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    Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria
    Studniberg, S ; Ioannidis, LJ ; Utami, RAS ; Trianty, L ; Liao, Y ; Abeysekera, W ; Li-Wai-Suen, CSN ; Pietrzak, HM ; Healer, J ; Puspitasari, AM ; Apriyanti, D ; Coutrier, F ; Poespoprodjo, JR ; Kenangalem, E ; Andries, B ; Prayoga, P ; Sariyanti, N ; Smyth, GK ; Cowman, AF ; Price, RN ; Noviyanti, R ; Shi, W ; Garnham, AL ; Hansen, DS (WILEY, 2022-04-01)
    Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.
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    RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes
    Pasternak, M ; Verhoef, JMJ ; Wong, W ; Triglia, T ; Mlodzianoski, MJ ; Geoghegan, N ; Evelyn, C ; Wardak, AZ ; Rogers, K ; Cowmarc, AF (NATURE PORTFOLIO, 2022-04-07)
    RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.
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    Safety, infectivity and immunogenicity of a Chick for genetically attenuated blood-stage malaria updates vaccine
    Webster, R ; Sekuloski, S ; Odedra, A ; Woolley, S ; Jennings, H ; Amante, F ; Trenholme, KR ; Healer, J ; Cowman, AF ; Eriksson, EM ; Sathe, P ; Penington, J ; Blanch, AJ ; Dixon, MWA ; Tilley, L ; Duffy, MF ; Craig, A ; Storm, J ; Chan, J-A ; Evans, K ; Papenfuss, AT ; Schofield, L ; Griffin, P ; Barber, BE ; Andrew, D ; Boyle, MJ ; Rivera, FDL ; Engwerda, C ; McCarthy, JS (BMC, 2021-11-22)
    BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).
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    AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver
    Yang, ASP ; Lopaticki, S ; O'Neill, MT ; Erickson, SM ; Douglas, DN ; Kneteman, NM ; Boddey, JA (WILEY, 2017-09-01)
    The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA-like protein, merozoite apical erythrocyte-binding ligand (MAEBL). MAEBL-deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.
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    High-dimensional mass cytomet identifies T cell and B cell signatures predicting reduced risk of Plasmodium vivax malaria
    Ioannidis, LJ ; Pietrzak, HM ; Ly, A ; Utami, RA ; Eriksson, EM ; Studniberg, S ; Abeysekera, W ; Li-Wai-Suen, CSN ; Sheerin, D ; Healer, J ; Puspitasari, AM ; Apriyanti, D ; Coutrier, FN ; Poespoprodjo, JR ; Kenangalem, E ; Andries, B ; Prayoga, P ; Sariyanti, N ; Smyth, GK ; Trianty, L ; Cowman, AF ; Price, RN ; Noviyanti, R ; Hansen, DS (AMER SOC CLINICAL INVESTIGATION INC, 2021-07-22)
    IFN-γ-driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence of their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single-cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax (P. vivax) malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that, whereas class-switched but not IgM+ MBCs were associated with a reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells, and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell-mediated immunity might also be required to control persistent P. vivax infection with low parasite burden.
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    4D analysis of malaria parasite invasion offers insights into erythrocyte membrane remodeling and parasitophorous vacuole formation
    Geoghegan, ND ; Evelyn, C ; Whitehead, LW ; Pasternak, M ; McDonald, P ; Triglia, T ; Marapana, DS ; Kempe, D ; Thompson, JK ; Mlodzianoski, MJ ; Healer, J ; Biro, M ; Cowman, AF ; Rogers, KL (NATURE RESEARCH, 2021-06-15)
    Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens.
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    Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle
    Favuzza, P ; Ruiz, MDL ; Thompson, JK ; Triglia, T ; Ngo, A ; Steel, RWJ ; Vavrek, M ; Christensen, J ; Healer, J ; Boyce, C ; Guo, Z ; Hu, M ; Khan, T ; Murgolo, N ; Zhao, L ; Penington, JS ; Reaksudsan, K ; Jarman, K ; Dietrich, MH ; Richardson, L ; Guo, K-Y ; Lopaticki, S ; Tham, W-H ; Rottmann, M ; Papenfuss, T ; Robbins, JA ; Boddey, JA ; Sleebs, BE ; Sabroux, HJ ; McCauley, JA ; Olsen, DB ; Cowman, AF (CELL PRESS, 2020-04-08)
    Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.
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    A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial
    Pawliw, R ; Farrow, R ; Sekuloski, S ; Jennings, H ; Healer, J ; Thuan, P ; Sathe, P ; Pasay, C ; Evans, K ; Cowman, AF ; Schofield, L ; Chen, N ; McCarthy, J ; Trenholme, K (BMC, 2018-08-06)
    BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)-compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.
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    Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum
    Voss, TS ; Tonkin, CJ ; Marty, AJ ; Thompson, JK ; Healer, J ; Crabb, BS ; Cowman, AF (WILEY-BLACKWELL, 2007-10-01)
    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.
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    RAPID UP-REGULATION OF MDR1 EXPRESSION BY ANTHRACYCLINES IN A CLASSICAL MULTIDRUG-RESISTANT CELL-LINE
    HU, XF ; SLATER, A ; WALL, DM ; KANTHARIDIS, P ; PARKIN, JD ; COWMAN, A ; ZALCBERG, JR (STOCKTON PRESS, 1995-05-01)
    Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp). The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin. The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.