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Interferon-gamma inhibits the intrahepatocytic development of malaria parasites
Abstract
In this study, we examined the activity of recombinant interferon (IFN)-gamma against Plasmodium berghei exoerythrocytic forms (EEF) grown in vitro within the highly differentiated human hepatoma cell line HEPG2. We assayed the effect of IFN-gamma on parasite growth by DNA hybridization using a P. berghei specific DNA probe. The specific activity of IFN-gamma against EEF is very high, and depends upon the time of lymphokine addition. When IFN-gamma is added to HEPG2 cells containing intracellular EEF, 6 hr after sporozoite invasion, parasite DNA replication is inhibited by approximately 75% at 10(3) U/ml and 50% at 1 U/ml. This treatment can either abolish or greatly reduce the infectivity of EEF for mice. When added earlier, 3 hr after completion of sporozoite invasion, IFN-gamma inhibits parasite replication to an even greater degree. The highest levels of inhibition were obtained when IFN-gamma was added 6 hr prior to sporozoite invasion (100% inhibition at 10(2) U/ml, approximately 55% inhibition at 0.1 U/ml, and 17% inhibition at 0.001 U/ml). We found that HEPG2 cells express approximately 44,000 surface receptors for IFN-gamma. These data are consistent with the view that IFN-gamma exerts its antimalarial activity by binding to surface receptors on hepatocytes and inducing intracellular changes unfavorable for parasite development. Tryptophan starvation does not appear to be involved in this process. These findings also support the idea that IFN-gamma, released from immune T cells upon encountering sporozoite antigen, may be an important effector mechanism in sterile immunity to sporozoite challenge.
... Mice have been instrumental in our understanding of malaria pathogenesis and have driven most of the progress made in the field. The initial studies using mouse models were on inbred A/J mice (10,11,84,85), BALB/c (28,86), C57BL/6 (87, 88), as well as outbred Theiler's Original (T.O.) mice (89). Experiments in mice revealed that CD8 + T cells are required for protection against a RAS challenge, while CD4 + T cells are dispensable, by depleting CD8 + and CD4 + T cells respectively, in immunocompetent mice (84). ...
... The initial studies using mouse models were on inbred A/J mice (10,11,84,85), BALB/c (28,86), C57BL/6 (87, 88), as well as outbred Theiler's Original (T.O.) mice (89). Experiments in mice revealed that CD8 + T cells are required for protection against a RAS challenge, while CD4 + T cells are dispensable, by depleting CD8 + and CD4 + T cells respectively, in immunocompetent mice (84). Additionally, IFN-γ and antibodies were also shown to be required for inhibition of the development of EEFs during protection induced by vaccination with RAS (84). ...
... Experiments in mice revealed that CD8 + T cells are required for protection against a RAS challenge, while CD4 + T cells are dispensable, by depleting CD8 + and CD4 + T cells respectively, in immunocompetent mice (84). Additionally, IFN-γ and antibodies were also shown to be required for inhibition of the development of EEFs during protection induced by vaccination with RAS (84). ...
Plasmodium spp.-infected mosquitos inject sporozoites into the skin of a mammalian host during a blood meal. These enter the host's circulatory system and establish an infection in the liver. After a silent metamorphosis, merozoites invade the blood leading to the symptomatic and transmissible stages of malaria. The silent pre-erythrocytic malaria stage represents a bottleneck in the disease which is ideal to block progression to clinical malaria, through chemotherapeutic and immunoprophylactic interventions. RTS,S/AS01, the only malaria vaccine close to licensure, although with poor efficacy, blocks the sporozoite invasion mainly through the action of antibodies against the CSP protein, a major component of the pellicle of the sporozoite. Strikingly, sterile protection against malaria can be obtained through immunization with radiation-attenuated sporozoites, genetically attenuated sporozoites or through chemoprophylaxis with infectious sporozoites in animals and humans, but the deployability of sporozoite-based live vaccines pose tremendous challenges. The protection induced by sporozoites occurs in the pre-erythrocytic stages and is mediated mainly by antibodies against the sporozoite and CD8⁺ T cells against peptides presented by MHC class I molecules in infected hepatocytes. Thus, the identification of malaria antigens expressed in the sporozoite and liver-stage may provide new vaccine candidates to be included, alone or in combination, as recombinant protein-based, virus-like particles or sub-unit virally-vectored vaccines. Here I review the efforts being made to identify Plasmodium falciparum antigens expressed during liver-stage with focus on the development of parasite, hepatocyte, mouse models, and resulting rate of infection in order to identify new vaccine candidates and to improve the efficacy of the current vaccines. Finally, I propose new approaches for the identification of liver-stage antigens based on immunopeptidomics.
... It has been known for a long time that IFN- can prevent liver stage infection (87). IFN- also inhibits the intrahepatocytic development of malaria parasites in vitro, but the mechanisms were unclear (87). ...
... It has been known for a long time that IFN- can prevent liver stage infection (87). IFN- also inhibits the intrahepatocytic development of malaria parasites in vitro, but the mechanisms were unclear (87). IFN- induces LC3 lipidation and formation of LC3 puncta, suggesting a role for LC3 in clearance of Plasmodium (88). ...
The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes.
... Plasmodium in the liver can trigger a type I interferon (IFN) response and recruit CD1d-restricted NKT cells (5). IFN-g, predominately secreted by recruited NKT cells, is considered to be not only the major innate immune effector to suppress the development of the liver stage in vivo (5,13), but also the critical effector to inhibit the EEFs in the PV of hepatocytes in vitro (14,15). Altogether, these findings strongly suggest that IFN-g should be included in our investigation of the effects of CSP on EEF development both in vitro and in vivo. ...
... However, rapamycin-enhanced autophagy significantly reduced the EEF number, parasite load, and EEF size in HepG2 cells in the presence of 0.2 U/mL IFN-g (Figure 2A and Supplementary Figure S5A). We found that rapamycin could not further reduce the parasite number and load, as well as the size, when treated with 0.5 U/mL or 1 U/mL IFN-g (Figure 2A and Supplementary Figure S5A), possibly because most of the EEFs were already killed by IFN-g at these higher concentrations (14), and enhanced autophagy could not further increase the effect of IFN-g. Meanwhile, the autophagy inhibitor LY294002 considerably reverted the killing effect of 0.5 U/mL and 1 U/ mL IFN-g on EEFs ( Figure 2B and Supplementary Figure S5B), and a similar result was also obtained using another autophagy inhibitor, wortmannin ( Figure 2C). ...
Although exo-erythrocytic forms (EEFs) of liver stage malaria parasite in the parasitophorous vacuole (PV) are encountered with robust host innate immunity, EEFs can still survive and successfully complete the infection of hepatocytes, and the underlying mechanism is largely unknown. Here, we showed that sporozoite circumsporozoite protein (CSP) translocated from the parasitophorous vacuole into the hepatocyte cytoplasm significantly mediated the resistance to the killing of EEFs by interferon-gamma (IFN-γ). Attenuation of IFN-γ-mediated killing of EEFs by CSP was dependent on its ability to reduce the levels of autophagy-related genes (ATGs) in hepatocytes. The ATGs downregulation occurred through its enhanced ubiquitination mediated by E3 ligase NEDD4, an enzyme that was upregulated by CSP when it translocated from the cytoplasm into the nucleus of hepatocytes via its nuclear localization signal (NLS) domain. Thus, we have revealed an unrecognized role of CSP in subverting host innate immunity and shed new light for a prophylaxis strategy against liver-stage infection.
... Whole organism approaches to vaccine development have also shown significant promise with near complete protection in experimental challenge studies [3,29], although efficacy in the target population in malaria endemic areas has been between 30 and 70% [30,31]. In human challenge models [6,32,33] and especially in animal models [9,[34][35][36], protection by whole parasite vaccines has been associated with the induction of IFN-c and granzyme B by parasite antigen targets. Identifying these parasite targets will aid the rapid development of more cost-effective and efficacious multi-epitope subunit vaccines. ...
... Vaccine xxx (xxxx) xxx two immune mediators have been variously associated with protection against liver stage Plasmodium infection [9,10,35]. PBMCs from a total of 125 subjects made positive IFN-c responses to at least one of the 31 peptide pools tested, compared to 22 subjects who made positive granzyme B responses. Twenty subjects made responses to both IFN-c and granzyme B, but these responses were to different peptide pools. ...
Sterile protection against clinical malaria has been achieved in animal models and experimental human challenge studies involving immunization with radiation attenuated Plasmodium falciparum sporozoite vaccines as well as by live sporozoites under chloroquine prophylaxis. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection. Although the exact parasite targets of protective CD8 + T cell responses are not fully defined, responses against a handful of vaccine candidate antigens have been associated with protection. Identifying the T cell targets in these antigens will facilitate the development of simpler, cost-effective, and efficacious next generation multi-epitope vaccines. The aim of this study was to identify immunodominant portions of four malaria vaccine candidate antigens using peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites. Cryopreserved PBMCs from 291 HLA-typed subjects were stimulated with pools of overlapping 15mer peptides spanning the entire sequences of P. falciparum circumsporozoite protein (CSP, 9 pools), apical membrane antigen 1 (AMA1, 12 pools), thrombospondin related anonymous protein (TRAP, 6 pools) and cell traversal for ookinetes and sporozoites (CelTOS, 4 pools) in FluoroSpot assays. 125 of 291 subjects made IFN-γ responses to 30 of the 31 peptide pools tested and 22 of 291 made granzyme B responses, with 20 making dual responses. The most frequent responses were to the CSP C-terminal region and the least frequent responses were to TRAP and CelTOS. There was no association between FluoroSpot responses and active malaria infection, detected by either microscopy, RDT, or PCR.
In conclusion, CSP and AMA1 have relatively higher numbers of epitopes that trigger IFN-γ and granzyme B-secreting T cells in adults with life-long malaria parasite exposure compared to the other two antigens tested, and highlights the continued relevance of these two antigens as vaccine candidates.
