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

Abstract

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.

Prime-and-Trap Malaria Vaccination To Generate Protective CD8+ Liver-Resident Memory T Cells

“…A single dose of 5 3 10 4 purified RAS protected most but not all animals (10 out of 13 mice), whereas lower single doses of RAS (2 3 10 4 ) were less protective (2 out of 5 mice, data not shown). Our experiments rely on gradient-purified RAS, which are more immunogenic than unpurified spz because mosquito Ags are largely eliminated (37). Despite increased RAS immunogenicity, sterile protection in all animals was never seen, even for a single high dose of purified RAS, which is consistent with earlier literature reported for unpurified RAS (40, 42, 44).…”

“…Luc-based in vivo imaging of liver burden was performed as described previously (37). Briefly, mice were gene gun immunized, and 5 d after boosting, they were infected with 2 3 10 4 luc-expressing purified P. yoelii spz.…”

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections

“… 29,30 qRT-PCR was subsequently performed using a multiplex assay for P. falciparum and pan- Plasmodium -specific 18S rRNA amplicons using a SensiFAST ™ Probe Lo-ROX One-Step Kit (Bioline, Taunton, MA). 31 Absolute copy number quantification was performed against an in vitro-transcribed RNA standard curve in a malaria-negative whole blood eluate and copy numbers were converted to estimated p/mL as described 30 parasite density estimates are based on the 18S rRNA content of synchronized ring-stage P. falciparum parasites and the 18S rRNA-to-parasite conversion factor may vary for non- P. falciparum species. qRT-PCR-positive specimens were classified as P. falciparum , P. falciparum or mixed, non- P. falciparum , and Plasmodium spp.…”

Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice

“… 43 Plasmodium 18S rRNA qRT-PCR was performed as described 65 although newly described pan- Plasmodium 18S rRNA-specific reagents were used herein. 66 Mice were considered positive if qRT-PCR was above the undetectable qRT-PCR signal obtained on day 6 post challenge.…”