Blood-stage Infection Research Articles

The immunological perturbations induced by ongoing blood-stage infection during Plasmodium GAP (Genetically Attenuated Parasites) vaccination

Abstract Plasmodium sporozoites (SPZ) are transmitted by mosquito bite, infect the liver, grow and differentiate as intrahepatocytic liver stages (LS) and ultimately form the first generation of red blood cell-infectious merozoites, which initiate the symptomatic blood stage infection. Immunization with replication-deficient radiation attenuated SPZ vaccine has shown to confer sterilizing immunity in animal models by preventing parasite growth into the liver before onset of symptomatic blood stage infection. This attenuated SPZ vaccine has undergone clinical testing and it was observed that sterilizing immunity was achieved high in malaria naïve subjects but lower in malaria pre-exposed subjects residing in malaria-endemic regions. Previous studies in rodent models indicate that the ongoing blood stage infection suppresses protection after immunization with replication deficient irradiated SPZ. We have shown that replication competent late liver stage arresting genetically attenuated parasites (RC-GAP) immunization confer superior protection when compared to irradiated SPZ. However, whether the blood stage infection would also affect the RC-GAP parasite vaccine mediated protective immunity by altering specific immune responses remains unknown. Here, we show that ongoing homologous blood stage infection during Plasmodium yoelii RC-GAP immunization suppress the vaccine mediated immunity in Balb/c mice. Our data suggest that the reduction in protection was associated with the perturbations of long-lived Ag specific antibody as well as tissue resident memory CD8 T cell (TRMs) responses generated against pre-erythrocytic SPZ and LS stages.

CYP2D6 Genetic Variation and Its Implication for Vivax Malaria Treatment in Madagascar.

Plasmodium vivax is one of the five human malaria parasite species, which has a wide geographical distribution and can cause severe disease and fatal outcomes. It has the ability to relapse from dormant liver stages (hypnozoites), weeks to months after clearance of the acute blood-stage infection. An 8-aminoquinoline drug primaquine (PQ) can clear the hypnozoites, and thus can be used as an anti-relapse therapeutic agent. Recently, a number of studies have found that its efficacy is compromised by polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene; decreased or absence of CYP2D6 activity contributes to PQ therapeutic failure. The present study sought to characterize CYP2D6 genetic variation in Madagascar, where populations originated from admixture between Asian and African populations, vivax malaria is endemic, and PQ can be deployed soon to achieve national malaria elimination. In a total of 211 samples collected from two health districts, CYP2D6 decreased function alleles CYP2D6*10, *17, *29, *36+*10, and *41 were observed at frequencies of 3.55–17.06%. In addition, nonfunctional alleles were observed, the most common of which were CYP2D6*4 (2.13%), *5 (1.66%), and the *4x2 gene duplication (1.42%). Given these frequencies, 34.6% of the individuals were predicted to be intermediate metabolizers (IM) with an enzyme activity score (AS) ≤ 1.0; both the IM phenotype and AS ≤ 1.0 have been found to be associated with PQ therapeutic failure. Furthermore, the allele and genotype frequency distributions add to the archaeological and genomic evidence of Malagasy populations constituting a unique, Asian-African admixed origin. The results from this exploratory study provide fresh insights about genomic characteristics that could affect the metabolism of PQ into its active state, and may enable optimization of PQ treatment across human genetic diversity, which is critical for achieving P. vivax elimination.

Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

SummaryBackgroundDevelopment of an effective vaccine against the pathogenic blood-stage infection of human malaria has proved challenging, and no candidate vaccine has affected blood-stage parasitemia following controlled human malaria infection (CHMI) with blood-stage Plasmodium falciparum.MethodsWe undertook a phase I/IIa clinical trial in healthy adults in the United Kingdom of the RH5.1 recombinant protein vaccine, targeting the P. falciparum reticulocyte-binding protein homolog 5 (RH5), formulated in AS01B adjuvant. We assessed safety, immunogenicity, and efficacy against blood-stage CHMI. Trial registered at ClinicalTrials.gov, NCT02927145.FindingsThe RH5.1/AS01B formulation was administered using a range of RH5.1 protein vaccine doses (2, 10, and 50 μg) and was found to be safe and well tolerated. A regimen using a delayed and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly improved antibody response longevity over ∼2 years of follow-up. Following primary and secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. We show that growth inhibition activity measured in vitro using purified immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth rate and also identify other antibody feature sets by systems serology, including the plasma anti-RH5 IgA1 response, that are associated with challenge outcome.ConclusionsOur data provide a new framework to guide rational design and delivery of next-generation vaccines to protect against malaria disease.FundingThis study was supported by USAID, UK MRC, Wellcome Trust, NIAID, and the NIHR Oxford-BRC.

Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

Background: Exagerated immune activation has a key role in the pathogenesis of malaria. During blood-stage infection, Plasmodium falciparum can interact directly with host immune cells through infected red blood cells (PfiRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of PfiRBCs and P. falciparum small-sized EVs (PfsEVs, also known as exosomes) from a Kenyan clinical isolate (PfKE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC). Methods: PfsEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite PfiRBCs were purified by magnetic column separation. The PfsEVs and the PfiRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant. Results: Relative to negative control conditions, PfsEVs induced CD25 expression on CD4+, CD19+ and CD14+ cells, while PfiRBCs induced on CD19+ and CD14+ cells. Both PfsEVs and PfiRBCs induced CD69 on CD4+, CD8+ and CD19+ cells. In addition, PfiRBCs induced higher expression of CD69 on CD14+ cells. CD69 induced by PfiRBCs on CD4+ and CD19+ cells was significantly higher than that induced by PfsEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both PfsEVs and PfiRBCs whereas MCP-1, IL-10, IL-17α were preferentially induced by PfsEVs and IP-10 and IFN-γ by PfiRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to PfsEVs. Conclusions: PfsEVs and PfiRBCs showed differential abilities to induce secretion of IL-17α and IFN-γ, suggesting that the former are better at inducing Th17, whilst the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of PfsEVs to activate monocytes, suggesting immune tolerance to PfsEVs may play a role in naturally acquired anti-disease immunity.

Plasmodium Ape1 is a multifunctional enzyme in mitochondrial base excision repair and is required for efficient transition from liver to blood stage infection

The malaria parasite has a single mitochondrion which carries multiple tandem repeats of its 6 kb genome encoding three proteins of the electron transport chain. There is little information about DNA repair mechanisms for mitochondrial genome maintenance in Plasmodium spp. Of the two AP-endonucleases of the BER pathway encoded in the parasite nuclear genome, the EndoIV homolog PfApn1 has been identified as a mitochondrial protein with restricted functions. We explored the targeting and biochemical properties of the ExoIII homolog PfApe1. PfApe1 localized in the mitochondrion and exhibited AP-site cleavage, 3′-5′ exonuclease, 3′-phosphatase, nucleotide incision repair (NIR) and RNA cleavage activities indicating a wider functional role than PfApn1. The parasite enzyme differed from human APE1 in possessing a large, disordered N-terminal extension. Molecular modelling revealed conservation of structural domains but variations in DNA-interacting residues and an insertion in the α-8 loop suggested differences with APE1. Unlike APE1, where AP-site cleavage and NIR activities could be mutually exclusive based on pH and Mg2+ ion concentration, PfApe1 was optimally active under similar conditions suggesting that it can function both as an AP-endonuclease in BER and directly cleave damaged bases in NIR under similar physiological conditions. To investigate the role of Ape1 in malaria life cycle, we disrupted the gene by double-cross-over homologous recombination. Ape1 knockout (KO) P. berghei parasites showed normal development of blood and mosquito stages. However, inoculation of mice with Ape1 KO salivary gland sporozoites revealed a reduced capacity to initiate blood stage infection. Ape1 KO parasites underwent normal liver stage development until merozoites egressed from hepatocytes. Our results indicated that the delay in pre-patent period was due to the inability of Ape1 KO merosomes to infect erythrocytes efficiently.

