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An abstract of the article "Interplay between AMA1 immunisation and parasite challenge leads to protection against malaria infection in rhesus macaques" that was presented at a conference titled "From Parasite to Prevention: Advances in the understanding of malaria" held in Edinburgh, Great Britain on October 20-22, 2010, is presented.

Apical Membrane Antigen 1 (AMA1) plays a vital role in the invasion of the host erythrocyte by the malaria parasite, Plasmodium . It is thus an important target for vaccine and anti-malaria therapeutic strategies that block the invasion process. AMA1, present on the surface of the parasite, interacts with RON2, a component of the parasite's rhoptry neck (RON) protein complex, which is transferred to the erythrocyte membrane during invasion. The D2 loop of AMA1 plays an essential role in invasion as it partially covers the RON2-binding site and must therefore be displaced for invasion to proceed. Several structural studies have shown that the D2 loop is very mobile, a property that is probably important for the function of AMA1. Here we present three crystal structures of AMA1 from P. falciparum (strains 3D7 and FVO) and P. vivax (strain Sal1), in which the D2 loop could be largely traced in the electron density maps. The D2 loop of PfAMA1-FVO and PvAMA1 (as a complex with a monoclonal antibody Fab) has a conformation previously noted in the P. knowlesi AMA1 structure. The D2 loop of PfAMA1-3D7, however, reveals a novel conformation. We analyse the conformational variability of the D2 loop in these structures, together with those previously reported. Three different conformations can be distinguished, all of which are highly helical and show some similarity in their secondary structure organisation. We discuss the significance of these observations in the light of the flexible nature of the D2 loop and its role in AMA1 function., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hypnozoites are dormant liver-stage parasites unique to relapsing malarial species, including the important human pathogen Plasmodium vivax , and pose a barrier to the elimination of malaria. Little is known regarding the biology of these stages, largely due to their inaccessible location. Hypnozoites can be cultured in vitro but these cultures always consist of a mixture of hepatocytes, developing forms, and hypnozoites. Here, using a GFP-expressing line of the hypnozoite model parasite Plasmodium cynomolgi , we describe a protocol for the FACS-based isolation of malarial hypnozoites. The purified hypnozoites can be used for a range of '-omics' studies to dissect the biology of this cryptic stage of the malarial life cycle., Competing Interests: Competing interestsThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis -derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Competing Interests: E.R., E.P., N.A., and C.M. are co-inventors on a patent on a tuberculosis vaccine held by the University of Zaragoza and Biofabri. E.R. and E.P. are employees of Biofabri. J.T. is an employee of the TuBerculosis Vaccine Initiative and an advisor to Biofabri. M.G.N. holds a patent on the inhibition of trained immunity with nanobiologics, and a patent on the stimulation of trained immunity with nanobiologics. M.G.N. is also a scientific founder of Trained Therapeutix and Discovery (TTxD). The remaining authors of the Radboud UMC and the authors from the BPRC declare no competing interests., (© 2020 The Author(s).)

To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis -derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies., Competing Interests: E.R., E.P., N.A., and C.M. are co-inventors on a patent-entitled tuberculosis vaccine held by the University of Zaragoza and Biofabri. E.R. and E.P. are employees of Biofabri. J.T. is an employee of TuBerculosis Vaccine Initiative and advisor to Biofabri., (© 2020 The Author(s).)

Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: In this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity., Results: AMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 - homologous to FVO AMA1 protein and NF54 - heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups., Conclusions: In brief, all seven vaccine - adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.

The recent research efforts to establish a Plasmodium vivax continuous, long-term blood-stage culture have focused on the ideal host cell type. However, this is only part of the story, as the P. vivax intraerythrocytic life cycle is complex. A successful, long-term, robust culture system will depend on a multifaceted approach combining the ideal cell type and parasite isolates, and the culture conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The deployment of a safe and effective malaria vaccine will be an important tool for the control of malaria and the reduction in malaria deaths. With the launch of the 2030 Malaria Vaccine Technology Roadmap, the malaria community has updated the goals and priorities for the development of such a vaccine and is now paving the way for a second phase of malaria vaccine development. During a workshop in Brussels in November 2014, hosted by the European Vaccine Initiative, key players from the European, North American and African malaria vaccine community discussed European strategies for future malaria vaccine development in the global context. The recommendations of the European malaria community should guide researchers, policy makers and funders of global health research and development in fulfilling the ambitious goals set in the updated Malaria Vaccine Technology Roadmap., (Copyright © 2015.)

