Stefan H. I. Kappe

Upregulated in infectious sporozoites gene 4 ( UIS4 ) encodes a parasitophorous vacuole membrane protein expressed in the sporozoite and liver stages of rodent malaria parasites. Parasites that lack UIS4 arrest in early liver-stage development, and vaccination of mice with uis4 ⁻ sporozoites confers sterile protection against challenge with infectious sporozoites. Currently, it remains unclear whether an ortholog of UIS4 is carried in the human malaria parasite Plasmodium falciparum , although the gene PF10_0164 has been identified as a candidate ortholog for UIS4 on the basis of synteny and structural similarity of the encoded protein. We show that PF10_0164 is expressed in sporozoites and blood stages of P. falciparum , where it localizes to the parasitophorous vacuole, and is also exported to the host erythrocyte. PF10_0164 is refractory to disruption in asexual blood stages. Functional complementation was tested in Plasmodium yoelii by replacing the endogenous copy of UIS4 with PF10_0164 . PF10_0164 localized to the parasitophorous vacuole membrane of liver stages, but transgenic parasites did not complete liver-stage development in mice. We conclude that PF10_0164 is a parasitophorous vacuole protein that is essential in asexual blood stages and that does not complement P. yoelii UIS4, and it is thus likely not a functional ortholog of UIS4.

To the Editor—We have read with interest the excellent recent report by Putrianti et al [1], in which infection treatment vaccination (ITV), with use of repeated vaccinations of mice with wildtype (WT) rodent malaria sporozoites followed by prophylactic primaquine (PQ) treatment, was used to induce sterile, lasting protective immunity against sporozoite challenge. PQ is a drug that kills parasite liver stages and, therefore, the authors concluded that persistence of liver stage parasites is not necessary to induce and maintain protective immunity. Furthermore, the authors noted that this differs fundamentally from vaccinations with irradiated sporozoites or genetically attenuated parasite (GAP) sporozoites, in which generation of an effective, lasting immune response depends on persistence of the parasite as liver stages. We respectfully disagree with the latter conclusion regarding GAPs.

Repeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine-induced pathology and death. Herein, we generated a novel, completely blood stage-attenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1(-) blood stages did not induce any patent infections and conferred complete sterile protection against lethal heterologous blood stage and sporozoite challenges. Partial protection was observed against lethal challenges with another parasite species, P. berghei. Importantly, subcutaneous immunization with Pynt1(-) conferred sterile protection against lethal blood stage challenges. We show that cellular and humoral immune responses are both essential for sterile protection. The study demonstrates that genetic manipulation provides a platform for the designed, complete attenuation of malaria parasite blood stages and suggests testing the safety and efficacy of P. falciparum NT1 knockout strains in humans.

The quest for an efficacious malaria vaccine has been ongoing for nearly a century with limited success. Malaria remains one of the most significant infectious diseases affecting human populations ...

Plasmodium sporozoites are the product of a complex developmental process in the mosquito vector and are destined to infect the mammalian liver. Attention has been drawn to the mosquito stages and pre-erythrocytic stages owing to recognition that these are bottlenecks in the parasite life cycle and that intervention at these stages can block transmission and prevent infection. Parasite progression in the Anopheles mosquito, sporozoite transmission to the mammalian host by mosquito bite, and subsequent infection of the liver are characterized by extensive migration of invasive stages, cell invasion, and developmental changes. Preparation for the liver phase in the mammalian host begins in the mosquito with an extensive reprogramming of the sporozoite to support efficient infection and survival. Here, we discuss what is known about the molecular and cellular basis of the developmental progression of parasites and their interactions with host tissues in the mosquito and during the early phase of mammalian infection.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36 . Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

Malaria remains one of the most devastating infectious diseases that threaten humankind. Human malaria is caused by five different species ofPlasmodiumparasites, each transmitted by the bite of femaleAnophelesmosquitoes. Plasmodia are eukaryotic protozoans with more than 5000 genes and a complex life cycle that takes place in the mosquito vector and the human host. The life cycle can be divided into pre-erythrocytic stages, erythrocytic stages and mosquito stages. Malaria vaccine research and development faces formidable obstacles because many vaccine candidates will probably only be effective in a specific species at a specific stage. In addition,Plasmodiumactively subverts and escapes immune responses, possibly foiling vaccine-induced immunity. Although early successful vaccinations with irradiated, live-attenuated malaria parasites suggested that a vaccine is possible, until recently, most efforts have focused on subunit vaccine approaches. Blood-stage vaccines remain a primary research focus, but real progress is evident in the development of a partially efficacious recombinant pre-erythrocytic subunit vaccine and a live-attenuated sporozoite vaccine. It is unlikely that partially effective vaccines will eliminate malaria; however, they might prove useful in combination with existing control strategies. Elimination of malaria will probably ultimately depend on the development of highly effective vaccines.

Background: Plasmodium sporozoites migrate to the liver where they traverse several hepatocytes before invading the one inside which they will develop and multiply into thousands of merozoites. Although this constitutes an essential step of malaria infection, the requirements of Plasmodium parasites in liver cells and how they use the host cell for their own survival and development are poorly understood. Results: To gain new insights into the molecular host-parasite interactions that take place during malaria liver infection, we have used high-throughput microarray technology to determine the transcriptional profile of P. berghei-infected hepatoma cells. The data analysis shows differential expression patterns for 1064 host genes starting at 6 h and up to 24 h post infection, with the largest proportion correlating specifically with the early stages of the infection process. A considerable proportion of those genes were also found to be modulated in liver cells collected from P. yoelii- infected mice 24 and 40 h after infection, strengthening the data obtained with the in vitro model and highlighting genes and pathways involved in the host response to rodent Plasmodium parasites. Conclusion: Our data reveal that host cell infection by Plasmodium sporozoites leads to a coordinated and sequential set of biological events, ranging from the initial stage of stress response up to the engagement of host metabolic processes and the maintenance of cell viability throughout infection.

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.