This study shows that PbGFP-Luccon parasites, can provide real-time quantitative information on the relation between in vivo parasite liver load and immunity against malaria. In vitro use of these parasites in the adapted TSNA allows for an easy and fast assessment of the functional sporozoite invasion inhibition by antibodies.
Previously, Mwakingwe et al. applied bioluminescent imaging and qRT-PCR to analyse parasite prevalence in the liver of immunized mice, using luciferase expressing P. yoelii . They did not, however, report on the direct quantitative relation between bioluminescent imaging and parasite (liver) load determined by qRT-PCR and the extent to which the transgenic parasite had similar characteristics as the WT parasite throughout the whole life cycle . In the future, a Py-Luc parasite that meets these requisites for assessment of the liver load upon challenge, can be used beside PbGFP-Luccon parasites, allowing for a direct comparison of the characteristics of P. yoelii and P. berghei in vivo.
Evaluation of immunity against malaria by bioluminescent imaging offers many advantages over conventional qRT-PCR analysis. This analysis technique is more simple, rapid and reduces the amount of mice needed. As an added value, expression of the reporter protein luciferase is restricted to live parasites and therefore allows specific detection of live parasites. This avoids detection of dead liver parasites, as may occur by the qRT-PCR assay . Measurement of parasite liver load upon challenge can be performed in vivo without the need for any invasive liver resection or biopsy. It does not require sacrificing animals and thereby reduces the number of animals and costs required for experimentation. Moreover, multiple measurements can be carried out in the same animal over time, linking the parasite liver load with protection and minimizing the effect of biological variation [6, 7]. While the use of PbGFP-Luccon over qRT-PCR has its clear benefits, there are limitations. The expression of luciferase in the parasite is relatively low and cannot be visualized earlier than 20 hours post-infection . Moreover, due to limitations in sensitivity, a low number of developing liver parasites may be missed which might mature into asexual parasites . Negative results of in vivo imaging of liver stage development can therefore not be used to claim sterile protection, which eventually requires sub-inoculation of blood from challenged mice into naive mice . Nonetheless, challenge of mice with a high number of PbGFP-Luccon sporozoites administered i.v does offer information on the parasite liver load in real time without sacrifying the mice. In the CPS model the overwhelming majority of the parasites do not develop in the liver beyond 30 hours. The presence of effector mechanisms that target early developing parasite stages can however, not formally be excluded. At least in this model with complete liver stage development, a high degree of immunity to late liver stage parasites can clearly be inferred.
Finally, the application of PbGFP-Luccon parasites is not restricted to the described assays. In a recent publication describing the host mediated factors regulating the inhibition of liver stage infection upon superinfection, luciferase expressing parasites were used to enable distinction between the parasites from the original infection and the superinfection .