A rapid and scalable density gradient purification method for Plasmodium sporozoites

Kennedy, Mark; Fishbaugher, Matthew; Vaughan, Ashley M.; Patrapuvich, Rapatbhorn; Boonhok, Rachasak; Yimamnuaychok, Narathatai; Rezakhani, Nastaran; Metzger, Péter; Ponpuak, Marisa; Sattabongkot, Jetsumon; Kappe, Stefan H. I.; Hume, Jen C. C.; Lindner, Scott E.

doi:10.1186/1475-2875-11-421

Cited by 90 publications

(108 citation statements)

References 27 publications

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“…In these purifications, we achieved ϳ50% recovery of sporozoites, less than the near-perfect recovery originally reported (33). The procedure provided highly purified sporozoites that were microscopically free of the debris present in unpurified sporozoite samples.…”

Section: Resultsmentioning

confidence: 86%

“…Unpurified sporozoites were diluted in Schneider's insect medium and injected without further purification after dissection. Where indicated, sporozoite purification was conducted prior to irradiation using the Accudenz gradient method (33). After layering one part salivary gland sporozoites over three parts 17% (wt/vol) Accudenz and centrifugation as reported previously (33), the top one-third of the gradient was transferred to a fresh tube, diluted with at least four parts Schneider's medium, aliquoted into 1.6-ml tubes, and centrifuged at 17,000 ϫ g for 4 min.…”

Section: Methodsmentioning

confidence: 99%

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Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses

2016

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cMalaria infection caused by Plasmodium parasites continues to cause enormous morbidity and mortality in areas where it is endemic, and there is no licensed vaccine capable of inducing sterile protection. Hyperimmunization with attenuated whole sporozoites can induce sterile protective immune responses targeting preerythrocytic antigens. Most animal models of hyperimmunization rely on sporozoites dissected from mosquito salivary glands and injected without further purification. In BALB/c mice, repeated small doses of P. yoelii sporozoites progressively expand the population of sporozoite-specific CD8 ؉ T cells. In this study, large secondary doses of unpurified sporozoites unexpectedly led to contraction of sporozoite-specific CD8 ؉ T cell responses in sporozoite-primed mice. While sporozoite-primed CD8 ؉ T cells alternatively can be expanded by secondary exposure to Listeria monocytogenes expressing recombinant Plasmodium antigens, such expansion was potently inhibited by coinjection of large doses of unpurified sporozoites and by uninfected salivary glands alone. Purification of sporozoites away from mosquito salivary gland debris by density gradient centrifugation eliminated salivary gland-associated inhibition. Thus, the inhibitory effect appears to be due to exposure to uninfected mosquito salivary glands rather than sporozoites. To further assess the effect of salivary gland exposure on later sporozoite vaccinations, mice were immunized with uninfected salivary glands from a single mosquito. Compared to naive mice, salivary gland presensitization reduced subsequent liver burdens by 71%. These data show that a component(s) in mosquito salivary glands reduces liver infection, thereby limiting antigen dose and contributing to lower-magnitude T cell responses. These findings suggest that sporozoite immunogenicity studies be performed using purified sporozoites whenever feasible.

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Section: Resultsmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses

2016

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“…Infection by mosquito bite or i.v. injection lends significant adaptability to the system, potentially allowing for the use of i.v.-administered cryopreserved sporozoites (31) in laboratory settings where there is no access to P. falciparum-infected mosquitoes. While there was no statistically significant difference in parasite liver burden between mosquito bite and i.v.…”

Section: Discussionmentioning

confidence: 99%

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo

et al. 2018

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“…The method used for purification of salivary gland sporozoites was based on a published method (14). The salivary glands of A. stephensi mosquitoes infected with P. falciparum NF54 were dissected into phosphate-buffered saline (PBS) containing 1% BSA in a volume of 1 ml.…”

Section: Methodsmentioning

confidence: 99%

Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

et al. 2015

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fThe extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N-and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.T he development of a vaccine to aid in the control of malaria is critical, as Plasmodium falciparum has evolved resistance to all antimalarial drugs deployed so far, including artemisinin (1). The leading malaria vaccine (RTS,S), currently in phase 3 trials, contains a formulated virus-like particle that encompasses the central and carboxyl-terminal domains of the circumsporozoite protein (CSP) fused to the hepatitis B virus surface antigen (2) and protects approximately 30% to 50% of infants or children from clinical disease for a limited duration (3, 4). Naturally derived human antibodies against a portion of the N-terminal region, including region 1, are associated with a reduced risk of disease (5), providing a basis to design new CSP vaccines. This N-terminal region of the CSP is absent from RTS,S.The importance of understanding protein structure because of its impact on the induction of broadly neutralizing antibodies and subsequent vaccine design continues to be revealed in the HIV arena (6, 7). In malaria, the importance of protein conformation for the induction of neutralizing antibodies was recently shown in vivo for an orthologue of the leading asexual-stage malaria vaccine antigen apical membrane antigen-1 (AMA-1). Only a recombinant AMA-1 forming a stable complex with a constrained synthetic rhoptry neck protein-2 peptide induced protective antibodies in vivo against a lethal blood-stage challenge malaria parasite infection (8). When developing a novel CSP vaccine, these more recent developments need to be considered with regard to the potential for changes within the CSP, such as through in vivo processing or conformational changes (9, 10) in a protein with a known extended rod-like structure (11), that could mask the adhesion domains located at the N-and C-terminal domains (9).To address these questions, a panel of CSP-speci...

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A rapid and scalable density gradient purification method for Plasmodium sporozoites

Cited by 90 publications

References 27 publications

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo

Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

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A rapid and scalable density gradient purification method for Plasmodium sporozoites

Cited by 90 publications

References 27 publications

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 + T Cell Responses

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 + T Cell Responses

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo

Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

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Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses

Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8 ⁺ T Cell Responses