Proteomes of plasmodium knowlesi early and late ring-stage parasites and infected host erythrocytes.

The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins.

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Systems biology of malaria explored with nonhuman primates.

"The Primate Malarias" book has been a uniquely important resource for multiple generations of scientists, since its debut in 1971, and remains pertinent to the present day. Indeed, nonhuman primates (NHPs) have been instrumental for major breakthroughs in basic and pre-clinical research on malaria for over 50 years. Research involving NHPs have provided critical insights and data that have been essential for malaria research on many parasite species, drugs, vaccines, pathogenesis, and transmission, leading to improved clinical care and advancing research goals for malaria control, elimination, and eradication. Whilst most malaria scientists over the decades have been studying Plasmodium falciparum, with NHP infections, in clinical studies with humans, or using in vitro culture or rodent model systems, others have been dedicated to advancing research on Plasmodium vivax, as well as on phylogenetically related simian species, including Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium knowlesi. In-depth study of these four phylogenetically related species over the years has spawned the design of NHP longitudinal infection strategies for gathering information about ongoing infections, which can be related to human infections. These Plasmodium-NHP infection model systems are reviewed here, with emphasis on modern systems biological approaches to studying longitudinal infections, pathogenesis, immunity, and vaccines. Recent discoveries capitalizing on NHP longitudinal infections include an advanced understanding of chronic infections, relapses, anaemia, and immune memory. With quickly emerging new technological advances, more in-depth research and mechanistic discoveries can be anticipated on these and additional critical topics, including hypnozoite biology, antigenic variation, gametocyte transmission, bone marrow dysfunction, and loss of uninfected RBCs. New strategies and insights published by the Malaria Host-Pathogen Interaction Center (MaHPIC) are recapped here along with a vision that stresses the importance of educating future experts well trained in utilizing NHP infection model systems for the pursuit of innovative, effective interventions against malaria.

Malaria disrupts the rhesus macaque gut microbiome.

Previous studies have suggested that a relationship exists between severity and transmissibility of malaria and variations in the gut microbiome, yet only limited information exists on the temporal dynamics of the gut microbial community during a malarial infection. Here, using a rhesus macaque model of relapsing malaria, we investigate how malaria affects the gut microbiome. In this study, we performed 16S sequencing on DNA isolated from rectal swabs of rhesus macaques over the course of an experimental malarial infection with Plasmodium cynomolgi and analyzed gut bacterial taxa abundance across primary and relapsing infections. We also performed metabolomics on blood plasma from the animals at the same timepoints and investigated changes in metabolic pathways over time. Members of Proteobacteria (family Helicobacteraceae) increased dramatically in relative abundance in the animal's gut microbiome during peak infection while Firmicutes (family Lactobacillaceae and Ruminococcaceae), Bacteroidetes (family Prevotellaceae) and Spirochaetes amongst others decreased compared to baseline levels. Alpha diversity metrics indicated decreased microbiome diversity at the peak of parasitemia, followed by restoration of diversity post-treatment. Comparison with healthy subjects suggested that the rectal microbiome during acute malaria is enriched with commensal bacteria typically found in the healthy animal's mucosa. Significant changes in the tryptophan-kynurenine immunomodulatory pathway were detected at peak infection with P. cynomolgi, a finding that has been described previously in the context of P. vivax infections in humans. During relapses, which have been shown to be associated with less inflammation and clinical severity, we observed minimal disruption to the gut microbiome, despite parasites being present. Altogether, these data suggest that the metabolic shift occurring during acute infection is associated with a concomitant shift in the gut microbiome, which is reversed post-treatment.

Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesi.

BACKGROUND: Kra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype. METHODS: Kra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections during acute and chronic infections. RESULTS: As expected, the kra monkeys controlled acute infections and remained with low-level, persistent parasitaemias without anti-malarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopenia, and moderate to severe anaemia. Mathematical modelling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low. CONCLUSIONS: Kra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3-5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.

Dramatic transcriptomic differences in Macaca mulatta and Macaca fascicularis with Plasmodium knowlesi infections.

Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which also may be relevant across Plasmodium species. Here we contrast P. knowlesi sporozoite-initiated infections in Macaca mulatta and Macaca fascicularis using whole blood RNA-sequencing and transcriptomic analysis. These macaque hosts are evolutionarily close, yet malaria-naïve M. mulatta will succumb to blood-stage infection without treatment, whereas malaria-naïve M. fascicularis controls parasitemia without treatment. This comparative analysis reveals transcriptomic differences as early as the liver phase of infection, in the form of signaling pathways that are activated in M. fascicularis, but not M. mulatta. Additionally, while most immune responses are initially similar during the acute stage of the blood infection, significant differences arise subsequently. The observed differences point to prolonged inflammation and anti-inflammatory effects of IL10 in M. mulatta, while M. fascicularis undergoes a transcriptional makeover towards cell proliferation, consistent with its recovery. Together, these findings suggest that timely detection of P. knowlesi in M. fascicularis, coupled with control of inflammation while initiating the replenishment of key cell populations, helps contain the infection. Overall, this study points to specific genes and pathways that could be investigated as a basis for new drug targets that support recovery from acute malaria.

Dynamic Control Balancing Cell Proliferation and Inflammation is Crucial for an Effective Immune Response to Malaria.

Malaria has a complex pathology with varying manifestations and symptoms, effects on host tissues, and different degrees of severity and ultimate outcome, depending on the causative Plasmodium pathogen and host species. Previously, we compared the peripheral blood transcriptomes of two macaque species (Macaca mulatta and Macaca fascicularis) in response to acute primary infection by Plasmodium knowlesi. Although these two species are very closely related, the infection in M. mulatta is fatal, unless aggressively treated, whereas M. fascicularis develops a chronic, but tolerable infection in the blood. As a reason for this stark difference, our analysis suggests delayed pathogen detection in M. mulatta followed by extended inflammation that eventually overwhelms this monkey's immune response. By contrast, the natural host M. fascicularis detects the pathogen earlier and controls the inflammation. Additionally, M. fascicularis limits cell proliferation pathways during the log phase of infection, presumably in an attempt to control inflammation. Subsequent cell proliferation suggests a cell-mediated adaptive immune response. Here, we focus on molecular mechanisms underlying the key differences in the host and parasite responses and their coordination. SICAvar Type 1 surface antigens are highly correlated with pattern recognition receptor signaling and important inflammatory genes for both hosts. Analysis of pathogen detection pathways reveals a similar signaling mechanism, but with important differences in the glutamate G-protein coupled receptor (GPCR) signaling pathway. Furthermore, differences in inflammasome assembly processes suggests an important role of S100 proteins in balancing inflammation and cell proliferation. Both differences point to the importance of Ca2+ homeostasis in inflammation. Additionally, the kynurenine-to-tryptophan ratio, a known inflammatory biomarker, emphasizes higher inflammation in M. mulatta during log phase. Transcriptomics-aided metabolic modeling provides a functional method for evaluating these changes and understanding downstream changes in NAD metabolism and aryl hydrocarbon receptor (AhR) signaling, with enhanced NAD metabolism in M. fascicularis and stronger AhR signaling in M. mulatta. AhR signaling controls important immune genes like IL6, IFNγ and IDO1. However, direct changes due to AhR signaling could not be established due to complicated regulatory feedback mechanisms associated with the AhR repressor (AhRR). A complete understanding of the exact dynamics of the immune response is difficult to achieve. Nonetheless, our comparative analysis provides clear suggestions of processes that underlie an effective immune response. Thus, our study identifies multiple points of intervention that are apparently responsible for a balanced and effective immune response and thereby paves the way toward future immune strategies for treating malaria.

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.

Acute Plasmodium Infection Promotes Interferon-Gamma-Dependent Resistance to Ebola Virus Infection.

