In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species.

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Potential benefits of gene editing for the future of poultry farming.

The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Young Pigs Consuming Lysozyme Transgenic Goat Milk Are Protected from Clinical Symptoms of Enterotoxigenic Escherichia coli Infection.

Background: Diarrheal diseases in infancy and childhood are responsible for substantial morbidity and mortality in developing nations. Lysozyme, an antimicrobial component of human milk, is thought to play a role in establishing a healthy intestinal microbiota and immune system. Consumption of breast milk has been shown to prevent intestinal infections and is a recommended treatment for infants with diarrhea.Objective: This study aimed to examine the ability of lysozyme-rich goat milk to prevent intestinal infection.Methods: Six-week-old Hampshire-Yorkshire pigs were assigned to treatment groups balanced for weight, sex, and litter and were fed milk from nontransgenic control goats (GM group) or human lysozyme transgenic goats (hLZM group) for 2 wk before they were challenged with porcine-specific enterotoxigenic Escherichia coli (ETEC). Fecal consistency, complete blood counts, intestinal histology, and microbial populations were evaluated.Results: Pigs in the hLZM group had less severe diarrhea than did GM pigs at 24 and 48 h after ETEC infection (P = 0.01 and 0.05, respectively), indicating a less severe clinical disease state. Relative to baseline, postmilk hLZM pigs had 19.9% and 137% enrichment in fecal Bacteroidetes (P = 0.028) and Paraprevotellaceae (P = 0.003), respectively, and a 93.8% reduction in Enterobacteriaceae (P = 0.007), whereas GM pigs had a 60.9% decrease in Lactobacillales (P = 0.003) and an 83.3% enrichment in Burkholderiales (P = 0.010). After ETEC infection, hLZM pigs tended to have lower amounts (68.7% less) of fecal Enterobacteriaceae than did GM pigs (P = 0.058). There were 83.1% fewer bacteria translocated into the mesenteric lymph nodes of hLZM pigs than into those of GM pigs (P = 0.039), and hLZM pigs had 34% lower mucin 1 and 61% higher tumor necrosis factor-α expression in the ileum than did GM pigs (P = 0.046 and 0.034, respectively).Conclusion: Results of this study indicate that human lysozyme milk consumption before and during ETEC infection has a positive effect on clinical disease, intestinal mucosa, and gut microbiota in young pigs.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

Advances in genetic engineering of the avian genome: "Realising the promise".

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

Consumption of transgenic milk containing the antimicrobials lactoferrin and lysozyme separately and in conjunction by 6-week-old pigs improves intestinal and systemic health.

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

Consumption of transgenic cows' milk containing human lactoferrin results in beneficial changes in the gastrointestinal tract and systemic health of young pigs.

Lactoferrin is an antimicrobial and immunomodulatory protein that is produced in high quantities in human milk and aids in the gastrointestinal (GI) maturation of infants. Beneficial health effects have been observed when supplementing human and animal diets with lactoferrin. A herd of genetically engineered cattle that secrete recombinant human lactoferrin in their milk (rhLF-milk) have been generated which provide an efficient production system and ideal medium for rhLF consumption. The effects of consumption of rhLF-milk were tested on young pigs as an animal model for the GI tract of children. When comparing rhLF-milk fed pigs to non-transgenic milk fed pigs (control), we observed that rhLF-milk fed pigs had beneficial changes in circulating leukocyte populations. There was a significant decrease in neutrophils (p = 0.0036) and increase in lymphocytes (p = 0.0017), leading to a decreased neutrophil to lymphocyte ratio (NLR) (p = 0.0153), which is an indicator of decreased systemic inflammation. We also observed changes in intestinal villi architecture. In the duodenum, rhLF-milk fed pigs tended to have taller villi (p = 0.0914) with significantly deeper crypts (p < 0.0001). In the ileum, pigs consuming rhLF-milk had villi that were significantly taller (p = 0.0002), with deeper crypts (p < 0.0001), and a thinner lamina propria (p = 0.0056). We observed no differences in cytokine expression between rhLF-milk and control-milk fed pigs, indicating that consumption of rhLF-milk did not change cytokine signaling in the intestines. Overall favorable changes in systemic health and GI villi architecture were observed; indicating that consumption of rhLF-milk has the potential to induce positive changes in the GI tract.

Mapping of equine cerebellar abiotrophy to ECA2 and identification of a potential causative mutation affecting expression of MUTYH.