The immune system plays a central role in the defense against environmental threats - such as infection with viruses, parasites or bacteria - but can also be a cause of disease, such as in the case of allergic or autoimmune disorders. In the past decades the impressive development of biotechnology has provided scientists with biological tools for the development of highly selective treatments for the different types of disorders. However, despite some clear successes the translation of scientific discoveries into effective treatments has remained challenging. The often-disappointing predictive validity of the preclinical animal models that are used in the selection of the most promising vaccine or drug candidates is the Achilles heel in the therapy development process. This publication summarizes the relevance and usage of non-human primates as pre-clinical model in infectious and autoimmune diseases, in particular for biologicals, which due to their high species-specificity are inactive in lower species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

To overcome polymorphism in the malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1), fusion protein chimeras comprised of three diversity-covering (DiCo) PfAMA1 molecules (D1, D2, and D3) and two allelic variants of the C-terminal 19-kDa region of merozoite surface protein 1 (MSP119) (variants M1 and M2) were generated. A mixture of fusion proteins (D1M1/D2M2D3) and the D1M1D2M2D3 fusion were compared to a single-unit mixture (D1/D2/D3/M1) in an immunological study in groups of rabbits. Following immunization, titers of antibodies (Abs) against four naturally occurring PfAMA1 alleles were high for all groups, as were growth inhibition assay (GIA) levels against two antigenically distinct laboratory parasite strains. Fusion of AMA1 to MSP119 did not suppress levels of antibodies against the AMA1 component. In addition, the breadth of antibody responses was unaffected. Anti-AMA1 antibodies were largely responsible for parasite growth inhibition, as shown in reversal-of-inhibition experiments by adding competing AMA1 antigen. For all groups, titration of the MSP119 antigen into the GIA led to only a small decrease in parasite inhibition, although titers of antibodies against MSP119 were increased 15-fold for the groups immunized with fusion proteins. GIA with affinity-purified anti-MSP119 antibodies showed that the 50% inhibitory concentrations of the anti-MSP119 antibody preparations were in the same order of magnitude for all animals tested, leading to the conclusion that fusing MSP119 to PfAMA1 leads to a small but significant increase in functional antibody levels. This study shows that combination of multiple vaccine candidates in fusion proteins may lead to improved characteristics of the vaccine.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

Long-term in vitro cultures of blood-stage parasites are so far feasible only for Plasmodium falciparum and P. knowlesi. In this chapter, we describe short-term ex vivo culturing of P. cynomolgi and P. vivax. We also describe long-term in vitro culturing of P. knowlesi as well as some techniques for synchronizing parasites. Cultured parasites can be used for a variety of purposes, e.g., for in vitro drug assays and antibody-mediated growth inhibition assays.

Background: Ivermectin (IVM) reduces the lifespan of malaria-transmitting mosquitoes after feeding on humans treated with IVM. If this effect is sufficiently long and strong, IVM could form part of a drug combination that not only treats malaria patients but also reduces onward transmission. Limited data are available on the exact duration of the mosquitocidal effect of IVM; daily mosquito feeding assays are required for this., Materials and Methods: We determined mortality rates of Anopheles stephensi mosquitoes that took a blood meal on Swiss mice, Wistar rats and Cynomolgus monkeys that received IVM orally at 200-400 μg/kg. Mosquito feeding assays were performed on five consecutive days after IVM administration. Mosquito mortality was determined in the first 72 hours after feeding., Results: Mosquito mortality was 70-100% when mosquitoes fed on any of the animals 1-2 days after the last IVM administration. After this time-point the mosquitocidal effect was still evident in some animals but became more variable., Conclusions: Our findings of a pronounced but short-lived mosquitocidal effect makes the timing of IVM administration crucial to form a useful addition to anti-malarial drugs., Competing Interests: Competing interests: No competing interests declared., (Copyright © 2012: Bastiaens et al.)

Background: Establishing antibody correlates of protection against malaria in human field studies and clinical trials requires, amongst others, an accurate estimation of antibody levels. For polymorphic antigens such as apical membrane antigen 1 (AMA1), this may be confounded by the occurrence of a large number of allelic variants in nature., Methods: To test this hypothesis, plasma antibody levels in an age-stratified cohort of naturally exposed children from a malaria-endemic area in Southern Ghana were determined by indirect ELISA. Titres against four single PfAMA1 alleles were compared with those against three different allele mixtures presumed to have a wider repertoire of epitope specificities. Associations of antibody levels with the incidence of clinical malaria as well as with previous exposure to parasites were also examined., Results: Antibody titres against PfAMA1 alleles generally increased with age/exposure while antibody specificity for PfAMA1 variants decreased, implying that younger children (≤ 5 years) elicit a more strain-specific antibody response compared to older children. Antibody titre measurements against the FVO and 3D7 AMA1 alleles gave the best titre estimates as these varied least in pair-wise comparisons with titres against all PfAMA1 allele mixtures. There was no association between antibody levels against any capture antigen and either clinical malaria incidence or parasite density., Conclusions: The current data shows that levels of naturally acquired antigen-specific antibodies, especially in infants and young children, are dependent on the antigenic allele used for measurement. This may be relevant to the interpretation of antibody titre data from measurements against single PfAMA1 alleles, especially in studies involving infants and young children who have experienced fewer infections.