Heterogeneity in response to serological exposure markers of recent Plasmodium vivax infections in contrasting epidemiological contexts.

Antibody responses as serological markers of Plasmodium vivax infection have been shown to correlate with exposure, but little is known about the other factors that affect antibody responses in naturally infected people from endemic settings. To address this question, we studied IgG responses to novel serological exposure markers (SEMs) of P. vivax in three settings with different transmission intensity. We validated a panel of 34 SEMs in a Peruvian cohort with up to three years' longitudinal follow-up using a multiplex platform and compared results to data from cohorts in Thailand and Brazil. Linear regression models were used to characterize the association between antibody responses and age, the number of detected blood-stage infections during follow-up, and time since previous infection. Receiver Operating Characteristic (ROC) analysis was used to test the performance of SEMs to identify P. vivax infections in the previous 9 months. Antibody titers were associated with age, the number of blood-stage infections, and time since previous P. vivax infection in all three study sites. The association between antibody titers and time since previous P. vivax infection was stronger in the low transmission settings of Thailand and Brazil compared to the higher transmission setting in Peru. Of the SEMs tested, antibody responses to RBP2b had the highest performance for classifying recent exposure in all sites, with area under the ROC curve (AUC) = 0.83 in Thailand, AUC = 0.79 in Brazil, and AUC = 0.68 in Peru. In low transmission settings, P. vivax SEMs can accurately identify individuals with recent blood-stage infections. In higher transmission settings, the accuracy of this approach diminishes substantially. We recommend using P. vivax SEMs in low transmission settings pursuing malaria elimination, but they are likely to be less effective in high transmission settings focused on malaria control.

Transdermal Immunization of Elastic Liposome-Laden Recombinant Chimeric Fusion Protein of P. falciparum (PfMSP-Fu24) Mounts Protective Immune Response.

Transdermal immunization exhibits poor immunogenic responses due to poor permeability of antigens through the skin. Elastic liposomes, the ultradeformable nanoscale lipid vesicles, overcome the permeability issues and prove a versatile nanocarrier for transcutaneous delivery of protein, peptide, and nucleic acid antigens. Elastic liposome-mediated subcutaneous delivery of chimeric fusion protein (PfMSP-Fu24) of Plasmodium falciparum exhibited improved immunogenic responses. Elastic liposomes-mediated immunization of PfMSP-Fu24 conferred immunity to the asexual blood-stage infection. Present study is an attempt to compare the protective immune response mounted by the PfMSP-Fu24 upon administered through transdermal and intramuscular routes. Humoral and cell-mediated immune (CMI) response elicited by topical and intramuscularly administered PfMSP-Fu24-laden elastic liposomes (EL-PfMSP-Fu24) were compared and normalized with the vehicle control. Sizeable immune responses were seen with the transcutaneously immunized EL-PfMSP-Fu24 and compared with those elicited with intramuscularly administered antigen. Our results show significant IgG isotype subclass (IgG1and IgG3) response of specific antibody levels as well as cell-mediated immunity (CMI) activating factor (IFN-γ), a crucial player in conferring resistance to blood-stage malaria in mice receiving EL-PfMSP-Fu24 through transdermal route as compared to the intramuscularly administered formulation. Heightened immune response obtained by the vaccination of EL-PfMSP-Fu24 was complemented by the quantification of the transcript (mRNA) levels cell-mediated (IFN-γ, IL-4), and regulatory immune response (IL-10) in the lymph nodes and spleen. Collectively, elastic liposomes prove their immune-adjuvant property as they evoke sizeable and perdurable immune response against PfMSP-Fu24 and justify its potential for the improved vaccine delivery to inducing both humoral and CM immune response.

Extrafollicular CD4 T cell-derived IL-10 functions rapidly and transiently to support anti-Plasmodium humoral immunity.

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria.

The spleen: "epicenter" in malaria infection and immunity.