During the 2013-2016 Ebola virus (EBOV) epidemic, a significant number of patients admitted to Ebola treatment units were co-infected with Plasmodium falciparum, a predominant agent of malaria. However, there is no consensus on how malaria impacts EBOV infection. The effect of acute Plasmodium infection on EBOV challenge was investigated using mouse-adapted EBOV and a biosafety level 2 (BSL-2) model virus. We demonstrate that acute Plasmodium infection protects from lethal viral challenge, dependent upon interferon gamma (IFN-γ) elicited as a result of parasite infection. Plasmodium-infected mice lacking the IFN-γ receptor are not protected. Ex vivo incubation of naive human or mouse macrophages with sera from acutely parasitemic rodents or macaques programs a proinflammatory phenotype dependent on IFN-γ and renders cells resistant to EBOV infection. We conclude that acute Plasmodium infection can safeguard against EBOV by the production of protective IFN-γ. These findings have implications for anti-malaria therapies administered during episodic EBOV outbreaks in Africa.

Humoral immunity prevents clinical malaria during Plasmodium relapses without eliminating gametocytes.

Plasmodium relapses are attributed to the activation of dormant liver-stage parasites and are responsible for a significant number of recurring malaria blood-stage infections. While characteristic of human infections caused by P. vivax and P. ovale, their relative contribution to malaria disease burden and transmission remains poorly understood. This is largely because it is difficult to identify 'bona fide' relapse infections due to ongoing transmission in most endemic areas. Here, we use the P. cynomolgi-rhesus macaque model of relapsing malaria to demonstrate that clinical immunity can form after a single sporozoite-initiated blood-stage infection and prevent illness during relapses and homologous reinfections. By integrating data from whole blood RNA-sequencing, flow cytometry, P. cynomolgi-specific ELISAs, and opsonic phagocytosis assays, we demonstrate that this immunity is associated with a rapid recall response by memory B cells that expand and produce anti-parasite IgG1 that can mediate parasite clearance of relapsing parasites. The reduction in parasitemia during relapses was mirrored by a reduction in the total number of circulating gametocytes, but importantly, the cumulative proportion of gametocytes increased during relapses. Overall, this study reveals that P. cynomolgi relapse infections can be clinically silent in macaques due to rapid memory B cell responses that help to clear asexual-stage parasites but still carry gametocytes.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3α and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M.

BACKGROUND: Complement-fixing antibodies are important mediators of protection against Plasmodium falciparum malaria. However, complement-fixing antibodies remain uncharacterized for Plasmodium vivax malaria. P. vivax merozoite surface protein 3α (PvMSP3α) is a target of acquired immunity and a potential vaccine candidate. METHODS: Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay. RESULTS: The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28. CONCLUSION: Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.

Functional genomics of simian malaria parasites and host-parasite interactions.

Two simian malaria parasite species, Plasmodium knowlesi and Plasmodium cynomolgi, cause zoonotic infections in Southeast Asia, and they have therefore gained recognition among scientists and public health officials. Notwithstanding, these species and others including Plasmodium coatneyi have served for decades as sources of knowledge on the biology, genetics and evolution of Plasmodium, and the diverse ramifications and outcomes of malaria in their monkey hosts. Experimental analysis of these species can help to fill gaps in knowledge beyond what may be possible studying the human malaria parasites or rodent parasite species. The genome sequences for these simian malaria parasite species were reported during the last decade, and functional genomics research has since been pursued. Here research on the functional genomics analysis involving these species is summarized and their importance is stressed, particularly for understanding host-parasite interactions, and potentially testing novel interventions. Importantly, while Plasmodium falciparum and Plasmodium vivax can be studied in small New World monkeys, the simian malaria parasites can be studied more effectively in the larger Old World monkey macaque hosts, which are more closely related to humans. In addition to ex vivo analyses, experimental scenarios can include passage through Anopheline mosquito hosts and longitudinal infections in monkeys to study acute and chronic infections, as well as relapses, all in the context of the in vivo host environment. Such experiments provide opportunities for understanding functional genomic elements that govern host-parasite interactions, immunity and pathogenesis in-depth, addressing hypotheses not possible from in vitro cultures or cross-sectional clinical studies with humans.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria.

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

Plasmodium vivax Parasite Load Is Associated With Histopathology in Saimiri boliviensis With Findings Comparable to P vivax Pathogenesis in Humans.

