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.     

Modeling the Dynamics of Plasmodium vivax Infection and Hypnozoite Reactivation In Vivo

The dynamics of Plasmodium vivax infection is characterized by reactivation of hypnozoites at varying time intervals. The relative contribution of new P. vivax infection and reactivation of dormant liver stage hypnozoites to initiation of blood stage infection is unclear. In this study, we investigate the contribution of new inoculations of P. vivax sporozoites to primary infection versus reactivation of hypnozoites by modeling the dynamics of P. vivax infection in Thailand in patients receiving treatment for either blood stage infection alone (chloroquine), or the blood and liver stages of infection (chloroquine + primaquine). In addition, we also analysed rates of infection in a study in Papua New Guinea (PNG) where patients were treated with either artesunate, or artesunate + primaquine. Our results show that up to 96% of the P. vivax infection is due to hypnozoite reactivation in individuals living in endemic areas in Thailand. Similar analysis revealed the around 70% of infections in the PNG cohort were due to hypnozoite reactivation. We show how the age of the cohort, primaquine drug failure, and seasonality may affect estimates of the ratio of primary P. vivax infection to hypnozoite reactivation. Modeling of P. vivax primary infection and hypnozoite reactivation provides important insights into infection dynamics, and suggests that 90–96% of blood stage infections arise from hypnozoite reactivation. Major differences in infection kinetics between Thailand and PNG suggest the likelihood of drug failure in PNG.     

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections

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.     

Role of Plasmodium berghei cGMP-dependent Protein Kinase in Late Liver Stage Development

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.     

Potential role of vaccines in elimination of Plasmodium vivax

The unique biology of Plasmodium vivax, with its ability to form latent hypnozoites in the liver stage and the early appearance of gametocytes during blood stage infection, makes it difficult to target for elimination with standard malaria control tools. Here, we use modelling studies to demonstrate that vaccines that target different stages of P. vivax could greatly assist efforts to eliminate P. vivax. Combination of vaccines that target different P. vivax life cycle stages may be required to achieve high efficacy. Our simulations demonstrate that repeated rounds of mass vaccination with multi-stage vaccines can help achieve pre-elimination levels of P. vivax in both low and high transmission settings. We review the status of global efforts to develop vaccines for P. vivax malaria. We describe the status of the leading P. vivax vaccine candidates and share some thoughts on the prospects for availability of an effective vaccine for P. vivax malaria.     

Determinants of relapse periodicity in Plasmodium vivax malaria

Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa. Plasmodium vivax infections are characterized by relapses of malaria arising from persistent liver stages of the parasite (hypnozoites) which can be prevented only by 8-aminoquinoline anti-malarials. Tropical P. vivax relapses at three week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in temperate regions and parts of the sub-tropics P. vivax infections are characterized either by a long incubation or a long-latency period between illness and relapse - in both cases approximating 8-10 months. The epidemiology of the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria control and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant of both the timing and the number of relapses. The intervals between relapses display a remarkable periodicity which has not been explained. Evidence is presented that the proportion of patients who have successive relapses is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax malaria is the systemic febrile illness itself. It is proposed that in endemic areas a large proportion of the population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and more prevalent than currently thought. These observations have important implications for the assessment of radical treatment efficacy and for malaria control and elimination.     

Neither the HIV Protease Inhibitor Lopinavir-Ritonavir nor the Antimicrobial Trimethoprim-Sulfamethoxazole Prevent Malaria Relapse in Plasmodium cynomolgi-Infected Non-Human Primates

Plasmodium vivax malaria causes significant morbidity and mortality worldwide, and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax relapses. HIV and P. vivax malaria geographically overlap in many areas of the world, including South America and Asia. Despite the increasing body of knowledge regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no data regarding the effects of these treatments on P. vivax''s hypnozoite form and clinical relapses of malaria. We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remains untested. Separately, although other antifolates have been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole, commonly used in HIV infection and exposure management, has not been evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we investigated the antimalarial activity of these drugs on Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested. Because HIV and malaria geographically overlap, and more patients are being managed for HIV infection and exposure, understanding HIV drug impact on malaria infection is important.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.     

Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery

Background

Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites.

Methodology

P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured.

Results

The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine.

Conclusion

Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Lymph-Node Resident CD8α+ Dendritic Cells Capture Antigens from Migratory Malaria Sporozoites and Induce CD8+ T Cell Responses

Malaria infection begins when a female Anopheles mosquito injects Plasmodium sporozoites into the skin of its host during blood feeding. Skin-deposited sporozoites may enter the bloodstream and infect the liver, reside and develop in the skin, or migrate to the draining lymph nodes (DLNs). Importantly, the DLN is where protective CD8⁺ T cell responses against malaria liver stages are induced after a dermal route of infection. However, the significance of parasites in the skin and DLN to CD8⁺ T cell activation is largely unknown. In this study, we used genetically modified parasites, as well as antibody-mediated immobilization of sporozoites, to determine that active sporozoite migration to the DLNs is required for robust CD8⁺ T cell responses. Through dynamic in vivo and static imaging, we show the direct uptake of parasites by lymph-node resident DCs followed by CD8⁺ T cell-DC cluster formation, a surrogate for antigen presentation, in the DLNs. A few hours after sporozoite arrival to the DLNs, CD8⁺ T cells are primed by resident CD8α⁺ DCs with no apparent role for skin-derived DCs. Together, these results establish a critical role for lymph node resident CD8α⁺ DCs in CD8⁺ T cell priming to sporozoite antigens while emphasizing a requirement for motile sporozoites in the induction of CD8⁺ T cell-mediated immunity.     

Plasmodium vivax – How hidden reservoirs hinder global malaria elimination

Plasmodium vivax is the most geographically widespread human malaria parasite. Global malaria efforts have been less successful at reducing the burden of P. vivax compared to P. falciparum, owing to the unique biology and related treatment complexity of P. vivax. As a result, P. vivax is now the dominant malaria parasite throughout the Asia-Pacific and South America causing up to 14 million clinical cases every year and is considered a major obstacle to malaria elimination. Key features circumventing existing malaria control tools are the transmissibility of asymptomatic, low-density circulating infections and reservoirs of persistent dormant liver stages (hypnozoites) that are undetectable but reactivate to cause relapsing infections and sustain transmission. In this review we summarise the new knowledge shaping our understanding of the global epidemiology of P. vivax infections, highlighting the challenges for elimination and the tools that will be required achieve this.     

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite

Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2(-) sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

CD81 is required for rhoptry discharge during host cell invasion by Plasmodium yoelii sporozoites

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and first infect the liver of their mammalian host, where they develop as liver stages before the onset of erythrocytic infection and malaria symptoms. Sporozoite entry into hepatocytes is an attractive target for anti‐malarial prophylactic strategies but remains poorly understood at the molecular level. Apicomplexan parasites invade host cells by forming a parasitophorous vacuole that is essential for parasite development, a process that involves secretion of apical organelles called rhoptries. We previously reported that the host membrane protein CD81 is required for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 acts at an early stage of infection, possibly at the entry step, but the mechanisms involved are still unknown. To investigate the role of CD81 during sporozoite entry, we generated transgenic P. yoelii parasites expressing fluorescent versions of three known rhoptry proteins, RON2, RON4 and RAP2/3. We observed that RON2 and RON4 are lost following rhoptry discharge during merozoite and sporozoite entry. In contrast, our data indicate that RAP2/3 is secreted into the parasitophorous vacuole during infection. We further show that sporozoite rhoptry discharge occurs only in the presence of CD81, providing the first direct evidence for a role of CD81 during sporozoite productive invasion.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.