Local Immune Response to Injection of Plasmodium Sporozoites into the Skin

Abstract

Malarial infection is initiated when the sporozoite form of the Plasmodium parasite is inoculated into the skin by a mosquito. Sporozoites invade hepatocytes in the liver and develop into the erythrocyte-infecting form of the parasite, the cause of clinical blood infection. Protection against parasite development in the liver can be induced by injection of live attenuated parasites that do not develop in the liver and thus do not cause blood infection. Radiation-attenuated sporozoites (RAS) and genetically attenuated parasites are now considered as lead candidates for vaccination of humans against malaria. Although the skin appears as the preferable administration route, most studies in rodents, which have served as model systems, have been performed after i.v. injection of attenuated sporozoites. In this study, we analyzed the early response to Plasmodium berghei RAS or wild-type sporozoites (WTS) injected intradermally into C57BL/6 mice. We show that RAS have a similar in vivo distribution to WTS and that both induce a similar inflammatory response consisting of a biphasic recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the skin injection site and proximal draining lymph node (dLN). Both WTS and RAS associate with neutrophils and resident myeloid cells in the skin and the dLN, transform inside CD11b+ cells, and induce a Th1 cytokine profile in the dLN. WTS and RAS are also similarly capable of priming parasite-specific CD8+ T cells. These studies delineate the early and local response to sporozoite injection into the skin, and suggest that WTS and RAS prime the host immune system in a similar fashion.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated <em>Plasmodium</em> Sporozoites

“…We recently demonstrated that needle-syringe injection of immunizing doses of P. berghei sporozoites in the mouse skin induces a successive recruitment of polymorphonuclear neutrophils followed by inflammatory monocytes in the skin and dLN 19 . The Protocol Section described above details the procedure used to successfully isolate live myeloid cells from both tissues following multiple injections of large number of sporozoites in the ear dermis (Figures 1 and 2).…”

“…In our experimental system (C57BL/6 mouse -P. berghei), complete protection was achieved after multiple injections in the ear dermis of highly concentrated suspension of parasites (50,000 RAS in 0.6 µl, 4 injection sites) 19 . In this context, we established the protocol described above to analyze more precisely the local host immune response to immunizing doses of RAS that allowed us to identify 2 major myeloid cell subsets recruited in the skin and dLN in response to the parasite 19 .…”

“…Multi-parametric flow cytometry allows accurate identification and quantification of immune cell changes in the context of host response to infection 19,20 . Isolation of large number of viable cells is therefore critical to perform reproducible phenotypic analysis.…”

“…Finally, an important parameter to take into account when analysing the host response to ID injection of sporozoites is the level of inflammation induced by both needle insertion and mosquito salivary gland extract deposition in the skin 19,20 . We therefore suggest to inject additional mice with either 1x PBS alone or salivary gland extracts from the same number of non-infected mosquitoes to evaluate the immune response specifically induced by the parasite.…”

“…We aimed to identify and quantify more precisely the leukocyte subsets recruited in the skin and corresponding dLN as well as those interacting with the parasite following intradermal injection of immunizing doses of RAS 19 . In this context, we isolated myeloid cells (CD45 We describe here a protocol for isolating myeloid cells from the mouse skin and dLN following intradermal injection of immunizing doses of RAS extracted from infected mosquito salivary glands.…”