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Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. Passive immunization of mice with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers significant protection against Plasmodium infection of mice. Furthermore, passive transfer of both AgTRIO antiserum and an anti-circumsporozoite protein monoclonal antibody provides synergistic protection. In this study, we generated monoclonal antibodies against AgTRIO to delineate the regions of AgTRIO associated with protective immunity. Monoclonal antibody 13F-1 markedly reduced Plasmodium infection in mice and recognized a region (VDDLMAKFN) in the carboxyl terminus of AgTRIO. 13F-1 is an IgG2a isotype monoclonal antibody, and the Fc region is required for protection. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a severe complication of malaria that occurs despite effective antimalarial treatment. Currently, noninvasive imaging procedures such as chest X-rays are used to assess edema in established MA-ARDS, but earlier detection methods are needed to reduce morbidity and mortality. The early stages of MA-ARDS are characterized by the infiltration of leukocytes, in particular monocytes/macrophages; thus, monitoring of immune infiltrates may provide a useful indicator of early pathology. In this study, Plasmodium berghei ANKA-infected C57BL/6 mice, a rodent model of MA-ARDS, were longitudinally imaged using the 18-kDa translocator protein (TSPO) imaging agent [(18)F]FEPPA as a marker of macrophage accumulation during the development of pathology and in response to combined artesunate and chloroquine diphosphate (ART+CQ) therapy. [(18)F]FEPPA uptake was compared to blood parasitemia levels and to levels of pulmonary immune cell infiltrates by using flow cytometry. Infected animals showed rapid increases in lung retention of [(18)F]FEPPA, correlating well with increases in blood parasitemia and pulmonary accumulation of interstitial inflammatory macrophages and major histocompatibility complex class II (MHC-II)-positive alveolar macrophages. Treatment with ART+CQ abrogated this increase in parasitemia and significantly reduced both lung uptake of [(18)F]FEPPA and levels of macrophage infiltrates. We conclude that retention of [(18)F]FEPPA in the lungs is well correlated with changes in blood parasitemia and levels of lung-associated macrophages during disease progression and in response to ART+CQ therapy. With further development, TSPO biomarkers may have the potential to accurately assess the early onset of MA-ARDS.

Malaria infects millions of people every year, and despite recent advances in controlling disease spread, such as vaccination, it remains a global health concern. The circumsporozoite protein (CSP) has long been acknowledged as a key target in antimalarial immunity. Leveraging the DNA vaccine platform against this formidable pathogen, the following five synthetic DNA vaccines encoding variations of CSP were designed and studied: 3D7, GPI1, ΔGPI, TM, and DD2. Among the single CSP antigen constructs, a range of immunogenicity was observed with ΔGPI generating the most robust immunity. In an intravenous (i.v.) sporozoite challenge, the best protection among vaccinated mice was achieved by ΔGPI, which performed almost as well as the monoclonal antibody 311 (MAb 311) antibody control. Further analyses revealed that ΔGPI develops high-molecular-weight multimers in addition to monomeric CSP. We then compared the immunity generated by ΔGPI versus synDNA mimics for the antimalaria vaccines RTS,S and R21. The anti-CSP antibody responses induced were similar among these three immunogens. T cell responses demonstrated that ΔGPI induced a more focused anti-CSP response. In an infectious mosquito challenge, all three of these constructs generated inhibition of liver-stage infection as well as immunity from blood-stage parasitemia. This study demonstrates that synDNA mimics of complex malaria immunogens can provide substantial protection as can a novel synDNA vaccine ΔGPI.

The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8+ T cell responses that are capable of eliminating developing parasites in hepatocytes resulting in protective immunity. In this study, we characterised the importance of the immunodominant CSP-derived epitope, SYIPSAEKI, of Plasmodium berghei in both sporozoite- and vaccine-induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-2-Kd, we established that epitope-specific CD8+ T cell responses contribute to parasite killing following sporozoite immunisation. Yet, sterile protection was achieved in the absence of this epitope substantiating the concept that other antigens can be sufficient for parasite-induced protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8+ T cell responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based vaccines did not confer complete protection, despite the induction of high levels of CSP-specific antibodies. These findings underscore the significance of CSP in protection against malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection against the parasite is mediated by CD8+ T cells specific for the immunodominant CSP-derived epitope.

The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8(+) T cell responses that are capable of eliminating developing parasites in hepatocytes, resulting in protective immunity. In this study, we characterized the importance of the immunodominant CSP-derived epitope SYIPSAEKI of Plasmodium berghei in both sporozoite- and vaccine-induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-2-K(d), we established that epitope-specific CD8(+) T cell responses contribute to parasite killing following sporozoite immunization. Yet, sterile protection was achieved in the absence of this epitope, substantiating the concept that other antigens can be sufficient for parasite-induced protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8(+) T cell responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based vaccines did not confer complete protection, despite the induction of high levels of CSP-specific antibodies. These findings underscore the significance of CSP in protection against malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection against the parasite is mediated by CD8(+) T cells specific for the immunodominant CSP-derived epitope.

Cytoadherence-linked asexual gene 9 (Clag9), a conserved Plasmodium protein expressed during the asexual blood stages, is involved in the cytoadherence of infected red blood cells (RBCs) to the endothelial lining of blood vessels. Here, we show that Plasmodium falciparum Clag9 (PfClag9) is a component of the PfClag9-RhopH complex that is involved in merozoite binding to human erythrocytes. To characterize PfClag9, we expressed four fragments of PfClag9, encompassing the entire protein. Immunostaining analysis using anti-PfClag9 antibodies showed expression and localization of PfClag9 at the apical end of the merozoites. Mass spectrometric analysis of merozoite extracts after immunoprecipitation using anti-PfClag9 antibody identified P. falciparum rhoptry-associated protein 1 (PfRAP1), PfRAP2, PfRAP3, PfRhopH2, and PfRhopH3 as associated proteins. The identified rhoptry proteins were expressed, and their association with PfClag9 domains was assessed by using protein-protein interaction tools. We further showed that PfClag9 binds human RBCs by interacting with the glycophorin A-band 3 receptor-coreceptor complex. In agreement with its cellular localization, PfClag9 was strongly recognized by antibodies generated during natural infection. Mice immunized with the C-terminal domain of PfClag9 were partially protected against a subsequent challenge infection with Plasmodium berghei, further supporting a biological role of PfClag9 during natural infection. Taken together, these results provide direct evidence for the existence of a PfRhopH-Clag9 complex on the Plasmodium merozoite surface that binds to human RBCs.

