3Rs of murine models of malaria infection: immunology meets experimental genetics
Lead Research Organisation:
London School of Hygiene & Tropical Medicine
Department Name: Infectious and Tropical Diseases
Abstract
Malaria is a mosquito-bourne, infectious disease that is caused by Plasmodium parasites. According the the World Health Organization's recent estimates, malaria accounts for 250 million cases of clinical disease and 0.6-1.2 million deaths worldwide, particularly in Africa and other developing nations. As such, malaria remains a global health priority, and research efforts have been aimed at developing more effective vaccines and treatments. Toward this goal, mouse models have been essential in the drive to understand the immune system and how it responds to malaria and confers protection against future infection. Similarly, mouse models are vital to identify potential targets of intervention.
Recently, we identified novel protein targets of potentially protective malaria-specific immune white blood cells. This finding now enables us to address fundamental parasitological and immunological questions: What are the functions of these proteins in the life cycle of the malaria parasite? Are they essential for the successful infection of the host? Do immune responses to these proteins contribute to the ability of an individual to be protected against infection? How are these proteins presented to immune cells? What allows a malaria-specific protein to be efficiently recognised by the immune system? The answer to these and similar questions can improve our understanding of the immune processes involved, and importantly, inform the design of new generation vaccines and therapeutic agents for malaria. We will begin to answer these questions by generating a series of modified malaria parasites that harbour certain mutations or features, thereby allowing us to characterise the molecular and immunological properties of these protein targets or antigens. In doing so, we will combine and apply tools that support the 3Rs framework of "conducting scientific experiments using animals humanely."
Currently, standard protocols require that large numbers of animals are employed to obtain meaningful data in malaria research. In this project, we will integrate recent advances in the generation of modified parasites into a single novel strategy. Our objective is to drastically reduce the number of animals required to generate these parasites (Reduction), to allow the non-invasive administration of drugs during the selection of parasites and to perform non-invasive detection of parasite development within intact animals in real time using bioluminescent parasites (Refinement). In addition, using the parasites that we have generated with our optimized strategy, we aim to develop in vitro assays to understand the interactions between the immune cells and the protein targets (Replacement). The tools and findings of this research will inform research both into the basic biology of malaria parasites and vaccine development whilst promoting the 3Rs framework in the context of infectious disease research.
Recently, we identified novel protein targets of potentially protective malaria-specific immune white blood cells. This finding now enables us to address fundamental parasitological and immunological questions: What are the functions of these proteins in the life cycle of the malaria parasite? Are they essential for the successful infection of the host? Do immune responses to these proteins contribute to the ability of an individual to be protected against infection? How are these proteins presented to immune cells? What allows a malaria-specific protein to be efficiently recognised by the immune system? The answer to these and similar questions can improve our understanding of the immune processes involved, and importantly, inform the design of new generation vaccines and therapeutic agents for malaria. We will begin to answer these questions by generating a series of modified malaria parasites that harbour certain mutations or features, thereby allowing us to characterise the molecular and immunological properties of these protein targets or antigens. In doing so, we will combine and apply tools that support the 3Rs framework of "conducting scientific experiments using animals humanely."
Currently, standard protocols require that large numbers of animals are employed to obtain meaningful data in malaria research. In this project, we will integrate recent advances in the generation of modified parasites into a single novel strategy. Our objective is to drastically reduce the number of animals required to generate these parasites (Reduction), to allow the non-invasive administration of drugs during the selection of parasites and to perform non-invasive detection of parasite development within intact animals in real time using bioluminescent parasites (Refinement). In addition, using the parasites that we have generated with our optimized strategy, we aim to develop in vitro assays to understand the interactions between the immune cells and the protein targets (Replacement). The tools and findings of this research will inform research both into the basic biology of malaria parasites and vaccine development whilst promoting the 3Rs framework in the context of infectious disease research.
Technical Summary
Our recent identification of epitopes recognised by CD8+ T cells, which are evoked by malaria liver stages, provides the basis to address fundamental parasitological and immunological questions: Are these proteins essential for the parasite life cycle? Do responses to these targets contribute to protection? How are these targets efficiently presented to CD8+ T cells? We will generate a series of Plasmodium berghei transgenic parasites as tools to answer these questions.
We propose a novel strategy that combines methods for positive/negative selection and flow cytometry-mediated sorting of fluorescent transgenic parasites to mutate candidate genes, along with integration of a green fluorescent protein and luciferase expression cassette in the parasite genome. While 'traditional' protocols require up to 30 mice for each cloning step, our strategy will allow the rapid generation of transgenic parasites using as few as three to five mice (Reduction). During the generation of the transgenic parasites, we will administer pyr and 5C (selection drugs) in the drinking water, not by injection (Refinement).
The various transgenic parasites will be assessed for their ability to complete the life cycle in both Anopheles stephensi mosquitoes and mice. Since the transgenic parasites express luciferase, their development in the liver can be evaluated by in vivo imaging (Refinement and Reduction). This method permits the non-invasive detection of parasites in intact animals and allows for the observation of the same animal as infection progresses. Gene-deficient (KO) parasites will be characterised based on their interactions with hepatocytes. Parasites with mutated epitopes (EM) will be used to immunise mice to determine the contribution of specific epitopes in protective immunity. Parasites expressing the ovalbumin epitope (OvaEpiSwaps) will be used to develop in vitro assays (Replacement) for antigen processing and presentation.
