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Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs. M. G.-L. was a postdoctoral fellow supported by the Plan Estratégico de Investigación e Innovación en Salud (PERIS, SLT002/16/00179) of the Generalitat de Catalunya, Spain. M. D.-V. was a predoctoral fellow supported by Secretaria d’Universitats i Recerca del Departament d’Economia i Creixement, Generalitat de Catalunya (2017 FI_B2_00029). I. A.-H. is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657). C. S. was funded by the German Research Foundation (DFG; fellowship SCHA2047/1-1). We acknowledge support from the National Institute of Health R21 program (1R21AI135680-01). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is supported by “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595) and acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership. We also acknowledge support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the CERCA Program. This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces. Work in the laboratory of Carmen Fernandez-Becerra and Hernando A. del Portillo is funded by the Ministerio Español de Economía y Competitividad (SAF2016 80655-R) and by and by the Ministerio de Ciencia e Innovación (PID2019-111795RB-I00). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Summary 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.

Summary The human malaria parasite Plasmodium vivax remains vastly understudied, mainly due to the lack of suitable laboratory models. Here, we report a humanized mouse model to test interventions that block P. vivax parasite transition from liver stage infection to blood stage infection. Human liver-chimeric FRGN huHep mice infected with P. vivax sporozoites were infused with human reticulocytes, allowing transition of exo-erythrocytic merozoites to reticulocyte infection and development into all erythrocytic forms, including gametocytes, in vivo. In order to test the utility of this model for preclinical assessment of interventions, the invasion blocking potential of a monoclonal antibody targeting the essential interaction of the P. vivax Duffy Binding Protein with the Duffy antigen receptor was tested by passive immunization. This antibody inhibited invasion by over 95%, providing unprecedented in vivo evidence that PvDBP constitutes a promising blood stage vaccine candidate and proving our model highly suitable to test blood stage interventions., Graphical Abstract, Highlights • A humanized mouse model allows P. vivax liver stage to blood stage transition • This model is highly suitable to test P. vivax blood stage interventions in vivo • P. vivax parasites can commit to sexual development as they emerge from the liver, Microbiology; Model Organism; Parasitology

Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage–arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage–arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2(–). Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2(–) liver stages in human liver-chimeric mice. However, P. falciparum mei2(–) liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2(–) is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.

Background Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available. Results Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite. Conclusions The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Malaria parasite infection continues to inflict extensive morbidity and mortality in resource-poor countries. The insufficiently understood parasite biology, continuously evolving drug resistance and the lack of an effective vaccine necessitate intensive research on human malaria parasites that can inform the development of new intervention tools. Humanized mouse models have been greatly improved over the last decade and enable the direct study of human malaria parasites in vivo in the laboratory. Nevertheless, no small animal model developed so far is capable of maintaining the complete life cycle of Plasmodium parasites that infect humans. The ultimate goal is to develop humanized mouse systems in which a Plasmodium infection closely reproduces all stages of a parasite infection in humans, including pre-erythrocytic infection, blood stage infection and its associated pathology, transmission as well as the human immune response to infection. Here, we discuss current humanized mouse models and the future directions that should be taken to develop next-generation models for human malaria parasite research.

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.

Graphical abstract, Highlights • Functional cloning identifies a Leishmania-specific 58 kD antimony resistance marker, ARM58. • ARM58 overexpression confers antimony resistance. • A neighbouring, structurally related gene lacks antimony resistance function. • ARM58 comprises 4 related domains of unknown function, classified as DUF1935. • Cosmid-based functional cloning is feasible in L. braziliensis in spite of RNAi., Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.

HIV-1 integrates into the host chromosome and persists as a provirus flanked by long terminal repeats (LTR). To date, treatment regimens primarily target the virus enzymes, virus attachment or virus-cell fusion, but not the integrated provirus. Thus, current antiretroviral therapies (i.e. cART) cannot eradicate HIV-1, a fact that highlights the urgency of pursuing new strategies to find a cure for HIV/AIDS. Previously, we engineered an experimental LTR-specific recombinase (Tre-recombinase) that can effectively excise integrated HIV-1 proviral DNA from infected human cell cultures (Sarkar et al. 2007 Science 316:1912). Subsequently, we demonstrated highly significant antiviral activity of this HIV-1 subtype A-specific Tre in humanized mice (Hauber et al. 2013 PLOS pathogens 9:e1003587). Broad clinical application, however, requires availability of a tre-recombinase that recognizes a majority of clinical HIV-1 isolates.

HIV research greatly benefits from the use of immunodeficient mouse systems that are susceptible to HIV target cells. Usually, immunodeficient mice are transplanted with primary human cells to develop a “human” immune system. Following engraftment, the animals are subsequently infected with HIV. Here, we applied a different approach and personalized mice by directly transplanting cells from an HIV-infected patient.

SummaryIn-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.