... Mice treated with IFN-γ prior to Plasmodium sporozoite injection display a reduction in the ensuing liver infection [22][23][24]39]. Since these studies have only assessed Plasmodium liver burden at 46 hpi, it remains unknown which stage of the Plasmodium liver infection was inhibited by IFN-γ. ...
... Treatment with IFN-γ, both in vitro and in vivo, leads to a reduction in the number of P. berghei intrahepatic forms present at the late stages of P. berghei liver infection [22][23][24]39]. Our results reveal for the first time that IFN-γ reduces the number of P. berghei intrahepatic forms as early as 6 hours post-sporozoite injection. ...
Sleeping sickness and malaria are parasitic diseases with overlapping geographical distributions in sub-Saharan Africa. We hypothesized that the immune response elicited by an infection with Trypanosoma brucei, the etiological agent of sleeping sickness, would inhibit a subsequent infection by Plasmodium, the malaria parasite, decreasing the severity of its associated pathology. To investigate this, we established a new co-infection model in which mice were initially infected with T. brucei, followed by administration of P. berghei sporozoites. We observed that a primary infection by T. brucei significantly attenuates a subsequent infection by the malaria parasite, protecting mice from experimental cerebral malaria and prolonging host survival. We further observed that an ongoing T. brucei infection leads to an accumulation of lymphocyte-derived IFN-γ in the liver, limiting the establishment of a subsequent hepatic infection by P. berghei sporozoites. Thus, we identified a novel host-mediated interaction between two parasitic infections, which may be epidemiologically relevant in regions of Trypanosoma/Plasmodium co-endemicity.
... A series of studies performed by Nussenzweig and colleagues (3)(4)(5)(6) in 1986-1987 revealed that exogenously administered IFN-g effectively inhibits the development of liver-stage parasites in vitro and in vivo. Recently, Boonhok et al. (7) reported that IFN-g-mediated inhibition occurs at least partially in an autophagy-related, protein-dependent manner in infected hepatocytes. ...
... injection of 1000 Pb-conGFP sporozoites. BV administration eliminates liver-stage parasites completely Pathways stimulated by type I and II IFNs can lead to the killing of hepatocytes infected with liver-stage parasites (3)(4)(5)(6)(7)(8). Because BV is a potent inducer of type I and II IFNs (32,33), and we observed BV-mediated protection as described above, we next investigated whether BV-induced IFNs could kill liver-stage parasites in vivo. ...
Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-a and IFN-g were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-a neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.
... A prerequisite to this route of malaria control and the development of novel therapies is a better understanding of liver-stage Plasmodium and its interactions with host hepatocytes. IFN-γ was previously shown to exhibit antimalarial activity against the liver stages of the mouse malarial species Plasmodium berghei and Plasmodium yoelli, in both cultured hepatocytes and murine models (5)(6)(7). In chimpanzees, IFN-γ was shown to protect against liver-stage infection by human malarial P. vivax (8). ...
... IFN-γ-Mediated Control of Liver-Stage P. vivax in HC04 Cells Is Independent of NO. The above-described effect of IFN-γ on the liver stage of P. berghei and P. yoelli in cultured mouse hepatocytes and wholeanimal models (5,7), and the ability of this cytokine to limit the liver stage of human malarial P. vivax replication in chimpanzees (8), suggested a role for IFN-γ in liver-stage P. vivax in humans. Therefore, in this study we asked whether IFN-γ treatment of cultured human hepatocytes infected with P. vivax sporozoites would promote parasite clearance. ...
Significance
IFN-γ plays an important role in the elimination of liver-stage Plasmodium parasites, but the mechanism involved in this process is unclear. In this study, we demonstrate that IFN-γ treatment induces a noncanonical autophagy pathway in human hepatocytes dubbed an LC3-associated phagocytosis (LAP)-like process, in which the parasitophorous vacuole membrane of the parasites is decorated with LC3, resulting in the colocalization of parasite compartments with lysosomes. Downstream autophagy-related proteins are involved in this pathway, whereas the upstream autophagy-initiating protein is not. Our work shows that a LAP-like process serves as a previously unidentified downstream effector of IFN-γ in elimination of the liver-stage human malarial parasite Plasmodium vivax .
... Second, IFN-' r may exert its effect by binding to surface receptors and inducing intracellular changes that are unfavorable for development of Listeria . This possibility is supported by reports that receptors for IFN-y are present on various cells, includinghepatocytes (30), and that IFN-'r induces phenotypic and functional alterations (31) . Indeed, this mechanism has re-on October 17, 2015 jem.rupress.org ...
... cently been shown to operate in hepatocytes with malaria parasites (30). These two mechanisms are not mutually exclusive and could occur simultaneously. ...
Studies were made on the effects of in vivo administration of anti-CD4 mAb, anti-CD8 mAb, or a combination of both mAbs on multiplication of bacteria, the levels of serum transaminases, and mortality in mice infected with Listeria monocytogenes. Results showed that in sublethal infection, CD8+ cells enhanced the peak of bacterial multiplication and liver cell necrosis, and CD4+ cells suppressed CD8+ cell-mediated enhancement. Results also showed that either CD4+ or CD8+ cells were necessary for, and capable of, mediating clearance of the bacteria. CD8+ cells were more efficient than CD4+ cells, but for optimal clearance both were necessary. In lethal listeriosis, treatment of mice with anti-CD8 mAb or a combination of both anti-CD4 and anti-CD8 mAbs, but not anti-CD4 mAb only, protected mice from death by decreasing multiplication of bacteria in the liver and spleen after a peak of approximately 10(8) CFU, and lowering the elevated serum levels of transaminases. These findings indicated that CD8+ cells were responsible for causing irreversible systemic Listeria infection and severe liver necrosis. In lethal listeriosis, administration of rMuIFN-gamma markedly prolonged survival by decreasing multiplication of bacteria and promoting recovery from liver necrosis.
... The clinical presentation of malaria ranges from asymptomatic parasitemia and mild illness to cerebral malaria (CM), a condition with a mortality rate of ~15% [2]. Host immune response directly impacts the severity and outcome of disease in murine models of severe malaria [3][4][5][6][7], and appears to be similarly important in severe malaria in humans [8][9][10][11]. ...
... A balance between pro-inflammatory and anti-inflammatory properties appears to be necessary to combat P. falciparum parasitemia. Cytokines such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β), promote inflammation and reduce parasitemia [6,7,12,13]. However, an exaggerated proinflammatory response is associated with increased morbidity and mortality in severe malaria [10,13,14]. ...
Transforming growth factor beta-1 (TGF-β1) is an important regulator of inflammation. Platelets are a major source of TGF-β1, and are reduced in severe malaria. However, the relationships between TGF-β1concentrations and platelet counts, pro- and anti-inflammatory cytokineand chemokine concentrations, and disease severity in malariahave not been characterized.
Platelet counts andserumconcentrations of TGF-β1 and interleukin-1beta (IL-1β), IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and RANTES were measured at the time of presentation inUgandan children with cerebral malaria (CM, n=75),uncomplicated malaria (UM, n=67) and healthy community children (CC, n=62).
TGF-β1 concentrations decreased with increasing severity of disease (median concentrations (25, 75 percentile) in ng/ml in CC, 41.4 (31.6, 57.4), UM, 22.7 (14.1,36.4), CM, 11.8 (8,21), P for trend<0.0001). In children with CM or UM, TGF-β1 concentrations correlated positively with platelet count (CM,P<0.0001, UM, P=0.0015). In children with CM, TGF-β1 concentration correlated negatively with IFN-γ, IL-6, and IL-10 and positively with RANTES concentrations(all P<0.01).TGF-β1 concentrationwas not associated with death or adverse neurologic or cognitive outcomes in children with CM.
TGF-β1 concentrations decrease with increasing P. falciparum disease severity. In cerebral malaria,thrombocytopenia correlates with decreased TGF-β1, and decreased TGF-β1 correlates withcytokine/chemokine changes associated with increased disease severity and death. Thrombocytopenia may mediate disease severity in malariathrough reduced TGF-β1-mediated regulation of cytokines associated with severe disease.
... However, this response is critical for the recruitment of immune cells to the infected hepatocyte, which leads to Plasmodium elimination (19). As IFN-␥ has been identified as a cytokine that is able to mediate the killing of intrahepatic Plasmodium parasites (40)(41)(42), we hypothesized that the mechanism of parasite elimination in the liver after reinfection is IFN-␥ dependent. We monitored transcript expression of the gene encoding this cytokine in whole-liver extracts at different time points after infection, using the previously established infec-tion protocol and chloroquine treatment. ...
... Our report provides genetic evidence of an important role for the type I IFN response and IFN-␥ in the host defense against malaria liver-stage reinfection. IFN-␥ treatment is known to efficiently kill intrahepatic parasites (40)(41)(42), but we provide the first evidence of a biologically relevant IFN-␥ response in vivo after mosquito bite-transmitted malaria infection (P. berghei). ...
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 malaria endemic regions have been mainly explored by studying the resistance induced by the blood stage of infection. However, epidemiologic studies have suggested that in high transmission areas, pre-erythrocytic stages also activate host resistance against reinfection. This, along with the recent discovery that liver infection trigger a specific and effective type I interferon (IFN) response, prompted us to hypothesize that this pre-erythrocytic 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 IFN-γ. Importantly, mathematical simulations indicate that the predictions based on our experimental murine reinfection model fit available epidemiological data. Overall, our study reveals that liver stage-induced innate immunity may contribute to the pre-erythrocytic resistance observed in humans in hyperendemic regions.
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... It is the most widely studied interferon in malaria infection since it is primarily involved in host defense against intracellular pathogens. Its protective role as an immune mediator emerged as early as in the liver stage [126,[188][189][190][191][192][193]. In vitro study of human recombinant IFN-treatment on P. berghei sporozoites-infected murine hepatocytes [190] or human hepatoma cells [189] identified an inhibitory effect of IFN-on parasite multiplication. ...