We used a genome-wide screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mice to identify genes in which recessive loss-of-function mutations protect against pathological neuroinflammation. We identified an R367Q mutation in the ZBTB7B (ThPOK) protein in which homozygosity causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Zbtb7b(R367Q) homozygous mice show a defect in the lymphoid compartment expressed as severe reduction in the number of single-positive CD4 T cells in the thymus and in the periphery, reduced brain infiltration of proinflammatory leukocytes in P. berghei ANKA-infected mice, and reduced production of proinflammatory cytokines by primary T cells ex vivo and in vivo. Dampening of proinflammatory immune responses in Zbtb7b(R367Q) mice is concomitant to increased susceptibility to infection with avirulent (Mycobacterium bovis BCG) and virulent (Mycobacterium tuberculosis H37Rv) mycobacteria. The R367Q mutation maps to the first DNA-binding zinc finger domain of ThPOK and causes loss of base contact by R367 in the major groove of the DNA, which is predicted to impair DNA binding. Global immunoprecipitation of ThPOK-containing chromatin complexes coupled to DNA sequencing (ChIP-seq) identified transcriptional networks and candidate genes likely to play key roles in CD4(+) CD8(+) T cell development and in the expression of lineage-specific functions of these cells. This study highlights ThPOK as a global regulator of immune function in which alterations may affect normal responses to infectious and inflammatory stimuli.

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA(+) ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

CD47 engagement by the macrophage signal regulatory protein alpha (SIRPα) inhibits phagocytic activity and protects red blood cells (RBCs) from erythrophagocytosis. The role of CD47-SIRPα in the innate immune response to Plasmodium falciparum infection is unknown. We hypothesized that disruption of SIRPα signaling may enhance macrophage uptake of malaria parasite-infected RBCs. To test this hypothesis, we examined in vivo clearance in CD47-deficient mice infected with Plasmodium berghei ANKA and in vitro phagocytosis of P. falciparum -infected RBCs by macrophages from SHP-1-deficient ( Shp-1 −/− ) mice and NOD.NOR- Idd13.Prkdc scid (NS- Idd13 ) mice, as well as human macrophages, following disruption of CD47-SIRPα interactions with anti-SIRPα antibodies or recombinant SIRPα-Fc fusion protein. Compared to their wild-type counterparts, Cd47 −/− mice displayed significantly lower parasitemia, decreased endothelial activation, and enhanced survival. Using macrophages from SHP-1-deficient mice or from NS- Idd13 mice, which express a SIRPα variant that does not bind human CD47, we showed that altered SIRPα signaling resulted in enhanced phagocytosis of P. falciparum -infected RBCs. Moreover, disrupting CD47-SIRPα engagement using anti-SIRPα antibodies or SIRPα-Fc fusion protein also increased phagocytosis of P. falciparum -infected RBCs. These results indicate an important role for CD47-SIRPα interactions in innate control of malaria and suggest novel targets for intervention.

Cerebral malaria (CM) is the most severe complication of human infection with Plasmodium falciparum . The mechanisms predisposing to CM are still not fully understood. Proinflammatory immune responses are required for the control of blood-stage malaria infection but are also implicated in the pathogenesis of CM. A fine balance between pro- and anti-inflammatory immune responses is required for parasite clearance without the induction of host pathology. The most accepted experimental model to study human CM is Plasmodium berghei ANKA ( Pb ANKA) infection in C57BL/6 mice that leads to the development of a complex neurological syndrome which shares many characteristics with the human disease. We applied this model to study the outcome of Pb ANKA infection in mice previously infected with Mycobacterium tuberculosis , the causative agent of tuberculosis. Tuberculosis is coendemic with malaria in large regions in the tropics, and mycobacteria have been reported to confer some degree of unspecific protection against rodent Plasmodium parasites in experimental coinfection models. We found that concomitant M. tuberculosis infection did not change the clinical course of Pb ANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. The immunological environments in spleen and brain did not differ between singly infected and coinfected animals; instead, the overall cytokine and T cell responses in coinfected mice were comparable to those in animals solely infected with Pb ANKA. Our data suggest that M. tuberculosis coinfection is not able to change the outcome of Pb ANKA-induced disease, most likely because the inflammatory response induced by the parasite rapidly dominates in mice previously infected with M. tuberculosis .

The leading malaria vaccine candidate, RTS,S, based on the Plasmodium falciparum circumsporozoite protein (CSP), will likely be the first publicly adopted malaria vaccine. However, this and other subunit vaccines, such as virus-vectored thrombospondin-related adhesive protein (TRAP), provide only intermediate to low levels of protection. In this study, the Plasmodium berghei homologues of antigens CSP and TRAP are combined. TRAP is delivered using adenovirus- and vaccinia virus-based vectors in a prime-boost regime. Initially, CSP is also delivered using these viral vectors; however, a reduction of anti-CSP antibodies is seen when combined with virus-vectored TRAP, and the combination is no more protective than either subunit vaccine alone. Using an adenovirus-CSP prime, protein-CSP boost regime, however, increases anti-CSP antibody titers by an order of magnitude, which is maintained when combined with virus-vectored TRAP. This combination regime using protein CSP provided 100% protection in C57BL/6 mice compared to no protection using virus-vectored TRAP alone and 40% protection using adenovirus-CSP prime and protein-CSP boost alone. This suggests that a combination of CSP and TRAP subunit vaccines could enhance protection against malaria.