Adherence of parasite-infected red blood cells (irbc) to the vascular endothelium of organs plays a key role in the pathogenesis of Plasmodium falciparum malaria. The prevailing hypothesis of why irbc adhere and sequester in tissues is that this acts as a mechanism of avoiding spleen-mediated clearance. Irbc of the rodent parasite Plasmodium berghei ANKA sequester in a fashion analogous to P. falciparum by adhering to the host receptor CD36. To experimentally determine the significance of sequestration for parasite growth, we generated a mutant P. berghei ANKA parasite with a reduced CD36-mediated adherence. Although the cognate parasite ligand binding to CD36 is unknown, we show that nonsequestering parasites have reduced growth and we provide evidence that in addition to avoiding spleen removal, other factors related to CD36-mediated sequestration are beneficial for parasite growth. These results reveal for the first time the importance of sequestration to a malaria infection, with implications for the development of strategies aimed at reducing pathology by inhibiting tissue sequestration.

Background: Increasing the breadth of the functional antibody response through immunization with Plasmodium falciparum apical membrane antigen 1 (PfAMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three PfAMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a PfAMA1-MSP1₁₉ fusion protein formulated in Montanide ISA 51., Methods: Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and in vitro parasite growth inhibition assays with three culture-adapted P. falciparum strains., Results: These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons., Conclusions: The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.

Cytidine diphosphate diacylglycerol synthase (CDS) diverts phosphatidic acid towards the biosynthesis of CDP-DAG, an obligatory liponucleotide intermediate in anionic phospholipid biosynthesis. The 78kDa predicted Plasmodium falciparum CDS (PfCDS) is recovered as a 50 kDa conserved C-terminal cytidylyltransferase domain (C-PfCDS) and a 28kDa fragment that corresponds to the unusually long hydrophilic asparagine-rich N-terminal extension (N-PfCDS). Here, we show that the two fragments of PfCDS are the processed forms of the 78 kDa pro-form that is encoded from a single transcript with no alternate translation start site for C-PfCDS. PfCDS, which shares 54% sequence identity with Plasmodium knowlesi CDS (PkCDS), could substitute for PkCDS in P. knowlesi. Experiments to disrupt either the full-length or the N-terminal extension of PkCDS indicate that not only the C-terminal cytidylyltransferase domain but also the N-terminal extension is essential to Plasmodium spp. PkCDS and PfCDS introduced in P. knowlesi were processed in the parasite, suggesting a conserved parasite-dependent mechanism. The N-PfCDS appears to be a peripheral membrane protein and is trafficked outside the parasite to the parasitophorous vacuole. Although the function of this unusual N-PfCDS remains enigmatic, the study here highlights features of this essential gene and its biological importance during the intra-erythrocytic cycle of the parasite., (Copyright (c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Plasmodium knowlesi is a primate malaria parasite that is phylogenetically close to the major human parasite Plasmodium vivax. P. knowlesi causes life-threatening disease in humans, infects a wide range of non-human primates and is one of few malaria parasites amenable to cyclical in vitro propagation. A robust in vivo and in vitro genetic manipulation system has been developed for this parasite, enabling in vitro-in vivo shuttling of transgenes, which (together with recent characterization of its genome and that of its macaque experimental host) offers unique opportunities to gain insight in molecular function and parasite-host interactions.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.

A new type of piroplasm, phylogenetically closest to Babesia microti-like parasites previously detected in Eurasian red squirrels (Sciurus vulgaris orientis), was identified in a rhesus monkey (Macaca mulatta) imported from China. After challenge with Plasmodium cynomolgi M strain blood-stage parasites, the rhesus monkey repeatedly showed markedly reduced levels of Plasmodium parasitemia when compared with animals not infected with this organism.

Apical membrane antigen 1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. Immune responses to Plasmodium AMA1 can have profound parasite-inhibitory effects, both as measured in vitro and in animal challenge models, suggesting AMA1 as a potential vaccine component. However, AMA1 is polymorphic, probably as a result of immune selection operating on an important target of naturally occurring immunity. The current understanding of AMA1 will be presented, particularly in relation to the vaccine potential of AMA1 and the approaches being taken towards clinical development.

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.

