Liver-stage Parasites Research Articles

Disruption of the Plasmodium falciparum liver-stage antigen-1 locus causes a differentiation defect in late liver-stage parasites

The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver-stage parasites undergo pre-erythrocytic replication. Liver-stage antigen-1 (LSA-1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages. Yet, the importance of LSA-1 for liver-stage parasite development remains unknown. Here we deleted LSA-1 in the NF54 strain of P. falciparum and analysed the lsa-1(-) parasites throughout their life cycle. lsa-1(-) sporozoites had normal gliding motility and invasion into hepatocytes. Six days after infection of a hepatocytic cell line, lsa-1(-) parasites exhibited a moderate phenotype with an ~50% reduction of late liver-stage forms when compared with wild type. Strikingly, lsa-1(-) parasites growing in SCID/Alb-uPA mice with humanized livers showed a severe defect in late liver-stage differentiation and exo-erythrocytic merozoite formation 7 days after infection, a time point when wild-type parasites develop into mature merozoites. The lsa-1(-) parasites also showed aberrant liver-stage expression of key parasite proteins apical membrane antigen-1 and circumsporozoite protein. Our data show that LSA-1 plays a critical role during late liver-stage schizogony and is thus important in the parasite transition from the liver to blood. LSA-1 is the first P. falciparum protein identified to be required for this transitional stage of the parasite life cycle.

Plasmodium falciparum Gametocyte Carriage Is Associated with Subsequent Plasmodium vivax Relapse after Treatment

Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRR = 3.5, 95% CI 2.0–6.0, p<0.001). The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRR = 3.0, 95% CI 1.9–4.7, p<0.001). Polymerase chain reaction (PCR) confirmed that only a low proportion of subjects (5/55 or 9.1%) who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.

Dendritic Cells and Hepatocytes Use Distinct Pathways to Process Protective Antigen from Plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2Kb epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.

Use of a Selective Inhibitor To Define the Chemotherapeutic Potential of the Plasmodial Hexose Transporter in Different Stages of the Parasite's Life Cycle

During blood infection, malarial parasites use D-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of D-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasite's development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a D-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of D-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC₅₀) of 11 μM. This process was insensitive to the external D-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC₅₀s in the region of 250 μM (the latter in a D-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages.

The intradermal route for inoculation of sporozoites of rodent malaria parasites for immunological studies

Rodent malaria parasites are commonly used for investigations into the immunology of pre-erythrocytic stage malaria infection, as sporozoites can be easily produced in the laboratory. In the majority of past immunological studies using this system, sporozoites are inoculated into mice via the intravenous (IV) route. In natural situations, however, sporozoites are deposited into the skin by the bite of Anopheline mosquitoes, and it is likely that the immunological response to such natural intradermal (ID) inoculation will be different to that achieved through the IV route. Although infected mosquito bites are sometimes used during experimental induction of immunity in mice, this method is problematic because of the low numbers of sporozoites introduced to the skin and the large variation in sporozoite inoculation between individual mosquitoes. Here, we show that ID inoculation of dissected mosquito salivary gland sporozoites of Plasmodium yoelii allows the accurate introduction of known numbers of sporozoites into the skin and that these parasites successfully invade the liver. Furthermore, immunization of mice using ID inoculations of live sporozoites contemporaneously with mefloquine treatment induces an immune response that is protective against the development of liver stage parasites, and this protection does not differ significantly from that achieved with IV immunizations performed in the same manner.

Enhancing Blood-Stage Malaria Subunit Vaccine Immunogenicity in Rhesus Macaques by Combining Adenovirus, Poxvirus, and Protein-in-Adjuvant Vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.