The role of naturally occurring CD4 + CD25 + Foxp3 + regulatory T cells (nTreg) in the pathogenesis of cerebral malaria (CM), which involves both pathogenic T cell responses and parasite sequestration in the brain, is still unclear. To assess the contribution and dynamics of nTreg during the neuropathogenesis, we unbalanced the ratio between nTreg and naive CD4 + T cells in an attenuated model of Plasmodium berghei ANKA-induced experimental CM (ECM) by using a selective cell enrichment strategy. We found that nTreg adoptive transfer accelerated the onset and increased the severity of CM in syngeneic C57BL/6 (B6) P. berghei ANKA-infected mice without affecting the level of parasitemia. In contrast, naive CD4 + T cell enrichment prevented CM and promoted parasite clearance. Furthermore, early during the infection nTreg expanded in the spleen but did not efficiently migrate to the site of neuroinflammation, suggesting that nTreg exert their pathogenic action early in the spleen by suppressing the protective naive CD4 + T cell response to P. berghei ANKA infection in vivo in both CM-susceptible (B6) and CM-resistant (B6-CD4 −/− ) mice. However, their sole transfer was not sufficient to restore CM susceptibility in two CM-resistant congenic strains tested. Altogether, these results demonstrate that nTreg are activated and functional during P. berghei ANKA infection and that they contribute to the pathogenesis of CM. They further suggest that nTreg may represent an early target for the modulation of the immune response to malaria.

Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene (PBANKA_060330) from Plasmodium berghei and studied its potential for use as a TBV. This gene, referred to as pbg37, encodes a protein of 37 kDa with a signal peptide and multiple transmembrane domains and is preferentially expressed in gametocytes. A recombinant Pbg37 (rPbg37) protein targeting the N-terminal 63 amino acids (amino acids 26 to 88) expressed in bacteria elicited strong antibody responses in mice. Western blotting demonstrated Pbg37 expression in gametocytes, zygotes, and, to a lesser extent, ookinetes and its predominant association with the membranes of gametocytes. Indirect immunofluorescence assay showed an abundant surface localization of Pbg37 on gametes and zygotes but reduced amounts on retorts and ookinetes. Knockout of pbg37 (Δpbg37) led to a considerable reduction in gametocytemia, which translated into a ~92.1% decrease in the oocyst number in mosquitoes. Deletion of pbg37 had a more substantial influence on the development and maturation of microgametocytes. As a result, the Δpbg37 lines exhibited a higher female/male gametocyte ratio, fewer mature male gametocytes, and defects in the exflagellation of mature microgametocytes. To test the transmission-blocking potential of Pbg37, an in vitro ookinete assay showed that the major inhibitory effects of anti-Pbg37 antiserum were on the exflagellation and fertilization processes. Direct feeding of mosquitoes on mice immunized with rPbg37 or a control protein showed that rPbg37-immunized and P. berghei-infected mice had a significant reduction (49.1%) in oocyst density compared to the controls. The conservation of this gene in Plasmodium warrants further investigations in human malaria parasites.

Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivax could have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen., Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivax could have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen. Here, two leading P. vivax preerythrocytic vaccine candidate antigens, the P. vivax circumsporozoite protein (PvCSP) and the thrombospondin-related adhesion protein (PvTRAP) were delivered as a combined vaccine. This strategy provided a dose-sparing effect, with 100% sterile protection in mice using doses that individually conferred low or no protection, as with the unadjuvanted antigens PvTRAP (0%) and PvCSP (50%), and reached protection similar to that of adjuvanted components. Efficacy against malaria infection was assessed using a new mouse challenge model consisting of a double-transgenic Plasmodium berghei parasite simultaneously expressing PvCSP and PvTRAP used in mice immunized with the virus-like particle (VLP) Rv21 previously reported to induce high efficacy in mice using Matrix-M adjuvant, while PvTRAP was concomitantly administered in chimpanzee adenovirus and modified vaccinia virus Ankara (MVA) vectors (viral-vectored TRAP, or vvTRAP) to support effective induction of T cells. We examined immunity elicited by these vaccines in the context of two adjuvants approved for human use (AddaVax and Matrix-M). Matrix-M supported the highest anti-PvCSP antibody titers when combined with Rv21, and, interestingly, mixing PvCSP Rv21 and PvTRAP viral vectors enhanced immunity to malaria over levels provided by single vaccines.

In animal models of experimental cerebral malaria (ECM), the glycosylphosphatidylinositols (GPIs) and GPI anchors are the major factors that induce nuclear factor kappa B (NF-κB) activation and proinflammatory responses, which contribute to malaria pathogenesis. GPIs and GPI anchors are transported to the cell surface via a process called GPI transamidation, which involves the GPI transamidase (GPI-T) complex. In this study, we showed that GPI16, one of the GPI-T subunits, is highly conserved among Plasmodium species. Genetic knockout of pbgpi16 (Δpbgpi16) in the rodent malaria parasite Plasmodium berghei strain ANKA led to a significant reduction of the amounts of GPIs in the membranes of merozoites, as well as surface display of several GPI-anchored merozoite surface proteins. Compared with the wild-type parasites, Δpbgpi16 parasites in C57BL/6 mice caused much less NF-κB activation and elicited a substantially attenuated T helper type 1 response. As a result, Δpbgpi16 mutant-infected mice displayed much less severe brain pathology, and considerably fewer Δpbgpi16 mutant-infected mice died from ECM. This study corroborated the GPI toxin as a significant inducer of ECM and further suggested that vaccines against parasite GPIs may be a promising strategy to limit the severity of malaria.

Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration., Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration. In this study, we report a novel protein that possesses a Trx domain-like structure of the thioredoxin family and is expressed on the surface of merozoites of the malaria parasite Plasmodium falciparum. This protein, namely, PfTrx-mero protein, displayed a mutated sequence character at the Trx domain, but with a specific binding activity to human erythrocytes. Specific antibodies to the protein inhibited merozoite invasion into human erythrocytes. Immunization with a homologous protein of Plasmodium berghei strain ANKA also showed significant protection against lethal infection in mice. These results suggested that the novel PfTrx-like-mero protein expressed on the surface of merozoites is an important ligand participating in erythrocyte invasion and a potential vaccine candidate.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3 −/− but not Irgm1 −/− mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8 + T cells within the brain. Although antigen-specific proliferation of transferred CD8 + T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3 −/− recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3 −/− mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3 −/− mice occurs due to impaired generation of CD8 + effector function. This defect is nonintrinsic to CD8 + T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

© 2015 American Society for Microbiology. The authors have paid a fee to allow immediate free access to this article., Following transmission through a mosquito bite to the mammalian host, Plasmodium parasites first invade and replicate inside hepatocytes before infecting erythrocytes and causing malaria. The mechanisms limiting Plasmodium reinfections in humans living in regions of malaria endemicity have mainly been explored by studying the resistance induced by the blood stage of infection. However, epidemiologic studies have suggested that in high-transmission areas, preerythrocytic stages also activate host resistance to reinfection. This, along with the recent discovery that liver infections trigger a specific and effective type I interferon (IFN) response, prompted us to hypothesize that this pre-erythrocyte-stage-induced resistance is linked to liver innate immunity. Here, we combined experimental approaches and mathematical modeling to recapitulate field studies and understand the molecular basis behind such resistance. We present a newly established mouse reinfection model and demonstrate that rodent malaria liver-stage infection inhibits reinfection. This protection relies on the activation of innate immunity and involves the type I IFN response and the antimicrobial cytokine gamma IFN (IFN-γ). Importantly, mathematical simulations indicate that the predictions based on our experimental murine reinfection model fit available epidemiological data. Overall, our study revealed that liver-stage-induced innate immunity may contribute to the preerythrocytic resistance observed in humans in regions of malaria hyperendemicity., This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) grants PTDC-SAU-MIC-117060-2010 (to Miguel Prudêncio) and EXCL/IMI-MIC/0056/2012 (to M.M.M.). P.L. was supported by Fondation pour la Recherche Médicale and FCT (fellowship SFRH/BPD/41547/2007). P.M. was supported by FCT (fellowship SFRH/BD/71098/2010). Miguel Prudêncio and M.P.D. are supported by an Australian Research Council Discovery Grant (DP120100064). M.P.D. is an NHMRC Senior Research Fellow.

Epidemiological observations have linked increased host iron with malaria susceptibility, and perturbed iron handling has been hypothesized to contribute to the potentially life-threatening anemia that may accompany blood-stage malaria infection. To improve our understanding of these relationships, we examined the pathways involved in regulation of the master controller of iron metabolism, the hormone hepcidin, in malaria infection. We show that hepcidin upregulation in Plasmodium berghei murine malaria infection was accompanied by changes in expression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway target genes, a key pathway involved in hepcidin regulation. We therefore investigated known agonists of the BMP/SMAD pathway and found that Bmp gene expression was not increased in infection. In contrast, activin B, which can signal through the BMP/SMAD pathway and has been associated with increased hepcidin during inflammation, was upregulated in the livers of Plasmodium berghei-infected mice; hepatic activin B was also upregulated at peak parasitemia during infection with Plasmodium chabaudi. Concentrations of the closely related protein activin A increased in parallel with hepcidin in serum from malaria-naive volunteers infected in controlled human malaria infection (CHMI) clinical trials. However, antibody-mediated neutralization of activin activity during murine malaria infection did not affect hepcidin expression, suggesting that these proteins do not stimulate hepcidin upregulation directly. In conclusion, we present evidence that the BMP/SMAD signaling pathway is perturbed in malaria infection but that activins, although raised in malaria infection, may not have a critical role in hepcidin upregulation in this setting.

CD8 + T cells play a pathogenic role in the development of murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice. Only a limited number of CD8 + epitopes have been described. Here, we report the identification of a new epitope from the bergheilysin protein recognized by PbA-specific CD8 + T cells. Induction and functionality of these specific CD8 + T cells were investigated in parallel with previously reported epitopes, using new tools such as tetramers and reporter cell lines that were developed for this study. We demonstrate that CD8 + T cells of diverse specificities induced during PbA infection share many characteristics. They express cytolytic markers (gamma interferon [IFN-γ], granzyme B) and chemokine receptors (CXCR3, CCR5) and damage the blood-brain barrier in vivo . Our earlier finding that brain microvessels in mice infected with PbA, but not with non-ECM-causing strains, cross-presented a shared epitope was generalizable to these additional epitopes. Suppressing the induction of specific CD8 + T cells through tolerization with a high-dose peptide injection was unable to confer protection against ECM, suggesting that CD8 + T cells of other specificities participate in this process. The tools that we developed can be used to further investigate the heterogeneity of CD8 + T cell responses that are involved in ECM.

Infection with Plasmodium falciparum may result in severe disease affecting various organs, including liver, spleen, and brain, resulting in high morbidity and mortality. Plasmodium berghei Anka infection of mice recapitulates many features of severe human malaria. The aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by ligands important in the modulation of the inflammatory response. We found that AhR-knockout (KO) mice infected with P. berghei Anka displayed increased parasitemia, earlier mortality, enhanced leukocyte-endothelial cell interactions in the brain microvasculature, and increased inflammation in brain (interleukin-17 [IL-17] and IL-6) and liver (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) compared to infected wild-type (WT) mice. Infected AhR-KO mice also displayed a reduction in cytokines required for host resistance, including TNF-α, IL-1β, and IFN-γ, in the brain and spleen. Infection of AhR-KO mice resulted in an increase in T regulatory cells and transforming growth factor β, IL-6, and IL-17 in the brain. AhR modulated the basal expression of SOCS3 in spleen and brain, and P. berghei Anka infection resulted in enhanced expression of SOCS3 in brain, which was absent in infected AhR-KO mice. These data suggest that AhR-mediated control of SOCS3 expression is probably involved in the phenotype seen in infected AhR-KO mice. This is, to our knowledge, the first demonstration of a role for AhR in the pathogenesis of malaria.