... Its protective role as an immune mediator emerged as early as in the liver stage [126,[188][189][190][191][192][193]. In vitro study of human recombinant IFN-treatment on P. berghei sporozoites-infected murine hepatocytes [190] or human hepatoma cells [189] identified an inhibitory effect of IFN-on parasite multiplication. Further in vivo study validated the importance of IFN-in protective immunity as it inhibits intracellular development of parasite within hepatocytes following challenge with P. berghei [194], P. yoelii [191], or P. vivax sporozoites [188] in mice and chimpanzee, respectively. ...
Malaria is one of the most serious infectious diseases in humans and responsible for approximately 500 million clinical cases and 500 thousand deaths annually. Acquired adaptive immune responses control parasite replication and infection-induced pathologies. Most infections are clinically silent which reflects on the ability of adaptive immune mechanisms to prevent the disease. However, a minority of these can become severe and life-threatening, manifesting a range of overlapping syndromes of complex origins which could be induced by uncontrolled immune responses. Major players of the innate and adaptive responses are interferons. Here, we review their roles and the signaling pathways involved in their production and protection against infection and induced immunopathologies.
... IFNg mediates the killing of liver-stage parasites in vitro (Mellouk et al., 1991;Schofield et al., 1987) and could be responsible for the parasite-induced innate immune inhibition of infection we have observed. Thus, we first sought to determine if lymphocytes were necessary for the innate reduction of liver-stage burden by performing infections in FRG triple-knockout mice (FAH À/À ; RAG2 À/À ; IL2Rg À/À ), which are deficient in B, T, NK, and NKT cells (Azuma et al., 2007). ...
... The ability of exogenous IFNg to inhibit development of Plasmodium liver stages has been described in vitro (Mellouk et al., 1991;Schofield et al., 1987), but the mechanisms by which IFNg is induced shortly following a primary liver-stage infection are not well understood. We show that Plasmodium pre-erythrocytic stages can induce an innate IFNg response and that this response is sufficient to inhibit both a primary and a subsequent liver-stage infection. ...
Mosquito-transmitted malaria parasites infect hepatocytes and asymptomatically replicate as liver stages. Using RNA sequencing, we show that a rodent malaria liver-stage infection stimulates a robust innate immune response including type I interferon (IFN) and IFNγ pathways. Liver-stage infection is suppressed by these infection-engendered innate responses. This suppression was abrogated in mice deficient in IFNγ, the type I IFN α/β receptor (IFNΑR), and interferon regulatory factor 3. Natural killer and CD49b(+)CD3(+) natural killer T (NKT) cells increased in the liver after a primary infection, and CD1d-restricted NKT cells, which secrete IFNγ, were critical in reducing liver-stage burden of a secondary infection. Lack of IFNAR signaling abrogated the increase in NKT cell numbers in the liver, showing a link between type I IFN signaling, cell recruitment, and subsequent parasite elimination. Our findings demonstrate innate immune sensing of malaria parasite liver-stage infection and that the ensuing innate responses can eliminate the parasite.
... In rodents, it has been shown that in vivo depletion of CD8+ T lymphocytes completely abolishes malaria immunity (Schofield et al., 1987;Weiss et al., 1988). In addition, passive transfer of cytotoxic T-cell clones recognising the Plasmodium berghei CS protein completely protects mice against sporozoite challenge (Romero et al., 1989). ...
... Moreover, a number of soluble immune mediators have been shown to be capable of blocking liver schizogony, e.g. cytokines IL-12 (Sedegah et al., 1994), IFN-γ (Ferreira et al., 1986;Mellouk et al., 1987;Schofield et al., 1987;Tsuji et al., 1995), TNF-α (Korner et al., 2010;Nussler et al., 1991), IL-1 (Mellouk et al., 1987) and IL-6 (Nussler et al., 1991;Pied et al., 1991). Additionally, reactive oxygen and nitrogen intermediates, O 2 − (Allison and Eugui, 1983) and nitric oxide (Mellouk et al., 1994;Nussler et al., 1991Nussler et al., , 1993Seguin et al., 1994), respectively, significantly contribute to protection. ...
Population studies show that individuals acquire immunity to Plasmodium vivax more quickly than Plasmodium falciparum irrespective of overall transmission intensity, resulting in the peak burden of P. vivax malaria in younger age groups. Similarly, actively induced P. vivax infections in malaria therapy patients resulted in faster and generally more strain-transcending acquisition of immunity than P. falciparum infections. The mechanisms behind the more rapid acquisition of immunity to P. vivax are poorly understood. Natural acquired immune responses to P. vivax target both pre-erythrocytic and blood-stage antigens and include humoral and cellular components. To date, only a few studies have investigated the association of these immune responses with protection, with most studies focussing on a few merozoite antigens (such as the Pv Duffy binding protein (PvDBP), the Pv reticulocyte binding proteins (PvRBPs), or the Pv merozoite surface proteins (PvMSP1, 3 & 9)) or the circumsporozoite protein (PvCSP). Naturally acquired transmission-blocking (TB) immunity (TBI) was also found in several populations. Although limited, these data support the premise that developing a multi-stage P. vivax vaccine may be feasible and is worth pursuing.
... T-cell depletion of mice renders the animals completely susceptible to sporozoite challenge (Weiss et al. 1988), whereas complete protection has been obtained by passive transfer of CS specific cloned T helper cells (Del Giudice et al. 1990) and cytotoxic T lymphocytes (CTL) (Romero et al. 1989, Weiss et al. 1990). Sporozoite specific CTL and g-IFN injection have shown to prevent the development of parasite inside the liver cells (Schofield et al. 1987, Kumar et al. 1988. ...
... It is very important that several MAPs particularly VI and VII induced a significant production of g-IFN in vitro. g-IFN has been related to malaria protection by preventing the intracellular development of parasite liver forms (Schofield et al. 1987) and the parasite multiplication in the circulation in animals immunized with P. falciparum MSP-1 . This cytokine seems to play its protective role through the induction of nitric oxide production which is capable of destroying intra-hepatic malaria parasites. ...
Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS (p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitroγ-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or γ-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitroγ-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.
... The lack of significance between protection and immune response may be due to the small size of the final groups, as well as to the highly variable immune response among participants, likely due to the population heterogeneity in terms of ethnicity, sex, and nutrition status among other potential factors. Moreover, the results are in agreement with a previous study 26 where no correlation was found when we analyzed antibody titers and IFN-γ responses but continues to be intriguing as both effector mechanisms have been experimentally shown to be associated with protection against Plasmodium pre-erythrocytic infection 33,34 . ...
A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo (adjuvant alone). Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11) (54%, 95% CI 0.25–0.84) and phase IIb (7/11) (64%, 95% CI 0.35–0.92). However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT 02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206).
... This route induces circulating PfCSP-specific antibodies as well as circulating and liver-resident T cell responses 6,7,9,10 . Although antibodies specific to PfCSP have been shown to mediate short-term VE [11][12][13] , numerous preclinical animal models of malaria infection have demonstrated that PfSPZ-specific T cells are required for durable sterilizing protection 8,14,15 . In malaria-naïve humans, multifunctional CD4 + T cell cytokine-producing cells in the blood have also been associated with protection by PfSPZ Vaccines 7,10 . ...
The radiation-attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine provides protection against P. falciparum infection in malaria-naïve adults. Preclinical studies show that T cell-mediated immunity is required for protection and is readily induced in humans after vaccination. However, previous malaria exposure can limit immune responses and vaccine efficacy (VE) in adults. We hypothesized that infants with less previous exposure to malaria would have improved immunity and protection. We conducted a multi-arm, randomized, double-blind, placebo-controlled trial in 336 infants aged 5–12 months to determine the safety, tolerability, immunogenicity and efficacy of the PfSPZ Vaccine in infants in a high-transmission malaria setting in western Kenya (NCT02687373). Groups of 84 infants each received 4.5 × 105, 9.0 × 105 or 1.8 × 106 PfSPZ Vaccine or saline three times at 8-week intervals. The vaccine was well tolerated; 52 (20.6%) children in the vaccine groups and 20 (23.8%) in the placebo group experienced related solicited adverse events (AEs) within 28 d postvaccination and most were mild. There was 1 grade 3-related solicited AE in the vaccine group (0.4%) and 2 in the placebo group (2.4%). Seizures were more common in the highest-dose group (14.3%) compared to 6.0% of controls, with most being attributed to malaria. There was no significant protection against P. falciparum infection in any dose group at 6 months (VE in the 9.0 × 105 dose group = −6.5%, P = 0.598, the primary statistical end point of the study). VE against clinical malaria 3 months after the last dose in the highest-dose group was 45.8% (P = 0.027), an exploratory end point. There was a dose-dependent increase in antibody responses that correlated with VE at 6 months in the lowest- and highest-dose groups. T cell responses were undetectable across all dose groups. Detection of Vδ2+Vγ9+ T cells, which have been correlated with induction of PfSPZ Vaccine T cell immunity and protection in adults, were infrequent. These data suggest that PfSPZ Vaccine-induced T cell immunity is age-dependent and may be influenced by Vδ2+Vγ9+ T cell frequency. Since there was no significant VE at 6 months in these infants, these vaccine regimens will likely not be pursued further in this age group. The PfSPZ Vaccine does not protect infants from infection with Plasmodium falciparum, the major cause of malaria.
... It was found to be safe and actively producing CSP specific B-and-T cell responses and also protection was not limited only to the NF54 strain of P. falciparum from which the vaccine has been made [62]. In subsequent studies, the involvement of CD4 + and CD8 + T cells, sentinels of cellular immunity against the LS infection and role of Abs specific to the sporozoites have revealed the significant role of T-and B cells against malaria infection, respectively [63]. ...
