult zebrafish (Danio rerio). In the present study we use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrafish from mycobacterial infection. Using a low-dose (∼20-30 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the amount of infected organs were reduced in the DNA vaccinated fish. Likewise, when infecting with a lethal infection dose (∼20,000-27,000 bacteria), vaccination significantly reduced both mortality and bacterial counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish is a promising new model for preclinical tuberculosis vaccine research.

Mycobacterium marinum causes a latent infection that can be reactivated by gamma irradiation in adult zebrafish.

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (≈ 35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5-10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.

Pirk is a negative regulator of the Drosophila Imd pathway.

NF-kappaB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. In this study we report that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. We conclude that Pirk is a negative regulator of the Imd pathway.