Malaria is a vector-borne disease caused by the single-cell eukaryote Plasmodium . The infectious parasite forms are sporozoites, which originate from midgut-associated oocysts, where they eventually egress and reach the mosquito hemocoel. Sporozoites actively colonize the salivary glands in order to be transmitted to the mammalian host. Whether residence in the salivary glands provides distinct and vital cues for the development of infectivity remains unsolved. In this study, we systematically compared the infectivity of Plasmodium berghei sporozoites isolated from the mosquito hemocoel and salivary glands. Hemocoel sporozoites display a lower proportion of gliding motility but develop into liver stages when added to cultured hepatoma cells or after intravenous injection into mice. Mice infected by hemocoel sporozoites had blood infections similar to those induced by sporozoites liberated from salivary glands. These infected mice display indistinguishable systemic inflammatory cytokine responses and develop experimental cerebral malaria. When used as metabolically active, live attenuated vaccine, hemocoel sporozoites elicit substantial protection against sporozoite challenge infections. Collectively, these findings show that salivary gland colonization does not influence parasite virulence in the mammalian host when sporozoites are administered intravenously. This conclusion has important implications for in vitro sporozoite production and manufacturing of whole-sporozoite vaccines.

Cerebral malaria (CM) is associated with excessive host proinflammatory responses and endothelial activation. The hematopoietic hormone erythropoietin (EPO) possesses neuroprotective functions in animal models of ischemic-hypoxic, traumatic, and inflammatory injuries. In the Plasmodium berghei ANKA model of experimental CM (ECM), recombinant human EPO (rhEPO) has shown evident protection against ECM. To elucidate the mechanism of EPO in this ECM model, we investigated the effect of rhEPO on host cellular immune responses. We demonstrated that improved survival of mice with ECM after rhEPO treatment was associated with reduced endothelial activation and improved integrity of the blood-brain barrier. Our results revealed that rhEPO downregulated the inflammatory responses by directly inhibiting the levels and functions of splenic dendritic cells. Conversely, rhEPO treatment led to significant expansion of regulatory T cells and increased expression of the receptor cytotoxic T lymphocyte antigen 4 (CTLA-4). The data presented here provide evidence of the direct effect of rhEPO on host cellular immunity during ECM.

Interleukin-27 (IL-27) is known to control primary CD4 + T cell responses during a variety of different infections, but its role in regulating memory CD4 + T responses has not been investigated in any model. In this study, we have examined the functional importance of IL-27 receptor (IL-27R) signaling in regulating the formation and maintenance of memory CD4 + T cells following malaria infection and in controlling their subsequent reactivation during secondary parasite challenge. We demonstrate that although the primary effector/memory CD4 + T cell response was greater in IL-27R-deficient (WSX-1 −/− ) mice following Plasmodium berghei NK65 infection than in wild-type (WT) mice, there were no significant differences in the size of the maintained memory CD4 + T population(s) at 20 weeks postinfection in the spleen, liver, or bone marrow of WSX-1 −/− mice compared with WT mice. However, the composition of the memory CD4 + T cell pool was slightly altered in WSX-1 −/− mice following clearance of primary malaria infection, with elevated numbers of late effector memory CD4 + T cells in the spleen and liver and increased production of IL-2 in the spleen. Crucially, WSX-1 −/− mice displayed significantly enhanced parasite control compared with WT mice following rechallenge with homologous malaria parasites. Improved parasite control in WSX-1 −/− mice during secondary infection was associated with elevated systemic production of multiple inflammatory innate and adaptive cytokines and extremely rapid proliferation of antigen-experienced T cells in the liver. These data are the first to demonstrate that IL-27R signaling plays a role in regulating the magnitude and quality of secondary immune responses during rechallenge infections.

Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8+ T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8+ T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8+ T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8+ T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8+ T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8+ T cells during liver-stage infection., Infection and Immunity, 81(10), pp.3825-3834; 2013

The development of vaccine candidates against Plasmodium vivax —the most geographically widespread human malaria species—is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax -infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I·C) [poly(I·C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4 + T cell responses. Moreover, E. coli -derived CS proteins in combination with poly(I·C)LC induced potent multifunctional (interleukin 2-positive [IL-2 + ], tumor necrosis factor alpha-positive [TNF-α + ], gamma interferon-positive [IFN-γ + ]) CD4 + effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [ Pb -CS( Pf )], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ∼50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4 + T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4 + T cell immunity that was significantly less potent than that with poly(I·C)LC. Overall, these data suggest that full-length CS proteins and poly(I·C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.

Lack of an adequate animal model of Plasmodium falciparum severe malarial anemia (SMA) has hampered the understanding of this highly lethal condition. We developed a model of SMA by infecting C57BL/6 mice with P. chabaudi followed after recovery by P. berghei infection. P. chabaudi / P. berghei -infected mice had an initial 9- to 10-day phase of relatively low parasitemia and severe anemia, followed by a second phase of hyperparasitemia, more profound anemia, reticulocytosis, and death 14 to 21 days after infection. P. chabaudi / P. berghei -infected animals had more intense splenic hematopoiesis, higher interleukin-10 (IL-10)/tumor necrosis factor alpha and IL-12/gamma interferon (IFN-γ) ratios, and higher antibody levels against P. berghei and P. chabaudi antigens than P. berghei -infected or P. chabaudi -recovered animals. Early treatment with chloroquine or artesunate did not prevent the anemia, suggesting that the bulk of red cell destruction was not due to the parasite. Red cells from P. chabaudi / P. berghei -infected animals had increased surface IgG and C3 by flow cytometry. However, C3 −/− mice still developed anemia. Tracking of red cells labeled ex vivo and in vivo and analysis of frozen tissue sections by immunofluorescence microscopy showed that red cells from P. chabaudi / P. berghei -infected animals were removed at an accelerated rate in the liver by erythrophagocytosis. This model is practical and reproducible, and its similarities with P. falciparum SMA in humans makes it an appealing system with which to study the pathogenesis of this condition and explore potential immunomodulatory interventions.