We propose a novel strategy that combines methods for positive/negative selection and flow cytometry-mediated sorting of fluorescent transgenic parasites to mutate candidate genes, along with integration of a green fluorescent protein and luciferase expression cassette in the parasite genome. While 'traditional' protocols require up to 30 mice for each cloning step, our strategy will allow the rapid generation of transgenic parasites using as few as three to five mice (Reduction). During the generation of the transgenic parasites, we will administer pyr and 5C (selection drugs) in the drinking water, not by injection (Refinement).
The various transgenic parasites will be assessed for their ability to complete the life cycle in both Anopheles stephensi mosquitoes and mice. Since the transgenic parasites express luciferase, their development in the liver can be evaluated by in vivo imaging (Refinement and Reduction). This method permits the non-invasive detection of parasites in intact animals and allows for the observation of the same animal as infection progresses. Gene-deficient (KO) parasites will be characterised based on their interactions with hepatocytes. Parasites with mutated epitopes (EM) will be used to immunise mice to determine the contribution of specific epitopes in protective immunity. Parasites expressing the ovalbumin epitope (OvaEpiSwaps) will be used to develop in vitro assays (Replacement) for antigen processing and presentation.
Planned Impact
Who will benefit from this research and how will they benefit from this research?
Academic impact:
The academic beneficiaries of this research include scientists working in the fields of cell biology, molecular biology and immunology of infectious diseases particularly malaria. This research combines these different scientific fields with animal welfare science, thus promoting multi-disciplinary synergies and complementary approaches to research. It is expected that all the experiments proposed will be conducted throughout the lifetime of the project.
A summary of the use of 3Rs in the project is outlined on page 7 of the Case for Support. It is expected that successful implementation of the project will lead to revision of our Standard Operating Procedures in the laboratory and protocols in our Home Office License.
The benefits of our research will be presented in local, European and international meetings and conferences. We will actively encourage other groups working on similar aspects (i.e. malaria and other infectious diseases) to embrace the 3Rs framework in their investigations.
Societal impact:
Researchers working on the project: In addition to gaining understanding of basic immunology, parasitology and molecular biology, staff working on the project will gain different complimentary training and skills that include research collaboration, research management, communication and presentation, and leadership. Thus, the research promotes investment in human resources.
The research will also have an impact outside its immediate academic environment. Since we expect that the results of this research will inform the development of more effective malaria vaccines, we hope that those greatly affected by malaria will ultimately benefit from this research through improved quality of life and health.
Researchers in industry interested in improvement of molecular and immunological methods will also benefit from this research.
The general public interested in scientists and their work and student considering careers in science and technology will benefit from this research. Parliamentarians and civil servants interested in animal science and welfare, human health and research policies, and scientific promotion and funding are also considered users. The theme of the project reinforces the contribution of basic science to both animal and human health research, and promotes biotechnology for health as a priority area of research in the UK.
Overall, the research promotes and strengthens scientific excellence in the UK and provides important insights to address a global health problem.
For an extensive discussion of how we plan to maximise impact, please see IMPACT SUMMARY AND PATHWAYS TO IMPACT.
Academic impact:
The academic beneficiaries of this research include scientists working in the fields of cell biology, molecular biology and immunology of infectious diseases particularly malaria. This research combines these different scientific fields with animal welfare science, thus promoting multi-disciplinary synergies and complementary approaches to research. It is expected that all the experiments proposed will be conducted throughout the lifetime of the project.
A summary of the use of 3Rs in the project is outlined on page 7 of the Case for Support. It is expected that successful implementation of the project will lead to revision of our Standard Operating Procedures in the laboratory and protocols in our Home Office License.
The benefits of our research will be presented in local, European and international meetings and conferences. We will actively encourage other groups working on similar aspects (i.e. malaria and other infectious diseases) to embrace the 3Rs framework in their investigations.
Societal impact:
Researchers working on the project: In addition to gaining understanding of basic immunology, parasitology and molecular biology, staff working on the project will gain different complimentary training and skills that include research collaboration, research management, communication and presentation, and leadership. Thus, the research promotes investment in human resources.
The research will also have an impact outside its immediate academic environment. Since we expect that the results of this research will inform the development of more effective malaria vaccines, we hope that those greatly affected by malaria will ultimately benefit from this research through improved quality of life and health.
Researchers in industry interested in improvement of molecular and immunological methods will also benefit from this research.
The general public interested in scientists and their work and student considering careers in science and technology will benefit from this research. Parliamentarians and civil servants interested in animal science and welfare, human health and research policies, and scientific promotion and funding are also considered users. The theme of the project reinforces the contribution of basic science to both animal and human health research, and promotes biotechnology for health as a priority area of research in the UK.
Overall, the research promotes and strengthens scientific excellence in the UK and provides important insights to address a global health problem.
For an extensive discussion of how we plan to maximise impact, please see IMPACT SUMMARY AND PATHWAYS TO IMPACT.