Transporters used by parasites as drug targets: tempting but underexploited

Transport proteins are required to obtain nutrients, remove waste products and regulate cellular volume and ion balances. They make a significant contribution to the relatively small number (< 350) of all known protein drug targets. Of the 186 human targets for FDA-approved oral drugs, 24 (13%) are transporters. However, they remain critically under-researched in the design of antimalarial interventions. As well as being excellent targets in their own right, transporters can provide delivery routes for drugs that target other essential parasite processes and are involved in mediating drug resistance. Therefore, there is ample scientific justification to increase research activity in this area. For parasite-encoded transporters, well over 100 known or proposed transport protein sequences have been identified in the P. falciparum genome (termed the Plasmodium permeome), with orthologues for the majority appearing in the other plasmodial genomes. Of these, many are considered to have high therapeutic potential such as 11 channels, 13 P-type ATPases and 11 ABC transporters. Yet, only a handful of the proteins within the Plasmodium permeome have been characterised in the detail required for assessment of therapeutic targeting. The P. falciparum hexose transporter, PfHT, is arguably the best studied parasite transporter. Here are presented recent studies on this potential antimalarial target, utilising the rodent P. berghei model to investigate the physiological importance of D-glucose transport in transmission and liver life cycle stages and to generate an in vivo model for testing inhibitors of PfHT. Plasmodium parasites may also utilise endogenous host transporters, offering further targeting opportunities. New evidence from liver stage parasites, using high-throughput microarray technology to determine the effect of P. berghei infection on the transcriptional profile of host liver cells, has identified over 60 host transport proteins or regulatory subunits that are differentially expressed during the course of infection. These proteins and the first characterisation of transport across the host plasma membrane in Plasmodium-infected liver cells will also be discussed.

Developmental transition of Plasmodium sporozoites into liver-stage forms is regulated by the RNA binding protein Pumilio 2

Developmental and cell fate decisions in many organisms are frequently made on a post-transcriptional level that involves RNA binding proteins and mechanisms of translational repression and regulation of translation of key mRNAs. In the unicellular eukaryote Plasmodium, one of the most dramatic life-cycle dependent changes in cell morphology and function occurs during transmission from the mosquito to the human host. In the mosquito salivary glands Plasmodium sporozoites remain slender, motile and infectious for several days/weeks. At this stage, Plasmodium parasites are on stand-by, awaiting injection onto the mammalian host. Immedietaly fter transmission and liver cell invasion, a remarkable differentiation process is initiated. During this process, each parasite rapidly transforms into a round, stationary exo-erythrocytic form (EEF) that expands into thousands of infectious merozoites to be released into the blood stream. Here we reveal a Plasmodium homolog of the RNA binding protein Pumilio as a key regulator of the sporozoite to EEF transition. In the absence of Pumilio-2 (Puf2), Plasmodium berghei sporozoites initiate early stage EEF development inside mosquito salivary glands, in the absence of the appropriate environmental cues. Mutant salivary gland sporozoites present reduced motility but also significantly reduced ability to invade and infect their mammalian host cells, both in vitro and in vivo. Global expression profiling confirmed that transgenic parasites exhibit genome-wide transcriptional adaptations that are typical for Plasmodium intra-hepatic development. Our data, demonstrates that Puf2 is a key player in the developmental control involved in the transition between sporozoite and EEF, implying that transformation of salivary gland-resident sporozoites into early liver stage parasites is in fact prevented by a post-translational mechanism.

Key factors regulating Plasmodium berghei sporozoite survival and transformation revealed by an automated visual assay

Malaria is transmitted to the host when Plasmodium sporozoites are injected by a mosquito vector. Sporozoites eventually enter hepatocytes, where they differentiate into liver-stage parasites. During the first hours after hepatocyte invasion, the crescent-shaped sporozoites transform into spherical intracellular exoerythrocytic parasites. This process, which precedes genome replication, can be mimicked in vitro in the absence of host cells. Here, we developed an automated method to follow transformation and cell death of sporozoites in vitro. This assay provides a rapid tool to test sporozoite survival and to screen for antiparasitic drugs. We found that extracellular bicarbonate and high temperature trigger transformation, whereas physiological serum albumin concentrations and media lacking bicarbonate delayed sporozoite death. Because bicarbonate also triggers ookinete transformation and exflagellation of gametocytes, we suggest that a common molecular mechanism regulates similar aspects of stage conversion in Plasmodium.

Natural products inhibiting the liver stage of the malaria parasite

Malaria parasite has a complex life cycle involving both human and mosquito as hosts. In human, the sporozoites inoculated by the Anopheles mosquito first invade the hepatocytes and develop the liver stage (LS) parasites from which the blood stage (BS) parasites originate. Antimalarial drug discovery is primarily directed against the asexual BS parasites that cause the clinical symptoms of the disease. The LS that has a longer duration, lower parasite loads and causal malaria prophylaxis effect, has not been truly exploited for drug purposes due to technical challenges. Primaquine is the only licensed drug used against LS parasites, but is highly toxic. Only two natural products with low selectivity and unknown targets were reported to kill LS parasites [1]. We investigated an in-house natural product library for LS activity by flow cytometry and immunofluorescence, both based on in vitro infections of Plasmodium yoelii in the hepatoma cells (HepG2:CD81). Recent studies [2,3] indicate that LS parasites exhibit an absolute requirement for de novo type II fatty acid biosynthesis (FAS-II) making this pathway an excellent target for LS drug discovery. Hence the compounds were also tested against recombinant FAS-II enzymes. Several lichen metabolites appeared to kill LS parasites, without toxicity on primary mammalian or hepatoma cells, and inhibited multiple crucial plasmodial FAS-II enzymes. Hence, these are the first natural products active against Plasmodium LS with a potential target. This presentation will cover our efforts to find natural products targeting the LS malaria parasite.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

BackgroundPlasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1.MethodsA recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes.ResultsFourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure.ConclusionsThis study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Extreme CD8 T Cell Requirements for Anti-Malarial Liver-Stage Immunity following Immunization with Radiation Attenuated Sporozoites

Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these “whole-parasite” vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.

Prolonged Antigen Presentation Is Required for Optimal CD8+ T Cell Responses against Malaria Liver Stage Parasites

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Transcriptional Analysis of the Pre-Erythrocytic Stages of the Rodent Malaria Parasite, Plasmodium yoelii

The molecular biology of the clinically silent pre-erythrocytic stages of mammalian Plasmodium spp, composed of both the sporozoite and liver stages, has remained largely uncharacterized. Improved understanding of the biological processes required for progression through the pre-erythrocytic stages could lead to the identification of novel drug and vaccine targets. To gain insights into the molecular events that occur during the pre-erythrocytic stages of Plasmodium, comparative transcriptional analysis was performed on radiation attenuated sporozoites (RAS), wild type sporozoites (wtSPZ) and liver stage parasites collected either 24 hours (24hrLS) or 48 hours (48hrLS) after mice were infected with Plasmodium yoelii. Our results revealed 1100 Plasmodium genes that were differentially expressed in one or more constituents of the pre-erythrocytic stages relative to the mixed blood stages. Overall, the transcriptional profile of P. yoelii gradually became more similar to the mixed blood stages as pre-erythrocytic stage development progressed into the mature liver stage schizont. The transcriptional profiles of RAS and wtSPZ were found to be nearly identical. Likewise, the transcriptional profile of 24hrLS was very similar to that of the 48hrLS parasites. The largest differences in gene expression were observed when comparing wtSPZ or RAS to either of the liver stage samples. Further characterization of the differentially expressed genes identified in this study could help elucidate the biological mechanisms employed by Plasmodium during the pre-erythrocytic stages.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites (137.16)

Abstract Immunization with irradiated sporozoites is the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization - a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate into liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite antigen.

Differential Effector Pathways Regulate Memory CD8 T Cell Immunity against Plasmodium berghei versus P. yoelii Sporozoites

Malaria results in >1,000,000 deaths per year worldwide. Although no licensed vaccine exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against Plasmodium parasite liver stage Ags. Multiple immune-effector molecules play a role in antimicrobial immunity mediated by memory CD8 T cells, including IFN-gamma, perforin, TRAIL, Fas ligand, and TNF-alpha. However, it is not known which pathways are required for memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. In this study, we used a novel immunization strategy to generate memory CD8 T cells in the BALB/c mouse model of P. berghei or P. yoelii sporozoite infection to examine the role of immune-effector molecules in resistance to the liver stage infection. Our studies reveal that endogenous memory CD8 T cell-mediated protection against both parasite species is, in part, dependent on IFN-gamma, whereas perforin was only critical in protection against P. yoelii. We further show that neutralization of TNF-alpha in immunized mice markedly reduces memory CD8 T cell-mediated protection against both parasite species. Thus, our studies identify IFN-gamma and TNF-alpha as important components of the noncytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Our studies also show that the effector pathways that memory CD8 T cells use to eliminate liver stage infection are, in part, Plasmodium species specific.

