Malaria is caused by several species of intracellular protozoan parasites of the Plasmodium genus and is responsible for nearly 500,000 deaths each year, primarily among young children. The first obligatory stage of malaria occurs in the liver, when sporozoites transmitted by infected mosquitoes invade hepatocytes. While liver stage malaria research has been relatively underexplored, recent technological advances have enabled the development of sophisticated assays to model liver infection, an attractive target for drug and vaccine development. Despite these advances, a major hurdle in liver stage research is the short shelf-life of ex vivo sporozoites, where decreased viability is observed within ~2h after dissection from mosquito salivary glands. Therefore, live mosquitoes must be maintained by researchers, creating many safety concerns when using human-infective species. Logistical challenges are also encountered for high-throughput screening efforts, where strict timelines prevent large pooling of parasites, limiting the standardization of large screening efforts, despite the development of robust assays. There is a clear need for the development of preservation methods for sporozoites. Here, we propose to develop a sporozoite preservation method based on our recent breakthrough in deep supercooling of aqueous solutions where we achieve very low temperatures (down to -20 oC) in the absence of ice crystallization. We hypothesize that at these supercooled temperatures, the diminished metabolic activity of sporozoites will prolong their functional viability, allowing their storage for up to 7-14 days. As ice crystallization is avoided, this may be a ?gentler? method to preserve parasites compared to standard cryopreservation approaches. The proposed stabilization period is sufficient time for global dissemination of parasites and would allow pooling of parasites over several days. We will accomplish this goal through two Specific Aims.
In Aim 1, we will prioritize cold storage solutions that lead to improved sporozoite viability and infectivity following 72 h storage under supercooled conditions (-4C), for both free sporozoites and whole glands. We will then optimize the solutions through supplementation with biomolecules known to mitigate cell damage under hypothermic storage conditions. Using these optimized storage solutions, in Aim 2 we will explore deeper supercooling temperatures (-20C) with a goal to preserve >50% infectivity following 7-14 d storage for free sporozoites and whole glands. We will further explore the preservation of whole mosquitoes to enable dissections at a later time. We expect that by the completion of this innovative exploratory project we will provide at least one solution towards the successful preservation of sporozoites for multiple species. As the methods to achieve supercooling are straightforward, this can be readily disseminated to other labs for immediate implementation.

Public Health Relevance

Vaccines and drugs that eliminate liver stage parasites are critical for malaria eradication, though research is hampered by the short ex vivo lifetime of sporozoites (~2h) once removed from the mosquito salivary glands. Here, we propose to develop a sporozoite preservation method based on our recent breakthrough in deep supercooling of aqueous solutions where we can achieve practically stable supercooling at very low temperatures (down to -20 oC) for extended periods of time. Our goal is to extend the ex vivo lifetime of sporozoites to 7-14 days, sufficient time to enable global dissemination of malaria parasites to researchers and facilitate the standardization of drug screening assays.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Cellular and Molecular Technologies Study Section (CMT)
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Mcgugan, Glen C
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Massachusetts General Hospital
United States
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