In vivo imaging in NHP models of malaria: challenges, progress and outlooks.

Animal models of malaria, mainly mice, have made a large contribution to our knowledge of host-pathogen interactions and immune responses, and to drug and vaccine design. Non-human primate (NHP) models for malaria are admittedly under-used, although they are probably closer models than mice for human malaria; in particular, NHP models allow the use of human pathogens (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi). NHPs, whether natural hosts or experimentally challenged with a simian Plasmodium, can also serve as robust pre-clinical models. Some simian parasites are closely related to a human counterpart, with which they may share a common ancestor, and display similar major features with the human infection and pathology. NHP models allow longitudinal studies, from the early events following sporozoite inoculation to the later events, including analysis of organs and tissues, particularly liver, spleen, brain and bone marrow. NHP models have one other significant advantage over mouse models: NHPs are our closest relatives and thus their biology is very similar to ours. Recently developed in vivo imaging tools have provided insight into malaria parasite infection and disease in mouse models. One advantage of these tools is that they limit the need for invasive procedures, such as tissue biopsies. Many such technologies are now available for NHP studies and provide new opportunities for elucidating host/parasite interactions. The aim of this review is to bring the malaria community up to date on what is currently possible and what soon will be, in terms of in vivo imaging in NHP models of malaria, to consider the pros and the cons of the various techniques, and to identify challenges.

Introduction

Malaria parasites infect a wide range of animals, including reptiles, birds, rodents and NHPs, in addition to humans. Four species, Plasmodium malariae, Plasmodium ovale, Plasmodium falciparum and Plasmodium vivax, are recognized as natural malaria parasites of humans. More recently, it has emerged that a fifth species, Plasmodium knowlesi, that morphologically resembles P. malariae and consequently has undoubtedly been misdiagnosed in the past, is a human pathogen [1]. The main simian Plasmodium parasites infecting Old World primates include P. knowlesi, Plasmodium coatneyi, Plasmodium fragile, Plasmodium cynomolgi, Plasmodium inui, Plasmodium fieldi, and Plasmodium simiovale, in South East Asia, as well as Plasmodium gonderi in Africa, while simian Plasmodium parasites infecting New World primates comprise Plasmodium brasilianum and Plasmodium simium in South America [2].

Simian Plasmodium parasites sharing similarities with human parasites were reported at the beginning of the XXth century [3]. Deciphering the evolutionary history of Plasmodium parasites and their primate hosts' restrictions is a matter of intensive research, to determine the origin of human Plasmodium parasites and when they emerged.

Plasmodium parasites show a large propensity to “jump” from NHPs to humans and the reverse. The origin of P. falciparum was recently determined. African Apes are likely a reservoir and the species has been found in Gorillas [4], [5], Bonobos [6] and Chimpanzees [7]. For P. vivax and P. malariae, which are genetically closely related to neotropical P. simium and P. brasilianum, the mechanisms of the host switch, from New World primates to humans (zoonosis) or the reverse (anthroponosis), have not been completely unraveled [8]. Some New World monkeys are susceptible to human P. falciparum, P. vivax and P. malariae. Current simian Plasmodium species known to naturally infect humans include not only P. knowlesi, but also, although very rarely, P. simium in South America and P. cynomolgi in Asia [3], [9].

This close relatedness between human and NHP hosts and between human and simian Plasmodium parasites, makes NHP models of choice for human malaria for the biomedical research community.

A plethora of animal models are available to study Plasmodium biology and pathogenesis, malaria immunity, and vaccine and drug efficacy

In contrast to other pathogens such as HIV, both in-field trials and experimental human challenges are feasible with Plasmodium [10]. Human subjects are mostly used to assess vaccine-induced protection. Nonetheless, animal models remain highly valuable, not only to avoid ethical problems with work on humans, but also to control the experimental conditions. Indeed the choice of the pathogen form (species, strain, isolate or clone) and the timing of infection allow the study of different aspects of the disease. Furthermore, the type of infection, using infected red blood cells (iRBCs) or sporozoites, leads to study more specifically the blood-stage or the transmission and the liver-stage, respectively.

Animal models allow longitudinal studies from the early events following sporozoite inoculation, analysis of clinically-silent stages and collections of organs and tissues. Confounding factors, such as previous infections, co-infections or malnutrition can be avoided. However, there is not a single animal model that perfectly reproduces the spectrum of human Plasmodium infection, malaria and immunity. Differences between parasite–host combinations may indeed be viewed as an asset, because they may reflect the heterogeneity of human malaria, as highlighted in a recent debate on animal models in malaria research [11]. Each model has different strengths, limitations and degree of relevance, and thus models should be carefully selected according to the scientific issues and hypotheses addressed.