Apical membrane antigen 1 (AMA1) is a membrane protein present in Plasmodium species and is probably common to all apicomplexan parasites. The recent crystal structure of the complete ectoplasmic region of AMA1 from Plasmodium vivax has shown that it comprises three structural domains and that the first two domains are based on the PAN folding motif. Here, we discuss the consequences of this analysis for the three-dimensional structure of AMA1 from other Plasmodium species and other apicomplexan parasites, and for the Plasmodium paralogue MAEBL. Many polar and apolar interactions observed in the PvAMA1 crystal structure are made by residues that are invariant or highly conserved throughout all Plasmodium orthologues; a subgroup of these residues is also present in other apicomplexan orthologues and in MAEBL. These interactions presumably play a key role in defining the protein fold. Previous studies have shown that the ectoplasmic region of AMA1 must be cleaved from the parasite surface for host-cell invasion to proceed. The cleavage site in the crystal structure is not readily accessible to proteases and we discuss possible consequences of this observation. The three-dimensional distribution of polymorphic sites in PfAMA1 shows that these are all on the surface and that their positions are significantly biased to one side of the ectoplasmic region. Of particular note, a flexible segment in domain II, comprising about 40 residues and devoid of polymorphism, carries an epitope recognized by an invasion-inhibitory monoclonal antibody and a T-cell epitope implicated in the human immune response to AMA1.

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.

Serum antibodies from 1071 people in two Kenyan villages were assayed using eight different recombinant Apical Membrane Antigen 1 (AMA1) protein constructs to investigate their role in naturally acquired immunity. In both communities, antibodies against the full-length ectodomain (both FVO and 3D7 allele constructs) prior to a malaria transmission season were significantly associated with protection from malaria in the following 6 months, even after adjusting for age and antibody reactivity to whole parasite (schizont) extract. However, these protective associations of antibodies were only seen among subjects that were parasite slide positive at the time of pre-season serum sampling. Competition ELISAs with the FVO and 3D7 allele constructs showed that antibodies can recognise either conserved or allele-specific epitopes in AMA1. Results encourage the development of an AMA1 vaccine based on the full-length ectodomain, and indicate that the function of human antibodies to allele-specific and conserved epitopes in AMA1 should be studied further.

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.

Plasmodium knowlesi provides a highly versatile transfection system for malaria, since it enables rapid genetic modification of the parasite both in vivo as well as in vitro. However, it is not possible to perform multiple genetic manipulations within one parasite line because of a lack of selectable markers. In an effort to develop additional selectable markers for this parasite, positive and negative selectable markers that have recently been successfully used in Plasmodium falciparum were tested. It was shown that the positive selectable markers human dihydrofolate reductase (hdhfr), blasticidin S deaminase (bsd) and neomycin phosphotransferase II (neo) all conferred drug resistance to P. knowlesi when introduced as episomes. The plasmid containing the hdhfr selectable marker was not only successfully introduced as circular form, but also as linear fragment, demonstrating for the first time single crossover integration in P. knowlesi. Thymidine kinase was tested for its potential as negative selectable marker and it was shown that recombinant P. knowlesi parasites expressing thymidine kinase from episomes were highly sensitive to ganciclovir compared to wild-type P. knowlesi. The availability of new positive selectable markers and a strong candidate for a negative selectable marker for P. knowlesi, in combination with the opportunity to perform targeted single crossover integration in P. knowlesi, significantly increases the flexibility of this transfection system, making it one of the most versatile systems available for Plasmodium.

Chemotherapy of apicomplexan parasites is limited by emerging drug resistance or lack of novel targets. PfHT1, the Plasmodium falciparum hexose transporter 1, is a promising new drug target because asexual-stage malarial parasites depend wholly on glucose for energy. We have performed a comparative functional characterization of PfHT1 and hexose transporters of the simian malarial parasite P. knowlesi (PkHT1), the rodent parasite P. yoelii (PyHT1) and the human apicomplexan parasite Toxoplasma gondii ( T. gondii glucose transporter 1, TgGT1). PkHT1 and PyHT1 share >70% amino acid identity with PfHT1, while TgGT1 is more divergent (37.2% identity). All transporters mediate uptake of D-glucose and D-fructose. PyHT1 has an affinity for glucose ( K (m) approximately 0.12 mM) that is higher than that for PkHT1 ( K (m) approximately 0.67 mM) or PfHT1 ( K (m) approximately 1 mM). TgGT1 is highly temperature dependent (the Q (10) value, the fold change in activity for a 10 degrees C change in temperature, was >7) compared with Plasmodium transporters ( Q (10), 1.5-2.5), and overall has the highest affinity for glucose ( K (m) approximately 30 microM). Using active analogues in competition for glucose uptake, experiments show that hydroxyl groups at the C-3, C-4 and C-6 positions are important in interacting with PkHT1, PyHT1 and TgGT1. This study defines models useful to study the biology of apicomplexan hexose permeation pathways, as well as contributing to drug development.