Genetically Attenuated Parasite Vaccines Induce Contact-Dependent CD8+ T Cell Killing of Plasmodium yoelii Liver Stage-Infected Hepatocytes

The production of IFN-gamma by CD8(+) T cells is an important hallmark of protective immunity induced by irradiation-attenuated sporozoites against malaria. Here, we demonstrate that protracted sterile protection conferred by a Plasmodium yoelii genetically attenuated parasite (PyGAP) vaccine was completely dependent on CD8(+) T lymphocytes but only partially dependent on IFN-gamma. We used live cell imaging to document that CD8(+) CTL from PyGAP-immunized mice directly killed hepatocyte infected with a liver stage parasite. Immunization studies with perforin and IFN-gamma knockout mice also indicated that the protection was largely dependent on perforin-mediated effector mechanisms rather than on IFN-gamma. This was further supported by our observation that both liver and spleen CD8(+) T cells from PyGAP-immunized mice induced massive apoptosis of liver stage-infected hepatocytes in vitro without the release of detectable IFN-gamma and TNF-alpha. Conversely, CD8(+) T cells isolated from naive mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might be used by wild-type parasites to subvert host immune responses during natural infection. However, CTLs from wild-type sporozoite-challenged mice could recognize and kill infected hepatocytes that were pulsed with circumsporozoite protein. Additionally, protection in PyGAP-immunized mice directly correlated with the magnitude of effector memory CD8(+) T cells. Our findings implicate CTLs as key immune effectors in a highly protective PyGAP vaccine for malaria and emphasize the critical need to define surrogate markers for correlates of protection, apart from IFN-gamma.

Triclosan inhibit the growth of the late liver‐stage of Plasmodium

Annually, approximately two million human deaths are caused worldwide by malaria, most of them being children. Plasmodium falciparum is the leading cause of cerebral malaria, the most severe and fatal form of disease. Moreover, the emergence of resistant strains to the existing drugs has worsened the situation. Currently, primaquine is the only drug available for eliminating liver-stage parasites. Because of the emergence of resistant parasite strains, it becomes necessary to find new targets unique to the malaria parasites. In the Plasmodium species, the discovery of a distinct Type-II fatty-acid synthesis pathway has created an opportunity to target this pathway for the development of new inhibitors of malaria parasite growth. The present study explored the growth inhibition potential of triclosan in the case of liver-stage parasites. Liver-stage of Plasmodium is an excellent target for intervention due to very small parasite load as well as possibility of eliminating parasites before it can cause blood-stage infection. Here we report that triclosan inhibits the development of the Plasmodium liver-stage parasites.

Antigens for pre‐erythrocytic malaria vaccines: building on success

Immunization with attenuated pre-erythrocytic malaria parasites can confer sterile protection against malaria in humans and rodents, and a single pre-erythrocytic antigen incorporated in a subunit vaccine has substantially reduced clinical Plasmodium falciparum malaria episodes in African infants during phase 2 trials. Building upon this success has been hindered by technical obstacles that limit research on pre-erythrocytic parasites, especially the liver stage (LS) parasites, and by an incomplete understanding of the immune mechanisms that confer protection in humans. Recent improvements in growing and isolating LS parasites have allowed progress in defining the transcriptome and proteome of the LS parasite, although more work remains to be done particularly for the early LS parasite of P. falciparum. Next generation pre-erythrocytic antigens can be assessed and prioritized based on immunization studies in animals, and on models of immunity such as attenuated parasite vaccines that confer sterile protection or naturally acquired LS-specific immune responses that correlate with protection in endemic areas. Although mechanisms of protection in humans remain poorly understood, the availability of a human malaria challenge model for early clinical testing of candidate vaccines is a valuable tool to confirm which immunogens should move forward to larger field trials.

Viral vectors in malaria vaccine development

Traditional vaccine technologies have resulted in an impressive array of efficacious vaccines against a variety of infectious agents. However, several potentially deadly pathogens, including retroviruses and parasites, have proven less amenable to the application of traditional vaccine platforms, indicating the need for new approaches. Viral vectors represent an attractive way to deliver and present vaccine antigens that may offer advantages over traditional platforms. Due to their ability to induce strong cell-mediated immunity (CMI) in addition to antibodies, viral vectors may be suitable for infectious agents, such as malaria parasites, where potent CMI is required for protection. Poxvirus-vectored malaria vaccines have been the most extensively studied in the clinic, achieving significant reductions in liver-stage parasite burden. More recently, adenovirus-vectored malaria vaccines have entered clinical testing. The most promising approach - heterologous prime-boost regimens, in which different viral vectors are sequentially paired with each other or with DNA or recombinant protein vaccines - is now being explored, and could provide high-grade protection, if findings in animal models are translatable to humans. Significant barriers remain, however, such as pre-existing immunity to the vector particle and an unexplained safety signal observed in one trial suggesting an increased risk of HIV acquisition in volunteers with pre-existing immunity to the vector.