Limitations of mice and humanized mouse models for malaria

Mice are extensively used for modeling malaria [12], [13], [14], [15], [16]. Human Plasmodium parasites are unable to infect mice, so rodent malaria parasites may be used. The four most widely used species are Plasmodium berghei, Plasmodium yoelii, Plasmodium chabaudi and Plasmodium vinckei, and they are used with inbred or outbred mice. They were isolated from their natural hosts, such as the African rats for P. berghei, and then adapted to laboratory mice. Rodent Plasmodium infections in mice display some, but not all, of the main features of the human infection and disease.

Complex humanized mouse models have been developed to overcome the host species barriers to the use of P. falciparum in small laboratory animals [17]. Briefly, mice with a reconstituted human hematopoietic system, producing human RBCs, susceptible to several P. falciparum strains and supporting long-lasting parasitemia and gametocyte development, would be ideal for studying the blood-stage. However, no such perfect model is available. Only transient models have been reported, involving perfusion of infected RBCs into immunodeficient mouse strains, such as NOD/SCID/IL2Rγ- [18], [19], [20].

For transmission and liver-stage studies, mice with engrafted human hepatocytes have been generated and there is evidence that following infection with P. falciparum sporozoites, liver-stage schizonts develop [21], [22]. The complete development of liver-stage parasites was recently reported, with liver-stage schizonts exhibiting exoerythrocytic merozoite formation and merosome release, and even with the transition from P. falciparum liver-stage infection to a blood-stage infection with gametocyte development [23]. However, the main drawback of these humanized mice is clearly their low to medium throughput. They are also inappropriate for the study of P. vivax, which requires human reticulocytes and thus human erythropoiesis. Finally, these mouse models are not adapted for the assessment of specific immunity which requires a mature and fully competent human immune system.

Advantages of NHPs over mice and humans for the study of parasite biology, infection dynamics and pathogenesis

There are also several well-established NHP models available, although they are not widely used. This is probably because NHP studies require appropriate specific facilities (possibly with associated mosquito facilities), and more rigorous ethical justification than other animal models. They also require a close collaborative work between primatologists and malariologists, and specially trained personnel.

Safety issues are raised and BSL2 or BSL3 animal facilities are required when using mosquito-borne Plasmodium species infectious for humans, such as human Plasmodium parasites and some of the macaque parasites, including P. cynomolgi and P. inui.

NHP studies are also particularly costly. Nevertheless, many believe that such models are worth the effort and they are committed to promote the use of NHPs, because these models of malaria closely reproduce human malaria. Indeed, continued investment in NHP systems was recognized as a priority at a recent meeting on animal models for research on severe malaria [15].

According to the molecular timescale for Vertebrate evolution, mice and humans are separated by ~ 40 million years. Over this period, their immune systems have been subjected to very different selection pressures and there have been divergent host/pathogen adaptations. The Macaca genus is more closely related to humans, the most recent common ancestor being ~ 25 million years ago [24]. The phylogenetic proximity of humans and NHPs is associated with, for instance, comparable dendritic cell subsets and similar panoplies of pattern recognition receptors [25], [26], [27]. Both are central to the study of malaria immunology and to vaccine development.

In vivo malaria liver-stage studies in humans are virtually impossible, whereas NHPs provide access to asymptomatic stages, including the journey of sporozoites from skin to liver and the dormant hypnozoite forms in the liver, as seen with P. vivax or P. cynomolgi. In addition, various genes are conserved among primate parasites (P. falciparum, P. vivax and P. knowlesi) but are absent from rodent parasites (P. berghei, P. yoelii and P. chabaudi) [28]. The potential protective efficacy of antigens corresponding to these genes or of new genetically attenuated sporozoites cannot be assessed in mice. The degree of attenuation resulting from the deletion of any particular gene may also differ between Plasmodium species [29].

The recent whole-genome sequencing of rhesus macaque (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) [30], [31], [32] makes these species even more valuable for modeling human diseases. Also, as spin-offs of the extensive use of these NHP species in HIV/AIDS research, numerous tools for detailed characterization of their physiology and immune responses, in characterized genetic backgrounds (MHC, restriction factors, and KIR genetics) are available [33], [34], [35], [36], [37]. In rhesus, cynomolgus and pigtailed macaques (Macaca nemestrina), tetramer staining to quantify antigen-specific CD8+T cells is feasible, since MHC class I typing is possible.

This drive for progress is well illustrated by Primate Info Net, a library and information service which gathers key information about various primate species and provides links to resources about NHPs in biomedical research (http://pin.primate/wisconsin.edu), or the Nonhuman Primate Reagent Resource whose goals are to facilitate the use of NHPs by producing specific reagents such as antibodies and recombinant cytokines, and by sharing information on cross-reactivity of immunological reagents and assays (http://nhpreagents.org). Similarly, a new research center dedicated to preclinical research programs on host–pathogen interactions, human vaccines and antimicrobial treatments in NHPs was recently launched in France: the Center for Infectious Disease Models and Innovative Therapies (IDMIT) (http://idmitcenter.fr) provides the scientific community with a NHP facility, with the cynomolgus macaques/P. cynomolgi model currently being implemented, combined with the most recent technologies, including imaging technologies (in vivo fluorescent microscopy and MRI are already available and PET–CT will be in 2017). To optimize the use of NHPs, particular emphasis is given to assay standardization and harmonization, and both a reagent repository and a biobank have been created.