Plasmodium sporozoites are inoculated into the skin of the mammalian host as infected mosquitoes probe for blood. A proportion of the inoculum enters the bloodstream and goes to the liver, where the sporozoites invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic, or liver, stage. Here, we show that a small fraction of the inoculum remains in the skin and begins to develop into exoerythrocytic forms that can persist for days. Skin exoerythrocytic forms were observed for both Plasmodium berghei and Plasmodium yoelii , two different rodent malaria parasites, suggesting that development in the skin of the mammalian host may be a common property of plasmodia. Our studies demonstrate that skin exoerythrocytic stages are susceptible to destruction in immunized mice, suggesting that their aberrant location does not protect them from the host's adaptive immune response. However, in contrast to their hepatic counterparts, they are not susceptible to primaquine. We took advantage of their resistance to primaquine to test whether they could initiate a blood-stage infection directly from the inoculation site, and our data indicate that these stages are not able to initiate malaria infection.

Vaccines have been at the forefront of global research efforts to combat malaria, yet despite several vaccine candidates, this goal has yet to be realized. A potentially effective approach to disrupting the spread of malaria is the use of transmission-blocking vaccines (TBV), which prevent the development of malarial parasites within their mosquito vector, thereby abrogating the cascade of secondary infections in humans. Since malaria is transmitted to human hosts by the bite of an obligate insect vector, mosquito species in the genus Anopheles , targeting mosquito midgut antigens that serve as ligands for Plasmodium parasites represents a promising approach to breaking the transmission cycle. The midgut-specific anopheline alanyl aminopeptidase N (AnAPN1) is highly conserved across Anopheles vectors and is a putative ligand for Plasmodium ookinete invasion. We have developed a scalable, high-yield Escherichia coli expression and purification platform for the recombinant AnAPN1 TBV antigen and report on its marked vaccine potency and immunogenicity, its capacity for eliciting transmission-blocking antibodies, and its apparent lack of immunization-associated histopathologies in a small-animal model.

Dendritic cells are the most potent antigen-presenting cells, but their roles in blood-stage malaria infection are not fully understood. We examined the effects of Flt3 ligand, a cytokine that induces dendritic cell production, in vivo on the course of infection with Plasmodium berghei ANKA. Mice treated with Flt3 ligand showed preferential expansion of CD8+ dendritic cells and granulocytes, as well as lower levels of parasitemia, and were protected from the development of lethal experimental cerebral malaria (ECM). Rag2 knockout mice treated with Flt3 ligand also showed inhibition of parasitemia, suggesting that this protection was due, at least in part, to the stimulation of innate immunity. However, it was unlikely that the inhibition of ECM was due simply to the reduction in the level of parasitemia. In the peripheral T cell compartment, CD8+ T cell levels were markedly increased in Flt3 ligand-treated mice after infection. These CD8+ T cells expressed CD11c and upregulated CXCR3, while the expression of CD137, CD25, and granzyme B was reduced. In the brain, the number of sequestered CD8+ T cells was not significantly different for treated versus untreated mice, while the proportion of CD8+ T cells that produce gamma interferon (IFN-γ) and granzyme B was significantly reduced in treated mice. In addition, sequestration of parasitized red blood cells (RBCs) in the brain was reduced, suggesting that altered CD8+ T cell activation and reduced sequestration of parasitized RBCs culminated in inhibition of ECM development. These results suggest that the quantitative and qualitative changes in the dendritic cell compartment are important for the pathogenesis of ECM., Infection and Immunity, 79(10), pp.3947-3956; 2011

Malaria infection is initiated by sporozoite invasion of hepatocytes and asexual reproduction of liver stages, processes that are regarded to be “clinically and diagnostically silent.” Merozoites, which egress from hepatocytes, infect erythrocytes in periodic cycles and induce disease. How the host innate immune system contributes to disease outcomes and to the induction of effector cells during malaria remains unclear. Likewise, how the initial liver stages may shape responses to blood-stage parasites is unknown. Here, using both sporozoite- and blood-stage-induced infections with the rodent malaria parasite Plasmodium berghei ANKA, we show that the MyD88 and Toll-like receptor 2/4 (TLR2/4) pathways play critical roles in the development of experimental cerebral malaria (ECM). Strikingly, an absolute dependence on MyD88 and TLR2/4 was observed when infections were initiated with sporozoites. In addition, we show that caspase-1 activation of interleukin-1β (IL-1β) and IL-18, which is associated with the inflammasome pathway, does not contribute to P. berghei ANKA-induced immunopathology. Consistent with these data, prophylactic cover with the IL-1β antagonist anakinra did not reduce the incidence of ECM. Therefore, we propose that protection against ECM due to loss of TLR signaling functions is caused by effector mechanisms other than IL-1β activation.

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ −/− ) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ −/− mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Infection of C57BL/6 mice with Plasmodium berghei ANKA induces a fatal neurological disease commonly referred to as experimental cerebral malaria. The onset of neurological symptoms and mortality depend on pathogenic CD8 + T cells and elevated parasite burdens in the brain. Here we provide clear evidence of liver damage in this model, which precedes and is independent of the onset of neurological symptoms. Large numbers of parasite-specific CD8 + T cells accumulated in the liver following P. berghei ANKA infection. However, systemic depletion of these cells at various times during infection, while preventing neurological symptoms, failed to protect against liver damage or ameliorate it once established. In contrast, rapid, drug-mediated removal of parasites prevented hepatic injury if administered early and quickly resolved liver damage if administered after the onset of clinical symptoms. These data indicate that CD8 + T cell-mediated immune pathology occurs in the brain but not the liver, while parasite-dependent pathology occurs in both organs during P. berghei ANKA infection. Therefore, we show that P. berghei ANKA infection of C57BL/6 mice is a multiorgan disease driven by the accumulation of parasites, which is also characterized by organ-specific CD8 + T cell-mediated pathology.

