Structural, Functional and Computational Studies of Membrane Recognition by Plasmodium Perforin-Like Proteins 1 and 2.

Perforin-like proteins (PLPs) play key roles in mechanisms associated with parasitic disease caused by the apicomplexan parasites Plasmodium and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1. Determination of the crystal structure of the membrane-binding APCß domain of PvPLP1 reveals notable differences with TgPLP1, reflected in its inability to bind lipid bilayers as TgPLP1 and PvPLP2 do. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal similar tropisms for lipids enriched in the inner leaflet of the mammalian plasma membrane. In addition PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCß domains of apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins in parasitic life cycles and disease.

Six-year trend analysis of malaria prevalence at University of Gondar Specialized Referral Hospital, Northwest Ethiopia, from 2014 to 2019.

Globally, malaria is the major public health disease caused by plasmodium species and transmitted by the bite of the female anopheles mosquito. Assessment of the trend of malaria prevalence is important in the control and prevention of the disease. Therefore, the objective of this study was to assess the six year trend of malaria prevalence at the University of Gondar Comprehensive Specialized Hospital, northwest Ethiopia, from 2014 to 2019. A retrospective laboratory registration logbook review study was conducted on the malaria blood film examination results at the University of Gondar Comprehensive Specialized Hospital. The data was collected by using a data extraction tool and entered into SPSS version 20 for analysis. Descriptive statistics were used to summarize the socio-demographic characteristics of study participants and presented by graphs, tables and texts. The binary logistic regression was also used to test the association the trend of malaria prevalence and different factors like sex, age, year, and season. From a total of 17,500 malaria blood film examinations, 1341 (7.7%) were confirmed for malaria parasites. Of the confirmed malaria cases, 47.2%, 45.6% and 7.2% were P. vivax, P. falciparum and mixed infection, respectively. The proportion of P. vivax was the predominant species in the first three study years (2014-2016) and P. falciparum became the predominant species in the last three study years (2017-2019). The odds of malaria prevalence was lower by 68%, 60% and 69% in the year 2017, 2018 and 2019 compared to 2014, respectively. It was also 1.41 times higher in males than in females. Moreover, the odds of malaria prevalence were 1.60, 1.64, 2.45 and 1.82 times higher in the age group of < 5, 5-14, 15-24 and 25-54 years old compared to the older age groups (> 54 years old), respectively. Even there was a significant declining in prevalence trend; malaria is still a major public health problem. The study showed that there was high seasonal fluctuation from year to year. Moreover, males and the younger age groups were more affected than females and old age groups, respectively. Therefore, malaria prevention and control activities should be strengthened and require extra efforts by considering these variability.

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

Plasmodium vivax pre-erythrocytic stages and the latent hypnozoite.

Plasmodium vivax is the most geographically widespread malaria parasite on the planet. This is largely because after mosquito transmission, P. vivax sporozoites can invade hepatocytes and form latent liver stages known as hypnozoites. These persistent liver stages can activate weeks, months or even years after an infected individual suffers a primary clinical infection. Activation then leads to replication and liver stage schizont maturation that ultimately cause relapse of blood stage infection, disease, and onward transmission. Thus, the latent hypnozoite can lie in wait during times when onward transmission is unlikely due to conditions that do not favor the mosquito. For example, in temperate climates where mosquito prevalence is only seasonal. Furthermore, the elimination of hypnozoites is challenging since the hypnozoite reservoir is currently undetectable and not killed by most antimalarial drugs. Here, we review our current knowledge of the pre-erythrocytic stages of the malaria parasite - the sporozoite and liver stages, including the elusive and enigmatic hypnozoite. We focus on our understanding of sporozoite biology, the novel animal models that are available to study the hypnozoite and hypnozoite activation and the ongoing efforts to understand the biological makeup of the hypnozoite that allow for its persistence in the human host.

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities.

BACKGROUND: Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission. METHODOLOGY/PRINCIPAL FINDINGS: Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44-94% for P. falciparum and from 23-72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37-100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398). CONCLUSIONS/SIGNIFICANCE: Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.

Diversity and natural selection of Merozoite surface Protein-1 in three species of human malaria parasites: Contribution from South-East Asian isolates.

The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.

Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India.

BACKGROUND: Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. METHODS: Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. RESULTS: Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. CONCLUSIONS: Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

Partial protection against P. vivax infection diminishes hypnozoite burden and blood-stage relapses.