The spleen is a complex secondary lymphoid organ that plays a crucial role in controlling blood‐stage infection with Plasmodium parasites. It is tasked with sensing and removing parasitized RBCs, erythropoiesis, the activation and differentiation of adaptive immune cells, and the development of protective immunity, all in the face of an intense inflammatory environment. This paper describes how these processes are regulated following infection and recognizes the gaps in our current knowledge, highlighting recent insights from human infections and mouse models.

Tissue macrophages and interferon-gamma signalling control blood-stage Plasmodium chabaudi infections derived from mosquito-transmitted parasites

Natural infection with Plasmodium parasites, the causative agents of malaria, occurs via mosquito vectors. However, most of our knowledge of the immune response to the blood stages of Plasmodium is from infections initiated by injection of serially blood-passaged infected red blood cells, resulting in an incomplete life cycle in the mammalian host. Vector transmission of the rodent malaria parasite, Plasmodium chabaudi chabaudi AS has been shown to give rise to a more attenuated blood-stage infection in C57Bl/6J mice, when compared to infections initiated with serially blood-passaged P. chabaudi-infected red blood cells. In mouse models, the host immune response induced by parasites derived from natural mosquito transmission is likely to more closely resemble the immune responses to Plasmodium infections in humans. It is therefore important to determine how the host response differs between the two types of infections.As the spleen is considered to be a major contributor to the protective host response to P. chabaudi, we carried out a comparative transcriptomic analysis of the splenic response to recently mosquito-transmitted and serially blood-passaged parasites in C57Bl/6J mice. The attenuated infection arising from recently mosquito-transmitted parasites is characterised by an earlier and stronger myeloid- and IFNγ-related response. Analyses of spleen lysates from the two infections similarly showed stronger or earlier inflammatory cytokine and chemokine production in the recently mosquito-transmitted blood-stage infections. Furthermore, tissue macrophages, including red pulp macrophages, and IFNγ-signalling in myeloid cells, are required for the early control of P. chabaudi recently mosquito-transmitted parasites, thus contributing to the attenuation of mosquito-transmitted infections.The molecules responsible for this early activation response to recently-transmitted blood-stage parasites in mice would be important to identify, as they may help to elucidate the nature of the initial interactions between blood-stage parasites and the host immune system in naturally transmitted malaria.

A Single-Cell Liver Atlas of Relapsing Human Malaria

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell dependencies and responses to them. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites in primary human hepatocytes and conduct transcriptional profiling. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.

Fluorescence Lifetime Imaging as a Noninvasive Tool to Study Plasmodium Falciparum Metabolism.

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.

Determining the prevalence and density of asymptomatic Plasmodium falciparum infections in low and high transmission settings in Western Kenya

Background/Objective: Malaria remains a global health burden as 2018 saw 228 million cases worldwide. Pre-elimination efforts aim to decrease disease transmission, which depends on development of asexual parasites to sexual gametocytes within the human host. To better determine the relationship between asexual parasites and gametocytes, we aim to quantify both stages within the same blood sample by developing a multiplex RT-qPCR assay. We hypothesize that gametocytemia decreases linearly with asexual parasitemia at higher parasitemias but increases at lower parasitemia to improve transmission probability. 
 
 Methods: We conducted a cross-sectional survey of healthy participants aged 0 to 92 years at four sites in Western Kenya (Ajigo, Webuye, Kipsamoite, Kapsisywa) with varying levels of transmission to determine prevalence of asymptomatic P. falciparum blood-stage infections. Parasite density was quantified by light microscopy and by quantitative PCR of genomic DNA isolated from dried blood spots using varATS primers. For varATS-positive samples, we isolated RNA from whole-blood and performed stage-specific quantification by multiplex Taqman RT-qPCR targeting PfPIESP2 (asexual), Pfs25 (female), and Pfs230p (male). 
 
 Results: Among all participants, 127 of 1355 (9.37%) were positive for asymptomatic P. falciparum infection by varATS-qPCR. Using varATS positivity as the standard, multiplex RT-qPCR targeting PfPIESP2 yielded a sensitivity of 78%. Across sites, overall parasite prevalence and gametocyte prevalence were highest in Ajigo (42.1%; 36%) followed by Webuye (10.5%; 9%) and Kipsamoite/Kapsisywa (0.873%; 0.1%). Pfs25-quantified gametocytemia decreased with decreasing varATS-quantified asexual parasitemia; however, the data poorly fit a linear model (R2 = 0.088). 
 