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection, particularly in children. The pathogenesis of cerebral malaria involves parasitized red blood cell (RBC)-mediated vascular inflammation, immune stimulation, loss of blood-brain barrier integrity, and obstruction of cerebral capillaries. Therefore, blunting vascular inflammation and immune cell recruitment is crucial in limiting the disease course. Beta interferon (IFN-β) has been used in the treatment of diseases, such as multiple sclerosis (MS) but has not yet been explored in the treatment of CM. Therefore, we sought to determine whether IFN-β also limits disease progression in experimental cerebral malaria (ECM). Plasmodium berghei -infected mice treated with IFN-β died later and showed increased survival, with improved blood-brain barrier function, compared to infected mice. IFN-β did not alter systemic parasitemia. However, we identified multiple action sites that were modified by IFN-β administration. P. berghei infection resulted in increased expression of chemokine (C-X-C motif) ligand 9 (CXCL9) in brain vascular endothelial cells that attract T cells to the brain, as well as increased T-cell chemokine (C-X-C motif) receptor 3 (CXCR3) expression. The infection also increased the cellular content of intercellular adhesion molecule 1 (ICAM-1), a molecule important for attachment of parasitized RBCs to the endothelial cell. In this article, we report that IFN-β treatment leads to reduction of CXCL9 and ICAM-1 in the brain, reduction of T-cell CXCR3 expression, and downregulation of serum tumor necrosis factor alpha (TNF-α). In addition, IFN-β-treated P. berghei -infected mice also had fewer brain T-cell infiltrates, further demonstrating its protective effects. Hence, IFN-β has important anti-inflammatory properties that ameliorate the severity of ECM and prolong mouse survival.

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S -transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum -infected young African children as diagnostic markers of CM.

Protein kinase C θ (PKCθ) functions as a core component of the immunological synapse and serves as a key protein in the integrated T-cell antigen receptor (TCR)/CD28-induced signaling cascade leading to T-cell activation. However, the involvement of PKCθ in host-mediated immune responses to pathogens has not been thoroughly investigated. We tested the consequences of PKCθ ablation on the host response to infection by Plasmodium berghei ANKA (PbA). We found that both PKCθ +/+ and PKCθ −/− C57BL/6J mice are susceptible to infection with PbA. However, despite a similar parasite burden, PKCθ +/+ mice had an earlier onset of neurological signs, characteristics of experimental cerebral malaria (ECM), resulting in an earlier death. These mice suffered from an early and pronounced splenomegaly with a concomitant increase in the total number of CD4 + splenic T cells. In contrast, a large proportion of PbA-infected PKCθ −/− mice overcame the acute phase characterized by neurological symptoms and survived longer than PKCθ +/+ mice. The partial resistance of PKCθ −/− mice to ECM was associated with an impaired production of Th1-type cytokines, including gamma interferon and tumor necrosis factor alpha/lymphotoxin-α, which are known to exacerbate symptoms leading to ECM. In addition, PbA infection-induced LFA-1 expression in CD8 + T cells was suppressed in PKCθ-deficient T cells, suggesting a diminished ability to adhere to endothelial cells and sequester in brain microvasculature, which may explain the decrease in neurological symptoms. These data implicate PKCθ in CD4 + Th1 + and CD8 + T-cell-mediated immune responses during PbA infection that contribute to the development of ECM.

Immunization with live Plasmodium sporozoites under chloroquine prophylaxis (Spz plus CQ) induces sterile immunity against sporozoite challenge in rodents and, more importantly, in humans. Full protection is obtained with substantially fewer parasites than with the classic immunization with radiation-attenuated sporozoites. The sterile protection observed comprised a massive reduction in the hepatic parasite load and an additional effect at the blood stage level. Differences in the immune responses induced by the two protocols occur but are as yet little characterized. We have previously demonstrated that in mice immunized with irradiated sporozoites, immune responses against the circumsporozoite protein (CSP), the major component of the sporozoite's surface and the leading malaria vaccine candidate, were not essential for sterile protection. Here, we have employed transgenic P lasmodium berghei parasites in which the endogenous CSP was replaced by that of P lasmodium yoelii , another rodent malaria species, to assess the role of CSP in the sterile protection induced by the Spz-plus-CQ protocol. The data demonstrated that this role was minor because sterile immunity was obtained irrespective of the origin of CSP expressed by the parasites in this model of protection. The immunity was obtained through a single transient exposure of the host to the immunizing parasites (preerythrocytic and erythrocytic), a dose much smaller than that required for immunization with radiation-attenuated sporozoites.

We have investigated the immunological basis of pregnancy-related Plasmodium berghei recrudescence in immune mice with substantial preexisting immunity. Specifically, we examined the relevance of this experimental model to the study of pregnancy-associated malaria (PAM) caused by P. falciparum in women with substantial preexisting protective immunity. We used mice with immunity induced prior to pregnancy and employed flow cytometry to assess their levels of immunoglobulin G (IgG) recognizing antigens on the surfaces of infected erythrocytes (IEs) in plasma. After immunization, the mice did not possess IgG specific for antigens on IEs obtained during pregnancy-related recrudescence but they acquired recrudescence-specific IgG over the course of several pregnancies and recrudescences. In contrast, levels of antibodies recognizing IEs from nonpregnant mice did not increase with increasing parity. Furthermore, maternal hemoglobin levels increased and pregnancy-related parasitemia decreased with increasing parity. Finally, parasitemic mice produced smaller litters and pups with lower weights than nonparasitemic mice. Taken together, these observations suggest that levels of antibodies specific for recrudescence-type IEs are related to the protection of pregnant mice from maternal anemia, low birth weight, and decreased litter size. We conclude that the model replicates many of the key parasitological and immunological features of PAM, although the P. berghei genome does not encode proteins homologous to the P. falciparum erythrocyte membrane protein 1 adhesins, which are of key importance in P. falciparum malaria. The study of P. berghei malaria in pregnant, immune mice can be used to gain significant new insights regarding malaria pathogenesis and immunity in general and regarding PAM in particular.