BACKGROUND: Plasmodium vivax can cause severe malaria with multisystem organ dysfunction and death. Clinical reports suggest that parasite accumulation in tissues may contribute to pathogenesis and disease severity, but direct evidence is scarce. METHODS: We present quantitative parasitological and histopathological analyses of tissue sections from a cohort of naive, mostly splenectomized Saimiri boliviensis infected with P vivax to define the relationship of tissue parasite load and histopathology. RESULTS: The lung, liver, and kidney showed the most tissue injury, with pathological presentations similar to observations reported from autopsies. Parasite loads correlated with the degree of histopathologic changes in the lung and liver tissues. In contrast, kidney damage was not associated directly with parasite load but with the presence of hemozoin, an inflammatory parasite byproduct. CONCLUSIONS: This analysis supports the use of the S boliviensis infection model for performing detailed histopathological studies to better understand and potentially design interventions to treat serious clinical manifestations caused by P vivax.

Comparative 3D genome organization in apicomplexan parasites.

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Analysis of erythrocyte dynamics in Rhesus macaque monkeys during infection with Plasmodium cynomolgi.

BACKGROUND: Malaria is a major mosquito transmitted, blood-borne parasitic disease that afflicts humans. The disease causes anaemia and other clinical complications, which can lead to death. Plasmodium vivax is known for its reticulocyte host cell specificity, but many gaps in disease details remain. Much less is known about the closely related species, Plasmodium cynomolgi, although it is naturally acquired and causes zoonotic malaria. Here, a computational model is developed based on longitudinal analyses of P. cynomolgi infections in nonhuman primates to investigate the erythrocyte dynamics that is pertinent to understanding both P. cynomolgi and P. vivax malaria in humans. METHODS: A cohort of five P. cynomolgi infected Rhesus macaques (Macaca mulatta) is studied, with individuals exhibiting a plethora of clinical outcomes, including varying levels of anaemia. A discrete recursive model with age structure is developed to replicate the dynamics of P. cynomolgi blood-stage infections. The model allows for parasitic reticulocyte preference and assumes an age preference among the mature RBCs. RBC senescence is modelled using a hazard function, according to which RBCs have a mean lifespan of 98 ± 21 days. RESULTS: Based on in vivo data from three cohorts of macaques, the computational model is used to characterize the reticulocyte lifespan in circulation as 24 ± 5 h (n = 15) and the rate of RBC production as 2727 ± 209 cells/h/µL (n = 15). Analysis of the host responses reveals a pre-patency increase in the number of reticulocytes. It also allows the quantification of RBC removal through the bystander effect. CONCLUSIONS: The evident pre-patency increase in reticulocytes is due to a shift towards the release of younger reticulocytes, which could result from a parasite-induced factor meant to increase reticulocyte availability and satisfy the parasite's tropism, which has an average value of 32:1 in this cohort. The number of RBCs lost due to the bystander effect relative to infection-induced RBC losses is 62% for P. cynomolgi infections, which is substantially lower than the value of 95% previously determined for another simian species, Plasmodium coatneyi.

Reproductive Longevity Predicts Mutation Rates in Primates.

Mutation rates vary between species across several orders of magnitude, with larger organisms having the highest per-generation mutation rates. Hypotheses for this pattern typically invoke physiological or population-genetic constraints imposed on the molecular machinery preventing mutations [1]. However, continuing germline cell division in multicellular eukaryotes means that organisms with longer generation times and of larger size will leave more mutations to their offspring simply as a byproduct of their increased lifespan [2, 3]. Here, we deeply sequence the genomes of 30 owl monkeys (Aotus nancymaae) from six multi-generation pedigrees to demonstrate that paternal age is the major factor determining the number of de novo mutations in this species. We find that owl monkeys have an average mutation rate of 0.81 × 10-8 per site per generation, roughly 32% lower than the estimate in humans. Based on a simple model of reproductive longevity that does not require any changes to the mutational machinery, we show that this is the expected mutation rate in owl monkeys. We further demonstrate that our model predicts species-specific mutation rates in other primates, including study-specific mutation rates in humans based on the average paternal age. Our results suggest that variation in life history traits alone can explain variation in the per-generation mutation rate among primates, and perhaps among a wide range of multicellular organisms.