 Conclusions: PfPIESP2 detected 78% of varATS-positive samples, suggesting it may be poorly sensitive at detecting low-density blood-stage infections. Assay design may improve with a more sensitive gene target and further optimization. Based on quantification results, the relationship between gametocytemia and asexual parasitemia appears direct but non-linear. Further work is necessary to better model the relationship between stage-specific parasitemia levels.

Linking nutrient sensing and gene expression in Plasmodium falciparum blood-stage parasites.

Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle of Plasmodium parasites is shared between two hosts, with infection of multiple cell types, and the parasite needs to adapt for survival and transmission through significantly different metabolic environments. Within the blood-stage alone, parasites encounter changing levels of key nutrients, including sugars, amino acids, and lipids, due to differences in host dietary nutrition, cellular tropism, and pathogenesis. In this review, we consider the mechanisms that the most lethal of malaria parasites, Plasmodium falciparum, uses to sense nutrient levels and elicit changes in gene expression during blood-stage infections. These changes are brought about by several metabolic intermediates and their corresponding sensor proteins. Sensing of distinct nutritional signals can drive P. falciparum to alter the key blood-stage processes of proliferation, antigenic variation, and transmission.

Plasmodium yoelii Uses a TLR3-Dependent Pathway to Achieve Mammalian Host Parasitism.

Malaria is associated with complicated immunopathogenesis. In this study, we provide evidence for an unexpected role of TLR3 in promoting the establishment of Plasmodium yoelii infection through delayed clearance of parasitemia in wild type C57BL/6jRj (B6) compared with TLR3 knockout mice. In this study, we confirmed an increased expression of Tlr3, Trif, Tbk1, and Irf7/Irf3 in the liver 42 h postinfection and the initiation of an early burst of proinflammatory response such as Ifng, NF-kB, and Tnfa in B6 mice that may promote parasite fitness. Interestingly, in the absence of TLR3, we showed the involvement of high IFN-γ and lower type I IFN response in the early clearance of parasitemia. In parallel, we observed an increase in splenic NK and NKT cells expressing TLR3 in infected B6 mice, suggesting a role for TLR sensing in the innate immune response. Finally, we find evidence that the increase in the frequency of CD19+TLR3+ B cells along with reduced levels of total IgG in B6 mice possibly suggests the initiation of TLR3-dependent pathway early during P. yoelii infection. Our results thus reveal a new mechanism in which a parasite-activated TLR3 pathway promotes blood stage infection along with quantitative and qualitative differences in Ab responses.

Gut Microbiota Composition Modulates the Magnitude and Quality of Germinal Centers during Plasmodium Infections

SUMMARYGut microbiota composition is associated with human and rodent Plasmodium infections, yet the mechanism by which gut microbiota affects the severity of malaria remains unknown. Humoral immunity is critical in mediating the clearance of Plasmodium blood stage infections, prompting the hypothesis that mice with gut microbiota-dependent decreases in parasite burden exhibit better germinal center (GC) responses. In support of this hypothesis, mice with a low parasite burden exhibit increases in GC B cell numbers and parasite-specific antibody titers, as well as better maintenance of GC structures and a more targeted, qualitatively different antibody response. This enhanced humoral immunity affects memory, as mice with a low parasite burden exhibit robust protection against challenge with a heterologous, lethal Plasmodium species. These results demonstrate that gut microbiota composition influences the biology of spleen GCs as well as the titer and repertoire of parasite-specific antibodies, identifying potential approaches to develop optimal treatments for malaria.

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.