Various complementary NHP models of human malaria

Several NHP/parasite combinations have been described. They are variously useful to study Plasmodium biology, transmission, immunology and/or pathology, acute and chronic infection, or severe malaria, cerebral malaria, placental malaria, and other malaria-associated pathologies (liver, lung, kidney or spleen involvement and thrombocytopenia, hemorrhage, and anemia). Very useful reviews have been published recently providing summaries of NHP models for the study of hepatic infections and pathogenesis [15], [38]. The various isolates/strains adapted to NHPs, their history and their characteristics (e.g. natural vs. experimental host, virulence, lethality, capacity to produce infective gametocytes, resistance to anti-malarial drugs, complete or partial development) have been exhaustively listed elsewhere [2], [39]. Here, we present only the most widely used models.

P. falciparum and P. vivax in neotropical primates

There are few in vivo models for human malaria using human parasites. The host specificity of malaria parasites is narrow such that the only suitable NHP species that can be infected with P. falciparum, P. vivax or P. malariae are some neotropical primates: South American owl monkeys (Aotus spp.) and squirrel monkeys (Saimiri spp.) [40], [41]. Furthermore, the parasites must be adapted to grow in these non-natural hosts. Only P. ovale cannot be adapted to these New World monkeys. The susceptibility to infection by sporozoites and/or iRBCs, the production of viable gametocytes, the characteristics of the blood-stage infection and the requirement for splenectomy all depend on the host/strain combination. The Colombian Night Monkey (Aotus lemurinus griseimembra) may emerge as one of the best NHP malaria models because of its high susceptibility to infection both by blood forms and by sporozoites of both P. falciparum and P. vivax [42]. Saimiri sciureus is used to investigate P. falciparum blood-stage human parasites, but splenectomy is required for reproducible high parasitemia after injection of parasitized RBCs [43]. Saimiri boliviensis is considered as the most reliable model for P. vivax [41].

Chimpanzees are also susceptible to P. falciparum and P. vivax, as well as P. malariae and P. ovale. However, their use is limited for ethical reasons. Indeed, the European Union has now banned the use of great apes for research purposes (Chimpanzees, Bonobos, Gorillas and Orangutans) and such research is also now highly restricted in the US [44].

The obvious advantage of New World primates over Old World primates as models for human malaria is that human parasites can be used. However, the advantages of using human parasites are counterbalanced by the limited supply of neotropical primates. There are fewer breeding colonies than for macaques, for example, and export of some of them from their countries of origin is prohibited. A recent workshop on neotropical primates in biomedical research [45] led to the conclusion that efforts should be pursued to develop new reagents and to support breeding colonies, including by improving colony and veterinary management. Finally, less blood for experimental purposes can be collected from these species than from macaques.

P. knowlesi in macaques

P. knowlesi is a primate malaria parasite endemic in some South East Asian species, for example cynomolgus and pig-tailed macaques. P. knowlesi is also the fifth malaria species naturally infecting humans and causing life-threatening disease [46]. Experimental infections have been described in a wide range of NHPs, from Old World primates rhesus macaques and baboon (Papio spp.) to New World primates, such as squirrel and Aotus monkeys and the common marmoset (Callithrix jacchus). Interestingly, there are two opposed asexual-blood stage outcomes in rhesus and cynomolgus macaques: P. knowlesi causes severe disease, inevitably lethal if untreated, after inoculation of rhesus macaques with sporozoites or iRBCs, whereas parasite development is controlled and chronic infection develops in its natural host, cynomolgus macaques. Parasitemia needs to be monitored daily because P. knowlesi has an asexual life cycle of about 24 h and the parasites can increase quickly to detrimental levels. The model of P. knowlesi in macaques is mainly used for investigating parasite biology, physiopathology and immunology, and for vaccine studies [47], [48], [49].

Simian Plasmodium species in macaques

Human Plasmodium species, other than P. knowlesi, do not infect Old World monkeys. However, there are many simian Plasmodium species naturally infecting macaques, and some are used as experimental models of human malaria.

P. coatneyi and P. fragile, which naturally infect cynomolgus macaques, and bonnet (Macaca radiata) and toque macaques (Macaca sinica), respectively, are used as models of P. falciparum in various macaques (M. mulatta, M. fascicularis, M. nemestrina or Macaca fuscata), with sequestration, rosetting, and severe disease, including cerebral malaria [50], [51], [52]. Both parasites are very virulent after sporozoite inoculation of rhesus macaques and Japanese macaques (M. fuscata) [53], [54].