Among the numerous infectious diseases, malaria still remains the main cause of morbidity and mortality across the world. Every year more than 200 million cases are registered and death toll is of around 4,00,000. The emergence of insecticide and drug resistance has surged an alarming situation to find an effective means to tackle it. From various approaches used for reducing the damage created by malaria to the society, developing effective vaccine has gained the attention of scientific community. The large genome size (24 MB), heterogeneity of the genes, complex life cycle in two different hosts, and expression of wide range of these genes are claimed to hinder the malaria vaccine development. It requires good understanding of the host-pathogen interaction and its correlation with the sterile protection. Recently, subunit vaccine have shown certain promising responses; however, the currently in use of RTS,S vaccine has failed to generate the long-term sterile protection as well as effector memory CD8+T cells. However, the success of sterile protection through vaccination has been proven long back by experimental approaches, where it could be achieved using irradiated sporozoites (RAS) in rodents and humans. Similarly, GAP (genetically attenuated parasite) and CPS (chloroquine chemoprophylaxis with Plasmodium sporozoites) have been shown to induce sterile immunity. Despite all the developments, generation of species and stage specific-CD8+ T cell responses has been modest. In order to generate long-lasting immune response, particularly, liver-stage specific-CD8+ T cells, it is indeed required to study the CD8+ T cell epitope repertoire and its implications on the host immune system. In this chapter we will discuss the current status of T cell-based vaccines and the challenges associated with it.
... In humans, vaccination correlates with an increase in the number of circulating CD8 + T cells that produce effector cytokines [9,10]. In mice, depletion of CD8 + T cells or neutralization of interferon-gamma after vaccination, but before challenge infection, results in a loss of sterile immunity [11,12]. Among all CD8 + T cells, intrahepatic memory CD8 + T cells are particularly important for providing long-lasting liver-stage immunity after intravenous RAS vaccination [13,14]. ...
Malaria remains a major cause of mortality in the world, and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of living radiation‐attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8⁺ memory T cells, with liver‐resident memory T cells (TRM) being particularly important. We have previously described a TCR transgenic mouse, termed PbT‐I, where all CD8⁺ T cells recognize a specific peptide from Plasmodium. PbT‐I form liver TRM cells upon RAS injection and are capable of protecting mice against challenge infection. Here we utilize this transgenic system to examine whether non‐living sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium‐specific liver TRM cells. We found that HKS vaccination induced the formation of memory CD8⁺ T cells in the spleen and liver, and importantly, liver TRM cells were fewer in number than that induced by RAS. Crucially, we showed the number of TRM cells was significantly higher when HKS were combined with the glycolipid α‐galactosylceramide as an adjuvant. In the future, this work could lead to development of an anti‐malaria vaccination strategy that does not require live sporozoites, providing greater utility.
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... L'IFNγ : produit majoritairement par les cellules NK et les LT (CD4 + & CD8 + ), il permet l'activation des PNN (Kumaratilake et al., 1991), des monocytes et des macrophages (Kumaratilake and Ferrante, 1994). Au stade pré-érythrocytaire (phase hépatique) de la maladie, l'IFNγ inhibe le développement des parasites (Schofield et al., 1987). Une étude a montré que des individus faisant un accès grave sécrétaient peu d'IFNγ comparés à ceux faisant un accès simple (Luty et al., 1999). ...
Le paludisme est une maladie tropicale causée par un parasite du genre Plasmodium. Chez l’Homme, un niveau élevé de phospholipase A2 sécrétée de groupe IIA humaine (hGIIA) est mesuré dans le plasma des patients impaludés. Cette enzyme est connue pour son rôle antibactérien et pro-inflammatoire. Cependant, son rôle dans le paludisme n’a jamais été exploré. Pour comprendre le rôle in vivo de la hGIIA dans cette pathologie, nous avons entrepris la caractérisation hématologique, histopathologique et immunohistochimique de l’infection de souris C57BL/6, transgéniques (Tg+) pour l’enzyme humaine, par l’espèce murine Plasmodium chabaudi chabaudi 864VD. Ce modèle reproduit un paludisme non létal. Nos résultats ont permis d’établir que les souris Tg+ ont un meilleur contrôle de l’infection au moment du pic de crise parasitaire (J14 post-inoculation), avec une diminution de 27% de la parasitémie, comparé aux souris « littermates » non transgéniques (Tg-). L’injection de hGIIA recombinante aux jours 12, 13 et 14 p.i. (0,125 mg/kg deux fois par jour) à des souris C57BL/6 wild-type (WT) infectées par P. c. chabaudi 864VD provoque une diminution d’environ 19% de la parasitémie à J14 p.i., démontrant un rôle direct de la hGIIA dans la diminution de la population parasitaire. Les données hématologiques montrent que l’infection chez la souris Tg+ provoque une anémie plus durable que chez la souris Tg- et une élévation nettement plus importante du nombre de leucocytes, en particulier des polynucléaires neutrophiles. Chez la souris Tg+ parasitée, on observe aussi l’activation d’un nombre important de lymphocytes et une activation spécifique des monocytes avant le pic de crise. Chez la souris Tg- infectée, les données histologiques mettent en avant une meilleure récupération des lésions histopathologiques du foie et une hyperplasie des lymphocytes B dans la rate, tandis que les souris Tg+ infectées présentent des lésions hépatiques tardives et une hématopoïèse extramédullaire splénique. Les résultats des analyses par RT-qPCR suggèrent que l’ARNm de la hGIIA augmente au pic parasitaire dans le foie des souris Tg+ infectées, mais diminue dans la rate et les cellules sanguines. L’injection de hGIIA recombinante au début de la phase patente est sans effet sur la parasitémie, ce qui laisse supposer que des événements plus tardifs dans l’infection sont nécessaires à l’activité antiparasitaire de l’enzyme. L’étude du rôle des lipoprotéines oxydées comme substrat potentiel de l’activité antiparasitaire de l’enzyme, basée sur des résultats in vitro, est abordée. En conclusion, nos études ont permis de dresser un tableau large de l’infection à Plasmodium chez la souris exprimant la hGIIA, et ouvrent de nouvelles perspectives dans l’analyse du rôle de l’enzyme dans la physiopathologie du paludisme.
... Since the discovery of type II fatty acid biosynthesis in Plasmodium, several synthetic compounds and natural products have been tested against plasmodial FAS-II enzymes ( The cultivation and maintenance of asexual blood stage parasites of P. falciparum in vitro has been estabhshed since the late 1970s and shortly afterwards protocols for the quantitative assessment of antimalarial activities were implemented (Trager & Jenson, 1978;Desjardins et a i, 1979 (Schofield et a l, 1987;Li et a l, 1991), an infrared fluorescence scanning system (Gego et a l, 2006) and indirect or direct immunofluorescent microscopy (HoUingdale et a l, 1983a;Carraz et a l, 2006). The development of transgenic rodent parasites expressing fluorescent reporter proteins enabled quantitative analysis by flow cytometry (Natarajan et a l, 2001;Tarun et a l, 2006). ...
Malaria is the number one parasitic disease worldwide with half of the world's population at risk and nearly one million death annually. Natural products have had an enormous impact in malaria chemotherapy as the majority of current antimalarial agents are natural products or derive from a natural product scaffold isolated from plants traditionally used against malaria. The development of resistance by the deadliest parasite species Plasmodium falciparum against many antimalarial agents, including the artemisinin combinations, has become a great concern for global public health. Hence, new antimalarial drugs for chemotherapy and prophylaxis are urgently needed. Type II fatty acid biosynthesis pathway (FAS-II) has been recently shown to be indispensable for the liver stage parasites. Fatty acid biosynthesis is a crucial pathway for all living organisms as fatty acids are essential for membrane formation and energy production. Plasmodium employs type II FAS with fundamental structural and organisational differences versus the type I human FAS. This renders plasmodial FAS-II to be an excellent target for liver stage parasites and causal malaria prophylaxis. Thus, the essential FAS-II enzymes FabI, FabG and FabZ were included in the screening for new malaria prophylactic agents. One approach in the search for natural antimalarial drugs was the screening of pure natural products. Selected secondary lichen metabolites (evernic acid, vulpic acid, psoromic acid and (+)-usnic acid) were assessed for their potency against P. falciparum blood stage parasites and P.yoelii liver stage parasites, plasmodial FAS-II enzymes and their cytotoxicity. Evernic acid was identified as first natural product with potential against liver stage parasites (IC50 19.5 μM) and the FAS-II enzyme FabZ as potential target, for which it is a competitive inhibitor. Another set of natural products tested in this study were 22 selected natural chalcones, a chemical group that acts as precursors for the well-known family of flavonoids. The assessment against P. falciparum blood stage parasites identified 2',6'-dihydroy-4,4'- dimethoxydihydrochalcone as chalcone with the best antiplasmodial activity (IC50 3.7 μM), next to the known inhibitor licochalcone A (IC50 1.0 μM). In addition butein, homobutein, eriodictyolchalcone and licochalcone A were identified as promising inhibitors of the FAS- II enzyme FabZ. Structure-activity relationship studies were performed. Another approach was the screening of selected Turkish plants Anthemis cretica subsp. anatolica, Anthemis pestalozzae (Asteraceae), Salvia virgata (Lamiaceae), Scrophularia lucida and Scrophularia pinardii (Scrophulariaceae). Previous studies have shown that members of the genera Anthemis, Salvia and Scrophularia displayed significant antiplasmodial potential. Hence aerial parts and roots were extracted separately with methanol (crude extracts), which was followed by liquid-liquid partitioning and yielded the hexane, chloroform and aqueous methanol subextracts. The crude extracts and subextracts were screened for their potential against P. falciparum blood stage parasites, FAS-II enzymes and for cytotoxicity. All species showed good to moderate antiplasmodial activity and inhibition against at least one FAS-II enzyme. The aerial parts of the completely unstudied endemic Turkish plant Anthemis pestalozzae (Asteraceae) showed the most interesting profile and were selected for bioactivity-guided fractionation using a variety of chromatographic methods. Four compounds could be isolated and were identified as the cyanogenic glycoside lucumin, the benzoic acid derivative 2,6-dihydroxybenzoic acid, the indole glycoside 3-carboxymethyl-indole-l-N-β-D-glucopyranoside and a sesquiterpene lactone. The first three compounds were identified for the first time in the genus Anthemis. The sesquiterpene lactone was identified to possess the structure which was postulated for sivasinolide, however comparison of NMR data revealed significant differences. Our data suggest that the compound originally declared to be sivasinolide is very likely a different compound. In addition, the presence of the flavonoid rutin in the aqueous methanol subextract could be shown by HPLC analysis. An extensive analysis of the fatty acid composition of several fractions from the aerial parts of Antbhemis pestalozzae showed the presence of 37 different fatty acids.
