Abstract
BackgroundMeasurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic Plasmodium berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques in vivo and in vitro.MethodsMice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with PbGFP-Luccon. The in vitro transgenic sporozoites neutralization assay (TSNA) was adapted by replacing PbCS(Pf) parasites for PbGFP-Luccon parasites.ResultsApplication of PbGFP-Luccon transgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses.ConclusionsPbGFP-Luccon parasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.
Highlights
Measurement of liver stage development is of key interest in malaria biology and vaccine studies
All mice immunized by chloroquine prophylaxis (CPS) (n = 10) or radiation attenuated sporozoites (RAS) (n = 10) with a dose regimen of three times 104 sporozoites and challenged by infectious mosquito bites, neither became parasitaemic nor displayed any bioluminescent signal originating from the liver (Figure 2a)
The robustness of protective immunity was explored by increasing the challenge level in mice that were immunized with CPS by a lower dose regimen of three times 4 × 103 sporozoites
Summary
Measurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic Plasmodium berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter luciferase. Plasmodium berghei parasites, expressing the bioluminescent reporter luciferase (PbGFP-Luccon) have been used to visualize and quantify parasite development in vitro in hepatic cells and in vivo in mice using real-time luminescence imaging [1]. Quantification of the number of parasites in hepatocytes is an important read-out to determine inhibitory activity This quantification of in vitro [2] and in vivo [3] parasite liver load is usually performed by (qRT)-PCR. This technique, is time-consuming and costly, since mice need to be sacrificed at each time point for in vitro quantification
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