P. cynomolgi, which naturally infects a wide variety of macaque hosts, including cynomolgus macaques, is a model for studying the biology, immunology and pathology of P. vivax. It mimics the biology and pathogenesis of P. vivax, with the dormant relapsing parasite forms, hypnozoites, and similar RBC infection features (reticulocytes) [48], [55], [56], [57].

Although P. fieldi and P. simiovale, which naturally infect cynomolgus and pigtailed macaques and toque macaques, respectively, share some characteristics with P. ovale, they are reproducing better features of P. vivax infection. Both are used as models of P. vivax in rhesus and cynomolgus macaques, but not as frequently as P. cynomolgi [49].

P. inui naturally infects a large variety of macaque species, and is considered as a macaque model for P. malariae, as it displays persistence and a similar kidney pathology [58].

Some macaque species are natural hosts, and consequently there has been evolutionary co-adaptation [59]. Note also that animals may have been exposed to malaria before experimental infection. It may be preferable to use naive animals: captive-born, or from areas where the prevalence of malaria is low, such as cynomolgus macaques from Mauritius.

In malaria endemic regions, there are other pathogens which may interfere with the host immune responses to Plasmodium. Macaques are also used to study a wide range of infections with human pathogens or their simian counterparts, as well as vaccines [60], [61]. Thus, it is possible to model co-infections in macaques, such as SIV/Plasmodium [62], [63], [64], [65] and Schistosoma/Plasmodium [66] and to study interference with vaccine-induced immune responses [67].

To conclude, NHP models of human malaria present several advantages over mouse models, which justify the efforts to facilitate and improve their use. This is especially true as in vivo imaging technologies, which have revolutionized our understanding of malaria in the last few years using mouse models, are actually not limited to small laboratory animals. However, insufficient access to adequate structures and equipment for imaging NHPs associated with suitable confinement for class 2 and 3 pathogens, in large part explains the relatively limited exploration of pathogen transmission and dissemination using in vivo imaging in NHPs. Nevertheless, in vivo imaging is entirely feasible in larger animals such as monkeys [68], [69]; even though adult male Aotus monkeys and rhesus macaques weigh about 1 and 8 kg on average, respectively, and are about 35 and 55 cm long, respectively [70] (mice weigh 20–30 g and are 6 cm long).

In vivo imaging and infectious diseases in NHPs

Early events in the transmission and dissemination of pathogens in the host organism through mucosal barriers are still poorly understood in most cases. A better characterization of these early steps of infection is needed to develop and improve prevention strategies and therapy.

In vivo imaging can be used for non-invasive and longitudinal studies, allowing the use of animals to be minimized and refined. Molecular imaging techniques such as optical imaging (fluorescence, bioluminescence), PET (positron emission tomography) and SPECT (single-photon emission tomography) can document molecular and cellular events. These methods are also complementary, as different techniques are appropriated for different biological investigations in different organs. The strengths and weaknesses of various imaging techniques have been reviewed elsewhere [71], [72].

Fluorescence imaging and bioluminescence imaging involve the detection of visible light and are therefore limited to the study of superficial organs; they have mainly been used in small animals. The feasibility of using bioluminescence to visualize infection by, and dissemination of, various viruses, such as murine herpes virus, mouse hepatitis virus, coronavirus, or Chikungunya virus, has been demonstrated in murine models [73], [74], [75]. The relative ease of use and the relatively low cost of these imaging techniques make them useful for the exploration of superficial organs even in large animals. Furthermore, with adapted endoscopic and intraoperative techniques, fluorescence could be used to study deeper organs. Traditional confocal microscopy can image at depths up to 100 μm. Intravital multiphoton imaging with multiphoton excitation can extend this limit up to a maximum of 1 mm in living animals [76], which is a huge advantage, especially when excision of the specimen is not possible. Recently, two-photon microscopy has been used for imaging and analyzing immune responses in single cells in intact tissues and for time-lapse imaging of living tissues. Indeed, the use of a pulsed infrared laser for fluorescent dye excitation was a major advance [77]: the high excitation wavelength allows deep tissue imaging and its relatively low energy, constrained to the focal plane, limits phototoxicity. As a consequence, two-photon microscopy has become the technique of choice to follow the dynamic behavior of immune cells in vivo; single cells can be studied in murine models of tumors, skin graft or infection [78], [79], [80]. For instance, intravital two-photon microscopy of the popliteal lymph nodes in mice was used to investigate how fluorescently labeled inactivated vesicular stomatitis virus (VSV) is captured from the lymph and transported into the B cell compartment to induce humoral immunity [81]. It has also been used to investigate the dynamic behavior of T cells infected with green fluorescent protein- (GFP-) expressing HIV in the stromal environment of lymph nodes [82]. Intravital microscopy of the skin or surgically exposed internal organs offer excellent resolution for studying individual cells or even subcellular structures and microorganisms [83], [84], although this is not strictly noninvasive. However, this technique has only been applied to murine models.