Latent forms of Plasmodium vivax, called hypnozoites, cause malaria relapses from the liver into the bloodstream and are a major obstacle to malaria eradication. To experimentally assess the impact of a partially protective pre-erythrocytic vaccine on reducing Plasmodium vivax relapses, we developed a liver-humanized mouse model that allows monitoring of relapses directly in the blood. We passively infused these mice with a suboptimal dose of an antibody that targets the circumsporozoite protein prior to challenge with P. vivax sporozoites. Although this regimen did not completely prevent primary infection, antibody-treated mice experienced 62% fewer relapses. The data constitute unprecedented direct experimental evidence that suboptimal efficacy of infection-blocking antibodies, while not completely preventing primary infection, has a pronounced benefit in reducing the number of relapses. These findings suggest that a partially efficacious pre-erythrocytic Plasmodium vivax vaccine can have a disproportionately high impact in positive public health outcomes.

Keras R-CNN: library for cell detection in biological images using deep neural networks.

BACKGROUND: A common yet still manual task in basic biology research, high-throughput drug screening and digital pathology is identifying the number, location, and type of individual cells in images. Object detection methods can be useful for identifying individual cells as well as their phenotype in one step. State-of-the-art deep learning for object detection is poised to improve the accuracy and efficiency of biological image analysis. RESULTS: We created Keras R-CNN to bring leading computational research to the everyday practice of bioimage analysts. Keras R-CNN implements deep learning object detection techniques using Keras and Tensorflow ( https://github.com/broadinstitute/keras-rcnn ). We demonstrate the command line tool's simplified Application Programming Interface on two important biological problems, nucleus detection and malaria stage classification, and show its potential for identifying and classifying a large number of cells. For malaria stage classification, we compare results with expert human annotators and find comparable performance. CONCLUSIONS: Keras R-CNN is a Python package that performs automated cell identification for both brightfield and fluorescence images and can process large image sets. Both the package and image datasets are freely available on GitHub and the Broad Bioimage Benchmark Collection.

Altered Gut Microbiota and Immunity Defines Plasmodium vivax Survival in Anopheles stephensi.

Blood-feeding enriched gut-microbiota boosts mosquitoes' anti-Plasmodium immunity. Here, we ask how Plasmodium vivax alters gut-microbiota, anti-Plasmodial immunity, and impacts tripartite Plasmodium-mosquito-microbiota interactions in the gut lumen. We used a metagenomics and RNAseq strategy to address these questions. In naïve mosquitoes, Elizabethkingia meningitis and Pseudomonas spp. are the dominant bacteria and blood-feeding leads to a heightened detection of Elizabethkingia, Pseudomonas and Serratia 16S rRNA. A parallel RNAseq analysis of blood-fed midguts also shows the presence of Elizabethkingia-related transcripts. After, P. vivax infected blood-meal, however, we do not detect bacterial 16S rRNA until circa 36 h. Intriguingly, the transcriptional expression of a selected array of antimicrobial arsenal cecropins 1-2, defensin-1, and gambicin remained low during the first 36 h-a time frame when ookinetes/early oocysts invaded the gut. We conclude during the preinvasive phase, P. vivax outcompetes midgut-microbiota. This microbial suppression likely negates the impact of mosquito immunity which in turn may enhance the survival of P. vivax. Detection of sequences matching to mosquito-associated Wolbachia opens a new inquiry for its exploration as an agent for "paratransgenesis-based" mosquito control.

Parasite density in severe malaria in Colombia.

BACKGROUND: Colombia has officially adopted the parasite density levels of severe malaria established by the WHO (>50,000 parasites/µl). These values have been inferred from areas of high transmission in Africa and are not consistent with the dynamics of low and unstable transmission in Colombia. The objective of this study was therefore to determine the parasite density values observed in patients with severe malaria and their distribution in the different ecoepidemiological regions of Colombia. METHODS: A retrospective and descriptive study of confirmed cases of severe malaria was conducted in endemic areas of malaria in Colombia over the period 2014-2017. Data were collected from secondary sources of the Subnational Programs of Malaria Prevention and Control. Person, place, and time variables were selected. The official definition of severe malaria was adopted, and compliance with these criteria was determined. Univariate and bivariate analyses were conducted with absolute and relative frequency measures, and the relevant statistical tests were applied. RESULTS: The overall parasite density values in Colombia showed a geometric mean of 5,919 parasites/µl (95% CI: 5,608-6,248). By parasite species, the values were 6,151 (95% CI: 5,631-6,718) for Plasmodium falciparum and 5,815 (95% CI: 5,428-6,230) for Plasmodium vivax. The highest parasite density values were recorded in the Amazon ecoepidemiological region (8,177; 95% CI: 6,015-11,116), and the lowest values were recorded in the Andean region (5,026; 95% CI: 2,409-10,480). CONCLUSIONS: In endemic areas of low and unstable malaria transmission in the Colombian territory, the parasite density levels observed in populations with severe malaria are lower than the officially established values. The parasite density criterion is not really a relevant criterion for the definition of severe cases in Colombia and it certainly not be used to make a clinical decision about the severity of the disease.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Population Pharmacokinetics and Pharmacodynamics of Chloroquine in a Plasmodium vivax Volunteer Infection Study.

