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.
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the ...only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as ...the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene ...families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Apical membrane antigen 1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. Immune responses to Plasmodium AMA1 can have ...profound parasite-inhibitory effects, both as measured in vitro and in animal challenge models, suggesting AMA1 as a potential vaccine component. However, AMA1 is polymorphic, probably as a result of immune selection operating on an important target of naturally occurring immunity. The current understanding of AMA1 will be presented, particularly in relation to the vaccine potential of AMA1 and the approaches being taken towards clinical development.
Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a candidate malaria vaccine, is polymorphic. This polymorphism is believed to be generated predominantly under immune selection pressure and, ...as a result, may compromise attempts at vaccination. Alignment of 355 PfAMA1 sequences shows that around 10% of the 622 amino acid residues can vary between alleles and that linkages between polymorphic residues occur. Using this analysis, we have designed three diversity-covering (DiCo) PfAMA1 sequences that take account of these linkages and, when taken together, on average incorporate 97% of amino acid variability observed. For each of the three DiCo sequences, a synthetic gene was constructed and used to transform the methylotrophic yeast Pichia pastoris, allowing recombinant expression. All three DiCo proteins were reactive with the reduction-sensitive monoclonal antibody 4G2, suggesting the DiCo sequences had conformations similar to those of naturally occurring PfAMA1. Rabbits were immunized with FVO strain PfAMA1 or with the DiCo proteins either individually or as a mixture. Antibody titers and the ability to inhibit parasite growth in vitro were determined. Animals immunized with the DiCo mix performed similarly to animals immunized with FVO AMA1 when measured against FCR3 strain parasites but outperformed animals immunized with FVO AMA1 when assessed against other strains. The levels of growth inhibition (~70%) induced by the mix of three DiCo proteins were comparable for FVO, 3D7, and HB3, suggesting that a considerable degree of diversity in AMA1 is adequately covered. This suggests that vaccines based upon the DiCo mix approach provide a broader functional immunity than immunization with a single allele.
Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, ...which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, ...despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.
Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, ...Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our ...understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (
) and in vivo (
), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.
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