Positron emission tomography (PET), magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT) can also be used in both small and large animals. Indeed, they are routinely used for clinical purposes [72]. These techniques can reveal molecular and cellular events. The sensitivity of PET is high, in the range of 10− 11 to 10− 12  mol/L, and is independent of the location, or depth, of the reporter probe. Combining morphological/anatomical and molecular imaging methods, using multimodality hardware and/or co-registration post-acquisition processing, allows whole-body exploration of the early events in the transmission of pathogens through mucosal barriers, assessment of the dynamics of pathogen biodistribution, evaluation of the dynamics of early events following vaccinations, and determination of the pharmacokinetics and pharmacodynamics of new drugs in infected hosts. CT (computed tomography) and MRI can be combined with PET or SPECT to provide anatomical references [85]. MRI gives good spatial resolution allowing both anatomical imaging and molecular imaging, if associated with contrast agents, for cell tracking, characterization of inflammation or cancer cell detection [86], [87], [88]. [18F]-labeled fluoro-2-deoxy-2-d-glucose ([18F]FDG) has been widely used for PET imaging of activated cells (such cells show increased glucose metabolism). [18F]-labeled 3′-fluoro-3′-deoxy-thymidine ([18F]FLT) was designed as a tracer for cell proliferation and is increasingly being exploited in oncology.

The location and replication of pathogens can be followed by PET reporter gene imaging, for example using herpes simplex virus-1 thymidine kinase (HSV1-tk). HSV1-tk phosphorylates a broad range of nucleoside analogs, and its expression can be imaged with several substrate analogs including 2′-deoxy-2′-18F-fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil (18F-FEAU) and acycloguanosine derivatives, such as 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG). This strategy has been widely used in various animal models including NHPs, but not yet in the malaria context [89], [90], [91], [92]. PET imaging has also been used to detect immune activation of T cells in lymphoid organs in the context of antitumor immunity using an [18F]-labeled 2′-deoxycytidine analog in a mouse model and [18F]FLT in humans after anti-tumor vaccination [93], [94].

The pharmacokinetics, biodistribution, metabolism and toxicity of diagnostic doses of novel radiolabeled or contrast imaging agents have been studied in NHPs to facilitate translation to clinical applications [68], [95]. In vivo brain imaging by PET and/or MRI has been developed for diverse experimental models of neurodegenerative studies. For instance, imaging techniques, including PET, can directly visualize the concentration and localization of amyloid deposits, the pathological marker of Alzheimer disease, throughout the brain [96], [97], [98].

The immune response to viral infection in SHIV-infected macaques has also been explored by SPECT [99] and delivery of a botulism vaccine has been studied by a combination of PET and MRI [69]. Also, [18F]FDG PET/CT imaging has also been used to assess novel combinations of new drugs in NHP models of tuberculosis [100].

The variety of imaging technologies available for the examination of living material is increasing rapidly. The choice of imaging technology for a study should be based on the biological issues to be resolved and the organ(s) or tissues to be explored. For Plasmodium infection and malaria studies, the organs and tissues of interest (e.g. skin, blood, lymph, liver, spleen, lungs or brain) depend on the malaria stage, transmission, and liver- and blood-stages. Similarly, the forms of the pathogen differ during parasitic development. Mosquitoes inject sporozoites into the skin. Sporozoites are characterized by their size (10–15 μm in length and 1 μm in diameter), their mobility, and their characteristics of migration from skin to liver, including the speed, the gliding motility and the cell traversal. After productive invasion of hepatocytes, parasites develop in the parasitophorous vacuole until the egress of merosomes from liver cells, with or without dormant hypnozoite formation, depending on the Plasmodium spp. After merosomes burst in the microvasculature, merozoites (1.5–2.5 μm in length and 1.0–2.0 μm in diameter) are released and invade RBCs. The resulting iRBCs can bind non-infected RBCs (rosetting), and adhere to endothelial cells of the microvasculature (sequestration). The rupture of schizont-infected erythrocytes releases new merozoites. Some parasites eventually differentiate into male and female gametocytes (up to 10 μm in length and 2.5 μm in diameter) that can be ingested by mosquitoes. Therefore, the strategy for in vivo imaging of Plasmodium infection and malaria, and thus the choice of imaging techniques, should be adapted to the resolution, depth of penetration, and sensitivity required. Multi-modal imaging approaches are entirely feasible.

In vivo imaging of malaria in NHPs

Clinical severe malaria: imaging of brain and spleen in NHPs

In vivo imaging studies of malaria have mainly focused on clinical features of the disease using various animal models, including NHP models. In particular, the features of cerebral malaria in NHPs have been explored using in vivo imaging.

