TruLarv Galleria mellonella- a model host for infection studies, toxicity testing and drug development

Summary | Background | Current state of the art | What could your Solution be used for? | Collaboration | Information on IP | 3Rs impact | References

This Solution received CRACK IT Solutions funding for two projects. Visit our science pages for further information about the projects carried out in partnership with Demuris and Envigo


Summary

Galleria mellonella larvae have been shown to have utility for research into microbial infection, for antimicrobial drug screening, to test the toxicity of chemicals and to understand the host response to infection. TruLarv™ are research-grade G. mellonella larvae developed by BioSystems Technology Ltd. We now seek partners to explore the integration of TruLarv™ into drug screening platforms.  TruLarv™ could be used for early stage toxicity or efficacy screening to identify candidates for further testing in mammals. We also envisage that TruLarv™ would have applications in related areas such as chemical toxicity testing and the testing of environmental samples.


Background

Attrition rates in drug discovery programmes are a problem for the pharmaceutical industry, adding cost and time to drug development programmes. Failures of drugs to perform as expected, because of toxicity or lack of efficacy, often appear during costly human clinical studies highlighting the lack of translation of current preclinical models. There is an urgent need for models that bridge the gap between in vitro studies and mammalian studies to improve the predictivity of preclinical studies and reduce late stage attrition.

G. mellonella larvae are living organisms that have been used widely in the past five years as microbial infection models [1]; antimicrobial drug screening models [2]; models to test the toxicity of chemicals [3]; and models to understand the host response to infection [4]. The major advantages of G. mellonella larvae over other invertebrate models are the ability to carry out experiments at 37°C and to precisely dose individual larvae with drugs/microorganisms by injection into a defined site. Reported studies include the testing of over 30 antibiotics and antifungal compounds in Galleria for their abilities to control infections caused by a range of pathogens and there is good agreement between the results of studies in humans and in Galleria [5].

More recently there has been a shift towards using this model to screen new approaches to disease control. These studies range from the testing of novel combinations of existing drugs to the testing of completely new treatments including the use of nanoparticles to deliver drugs [6], bacteriophage therapies [7], chemicals such as gallium, silver and zinc [8, 9], antimicrobial peptides [10] and photodynamic therapy [11]. The diverse range of approaches to disease control, which have been tested at an early stage, indicates the potential value of Galleria as an in vivo screening model.

However, the G. mellonella model has not been adopted outside of academia because the larvae used are typically sold as feedstuffs for pet reptiles and fish. Whilst cheap to purchase, there are significant variations in the behaviour of larvae within batches and between batches [1]. This is a barrier to the further development and wider adoption of this model.


Current state of the art

TruLarv™ are research grade G. mellonella larvae developed by BioSystems Technology Ltd. TruLarv™ are weight and age defined, surface decontaminated and are bred without the use of antibiotics or other drugs. The genome sequence of the breeding colony is currently being determined. G. mellonella assays often use mortality as an endpoint, usually within 24 – 72 hours. However, disease progression in G. mellonella larvae can be scored on a numerical scale on the basis of change in mobility and colour, allowing simple and rapid scoring of test groups [1]. Scoring of larvae is currently performed by an individual at a series of defined timepoints.

End points in G. mellonella infection models include survival rate, which can be assessed up to five days post infection, facilitating the calculation of a maximum half lethal dose (LD50); expression of antimicrobial proteins in response to infection; production of lactate dehydrogenase as a marker of cell damage and biophotonic imaging to measure proliferation of bioluminescent microorganisms responsible for larval infection.  A pathological scoring system was recently proposed in which an assessment of larval mobility, cocoon formation, melanisation and survival was used to assess larval health.

In our trials we have shown that TruLarv™ behave more consistently and reproducibly than pet shop larvae allowing the statistical power of experiments to be markedly increased. In some studies differences between test and control groups which were not apparent using pet shop larvae were statistically different using TruLarv™ (data not published). These findings indicate the value of TruLarv™ and now open opportunities for this model to be developed for studies which bridge the gap between in vitro studies and mammalian studies. In addition, the potential to carry out high throughput studies now opens new opportunities to improve the effectiveness of drug dicovery programmes.


