Plasmodium species, the aetiological agents of malaria, cycle between a mosquito vector and vertebrate host to complete its life cycle. Mammals become infected with malaria parasites when bitten by an infected Anopheles mosquito, which deposits the sporozoite form of the parasite into the host's skin (reviewed in
). Sporozoites then actively seek the host vasculature and upon invading it, passively migrate to the liver where they ultimately infect a hepatocyte and replicate within it. This liver stage of development (which lasts two days for rodent-infective species or six to eight days for human-infective species) results in the release of tens of thousands of red blood cell-infectious exoerythrocytic merozoites. These merozoites then initiate the blood stage of malaria infection that is associated with all clinical symptoms of the disease.
The characterization of sporozoite infectivity has often been limited by the presence of bacteria and yeast. Because of these contaminating microbes, the duration of many in vitro infections of permissive hepatocytes is often limited to only a few days. In addition, immunocompromised mice with humanized livers have recently been developed to provide an in vivo model of liver stage infections for both Plasmodium falciparum and Plasmodium vivax (
[2, 3], Mikolajczak et al., personal communication). Infections of these mice with large numbers of unpurified sporozoites can also systemically introduce large amounts of these microbes, which may negatively impact the health of these mice and affect the innate immune response against the parasite during the 6–8 day liver stage infection. These contaminants can similarly confound investigations of basic immunological responses of non-humanized mice against rodent malaria, as well.
Biochemical studies of sporozoites have also been hampered by relatively large amounts of residual mosquito debris compared to the parasite mass. For instance, proteomic studies of various Plasmodium species have used unpurified sporozoites and this has resulted in the identification of only a limited number of parasite-specific peptide hits, presumably due to the large number of contaminating mosquito-derived peptides
[4–6]. Substantial reduction of mosquito proteins would substantially help such characterizations.
A previously published method of sporozoite purification requires that the parasites be first coated with bovine serum albumin (BSA) and then passed through ion exchange resin
. While this described procedure produces well-purified sporozoites, the fraction of sporozoites recovered is routinely low (30-40%) even with sporozoites obtained from isolated salivary glands (Lindner S., unpublished results). Moreover, this approach yields parasites coated with BSA, which is known to induce sporozoite apical organelle secretion and motility
[8, 9]. Another sporozoite purification method that employs BSA utilizes a discontinuous density gradient composed of diatrozoate sodium/Hypaque and several lengthy, high speed spins to purify sporozoites
. When injected into mice, these sporozoite preparations resulted in the death of significant numbers of the mice due to an immune response to BSA
. A revised protocol from the same authors that employed mouse serum in lieu of BSA did curtail mouse death, but did not reduce contaminating microbial burdens (ibid).
Ideally, an effective malaria vaccine will confer sterile protection prior to the onset of symptoms. The most promising subunit vaccine candidate, RTS,S, is currently in Phase 3 trials by GlaxoSmithKline but has shown only partial protection from subsequent infections
. Recent efforts have also focused on developing malaria vaccines comprised of live sporozoites attenuated by irradiation or genetic disruption, including the first trial testing syringe delivery of irradiated sporozoites
[13–16]. These attenuated parasites are able to productively infect hepatocytes, but arrest at specific points within the liver stage of parasite development
. Interestingly, genetically attenuated parasites that arrest as late liver stage parasites (e.g. pyfab b/f
parasites) provide greater and broader CD8+ T-cell responses and higher levels of protection than do radiation attenuated parasites or early arresting genetically attenuated parasites (ibid). Further studies and large-scale implementation of these vaccine candidates would greatly benefit from the production of large numbers of sporozoites that have been purified away from mosquito-derived contaminants to facilitate their characterization and deployment.
To address all of the above problems, herein is described a discontinuous density gradient purification method that employs a dense layer composed of Accudenz dissolved in water. This strategy effectively removes mosquito debris and hemocoel lipids, does not require BSA or serum, greatly reduces bacterial contamination, and eliminates yeast contamination. Moreover, this approach is applicable across all species of Plasmodium tested, including rodent-infective Plasmodium yoelii and human-infective P. falciparum and P. vivax sporozoites. Importantly, this method is scalable, as >107 sporozoites can be routinely purified in a single gradient with excellent recovery rates. Lastly, this method is validated by demonstrating that these purified sporozoites are fully infectious in standard in vitro and/or in vivo infectivity assays for all three species. Taken together, this method provides a means to rapidly purify Plasmodium sporozoites for both basic research and vaccine production applications.