Cerebral malaria is a major complication of severe human malaria and is defined as acute encephalopathy caused by P. falciparum infection [101]. Imaging analysis of the brain is currently the preferred technique for the evaluation and diagnosis of impaired cerebral function. There have been numerous reports of brain imaging (CT, MRI) in patients with cerebral malaria [102], [103], [104], [105], revealing brain swelling, small hemorrhagic lesions, and focal lesions in the cerebrum and brainstem. However, the correlation between pathological changes and altered metabolic activity, which may directly reflect neurological symptoms in cerebral malaria patients, is not clearly understood [106].

MRI and magnetic resonance spectroscopy (MRS) have been used to study experimental cerebral malaria and to identify its early markers in mice infected with P. berghei ANKA [107], [108], [109]. Platelet accumulation, which occurs in the microvasculature of patients with cerebral malaria, has also been investigated by MRI in the P. berghei ANKA/murine model [110].

PET imaging with [18F]FDG has been used in a primate model of severe human malaria (Japanese macaques infected with P. coatneyi) to evaluate cerebral glucose metabolism [53], [54], [106], [111]. The value of the P. coatneyi Japanese macaque model for the study of cerebral malaria was demonstrated by the identification of cytoadherence of infected erythrocytes to brain endothelial cells within microvessels in vivo, similar to observations in human cerebral malaria [53], [54]. Indeed, FDG-PET imaging studies demonstrated that pathological findings in infected monkeys were similar to those in human cases of P. falciparum malaria [106].

The group that studied cerebral glucose metabolism in Japanese macaques infected with P. coatneyi also investigated the relationship between glucose uptake by the spleen determined by FDG-PET and histopathological changes in spleen tissue: FDG uptake was higher in infected than control animals. This may reflect the activation of host splenic clearance systems or glucose consumption by congested malarial parasites themselves, or both [112].

Imaging analysis of the progression of pathological processes in NHP models can be informative about the mechanisms of malaria. The use of in vivo imaging in NHP models of experimental malaria may help the development of imaging strategies that could be transferred to studies for humans. However, murine models of malaria, and particularly as concerns the transmission, liver-stage and blood stage of the parasite, have been much more extensively used than NHP models for in vivo imaging.

Transmission, liver- and blood-stages: imaging mainly in mice

The techniques of in vivo imaging can contribute to parasitology by providing dynamic images of host–pathogen interactions in vivo, thereby overcoming the limitations of post-mortem investigation and in vitro studies (reviewed in [113], [114], [115]). Most imaging studies in murine models of malaria have used fluorescence, including wide-field fluorescence microscopy, intravital microscopy, and bioluminescence. To analyze the migration of parasites, transgenic parasites expressing GFP [116], [117], red fluorescent protein (RFP) [118] or a GFP–luciferase fusion protein [119] were used. Fluorescent P. berghei sporozoites have been used to study mosquito and mammalian host interactions in the dermis with malaria parasites by in vivo microscopy [116], [117], [120], [121], [122]. Not all sporozoites reach the liver. Some enter lymph vessels and accumulate in the draining lymph node, where they appear to reside for some time before being degraded [120]; others remain in the skin. It is possible to observe and count the number of fluorescent parasites that are ejected from the mosquito over time [123]. This has revealed that there is a limited pool of Plasmodium sporozoites available for ejection within the vector's salivary glands.

RFP- and GFP-expressing parasites and various in vivo microscopy techniques have been used for in vivo investigations of the behavior of P. berghei parasites in the hepatic tissue of the murine host. These studies have provided useful information about the primary infection process and about parasite interactions with the host immune cells in the liver [118], [124], [125], [126].

The use of bioluminescence imaging has been described for in vitro screening of inhibitors and chemicals for antimalarial activity against blood stages of P. berghei [127], and for in vitro and in vivo assays to analyze Plasmodium liver stage development using transgenic P. berghei (PbGFP-Luccon) or P. yoelii (Py-GFP-luc) [128] parasites, which express the bioluminescent reporter protein, luciferase. In these assays, parasite development in hepatocytes can be visualized and quantified by real-time bioluminescence imaging both in culture and in live mice. The luminescence signal is a measure of the parasite load in the liver parasite [129], [130]. PbGFP-Luccon can therefore be used for the evaluation of protective immunity against malaria, following immunization with either radiation attenuated sporozoites or wild-type sporozoites under chloroquine prophylaxis [131].

These transgenic parasites are straightforward and valuable tools for investigating the biology and immunology underlying the mechanisms of pathogen transmission and dissemination, and the mechanisms of protection against malaria. However, findings in murine models require confirmation in NHP models. Rodents and primates differ in their sites and mechanisms of erythropoiesis, making NHP research for blood-stage malaria valuable. Furthermore, work with NHP models would contribute to a more complete understanding of the mechanisms of infection of some parasites, such as P. vivax. This species has a silent liver phase in humans which could not been explored in murine models since rodent parasites do not have a dormant liver-stage form.

