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In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53-68%) than IV-I (93-95%). Unprotected mice developed less sporozoite-specific CD8 + and CD4 + effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8 + dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel 'gene insertion/marker out' (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research., (© 2011 Lin et al.)