Engineering Galleria Mellonella as a model for infection, immunity and inflammation

Growing evidence supports the use of the larvae of the waxmoth, Galleria Mellonella, as an in vivo animal partial replacement model, particularly in the related fields of infection, immunity and inflammation.

Although they are insects, their immune system is very similar to humans. There is huge potential impact on the number of rodents used in scientific research, if Galleria can be optimised as a model organism. However, currently, the health of larvae after injection with bacteria or fungal pathogens is monitored only by a crude assessment of whether/when they turn black and die.

In the first part of this project, we will make transgenic larvae that glow (fluoresce) differentially under stress, infection, immune challenge or inflammatory status. We will light up their macrophages, the cells in the immune system that wander around, looking for foreign pathogens and "eating them". This will allow us to measure the movement of this important type of cell before and after infection, revolutionising the amount and type of information we can obtain about the way animals, and we, respond to infection.

The second aim of the project is to use these fluorescent Galleria larvae to understand how the most common cause of human fungal disease in the world, a pathogen called Candida albicans, hides itself from our immune system. We know that Candida has something on their cell surface that the immune system normally recognises but that, in some cases, their environment triggers a change that ends up masking this signal. We will expose Candida to many of these sorts of environments and ask how effective the glowing Galleria macrophages are at recognising the fungus.

Our work will therefore not only figure out the key molecular signals that mask Candida from the immune system, potentially identifying candidate molecules for new anti-fungal agents, but also has the potential to revolutionise the use of Galleria as mouse replacement model, saving many thousands of mice from being used in scientific research in the future.

The laboratory of the Lead applicant (Wakefield) has very recently optimised Galleria breeding facilities, developed a robust exogenous DNA injection protocol and used PBac-mediated germline transposition to generate the world's first transgenic Galleria.

In this 30 month NC3R application, we will first use existing DNA constructs that specifically drive the expression of fluorescent proteins in Drosophila embryonic and larval hemocytes, such as srpHemoH2A::3xmCherry, to generate a series of transgenic Galleria lines in which hemocytes specifically fluoresce. We will also make a series of new Galleria-specific constructs to generate endogenous transposon-based constructs and subsequent lines. We will modify existing protocols for imaging Drosophila hemocytes, to generate time-lapse movies of Galleria larval hemocytes. These will be analysed using tracking software, originally designed to track Drosophila hemocytes, in order to quantify directionality, velocity and polarity of hemocytes during normal development and after immune or apoptotic challenge.

These tools and techniques will be used in functional screens aimed to uncover the molecular pathways underlying evasion from host immune response by the important human fungal pathogen, Candida albicans. The co-Investigator (Brown) has recently shown that the availability of the cell surface epitope beta-glucan on C.albicans directly correlates with the ability of the host to phagocytose and clear the pathogen, but that host factors can alter the availability of the beta-glucan through pathogen pre-adaptation. Transgenic Galleria larvae will be injected with GFP-NAT1 labelled C. albicans cells, and the degree of phagocytic uptake, under a variety of fungal preadaptation conditions and in C. albicans mutants, will be quantified. This will determine the major host factors that contribute to epitope masking and therefore pathogenicity.

In vivo models for drug testing and disease study continue to increase, with ~50% of all procedures now involving genetically modified animals (UK Home-Office, 2017). The rapid rise in the number of animals, particularly mice, used in research (from 2.7m to 4.1m) runs contrary to regulators' efforts to 'reduce, refine or replace' mammalian in vivo studies. Recent evidence, including the current NC3R priority call itself, supports the increasing use of the larvae of the waxmoth, Galleria Mellonella, as an in vivo animal replacement model, particularly in the related fields of infection, immunity and inflammation.

Publications of studies using Galleria larvae have increased over 10-fold in the last 6 years and there is also growing industry decision-maker support for the use of the Galleria platform as a novel in vivo model, particularly in the field of anti-microbial research (please see letters of support). However, one major limitation is the lack of transgenic tools that can quantify infection status of Galleria larvae. As far as we aware, we are the only research group world-wide to have developed a successful methodology platform to generate transgenic Galleria; and we have done so in partnership with the only world-wide distributor of research grade, antibiotic-free, age and health-matched Galleria larvae (BioSystems Technologies - BST). We believe this would dramatically increase the use of Galleria in research, enabling more accurate reporting of larval-health and infection severity, enhancing system reliability, speed and potential throughput relative to current animal models.

As such, the work outlined in this proposal will significantly impact the established global Galleria user base, including academic researchers, CROs and pharmaceutical companies who use ~3.2m larvae/year (in a market that is increasing at ~10% year-on-year). Moreover, it will stimulate their use by other academic researchers, and the £2.5bn preclinical drug discovery and toxicology testing market that currently almost exclusively use rodent models. Based on current adoption patterns, it is likely that the incorporation of transgenic reporter Galleria into drug discovery/safety research flows will also contribute to faster, cheaper, statistically robust scale experimentation. The cost of a single mouse used for experimentation is in the region of £2-400. The cost of a single age and weight matched, sterile Galleria larvae is £1. The experiments to be undertaken in this proposal, for example, would amount to ~£150,000 if mice were used, as opposed to £750 for Galleria.

Importantly the research project that constitutes work package 3 of this proposal will further advance our understanding of the molecular, cellular and tissue-specific basis of host response to the important human fungal pathogen, Candida albicans. The co-investigator, Brown, currently uses rodent models. The screen planned to investigate the response of Galleria hemocytes to infection by C. albicans under a variety of conditions and mutant backgrounds would have required 840 mice.

Finally, we expect the work in this proposal to stimulate new, additional transgenic capability in Galleria. The demonstration of generating PBac-based transgenics in Galleria, will encourage BST and other research groups world-wide to generate reporter lines for specific immune defence pathways, transcriptional regulators and pathogen-specific responses. We also expect it to stimulate uptake of new technologies, such as CRISPR/Cas gene editing, in this organism,