Tools required facilitating in vivo imaging of malaria in NHP models

Progress in various areas would allow the use of NHP models of human malaria to be extended: improvement of techniques for culture and laboratory maintenance of Plasmodium, completion of their genome sequences and generation of transgenic strains [132], [133]. These issues are not specific to the in vivo imaging field. Advances in these areas are in line with the research agenda for malaria eradication (malERA), recently established by the consultative group on Basic Science and Enabling Technologies [134].

Currently, it is not possible to culture Plasmodium throughout its complete life cycle. Long-term in vitro blood-stage culture of P. vivax and P. cynomolgi is not possible, in contrast to P. falciparum and P. knowlesi [135]. If such culture techniques were available, it would be possible to study at least blood-stage parasites more easily, to screen drugs, and to feed female Anopheles mosquitoes, without having to use infected donor NHPs for a continuous source of parasites. Unfortunately, some in vitro-adapted strains lose their capacity to produce sexual stages, like for P. knowlesi [135].

Bites from infected mosquitoes or sporozoite injection can be used for experimental sporozoite inoculation. Sporozoites isolated from salivary glands of mosquitoes have a limited ex vivo viability [136], [137], and methods to improve their preservation would be useful.

Mosquito colonies need to be maintained near the NHP facilities to facilitate transmission and liver-stage studies in NHP models due to the lack of an effective in vitro sporozoite culture system, including for the transformation of gametocytes into ookinetes and the sporogonic development into sporozoites occurring in mosquitoes.

Whole genome sequences of P. falciparum [138], P. vivax [139] and P. knowlesi have been reported [140]. Draft sequences for three P. cynomolgi strains were published recently [141]. Improved and additional genomes for each species are in progress.

Genetic manipulations (gene disruption, mutation, tagging or introduction) were initially developed in P. berghei [142] and then in P. falciparum [143], [144], [145]. The generation of transgenic parasites that contain an imaging-reporter gene, either a fluorescent reporter or an enzymatic reporter, such as luciferase or tk, is essential for in vivo imaging. Exogenous genes can be introduced either on a plasmid or by homologous recombination of a linear DNA fragment into the parasite genome, targeted to a silent chromosomal locus without alteration of the phenotype of the parasite after disruption, such as the dispensable Pf47 locus in P. falciparum. Episomal plasmids containing origins of replication derived from bacteria do replicate in Plasmodium; they are not equally segregated into daughter cells during mitosis and they may be lost. Stable episomal transfection is however possible, and differs from transient episomal transfection by the presence of a selection marker in the transfected DNA. Multiple rounds of drug treatment allow selecting parasites with crossover integration of episomes. For comprehensive reviews and detailed protocols, see [146], [147], [148], [149]. Briefly, the asexual stage has a haploid genome and thus is the preferred stage for the generation of transgenic parasites because it requires modification of only a single allele. Optimal conditions have been defined for electroporation and for nucleofection ensuring the access of exogenous DNA to the parasite nucleus while preserving viability. Transfection efficiencies differ according to the developmental stage of the parasite and the Plasmodium species. Infected erythrocytes containing ring-stage parasites (P. falciparum) or mature schizonts (P. berghei) can be used for genetic manipulations, and transfected (e.g. resistant or fluorescent) parasites can be selected and cloned either in vivo or in vitro. Various promoters, both homologous and heterologous, with different strengths and timing of activity, e.g. stage-specific, conditional or constitutive, are available for use in these species.

The third Plasmodium parasite for which transfection has been reported is P. knowlesi [132], [150], [151], [152]. Stable transfection of P. vivax has not yet been described: only transient transfection of P. vivax blood-stage parasites using P. vivax-infected reticulocytes from splenectomized monkeys has been reported [153], [154]. Stable transfection of P. cynomolgi has been demonstrated [155], [156].

There is no standardization in generating and reporting genetically modified primate malaria parasites, in contrast to rodent transgenic parasites [157]. There are several transgenic P. falciparum expressing an imaging reporter gene during blood-stage. They are used for in vitro assays, likely because of the lack of gametocyte production. Theoretically, RBCs infected with these transgenic parasites could be used for in vivo imaging of blood-stage. Transgenic P. falciparum expressing an imaging reporter gene throughout the entire parasite life cycle include P. falciparum expressing GFP (3D7HT-GFP) [158], deposited at the Malaria Research and Reference Reagent Resource Center (MR4) and tested for liver-stage using ex vivo human primary hepatocytes infection; and P. falciparum expressing a luciferase–GFP fusion (NF54HT-GFP–Luc) tested for liver-stage using human liver chimeric mice [23]. They are available for in vivo imaging studies in NHP models of malaria. Recently, a transgenic P. cynomolgi with two expression cassettes for constitutive expression of GFP and mCherry was developed. Ex vivo infection of primary hepatocytes from rhesus monkey was performed and developing liver stage and hypnozoites forms were documented by live fluorescent microscopy [55]. Finally, the adaptation of a P. knowlesi line expressing GFP throughout the asexual blood-stage cycle to continuous culture in human erythrocytes has been recently demonstrated, providing potential tools for studying blood-stage in NHPs [159].