What could your Solution be used for?

Our solution is initially targeted towards organisations involved in the development of new antimicrobial drugs. TruLarv™ would be used to test compounds at an early stage both for toxicity and efficacy. However, we envisage that our larvae would have applications in related areas, such as the testing of drugs which modulate immune responses. The model would also have applications for chemical toxicity testing including the testing of environmental samples.


Need for collaboration

We seek partners who are interested in integrating TruLarv™ into antimicrobial drug discovery platforms.  We envisage that TruLarv™ would be used at an early stage for toxicity and or efficacy screening and would identify candidates that would be suitable for progression through the drug development pathway for further testing studies in mammals. We are also interested in connecting with organisations with expertise in imaging and tracking technologies, which would be used to automate the scoring of large groups of larvae, allowing high throughput screens to be developed.

For these collaborations we would contribute research grade larvae, expertise in handling larvae, and expertise in scoring disease in larvae. We seek partners who could provide compounds for efficacy or toxicity testing and who have an interest in integrating our model into test platforms in an industrial setting.


Information on IP

A patent application to protect breeding, colony management and quality control processes by BioSystems Technology Ltd has been filed.


3Rs impact assessment

Used as a preliminary screen, G. mellonella larvae facilitate the selection of promising leads for further testing in vertebrates, significantly reducing the number of vertebrate animals used in drug development. For example, use of G. mellonella larvae early in the development of probiotics has reduced the use of vertebrate model hosts (poultry) by 80% in some studies. Around 50 lactobacilli strains have been screened in the Galleria model in order to select 10 for further studies. These studies would normally be carried out in day old chicks, with five chicks being commonly used per lactobacilli strain. Using Galleria, it has been possible to reduce the number of chicks required for these studies by at least 200 (data not published – personal communication from Professor Roberto La Ragione, University of Surrey).


References

  1. Tsai CJ, Loh JM, and Proft T (2016). Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing. Virulence p. 1-16.
  2. Desbois AP and Coote PJ Coote (2012). Utility of Greater Wax Moth Larva (Galleria mellonella) for Evaluating the Toxicity and Efficacy of New Antimicrobial Agents. Adv Appl Microbiol 78: p. 25-53.
  3. Megaw J et al. (2015). Galleria mellonella as a novel in vivo model for assessment of the toxicity of 1-alkyl-3-methylimidazolium chloride ionic liquids. Chemosphere 139: p. 197-201.
  4. Harding CR et al. (2013). The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice. Infect Immun 81(7): p. 2598-605.
  5. Thomas RJ et al. (2013). Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei. Int J Antimicrob Agents 41(4): p. 330-6.
  6. Deacon J et al. (2015). Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: formulation, characterisation and functionalisation with dornase alfa (DNase). J Control Release 198: p. 55-61.
  7. Seed KD and Dennis, JJ (2009). Experimental bacteriophage therapy increases survival of Galleria mellonella larvae infected with clinically relevant strains of the Burkholderia cepacia complex. Antimicrob Agents Chemother 53(5): p. 2205-8.
  8. Antunes LC et al. (2012). In vitro and in vivo antimicrobial activities of gallium nitrate against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 56(11): p. 5961-70.
  9. Coughlan A et al. (2010). Zinc and silver glass polyalkenoate cements: an evaluation of their antibacterial nature. Biomed Mater Eng 20(2): p. 99-106.
  10. Dean SN, Bishop BM and van Hoek ML (2011). Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37. Front Microbiol 2: p. 128.
  11. Chibebe Junior J et al. (2013). Photodynamic and antibiotic therapy impair the pathogenesis of Enterococcus faecium in a whole animal insect model. PLoS One 8(2): p. e55926.


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