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected withPlasmodium falciparummalaria will progress to CM. We used thePlasmodium bergheiANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin α1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin α1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi . Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. We examined global gene expression patterns during fatal murine CM (FMCM) and noncerebral malaria (NCM) by microarray analysis. There was differential expression of a number of genes, including some not yet characterized in the pathogenesis of FMCM. Some gene induction was observed during Plasmodium berghei infection regardless of the development of CM, and there was a predominance of genes linked to interferon responses, even in NCM. However, upon real-time PCR validation and quantitation, these genes were much more highly expressed in FMCM than in NCM. The observed changes included genes belonging to pathways such as interferon signaling, major histocompatibility complex processing and presentation, apoptosis, and immunomodulatory and antimicrobial processes. We further characterized differentially expressed genes by examining the cellular source of their expression as well as their temporal expression patterns during the course of malaria infection. These data identify a number of novel genes that represent interesting candidates for further investigation in FMCM.

The number of malaria sporozoites delivered to a host by mosquitoes is thought to have a significant influence on the subsequent course of the infection in the mammalian host. We did studies with Anopheles stephensi mosquitoes with salivary gland infections of Plasmodium berghei sporozoites expressing a red fluorescent protein. After individual mosquitoes fed on an ear pinna or the ventral abdomen of a mouse, fluorescence microscopy was used to count numbers of sporozoites. Mosquitoes allowed to feed on the ear for periods of 3 versus 15 min deposited means of 281 versus 452 sporozoites, respectively, into the skin; this may have epidemiological implications because mosquitoes can feed for longer periods of time on sleeping hosts. Mosquitoes feeding on the ventral abdomen injected sporozoites not only into the skin but also into the underlying peritoneal musculature. Although mosquitoes injected fewer sporozoites into the abdominal tissues, more of these were reingested into the mosquito midgut, probably a consequence of easier access to blood intake from the abdominal area. The most consistent parameter of sporozoite transmission dynamics under all conditions of mosquito probing and feeding was the relatively slow release rate of sporozoites (∼1 to 2.5 per second) from the mosquito proboscis. The numbers of sporozoites introduced into the host by mosquitoes and the transmission efficiencies of sporozoite delivery are multifactorial phenomena that vary with length of probing time, skin site being fed upon, and numbers of sporozoites within the salivary glands.

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36 -deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36 -deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36 -deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36 -deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36 -deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36 -deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.

The infection of mice with Plasmodium berghei ANKA constitutes the best available mouse model for human Plasmodium falciparum -mediated cerebral malaria, a devastating neurological syndrome that kills nearly 2.5 million people every year. Experimental data suggest that cerebral disease results from the sequestration of parasitized erythrocytes within brain blood vessels, which is exacerbated by host proinflammatory responses mediated by cytokines and effector cells including T lymphocytes. Here, T cell responses to P. berghei ANKA were analyzed in cerebral malaria-resistant and -susceptible mouse strains. CD4 + T-cell proliferation and interleukin-2 (IL-2) production in response to parasite-specific and polyclonal stimuli were strongly inhibited in cerebral malaria-resistant mice. In vitro and in vivo depletion of CD4 + CD25 + regulatory T (T reg ) cells significantly reversed the inhibition of CD4 + T-cell proliferation and IL-2 production, indicating that this cell population contributes to the suppression of T-cell function during malaria. Moreover, in vivo depletion of T reg cells prevented the development of parasite-specific TH1 cells involved in the induction of cerebral malaria during a secondary parasitic challenge, demonstrating a regulatory role for this cell population in the control of pathogenic responses leading to fatal disease.

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8 + T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8 + T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-γ) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1rcongenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Understanding the protective mechanism in the liver induced by recombinant vaccines against the pre-erythrocytic stages of malaria is important for vaccine development. Most studies in mice have focused on splenic and peripheral blood T cells and identified gamma interferon (IFN-γ)-producing CD8+T cells as correlates of protection, which can be induced by prime-boost vaccination with recombinant poxviruses. Invariant natural killer T (Vα14iNKT) cells can also protect against liver stage malaria, when activated, and are abundant in the liver. Since poxviruses have nonspecific immunomodulating effects, which are incompletely understood, we investigated whether recombinant poxviruses affect the protective properties of hepatic Vα14iNKT cells and thus vaccine efficacy. We show that intradermal vaccination with recombinant poxviruses activated Vα14iNKT cells and NK cells in the livers of BALB/c mice while inducing IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing pre-erythrocytic stage antigen-specific CD8+T cells. Greater numbers of hepatic Vα14iNKT cells secreted interleukin-4 than IFN-γ. Vaccinated Vα14iNKT-cell-deficient mice had lower, but still protective levels of hepatic and splenic IFN-γ+and TNF-α+CD8+T cells and better protection rates later after challenge withPlasmodium bergheisporozoites. Therefore, vaccine-activated hepatic Vα14iNKT cells help in generating specific T cells but are not required for protection induced by recombinant poxviruses. Furthermore, double-positive INF-γ+/TNF-α+CD8+T cells were enriched in protected livers, suggesting that cells expressing both of these cytokines may be most relevant for protection.

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue ofPlasmodium falciparumMSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number ofPlasmodium yoeliiisolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13P. yoeliiisolates sequenced, even though many of the same isolates showed pronounced variability in their MSP119sequences. Nonsynonymous mutations were detected only for the isolatesPlasmodium yoelii nigeriensisN67 andPlasmodium yoelii killicki193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethalPlasmodium yoelii yoeliiYM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein fromPlasmodium bergheiANKA andPlasmodium chabaudi adamiDS and challenged withP. y. yoeliiYM. Interestingly, significant protection was afforded byP. bergheiANKA MSP4/5, which shows 81% sequence identity withP. y. yoeliiYM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinantP. c. adamiDS MSP4/5, which shows 55.7% sequence identity withP. y. yoeliiYM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP119and AMA1.

BALB/c interleukin-4 (IL-4−/−) or IL-4 receptor-α (IL-4rα−/−) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes ofPlasmodium berghei(ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4−/−and IL-4rα−/−mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-γ) production in the liver. These results suggest that IFN-γ-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule againstPlasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity againstP. bergheiinfection in these mice, with no major role for IL-13.