Toll-like receptor 4, Toll-like receptor 7 and Toll-like receptor 9 agonists enhance immune responses against blood-stage Plasmodium chabaudi infection in BALB/c mice

BackgroundToll-like receptor (TLR) signals play vital roles during the blood-stage of malaria infections. However, the roles of TLR agonists in the regulation of immune responses and the development of protective immunity to malaria remain poorly understood. MethodBALB/c mice were pre-treated with TLR4, TLR7 and TLR9 agonists, followed by infection with Plasmodium chabaudi. After infection, splenic dendritic cells (DCs), Th1 cells and programmed death-1 (PD-1) expressed on Th1 cells, as well as regulatory T cells (Tregs) were analyzed by flow cytometry. The levels of IFN-γ, TNF-α, TGF-β and IL-10 in splenocytes and IgG1 and IgG2a in serum were measured by ELISA. ResultAdministration of TLR4, TLR7 and TLR9 agonists prior to infection improved disease outcomes. All TLR agonists promoted DC activation, and the proportions of Th1 cells increased. In TLR4, TLR7 and TLR9 agonist treated groups the levels of pro-inflammatory cytokines IFN-γ and TNF-α were elevated, and IgG1 and IgG2a serum levels were also significantly increased. TLR4, TLR7 and TLR9 agonists diminished the activation of Tregs and down-regulated the anti-inflammatory cytokines TGF-β and IL-10. Finally, PD-1 expressed on Th1 cells were decreased in TLR4, TLR7 and TLR9 agonist treated groups compared with control groups. ConclusionTLR4, TLR7 and TLR9 agonists activated DC-mediated innate immune responses and adaptive immune response, which against the blood-stage of Plasmodium and might be applied to malaria protection and treatment.

Sleep Disturbance during Infection Compromises Tfh Differentiation and Impacts Host Immunity.

SummaryAlthough the influence of sleep quality on the immune system is well documented, the mechanisms behind its impact on natural host immunity remain unclear. Meanwhile, it has been suggested that neuroimmune interactions play an important role in this phenomenon. To evaluate the impact of stress-induced sleep disturbance on host immunity, we used a murine model of rapid eye movement sleep deprivation (RSD) integrated with a model of malaria blood-stage infection. We demonstrate that sleep disturbance compromises the differentiation of T follicular helper cells, increasing host susceptibility to the parasite. Chemical inhibition of glucocorticoid (Glcs) synthesis showed that abnormal Glcs production compromised the transcription of Tfh-associated genes resulting in impaired germinal center formation and humoral immune response. Our data demonstrate that RSD-induced abnormal activation of the hypothalamic-pituitary-adrenal axis drives host susceptibility to infection. Understanding the impact of sleep quality in natural resistance to infection may provide insights for disease management.

A Plasmodium cysteine protease required for efficient transition from the liver infection stage

The transitions between developmental stages are critical points in the Plasmodium life cycle. The development of Plasmodium in the livers of their mammalian hosts bridges malaria transmission and the onset of clinical symptoms elicited by red blood cell infection. The egress of Plasmodium parasites from the liver must be a carefully orchestrated process to ensure a successful switch to the blood stage of infection. Cysteine protease activity is known to be required for liver-stage Plasmodium egress, but the crucial cysteine protease(s) remained unidentified. Here, we characterize a member of the papain-like cysteine protease family, Plasmodium berghei serine repeat antigen 4 (PbSERA4), that is required for efficient initiation of blood-stage infection. Through the generation PbSERA4-specific antisera and the creation of transgenic parasites expressing fluorescently tagged protein, we show that PbSERA4 is expressed and proteolytically processed in the liver and blood stages of infection. Targeted disruption of PbSERA4 results in viable and virulent blood-stage parasites. However, upon transmission from mosquitoes to mice, Pbsera4(-) parasites displayed a reduced capacity to initiate a new round of asexual blood-stage replication. Our results from cultured cells indicate that this defect results from an inability of the PbSERA4-deficient parasites to egress efficiently from infected cells at the culmination of liver-stage development. Protection against infection with wildtype P. berghei could be generated in animals in which Pbsera4(-) parasites failed to establish infection. Our findings confirm that liver-stage merozoite release is an active process and demonstrate that this parasite-encoded cysteine protease contributes to parasite escape from the liver.