... RAS immunization induces CD8+ and CD4+ T cells that kill malaria-infected hepatocytes [11][12][13][14]. Priming of antigen-specific effector T-cells by RAS in human and mouse infection models plateaus after the first immunization, [7,15] suggesting that subsequent homologous RAS boost provides only minor gains in parasite-targeting T cell populations. ...
Background:
Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known.
Methods:
In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state.
Results:
These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold.
Conclusions:
This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.
... The results suggest that while both CD4 ϩ and CD8 ϩ T cells play a prominent role in immunity induced by an rPyCSP-adjuvanted vaccine, protection against pre-erythrocytic-stage malaria can be achieved in the absence of these T cell populations, albeit at a reduced level. In murine malaria parasite models where immunization with radiation-attenuated Plasmodium sporozoites or with CSP delivered as a DNA plasmid was tested, both CD8 ϩ T cells and IFN-␥ were found to be necessary to maintain immunity (20)(21)(22)(23)(24). To study the role of IFN-␥ in rPyCSP vaccine-induced immunity, we immunized IFN-␥-KO mice with rPyCSP and then challenged them with P. yoelii sporozoites. ...
Development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01 E vaccine in the phase 3 studies highlighted the urgency to design and test more efficacious next generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelli ( rPy CSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody-dependent, as evidenced by the complete loss of immunity in B-cell knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type antibody isotype IgG1 levels were associated with protective immunity. The fact that immunized IFN-γ KO mice and WT mice have similar levels of protective immunity and the absence of IFN-γ producing CD4 ⁺ and CD8 ⁺ T cells in protected mice in flow cytometry indicate that immunity is IFN-γ independent. Protection against sporozoite challenge correlated with the higher frequency of CD4 ⁺ T cells that express IL-2, IL-4 and TNF-α. In the RTS,S study, clinical immunity was associated with the higher IgG levels and frequency of IL-2 and TNF-α producing CD4 ⁺ T cells. The other hallmarks of immunity in our study included an increased number of the follicular B-cells, but a loss in Th follicular T cells. These resu-lts provide an excellent model system to evaluate the efficacy of novel adjuvants, vaccine dosage and determine the correlates of immunity in search for superior candidate malaria vaccines.
... 17 In Malian adults, VE against Pf infection during the 24 weeks after last vaccine dose was 52% by time to infection analysis and 29% by proportional analysis. 18 Protection by immunization with sporozoites is dependent on T cells in mice and nonhuman primates 13,[21][22][23][24] and thought to be T cell-dependent in humans. 13 The durable protection demonstrated in the Mali trial was associated with elevated gamma delta T-cell frequencies, providing support for this hypothesis. ...
In 2016, there were more cases and deaths caused by malaria globally than in 2015. An effective vaccine would be an ideal additional tool for reducing malaria's impact. Sanaria® PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) has been well tolerated and safe in malaria-naïve and experienced adults in the United States and Mali and protective against controlled human malaria infection with Pf in the United States and field transmission of Pf in Mali, but had not been assessed in younger age groups. We, therefore, evaluated PfSPZ Vaccine in 93 Tanzanians aged 45 years to 6 months in a randomized, double-blind, normal saline placebo-controlled trial. There were no significant differences in adverse events between vaccinees and controls or between dosage regimens. Because all age groups received three doses of 9.0 × 105 PfSPZ of PfSPZ Vaccine, immune responses were compared at this dosage. Median antibody responses against Pf circumsporozoite protein and PfSPZ were highest in infants and lowest in adults. T-cell responses were highest in 6-10-year olds after one dose and 1-5-year olds after three doses; infants had no significant positive T-cell responses. The safety data were used to support initiation of trials in > 300 infants in Kenya and Equatorial Guinea. Because PfSPZ Vaccine-induced protection is thought to be mediated by T cells, the T-cell data suggest PfSPZ Vaccine may be more protective in children than in adults, whereas infants may not be immunologically mature enough to respond to the PfSPZ Vaccine immunization regimen assessed.
... In fact, liver-stage infection results in the recruitment of natural killer T (NKT) cells in an IFNAR-dependent manner, that through an IFNγ-mediated mechanism are responsible for the control of Plasmodium liver infection [83]. Although IFNγ has been shown to directly kill liver-stage parasites [84,85], it is possible that an IFNγindependent killing mechanism within the hepatocyte could also take place. ...
The innate immune response to malaria has always attracted the interest of researchers trying to understand the basis for the high fevers observed in malaria patients during blood-stage infection and the lack of an apparent response to the liver-stage infection. Research targeting specific parts of the immune response has contributed to a basic understanding of the concepts that play a role in malaria-induced inflammation. Given the complexity of the immune response in general and to the parasite in particular, some findings have been contradictory. Here we summarize a large body of work including the host innate immune response to a Plasmodium liver and blood-stage infection, focusing on the different parasite- and host-derived molecules that trigger inflammation, the immune cell types involved, and the role of different cytokines in inflammation and pathology of malaria.
... 10,11 It has also been demonstrated that protection requires that irrad-spz remain sufficiently viable to invade hepatocytes and undergo partial development to induce stimulation of CD4+ and CD8+ T-cell cytotoxic, dendritic cells, and cytokine responses necessary for protection, such as interferon-gamma (IFN-γ) and interleukin-12 (IL-12). 9,[12][13][14][15][16][17][18][19][20][21][22] Moreover, IFN-γ production has been established as the primary effector immune response that contributes to long-lasting protection against pre-erythrocytic stages in the irrad-spz model. 14,23 In human volunteers immunized with P. falciparum irrad-spz, the presence of antibodies to the circumsporozoite protein (CSP) and increased levels of tumor necrosis factor α (TNF-α), IFN-γ, and IL-6 have been correlated with protection. 1 , 6, 24 Although the irrad-spz model was first described nearly 40 years ago, only a total of three volunteers have been vaccinated with Plasmodium vivax irrad-spz, from which only one was protected after two immunizations. 1 Similarly, although immunization of non-human primates with irrad-spz from human Plasmodium species followed by live challenge infection would be a useful model for characterizing protective immune mechanisms and for identifying novel malaria vaccine candidates, in the past three decades only a few trials have been conducted. ...
Abstract. A non-human primate model for the induction of protective immunity against the pre-erythrocytic stages of Plasmodium vivax malaria using radiation-attenuated P. vivax sporozoites may help to characterize protective immune mechanisms and identify novel malaria vaccine candidates. Immune responses and protective efficacy induced by vaccination with irradiated P. vivax sporozoites were evaluated in malaria-naive Aotus monkeys. Three groups of six monkeys received two, five, or ten intravenous inoculations, respectively, of 100,000 irradiated P. vivax sporozoites; control groups
received either 10 doses of uninfected salivary gland extract or no inoculations. Immunization resulted in the production
low levels of antibodies that specifically recognized P. vivax sporozoites and the circumsporozoite protein. Additionally,
immunization induced low levels of antigen-specific IFN-γ responses. Intravenous challenge with viable sporozoites
resulted in partial protection in a dose-dependent manner. These findings suggest that the Aotus monkey model may be
able to play a role in preclinical development of P. vivax pre-erythrocytic stage vaccines.
... Cerebral malaria (CM) is a deadly disease that affects more than 500,000 children in sub-Saharan Africa every year and kills ∼110,000 of these children. 1 The pathogenesis of CM is thought to involve both parasite sequestration in the cerebral microvasculature, with tissue hypoxia and ischemic damage, and immunologic responses to P. falciparum, including cytokine responses. 2 Cytokines and chemokines may protect from disease by direct and indirect effects on the parasite, 3,4 but they may also contribute to disease, through recruitment of inflammatory cells, 5 augmented production and activity of other cytokines, 5 and direct toxicity to cells and tissue. 6,7 Murine models of CM have clearly demonstrated involvement of brain parenchymal cells, with activation of microglial cells, 8,9 damage to astrocytes, 10 and increased mRNA expression of genes regulating tumor necrosis factor-a (TNF-␣) 11 and interferon-␥ (IFN-␥). ...
... In PbA infections, SOCS-2 À/À mice displayed resistance to malaria by limiting parasite growth. During malaria infection, IFN-c plays a critical role in the context of parasite replication (Schofield et al., 1987). These data indicate that the absence of SOCS2 alters the canonical response to PbA infection, characterized by a strong IFN-c response in the spleen as observed in infected WT animals. ...
Plasmodium falciparum infection results in severe malaria in humans, affecting various organs, including the liver, spleen and brain, and resulting in high morbidity and mortality. The Plasmodium berghei ANKA (PbA) infection in mice closely recapitulates many aspects of human cerebral malaria (CM); thus, this model has been used to investigate the pathogenesis of CM. Suppressor of cytokine signaling 2 (SOCS2), an intracellular protein induced by cytokines and hormones, modulates the immune response, neural development, neurogenesis and neurotrophic pathways. However, the role of SOCS2 during CM remains unknown. SOCS2 knockout (SOCS2−/−) mice infected with PbA show an initial resistance to infection with reduced parasitemia and production of TNF, TGF-β, IL-12 and IL-17 in the brain. Interestingly, in the late phase of infection, SOCS2−/− mice display increased parasitemia and reduced Treg cell infiltration, associated with enhanced levels of Th1 and Th17 cells and related cytokines IL-17, IL-6, and TGF-β in the brain. A significant reduction in protective neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), was also observed. Moreover, the molecular alterations in the brain of infected SOCS2−/− mice were associated with anxiety-related behaviors and cognition impairment. Mechanistically, these results revealed enhanced nitric oxide (NO) production in PbA-infected SOCS2−/− mice, and the inhibition of NO synthesis through l-NAME led to a marked decrease in survival, the disruption of parasitemia control and more pronounced anxiety-like behavior. Treatment with l-NAME also shifted the levels of Th1, Th7 and Treg cells in the brains of infected SOCS2−/− mice to the background levels observed in infected WT, with remarkable exception of increased CD8+IFN+ T cells and inflammatory monocytes. These results indicate that SOCS2 plays a dual role during PbA infection, being detrimental in the control of the parasite replication but crucial in the regulation of the immune response and production of neurotrophic factors. Here, we provided strong evidence of a critical relationship between SOCS2 and NO in the orchestration of the immune response and development of CM during PbA infection.