Equine Cerebellar Abiotrophy (CA) is a neurological disease found in Arabian horses. CA is characterized by post-natal degeneration of the Purkinje cells of the cerebellum. Signs of CA include ataxia, head tremors, and a lack of balance equilibrium. We have discovered a linkage of the CA phenotype to a microsatellite marker on ECA2 and identified a region of conserved homozygosity spanning approximately 142 kb. Complete sequencing of the four genes in this region identified one SNP found only in Arabian horses, located in exon 4 of TOE1 and approximately 1200 base pairs upstream of MUTYH, adjacent to a possible binding site for the transcription factor GATA2. qPCR analysis of cDNA from the cerebella of affected and unaffected horses suggested that MUTYH expression is down-regulated in affected horses. This SNP may therefore have a function effect on TOE1, or a regulatory effect on MUTYH by negatively affecting the binding affinity of GATA2.

A novel murine model of post-implantation malaria-induced preterm birth.

Despite major advances made in malaria treatment and control over recent decades, the development of new models for studying disease pathogenesis remains a vital part of malaria research efforts. The study of malaria infection during pregnancy is particularly reliant on mouse models, as a means of circumventing many challenges and costs associated with pregnancy studies in endemic human populations. Here, we introduce a novel murine model that will further our understanding of how malaria infection affects pregnancy outcome. When C57BL/6J (B6) mice are infected with Plasmodium chabaudi chabaudi AS on either embryonic day (E) 6.5, 8.5, or 10.5, preterm birth occurs in all animals by E16.5, E17.5, or E18.5 respectively, with no evidence of intrauterine growth restriction. Despite having the same outcome, we found that the time to delivery, placental inflammatory and antioxidant transcript upregulation, and the relationships between parasitemia and transcript expression prior to preterm birth differed based on the embryonic day of infection. On the day before preterm delivery, E6.5 infected mice did not experience significant upregulation of the inflammatory or antioxidant gene transcripts examined; however, peripheral and placental parasitemia correlated positively with Il1ß, Cox1, Cat, and Hmox1 placental transcript abundance. E8.5 infected mice had elevated transcripts for Ifnγ, Tnf, Il10, Cox1, Cox2, Sod1, Sod2, Cat, and Nrf2, while Sod3 was the only transcript that correlated with parasitemia. Finally, E10.5 infected mice had elevated transcripts for Ifnγ only, with a tendency for Tnf transcripts to correlate with peripheral parasitemia. Tumor necrosis factor deficient (TNF-/-) and TNF receptor 1 deficient (TNFR1-/-) mice infected on E8.5 experienced preterm birth at the same time as B6 controls. Further characterization of this model is necessary to discover the mechanism(s) and/or trigger(s) responsible for malaria-driven preterm birth caused by maternal infection during early pregnancy.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates.

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite's lifecycle.

Genetic approaches for increasing fitness in endangered species.

The global rate of wildlife extinctions is accelerating, and the persistence of many species requires conservation breeding programs. A central paradigm of these programs is to preserve the genetic diversity of the founder populations. However, this may preserve original characteristics that make them vulnerable to extinction. We introduce targeted genetic intervention (TGI) as an alternative approach that promotes traits that enable species to persist in the face of threats by changing the incidence of alleles that impact on fitness. The TGI toolkit includes methods with established efficacy in model organisms and agriculture but are largely untried for conservation, such as synthetic biology and artificial selection. We explore TGI approaches as a species-restoration tool for intractable threats including infectious disease and climate change.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.

Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology.

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-ß1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-ß1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.

Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi.

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

A Phenotypic Screen for the Liver Stages of Plasmodium vivax.

Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite's liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype. Graphical abstract: Overview of P. vivax liver stage screening assay performed at the Institute Pasteur of Cambodia.

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.

A novel murine model for assessing fetal and birth outcomes following transgestational maternal malaria infection.

Plasmodium falciparum infection during pregnancy is a major cause of severe maternal illness and neonatal mortality. Mouse models are important for the study of gestational malaria pathogenesis. When infected with Plasmodium chabaudi chabaudi AS in early gestation, several inbred mouse strains abort at midgestation. We report here that outbred Swiss Webster mice infected with P. chabaudi chabaudi AS in early gestation carry their pregnancies to term despite high parasite burden and malarial hemozoin accumulation in the placenta at midgestation, with the latter associated with induction of heme oxygenase 1 expression. Infection yields reduced fetal weight and viability at term and a reduction in pup number at weaning, but does not influence postnatal growth prior to weaning. This novel model allows for the exploration of malaria infection throughout pregnancy, modeling chronic infections observed in pregnant women prior to the birth of underweight infants and enabling the production of progeny exposed to malaria in utero, which is critical for understanding the postnatal repercussions of gestational malaria. The use of outbred mice allows for the exploration of gestational malaria in a genetically diverse model system, better recapitulating the diversity of infection responses observed in human populations.