Chloroquine has been used for the treatment of malaria for > 70 years; however, chloroquine pharmacokinetic (PK) and pharmacodynamic (PD) profile in Plasmodium vivax malaria is poorly understood. The objective of this study was to describe the PK/PD relationship of chloroquine and its major metabolite, desethylchloroquine, in a P. vivax volunteer infection study. We analyzed data from 24 healthy subjects who were inoculated with blood-stage P. vivax malaria and administered a standard treatment course of chloroquine. The PK of chloroquine and desethylchloroquine was described by a two-compartment model with first-order absorption and elimination. The relationship between plasma and whole blood concentrations of chloroquine and P. vivax parasitemia was characterized by a PK/PD delayed response model, where the equilibration half-lives were 32.7 hours (95% confidence interval (CI) 27.4-40.5) for plasma data and 24.1 hours (95% CI 19.0-32.7) for whole blood data. The estimated parasite multiplication rate was 17 folds per 48 hours (95% CI 14-20) and maximum parasite killing rate by chloroquine was 0.213 hour-1 (95% CI 0.196-0.230), translating to a parasite clearance half-life of 4.5 hours (95% CI 4.1-5.0) and a parasite reduction ratio of 400 every 48 hours (95% CI 320-500). This is the first study that characterized the PK/PD relationship between chloroquine plasma and whole blood concentrations and P. vivax clearance using a semimechanistic population PK/PD modeling. This PK/PD model can be used to optimize dosing scenarios and to identify optimal dosing regimens for chloroquine where resistance to chloroquine is increasing.

Single-cell transcription analysis of Plasmodium vivax blood-stage parasites identifies stage- and species-specific profiles of expression.

Plasmodium vivax and P. falciparum, the parasites responsible for most human malaria worldwide, exhibit striking biological differences, which have important clinical consequences. Unfortunately, P. vivax, unlike P. falciparum, cannot be cultivated continuously in vitro, which limits our understanding of its biology and, consequently, our ability to effectively control vivax malaria. Here, we describe single-cell gene expression profiles of 9,215 P. vivax parasites from bloodstream infections of Aotus and Saimiri monkeys. Our results show that transcription of most P. vivax genes occurs during short periods of the intraerythrocytic cycle and that this pattern of gene expression is conserved in other Plasmodium species. However, we also identify a strikingly high proportion of species-specific transcripts in late schizonts, possibly associated with the specificity of erythrocyte invasion. Our findings provide new and robust markers of blood-stage parasites, including some that are specific to the elusive P. vivax male gametocytes, and will be useful for analyzing gene expression data from laboratory and field samples.

A Way Forward for Culturing Plasmodium vivax.

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish Plasmodium vivax in continuous culture have failed. Unlike P. falciparum, which can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth in vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is protection from oxidative stress by glucose-6-phosphate dehydrogenase (G6PD). Here, we also suggest supplements to the media and procedures that may reduce oxidative stress and, as a result, establish a system for the continuous culture of P. vivax.

Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle.

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity.

Main B-cell epitopes of PvAMA-1 and PvMSP-9 are targeted by naturally acquired antibodies and epitope-specific memory cells in acute and convalescent phases of vivax malaria.

Although antibodies are considered critical for malaria protection, little is known about the mechanisms/factors that maintain humoral immunity, especially regarding the induction and maintenance of memory B cells over time. In Brazilian endemic areas, this is the first time that the profile of antibody responses and the occurrence of antigen-specific memory B cells (MBC) against P vivax were investigated during acute malaria and up to six months after parasite clearance. For this, we selected two peptides, PvAMA-1(S290-K307) and PvMSP-9(E795-A808) , which represent the apical membrane antigen-1 and merozoite surface protein-9 of P vivax, respectively. Both peptides were previously described as containing linear B-cell epitopes. Our findings were as follows: 1-both peptides were recognized by IgG antibodies at a high frequency (between 24% and 81%) in all study groups; 2-in the absence of infection, the IgG levels remained stable throughout 6 months of follow-up; and 3-PvAMA-1(S290-K307) and PvMSP-9(E795-A808) -specific MBCs were detected in all individual groups in the absence of reinfection throughout the follow-up period, suggesting long-lived MBC. However, no positive association was observed between malaria-specific antibody levels and frequency of MBCs over time. Taken together, these results suggest that peptides can be, in the future, an alternative strategy to polypeptidic vaccine formulation.

K562 erythroleukemia line as a possible reticulocyte source to culture Plasmodium vivax and its surrogates.

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.

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites.

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.