... Cytokines and other factors such as nitric oxide contribute to protection at the pre -e ry t h ro c ytic level (52); however, some of these pro -i n f l a mm a t o ry cytokines (IL-1, IL-6 and TNF) are also involved in the induction of severe and complicated malaria (53)(54)(55). For example, while the injection of IFN-γ inhibits the development of parasite liver forms, the protective immunity induced by i rradiated sporozoites is eliminated by in vivo treatment of mice with anti-IFN-γ antibodies (56). In this case, one of the proposed parasite killing mechanisms is through an L-arg i n i n i n e -d e p e ndent pathway as well as through induction of reactive oxygen intermediates (57). ...
Malaria is currently considered one of the most health-threatening diseases of humans producing hundreds of million clinical cases every year in the tropical and subtropical regions of the world. Together Plasmodium falciparum and P vivax are responsible for most of the 500 millions cases produced yearly. Significant efforts are being made to identify the immune mechanisms involved both in protection and physio-pathogenesis of the disease as well as to develop protective vaccines. Both innate and acquired immune mechanisms participate in malaria protection and an important degree of immunity is developed in individuals permanently exposed to malaria in endemic areas, however it is not sufficient to induce sterile protection in the host. Several of the most important clinical complications such as cerebral malaria, are associated with the failure of host defences to control parasite replication, sequestration of parasitised red blood cells in several organs and the excessive secretion of pro-inflammatory cytokines. Although vaccination of animals and human volunteers has demonstrated the feasibility of the induction of sterile immunity, several vaccine candidates are intended to diminish the disease. We review here the current concepts about the immune responses induced by malaria, their potential in disease induction and some of the most relevant vaccine candidates.
... Blockage of IFN-γ within 2 days after transfer abrogates protection (Schofield et al., 1987b;Rodrigues et al., 1991;Weiss et al., 1992), a result that supports the concept that soluble factors are obligatory for protection when CTLs are unable to approach infected hepatocytes. A role of cytokines, in particular IFN-γ and TNF-α, in parasite killing is clearly documented, both in vivo and in vitro (Ferreira et al., 1986;Mellouk et al., 1987Mellouk et al., , 1991Schofield et al., 1987a;Rodrigues et al., 1991;Weiss et al., 1992;Seguin et al., 1994;Renggli et al., 1997;Doolan and Hoffman, 2000;Jobe et al., 2007;Mueller et al., 2007;Butler et al., 2010). For example, C57BL/6 mice deficient in both perforin and the CD95/CD95L pathway were protected after immunization with Pb-RAS suggesting that parasite-specific CD8 effector T cell-derived cytokines activate mechanisms responsible for the elimination of the intracellular LS (Renggli et al., 1997). ...
Plasmodium falciparum malaria remains one of the most serious health problems globally. Immunization with attenuated parasites elicits multiple cellular effector mechanisms capable of eliminating Plasmodium liver stages. However, malaria liver stage immunity is complex and the mechanisms effector T cells use to locate the few infected hepatocytes in the large liver in order to kill the intracellular liver stage parasites remain a mystery to date. Here, we review our current knowledge on the behavior of CD8+ effector T cells in the hepatic microvasculature, in malaria and other hepatic infections. Taking into account the unique immunological and lymphogenic properties of the liver, we discuss whether classical granule-mediated cytotoxicity might eliminate infected hepatocytes via direct cell contact or whether cytokines might operate without cell-cell contact and kill Plasmodium liver stages at a distance. A thorough understanding of the cellular effector mechanisms that lead to parasite death hence sterile protection is a prerequisite for the development of a successful malaria vaccine to protect the 40% of the world’s population currently at risk of Plasmodium infection.
... Although the contribution of cytokines and other inflammatory molecules during malarial erythrocytic infection is extensively studied, still little is known about host factors that control Plasmodium development in the liver. Work in rodents and cultured hepatocytes showed that several host cytokines strongly inhibit parasite development inside hepatocytes, including IFN-γ, TNF-α, IL-1 and IL-6 (Schofield et al., 1987;Maheshwari, 1990;Pied et al., 1992;Vreden et al., 1992). Both TNF-α-induced (Vreden et al., 1992) and IL1-induced (Nussler et al., 1991) inhibitions of liver-stage development are mediated by IL-6. ...
Plasmodium spp., which cause malaria, produce a histamine releasing factor (HRF), an orthologue of mammalian HRF. HRF produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF is sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. The target cells modulated by HRF for IL-6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down-regulate a cytokine with anti-parasite activity. Our data also highlight the link between a prolonged transition from liver to blood-stage infection and reduced incidence of experimental cerebral malaria (ECM).
... Complex, stage-specific networks of antibody-dependent and cell-mediated interactions provide immunity against Plasmodium spp., with clinical implications depending on the type and timing of cytokine release. Early type-1 responses, dominated by IFN-γ, IL-2 and TNF, have been reportedly associated with inhibition of liver stage development [26][27][28][29][30][31], resolution of acute malaria parasitaemias [32][33][34] and delay of reinfection [35], as confirmed by the absolute requirement of IFN-γ in the effector mechanism of sporozoite-induced protective immunity [35][36][37][38]. Release of these cytokines, initiated by the innate immune system (Natural killer (NK) cells, γδTand αβT-cells) [39][40][41] and sustained by Plasmodium-specific CD8 + and CD4 + cells [32,37,39], requires to be timely counterbalanced by a switch to type-2 responses which propagate humoral immunity against the erythrocytic stage, and limit the pathogenicity of pro-inflammatory cytokines [42,43]. ...
Background
The immune system plays a critical role in the development of co-infections, promoting or preventing establishment of multiple infections and shaping the outcome of pathogen-host interactions. Its ability to mediate the interplay between visceral leishmaniasis (VL) and malaria has been suggested, but poorly documented. The present study investigated whether concomitant infection with Leishmania donovani complex and Plasmodium falciparum in naturally co-infected patients altered the immunological response elicited by the two pathogens individually.
Results
Circulating levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IL-17A and tumor necrosis factor (TNF) were assessed in sera of patients infected with active VL and/or malaria and healthy individuals from Gedarif State, Sudan. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted significant differences in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum to mutually interact at the immunological level. Progressive polarization towards type-1 and pro-inflammatory cytokine patterns characterized the co-infected patients, whose response partly reflected the effect elicited by VL (IFN-γ, TNF) and malaria (IL-2, IL-13), and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Significantly reduced levels of P. falciparum parasitaemia (P <0.01) were detected in the co-infected group as opposed to the malaria-only patients, suggesting either a protective or a non-detrimental effect of the co-infection against P. falciparum infection.
Conclusions
These findings suggest that a new immunological scenario may occur when L. donovani and P. falciparum co-infect the same patient, with potential implications on the course and resolution of these diseases.
... spleen (SP), lamina propria (LP) and Peyer's patches (PP) of mouse model. Cell mediated immune responses, mainly CD4+ and CD8+ T-cells, are implicated in protection against the liver stage of infection (Schofield et al., 1987), and antibodies are involved in the protection against sporozoites. ...
T-cells play a critical role in resistance to malaria, not only because they function as helper cells for an antibody response, but also because they serve as effector cells. Such cellular immunity is directly implicated in protection from sporozoites as well as from blood stage parasites. The aim of this study was to induce cell mediated immune responses to peptide antigens of Plasmodium vivax co-encapsulated with CpG oligodeoxynucleotide (ODN) in microparticles. In the present study, we have investigated the immunomodulatory effects of two CpG adjuvants, CpG 1826 and CpG 2006 to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen (Pvs24) in microparticle delivery. The T-cell proliferation response study of the cells collected from spleen, lamina propria and peyer's patches showed significantly high (p<0.001) stimulation index when primed with peptide antigens in microparticles co-encapsulating CpG ODN adjuvant as compared to peptide alone primed mice. The cytokine measurement profile of IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-10 in culture supernatants of cells primed with peptide antigens in microparticles co-encapsulating CpG ODN showed higher levels of IFN- gamma followed by TNF-alpha and IL-2, with relatively low levels of IL-4 and IL-10.
... La Inmunidad natural contra los esporozoítos no es promovida eficientemente en el huésped (Beadle et al. 1975), pero se ha demostrado que la respuesta celular y humoral inducida por vacunas juega un papel importante en la protección contra la infección por esporozoitos (Nussenzweig et al. 1967). Una vez el esporozoito entra a las células hepáticas, el parásito es atacado directamente por los Linfocitos T citotóxicos (CTL) que pueden actuar directamente por citólisis de los hepatocitos infectados (Hoffman & Doolan 2000), o a través de la liberación de citoquinas como IFN-γ (Doolan & Hoffman 2000; Schofield et al. 1987; Sedegah et al. 1994) o IL-6 (Nussler et al. 1991; Pied et al. 1992; Pied et al. 1991) o por la inducción de la sintasa de óxido nítrico inducible (Seguin et al. 1994). Una vez el parásito abandona las células hepáticas para invadir a los eritrocitos, es interceptado por los anticuerpos que juegan un papel principal en la respuesta inmune contra estadios asexuales a través de algunos mecanismos como la inhibición de la invasión del parásito a los glóbulos rojos como ocurre con el bloqueo de la proteína Duffy Binding Protein (DBP) en el caso de infección por P. vivax (Michon et al. 2000), el bloqueo intra-eritrocítico del parásito, o la inhibición mediada por células mononucleares (Holder & Riley 1996). ...
