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Key Points Malaria is the most deadly parasitic infection of humans, killing up to 1 million people per year. No vaccine is currently available, and the development of drug-resistant Plasmodium spp. is of increasing concern. The first phase of infection, the pre-erythrocytic (PE) phase, is clinically asymptomatic. Only after parasite replication in the liver and infection of large numbers of erythrocytes do symptoms arise. The PE phase comprises sporozoites (the infectious stage) and the liver stages. Once injected by a mosquito, the sporozoites can remain in the skin, be transported in lymph vessels to draining lymph nodes or travel through the bloodstream to the liver. In the liver, sporozoites undergo an elaborate replication and developmental programme and transform into the merozoites that are released from the liver to infect erythrocytes. The PE phase of infection is a formidable window of opportunity for therapeutic interventions owing to the small number of parasites present. Thus, targeting this 'bottleneck' of Plasmodium spp. infection with vaccines is an attractive strategy. Live attenuated parasites mimicking the PE phase of infection can be used as vaccines. Attenuation is achieved by radiation, genetic alterations or drug-mediated developmental arrest. The symptoms of malaria are associated with the erythrocytic phase of Plasmodium spp. infection, but the pre-erythrocytic (PE) phase, which is clinically silent, has long been of interest as a potential vaccination target. Robert Ménard and colleagues review how our understanding of the PE phase has changed over the past decade and how this in turn has informed our understanding of the host immune response. Malaria, which is caused by Plasmodium spp., starts with an asymptomatic phase, during which sporozoites, the parasite form that is injected into the skin by a mosquito, develop into merozoites, the form that infects erythrocytes. This pre-erythrocytic phase is still the most enigmatic in the parasite life cycle, but has long been recognized as an attractive vaccination target. In this Review, we present what has been learned in recent years about the natural history of the pre-erythrocytic stages, mainly using intravital imaging in rodents. We also consider how this new knowledge is in turn changing our understanding of the immune response mounted by the host against the pre-erythrocytic forms.

Plasmodium , the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 10⁵ PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35–87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36–99%) remained without parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all vaccine recipients. Cytokine production by T cells from vaccinated subjects after in vitro stimulation with homologous (NF54) or heterologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZspecific T-cell responses in the blood peaked after the first immunization and were not enhanced by subsequent immunizations. Collectively, these data suggest durable protection against homologous and heterologous Pf parasites can be achieved with PfSPZ Vaccine. Ongoing studies will determine whether protective efficacy can be enhanced by additional alterations in the vaccine dose and number of immunizations.

Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombospondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin αvβ3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin αvβ3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors.

Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is culturing methodology, which has been described for several species, including the major disease-causing human malaria parasite, . Here, we review whole sporozoite vaccine immunogenicity and culturing platforms for sporozoite production.

Sanaria Inc. has developed methods to manufacture, purify and cryopreserve aseptic Plasmodium falciparum (Pf) sporozoites (SPZ), and is using this platform technology to develop an injectable PfSPZ-based vaccine that provides high-grade, durable protection against infection with Pf malaria. Several candidate vaccines are being developed and tested, including PfSPZ Vaccine, in which the PfSPZ are attenuated by irradiation, PfSPZ-CVac, in which fully infectious PfSPZ are attenuated in vivo by concomitant administration of an anti-malarial drug, and PfSPZ-GA1, in which the PfSPZ are attenuated by gene knockout. Forty-three research groups in 15 countries, organized as the International PfSPZ Consortium (I-PfSPZ-C), are collaborating to advance this program by providing intellectual, clinical, and financial support. Fourteen clinical trials of these products have been completed in the USA, Europe and Africa, two are underway and at least 12 more are planned for 2015–2016 in the US (four trials), Germany (2 trials), Tanzania, Kenya, Mali, Burkina Faso, Ghana and Equatorial Guinea. Sanaria anticipates application to license a first generation product as early as late 2017, initially to protect adults, and a year later to protect all persons >6 months of age for at least six months. Improved vaccine candidates will be advanced as needed until the following requirements have been met: long-term protection against natural transmission, excellent safety and tolerability, and operational feasibility for population-wide administration. Here we describe the three most developed whole PfSPZ vaccine candidates, associated clinical trials, initial plans for licensure and deployment, and long-term objectives for a final product suitable for mass administration to achieve regional malaria elimination and eventual global eradication.

Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition. Here, the authors report transcriptomes and proteomes of oocyst sporozoite and salivary gland sporozoite stages in rodent-infectious Plasmodium yoelii parasites and human infectious Plasmodium falciparum parasites and define two waves of translational repression during sporozoite maturation.

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.