Plasmodium sporozoite neutralization in the host skin – SporoSTOP

To determine the role of tissues in protection we have recently developed a method that allows quantifying the impact of passage of sporozoites through the skin and to compare it with the infection initiated by intravenous injection of parasites (Aliprandini et al, Nat Microbiol 2018). This method will allow determining the preferential action site of the different monoclonal antibodies (mAb) to be tested as well as their relative protective efficacy. Selected mAb will be used to be studied in details by in vivo imaging using luciferase, GFP, GFP-CSP expressing parasites to quantitatively observe sporozoite neutralization in real-time. In vitro, we are quantifying and developing assays to measure potential neutralizing activities mediated by these mAbs such as inhibition of motility and cell infection, cytotoxic activity, circumsporozoite precipitation reaction to correlate them with protection in vivo in the tissues.
Finally, other surface proteins candidate will be tested as potential targets of neutralizing antibodies using passive and active vaccination.

During these 18 initial months, we mainly focused on the objectives (1) and (2).
We showed that the monoclonal antibody (mAb) 3D11 which targets the Plasmodium berghei CSP also predominantly acts on sporozoites during the passage through the skin, as previously shown for P. yoelii. Additionally, in vivo imaging indicates a late neutralizing effect of 3D11 following the invasion of the hepatic parenchyma. This late effect was confirmed in vitro following the invasion of the host cells. Detailed characterization of this phenomenon will be performed in vitro and in vivo using the GFP-CSP sporozoites in the continuation of the project.
We also tested 14 human mAbs directed against the P. falciparum CSP. In vivo, all the protective human mAbs mainly targeted sporozoites in the host skin. At the concentration tested, the majority of human mAbs did not significantly protect mice when sporozoites were inoculated intravenously, indicating a low neutralizing activity in the blood/liver. Despite the predominant neutralization action in the skin, three of them also significantly neutralized sporozoites in the blood/liver like 3D11. In vitro, these 14 hmAbs were tested in assays of inhibition of parasite motility, cytotoxic activity, formation of circumsporozoite precipitation reaction (CSPR) and binding to sporozoites and each activity was correlated with protective activity in vivo. Cytotoxicity, inhibition of motility and CSPR were correlated with protection after sporozoite challenge in the skin. Therefore, with these results we confirmed the crucial role of the skin in the protection mediated by cytotoxic antibodies targeting the CSP.
In vivo imaging of the skin of mice transferred with protective hmAbs with low neutralizing activity on the blood, showed inhibition of sporozoite blood vessel invasion as expected. However, the level of inhibition was much inferior to the level of protection in vivo, indicating that these protective hmAbs were decreasing the fitness of the parasite during its migration through the skin.
We are now designing experiments to test the hypothesis that antibodies targeting the CSP are decreasing the parasite fitness when the sporozoite locomotes in 3D. This assay once fully established could be used to test and correlate the sera neutralizing activity of volunteers vaccinated with RTS,S with the protective status as proposed in the objective (4), and eventually, be used to optimize human vaccines based on PfCSP.
Finally, the identification of antibodies that potentiate anti-CSP neutralization in the skin will be pursued in the next years.

In this first period of studies, we showed the crucial and general role of the skin in the protection mediated by cytotoxic antibodies targeting the CSP using 14 anti-P. falciparum human monoclonal antibodies and one P.berghei monoclonal antibody. We also established some correlates of protection of human mAbs targeting the Pf CSP. We also successfully generated a GFP-CSP P.berghei transgenic parasite which will allow imaging in details the action of protective antibodies in the skin and liver and study the neutralizing effect of anti-CSP antibodies in the parasite intracellular development.
Finally, in the following years, we will focused on the development of an in vitro assay to quantify the loss of parasite infectivity using a 3D migratory environment to correlate this neutralizing activity with protection in vivo in vaccinated volunteers, as well as, on the test of protective activity of other surface protein candidates.

-

Plasmodium sporozoites are the highly motile forms of malaria-causing parasites transmitted by the mosquito into the dermis of the mammalian host. Sporozoite motility is essential to access the blood circulation and can be inhibited by antibodies against the major protein of the sporozoite surface, the circumsporozoite protein (CSP). Recently, the Amino lab discovered a novel mechanism by which anti-CSP antibodies directly kill sporozoites of rodent and human infecting Plasmodia. Cytotoxicity is dependent on parasite motility and secretion of a sporozoite perforin-like protein, both indispensable for the parasite progression in the host skin. Accordingly, protection in immunized mice was only observed following parasite inoculation into the skin by microinjection or mosquito bite, but not after intravascular injection of parasites. The Frischknecht lab has developed a range of new assays to investigate sporozoite motility including the deployment of laser tweezers and subtle mutagenesis of key motility proteins. Recently the Frischknecht lab has generated a series of parasite lines expressing different types of CSP as fusion proteins with GFP. Here we propose to combine our expertise, to (i) test the generality of skin-dependent protective mechanism of anti-CSP antibodies using sporozoites harboring the CSP of rodent or human infecting Plasmodium spp; (ii) image and characterize quantitatively the way protective antibodies are neutralizing sporozoites in the skin using parasites expressing GFP-CSP fusion proteins and biophysical methods; (iii) identify antibodies against other sporozoite surface proteins that potentiate anti-CSP neutralization in the skin and (iv) develop an in vitro 3D environment to rapidly test if levels of protection in individuals that are naturally exposed to mosquitoes or were vaccinated with RTS,S/AS01 correlate with cytotoxic activity. The expected results (i) will help our understanding of how CSP mediates sporozoite infectivity, (ii) should lead to a better understanding of the way antibodies can directly kill a parasite and (iii) could improve the efficacy of vaccination based on CSP, either by providing an immune correlate of protection and/or by the addition of novel protective antigens targeting sporozoites in the skin.