: La malaria induce anticuerpos que son capases de suprimir la
infectividad de gametocitos y gametos en el estómago del mosquito vector. Sin embargo se conoce poco sobre la duración de la respuesta mediada por anticuerpos, la especificidad de la respuesta contra diferentes aislados de Plasmodium y el papel del complemento.
En este estudio se reporta la actividad reductora de la transmisión (ART) de sueros obtenidos de 105 voluntarios infectados con P. vivax y de 44 individuos no infectados todos provenientes del municipio de Buenaventura, un área endémica de malaria y evaluados usando ensayos funcionales de alimentación artificial por membrana con mosquitos Anopheles albimanus.
En los voluntarios infectados, encontramos que la ART es variable con mayor frecuencia por encima del 50%, mientras que los voluntarios no infectados a pesar de que también es variable la mayor frecuencia se encontró por debajo del 50%; las diferencias entre los dos grupos son estadísticamente significativas. En este ultimo grupo se relacionó la ART y el titulo de anticuerpos.
La duración de la ART esta entre 2-4 meses después de la infección.
En el 70% de los casos se encontraron diferentes niveles de ART con los diferentes aislados. En algunos casos el nivel aumento y en otros disminuyo con respecto al obtenido con el aislado autologo.
También se encontró en varios ensayos que esta actividad es dependiente del complemento.
... Cellular immunogenicity in mice. Cellular immune responses play a pivotal role in conferring protection from P. falciparum infection at the PE stage (29)(30)(31)(32)(33). Prior to combining the CSP, LSA1, TRAP, and CelTOS plasmids into the single-dose MAV4 formulation, cellular immunogenicity induced by the individual pDNAs was assessed by IFN-␥ ELISpot assay (Fig. 3A). ...
A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able
to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical
assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP),
and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen
with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent
assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA
with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all
antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4+ and CD8+ T cell compartments. Furthermore, hepatic CD8+ lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model,
which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8+ granzyme B+ T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria
parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.
A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.
Malaria is one of the most important tropical diseases in the history of the world. This vector-borne disease has been a significant cause of morbidity and mortality in tropical countries of Africa, Asia, and Latin America. As such, this book provides updated information on epidemiological and public health research of malaria conducted in the last decade. Over four sections, chapters discuss such topics as diagnosis, epidemiology and surveillance, policy and prevention, and vector control and vaccines.
Malaria Impfstoffkandidaten, welche Sicherheit und Wirksamkeit gegen prä-erythrozytische Stadien bieten, sind nach wie vor in der Entwicklung. Experimentelle Immunisierungsstudien mit genetisch attenuierten Parasiten (GAP), welche die Entwicklung über das klinisch asymptomatische Leberstadium hinaus verhindern, erwiesen sich als sicher und effizient. ΔSLARP GAP-Sporozoiten arretieren vollständig in der Leber, bieten jedoch keinen langanhaltenden Schutz. Hingegen zeigen Immunisierungen mit ΔP36p/P36 Sporozoiten einen langanhaltenden Schutz, führen jedoch während der Immunisierung gelegentlich zu Blutstadieninfektionen. Diese Studie liefert eine systematische vorklinische Bewertung eines dreifachen KO GAP-Parasiten, durch die Kombination von ΔSLARP und ΔP36p/P36. KO Parasiten arretierten vollständig in vitro und in vivo, aber der zeitnahe Blutinfektionsbeginn nach einer Sporozoiteninfektion in Mäusen zeigte eine verminderte Wirksamkeit des Impfstoffs. Während ein besserer Schutz durch einen späten Leberstadien Entwicklungsstillstand erreicht werden kann, bleiben die zugrundeliegenden molekularen Mechanismen unklar. Eine Vorrausetzung für die Leberzellen Antigenpräsentation ist die Präsenz von parasitären Antigenen im hepatozyten Zytoplasma. Der Proteinexportkomplex PTEX ist in Leberstadien nicht vollständig funktionstüchtig, da das essentielle Hitzeschockprotein 101 (HSP101) nicht exprimiert wird. Um die Rolle von HSP101 für den Leberproteinexport zu klären, wurden transgene HSP101 exprimierende Parasiten erzeugt. Transgene Parasiten weisen in vitro und in vivo schwere Wachstumsstörungen im Leberstadium auf und bieten keinen Impfschutz. Die Ergebnisse legen nahe, dass die Expression von HSP101 streng kontrolliert wird und der Export im frühen Leberstadien nicht wiederhergestellt werden kann. Insgesamt können prä-klinische Studien und die Weiterentwicklung von GAP-basierten Impfstoffkandidaten die laufenden humanen Impfstoffstudien beeinflussen und vorantreiben.
This chapter discusses human malaria, which is caused by infection by one of the four species of Plasmodium, of which P. falciparum is the most pathogenic. The classic symptoms of malaria, such as fever and malaise with intermittent paroxysms, are typical of P. vivax, P. malariae, and P. ovale malaria. Human malaria infections tend to become synchronous, with large numbers of schizont-infected erythrocytes rupturing within a short space of time. Thus, the typical pattern of fever seen in malaria is associated with the cyclic maturation of asexual stages. Fever spikes coincide with schizont ruptures. In areas with stable endemic P. falciparum malaria, parasitemia is most common in young children, and the incidence of parasitemia declines steadily with age. The acquisition of antimalarial immunity is slow, developing gradually after years of exposure. The precise timing of the events depends on the local patterns of malaria transmission and levels of endemicity. Multiple malaria infections are necessary to achieve effective antiparasite immunity.
Histamine, a biogenic amine derived from the decarboxylation of amino acid histidine by an enzyme histidine decarboxylase. It involves the local immune responses as well as regulating physiological functions. As part of an immune response to foreign pathogen (including malarial parasite infection), histamine is produced by basophils and by mast cells found in nearby connective tissue. Elevation in immune mediators such as IL-1, IL-6, IL-8, TNF-α, NO and histamine have been associated with disease severity in malarial infection. Histamine releasing factor (HRF) is a peptide described in mice and humans, causes the release of histamine from basophils. HRF belongs to a class of protein called translationally controlled tumour protein (TCTP) homologs. Recently a Plasmodium falciparum TCTP is identified. This protein has a high homology to human HRF. The central nervous system signs and symptoms such as drowsiness, coma, multiple seizures, destruction of blood brain barrier etc are supposed to be due to histamine secretion in CNS during Plasmodium falciparum infection. In this chapter we will discuss the pathophysiological effects of histamine in severe malaria infection.
Malaria infection crisis, at which the parasitemia drops precipitously and the parasite loses infectivity to the mosquito vector, occurs in many natural malaria systems, and has not been explained. We demonstrate that in a simian malaria parasite (Plasmodium cynomolgi in its natural host, the toque monkey), the loss of infectivity during crisis is due to the death of circulating intraerythrocytic gametocytes mediated by crisis serum. These parasite-killing effects in crisis serum are due to the presence in the serum of cytokines tumor necrosis factor and interferon gamma, which are produced by the host as a result of the malaria infection. The killing activity of each cytokine is absolutely dependent upon the presence of additional, as yet unidentified factor(s) in the crisis serum.
Protozoan parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, trypanosomes, and Leishmania, are a major cause of disease in both humans and other animals, highlighting the need to understand the full spectrum of strategies used by the host immune system to sense and respond to parasite infection. Although type II interferon (IFN-γ) has long been recognized as an essential antiparasite immune effector, much less is known about the role of type I interferons (IFN-α and -β) in host defense, particularly in vivo. Recent studies are reviewed which collectively highlight that type I IFN can be induced in response to parasite infection and influence the outcome of infection.
The host response to infection requires an immune response to be strong enough to control the pathogen but restrained, to minimise immune-mediated pathology. The conflicting pressures of immune activation and immune suppression are particularly apparent in parasite infections, where co-evolution of host and pathogen has selected many different compromises between protection and pathology. Cytokine signals are critical determinants of both protective immunity and immunopathology and, in this review, we outline the key cytokines that are responsible for the protective and pathological immune responses elicited by parasite infections. We focus on the regulatory cytokine IL-10 and its role in protozoan and helminth infections. We discuss the sources and targets of IL-10 during parasite infection, the signals that initiate and reinforce its action, and its impact on the invading parasite, on the host tissue, and on coincident immune responses. This article is protected by copyright. All rights reserved.
Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280-288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.
Mice treated with dead Propionibacterium acnes (previously called Corynebacterium parvum), up to 30 days before infection with any of three strains of Trypanosoma brucei, were more able to limit the level of first-wave parasitaemia than untreated controls. Reduced parasitaemia was not due to enhanced phagocytosis of input parasites and was associated with a dramatic reduction in the proportion of multiplying T. brucei in the blood of treated as compared to control mice. For 4 days after P. acnes treatment, T. brucei growth-inhibitory molecules, assayed by their effect on T. brucei multiplication under axenic culture conditions, were detected in the serum of recipient mice. The molecules were released by macrophages collected from the peritoneal cavity of P. ac/ies-treated mice, and similar molecules were produced in vitro by macrophages from normal mice after incubation with P. acnes. Accessory studies suggested that the molecules were breakdown products of P. acnes and were unlikely to be responsible for the long-term in-vivo effects of the P. acnes treatment. It was also shown that monokines and lymphokines which are likely to be induced by in-vivo P. acnes treatment, i.e. IL-1, IL-2, TNFα, INFα, INFβ, INFy, PGE1, PGE2, PGF2, and biological mediators present in Con-A and LPS-induced spleen cell supernatants (collected 20, 40, 60 or 80 h after mitogen stimulation) had no influence on T. brucei growth under axenic culture conditions over a wide range of concentrations. The studies suggest that the P. acnes effect was not due to a direct interaction of these biological mediators with the T. brucei. We suggest that the reduction in T. brucei parasitaemia in P. acnes-treated mice reflects secondary physiological effects of one or more unidentified biological mediators.
Protective immune responses and the functional role of spleen cells in mice infected with Babesia rodhaini were examined with an in vitro proliferation assay systems and by in vivo passive transfer of spleen cells to uninfected mice. Mice that resolved primary babesial infection after chemotherapy (Babesia immune mice) had transient and low parasitemia after challenge infection and high rates (75%) of survival. Babesia hyperimmune mice, by contrast, had no detectable parasitemia after challenge and 100% survival. Proliferative response of spleen cells to Babesia lysate antigen (BLA) were determined for mice from both groups. This proliferative response was inhibited by treatment of spleen cells with anti-T cell serum and monoclonal antibody (MAb) to Lyt l antigen. Spleen cells of hyperimmime mice produced larger amount of IL-2 production than those of immune mice. Transfer of spleen cells from immune mice to nonimmune mice provided protection against babesial infection and recipient mice had high titers of anti-babesial antibody. When these spleen cells were treated with anti-T cell serum or anti-mouse Ig serum, protection against challenge was abolished. By contrast, transfer of hyperimmune spleen cells was capable of protecting recipient mice. Treatment of hyperimmune spleen cells with antiserum to mouse Ig or MAb against Lyt 1 and Lyt 2 antigens did not interfere with their ability to protect recipient mice against infection, even though recipient mice had low levels of antibody production. These results indicate that humoral immune response is important in establishing protection after primary infections while the panicipation of Lyt 1 + cells and Lyt 2 + cells and other aspects of the cell-mediated immune response is important in controlling secondary infections.
Previous studies haveshownthattheimmune-regulated cytokine gammainterferon (IFN-fy) activates host cells torestrict intracellular growth ofthebacterial pathogen Chlamydia trachomatis byinduction ofthe tryptophan-catabolizing enzymeindoleamine 2,3-dioxygenase (IDO). Recently, subinhibitory levels ofIFN-y wereusedtogenerate aninvitro persistent chlamydial infection characterized bylarge aberrant, noninfectious reticulate bodies fromwhichinfectious progeny could berecovered following theremoval ofIFN-fy. Studies weredonetodetermine ifthemechanism functioning toinduce chlamydiae toenter apersistent state inthe presence oflowlevels ofIFN-'y wassimilar tothatreported toinhibit chlamydial growth. Hostcells treated withlevels ofIFN-y required toinduce persistence wereassessed forIDOactivity byhigh-performance liquid chromatography analysis oftryptophan anditscatabolic products. Substantial tryptophan catabolism was detected inacid-soluble cellular pools, indicating that theintracellular availability ofthis essential aminoacid waslimited underthese conditions. Inaddition, amutantcell line responsive toIFN-'y butdeficient inIDO activity wasshowntosupport C.trachomatis growth, butaberrant organisms werenotinduced inresponse to IFN-ytreatment. Analyses ofinfected cells cultured inmediumwithincremental levels ofexogenous tryptophan indicated thatpersistent growth wasinduced byreducing theamountofthis essential aminoacid. These studies confirmed that nutrient deprivation byIDO-mediated tryptophan catabolism wasthemechanism bywhichIFN-'y mediates persistent growth ofC.trachomatis.
We describe simple and sensitive in vitro and in vivo assays to analyze Plasmodium liver stage development using transgenic P. berghei parasites (PbGFP-Luccon), which express the bioluminescent reporter protein, luciferase. In these assays, parasite development in hepatocytes is visualized and quantified by real-time bioluminescence imaging both in culture and in live mice. We also describe quantification of in vitro liver-stage development by measuring luminescence using a microplate reader. Reporter-parasite based quantification of liver-stage development is faster and correlates very well with established quantitative RT-PCR methods currently used to assess parasite development inside hepatocytes, both in live mice and in culture.
Ivan Morrison, Evans Taracha and Declan McKeever here summarize recent work that has provided direct evidence that cytotoxic T lymphocyte responses play an important role in mediating immunity to Theileria parva and consider observations on the responses to primary infections which suggest that T cells may also contribute to the pathogenesis of the disease.
Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes.
A 2.3 kb, 32P-labeled repetitive DNA probe of Plasmodium berghei was used to measure the amount of parasite DNA in the liver of Norway Brown rats and mice infected with sporozoites. Standard hybridization curves were obtained by probing different amounts (100 pg to 1 μg) of P. berghei DNA immobilized on nitrocellulose filters. Host DNA did not interfere with hybridization specificity and sensitivity. A 100-fold increase in hepatic parasite DNA was detected between 25 h post-infection and the peak of parasite proliferation, detected at 44 h. The amount of parasite DNA increased with the number of injected sporozoites. At 5 h post-infection, a large proportion of parasite DNA was found in the spleen. However, this diminished with time and was negligible in amount at 25 h. A significant number of viable sporozoites were probably cleared in the spleen, since considerably more parasite DNA was found in the livers of splenectomized rats than in sham-operated counterparts. Although older rats develop much lower parasitemias upon inoculation of sporozoites, no significant differences were observed in the amount of parasite DNA in rats, 43 and 152 days old, injected with equal numbers of sporozoites. The higher resistance to malaria displayed by older rats is probably controlled by post-hepatic events. The infectivity of sporozoites for A/J mice was calculated to be about 1/20th that of Norway Brown rats.
Natural killer (NK) cell activity and interferon levels have been measured in the peripheral blood of children acutely ill with Plasmodium falciparum infection. The NK cell levels were found to be raised in the malaria-infected children, with a positive correlation between the degree of parasitemia and lytic activity. Comparatively high titers of antiviral activity was discovered in sera from the majority of P. falciparum-infected children, again positively correlating with the degree of parasitemia and NK levels. The characteristics of the antiviral factor indicated alpha-type interferon to be the dominating agent involved. Addition of exogenous interferon in vitro potentiated the NK levels of PBL from normal children while having no significant impact on cells from malaria-infected children.
Interferon-gamma (IFN-gamma) induces many effector functions in macrophages, presumably through binding to cell surface receptors. Such receptors have been documented in murine macrophages. This report demonstrates that IFN-gamma interacts with a specific receptor on human monocytes and monocyte-like cell lines, U937 and HL60. Recombinant IFN-gamma (rIFN-gamma) was radioiodinated by using Bolton-Hunter reagent to high specific activity. 125I-rIFN-gamma bound in a specific and saturable manner. Saturation of binding sites occurred at 10(-9) M (300 U/ml); there were 4000 specific binding sites per cell for both monocytes and the cell lines. Purified lymphocyte-derived IFN-gamma as well as rIFN-gamma competed for binding sites with 125I-rIFN-gamma. There was no inhibition of binding of the 125I-rIFN-gamma by rIFN-alpha or rIFN-beta. The forward rate constant, k1, at 4 degrees C was about 8 X 10(5) M-1 sec-1 for each cell type studied. In the presence of excess ligand, the dissociation rate constant, k-1, was about 2 X 10(-4) sec-1. The Ka calculated from these constants (4 X 10(9) M-1) agreed closely with that calculated from experiments performed at equilibrium (8 X 10(9) M-1). Because the dissociation of rIFN-gamma from cells was enhanced in the presence of ligand, interactions between binding sites of a negative cooperative type could be operative. These studies demonstrate that rIFN-gamma binds in a specific and saturable manner and with high affinity to a receptor on human monocytes as well as monocyte-like cell lines.
The effect of recombinant murine interferon-gamma on the growth of Chlamydia trachomatis was analyzed in a mouse fibroblast cell line (McCoy cells). Murine interferon-gamma had a very potent anti-chlamydial activity, although minimally affecting cellular proliferation. Over 95% inhibition of chlamydial inclusions was obtained at a concentration of 1 U/ml of interferon. At a concentration of 1 U/ml of murine interferon-gamma, there was minimal inhibition of the proliferative capacity of McCoy cells. Approximately 50% inhibition of cell growth was obtained with a concentration of 10 U/ml of interferon. Varying concentrations of tryptophan in the medium did not alter either the anti-chlamydial or the anti-proliferative activity of the interferon.
Techniques for laboratory maintenance of the Plasmodium herghei-Anopheles stephensi system for rodent malaria are described. The system permits the regular production of sporozoite-induced blood infections in susceptible rodents, as well as the regular attainment of preerythrocytic schizonts in the livers of young white rats. The most sensitive measures of the infectivity of a pool of sporozoites are the time to patency of infected animals, and the percentage of sporozoites which become preerythrocytic schizonts. Three kinds of variability in this infectivity were noted: 1) a variability between different pools of sporozoites, 2) an interspecific variability with tree rats, young white rats, and mice being susceptible in that order, 3) an intraspecific variability, with some animals of a given species being less susceptible than others.
Protective immunity against malaria has been achieved in hosts ranging from birds to man by repeated inoculation of irradiated sporozoites. The main antigens involved in protective immunity to sporozoites are the circumsporozoite (CS) proteins, which are part of a family of proteins, covering the whole surface membrane of the parasite, and which have similar physico-chemical and antigenic properties. Monovalent fragments of monoclonal antibodies to CS proteins neutralize sporozoite infectivity. All monoclonal antibodies recognize a single immunodominant region within the various CS proteins, and this region contains repetitive epitopes. The recurrent immunodominant epitope of the CS protein of P. knowlesi has been identified, and shown to consist of 12 tandemly repeated subunits of 12 amino acids. The dimer of the dodecapeptide was coupled to protein carriers, emulsified in Freund's complete adjuvant, and injected into rodents and monkeys. All animals made anti-peptide antibodies, and most of the antisera reacted with P. knowlesi CS protein.
The attachment and entry of Plasmodium berghei sporozoites to cultured human lung fibroblast (WI38) or hepatoma (HepG2-A16) cells in vitro has been visualized using an immunoperoxidase technique coupled with light microscopy. Attachment and entry was substantially more frequent with HepG2-A16 cells, and appeared to be mediated by the Pb44 sporozoite surface protective antigen. When sporozoites were incubated with intact monoclonal antibodies to Pb44 or their monovalent Fab fragments, attachment was inhibited, suggesting that this technique may be an in vitro assay of protective immunity. Sporozoites appeared to enter cells actively, rather than by cell phagocytosis. Once within a membrane-bound vacuole, the sporozoite transformed into a trophozoite. It was suggested that Pb44 recognized a specific cell receptor, and that this technique may permit receptor characterization.
Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite.
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