Conclusions and perspectives

In vivo imaging is an enormously powerful approach to deciphering host/Plasmodium interactions in mouse models, and NHP models are extremely valuable models of human malaria. Consequently, it is unsurprising that in vivo imaging is increasingly linked to primate parasitology, particularly as it becomes clear that in vivo imaging is feasible with larger animals. However, so far, studies using in vivo imaging approaches in NHPs are rare. They have been limited to PET imaging to document inflammation and cerebral malaria in the M. fuscata/P. coatneyi model [106], [111], [112]. Admittedly, facilities where the dual NHP/malaria expertise is available, and have both the desired model up-and-running and appropriate in vivo imaging devices can be difficult to find (Table 1 ). Nevertheless, the list of transgenic parasites encoding an imaging reporter gene usable in NHP models is growing (Table 2 ). To our knowledge, they have not yet been tested in vivo.

Table 1

Several malaria NHP models have been developed to study host/pathogen interactions, disease pathogenesis, malaria immunity, drugs and vaccines. New World primates (Aotus and Saimiri) are used for experimental infections with either human malaria species (except P. ovale) or simian malaria species. They are the only animal models available for the study of vaccine efficacy or drug susceptibility of human malaria parasites P. falciparum and P. vivax. Macaques (Old World primates), when infected with simian malaria parasites, can also be used to study the biology of the human malaria and to explore mechanisms and treatments for severe pathology associated with malaria infections. Only the main models are listed below [2], [11], [38], [39].

	Plasmodium	NHP	Model	Remarks	References
Human Plasmodium	P. falciparuma	Aotus lemurinus griseimembra	Human infection with P. falciparum, severe malaria and vaccine efficacy	No cerebral malaria	[42]
	P. vivax	Aotus lemurinus griseimembra	Human infection with P. vivax, severe malaria and vaccine efficacy	For protective efficacy testing of sporozoite and liver stage vaccines but not blood-stage vaccines (unless strains further adapted in spleen-intact animals)	[41], [42]
		Saimiri boliviensis
	P. knowlesia	Macaca mulatta	Human infection with P. knowlesi, severe malaria and malaria immunity	Including cyto-adherence, parasite sequestration and Ag variation with Ag switching in vivo; renal failure	[47], [48], [49]
		Macaca fascicularis	Natural control of parasite development and chronic infection (natural host)
Simian Plasmodium	P. coatenyi	Macaca mulatta	Human infection with P. falciparum and severe malaria	Including cerebral malaria, cytoadherence, parasite sequestration and Ag variation, anemia, placental malaria	[50], [51], [53], [54], [66]
		Macaca fuscatab
	P. cynomolgia	Macaca mulatta	Human infection with P. vivax and malaria immunity	Including relapse-infections resulting from reactivation of hypnozoites and unique iRBC features	[48], [55], [56], [57], [64]
		Macaca fascicularis
	P. fragile	Macaca mulatta	Human infection with P. falciparum and severe malaria	Including cerebral malaria; cytoadherence, parasite sequestration and Ag variation	[52], [62], [63], [65]

The only transgenic parasites encoding an imaging reporter gene that could be used for in vivo fluorescent microscopy and bioluminescence are P. falciparum, P. knowlesi and P. cynomolgi. So far, they have only been used in vitro.

To date, only cerebral malaria in the NHP model of M. fuscata/P. coatneyi was studied using in vivo imaging (PET).

Table 2

Available tools for in vivo imaging of Plasmodium parasites and host response in NHPs. So far these transgenic parasites expressing an imaging reporter gene have only been used for in vitro studies, and should be developed for other parasites such as P. vivax to explore the biology of Plasmodium by in vivo imaging.

Imaging the pathogen
Available transgenic Plasmodium parasites	Imaging techniques	References
• Transgenic GFP and GFP–Luc P. falciparum with a constitutive expression	Fluorescence Bioluminescence	[145], [158], [23]
• Transgenic P. falciparum with a blood-stage expression
• Transgenic GFP–mCherry P. cynomolgi with a constitutive expression	Fluorescence	[55]
• Transgenic GFP P. knowlesi with a blood-stage expression	Fluorescence	[159]
Imaging the host
Model	Imaging techniques	References
Cerebral/splenic glucose metabolism in P. coatneyi/Japanese macaque models	[18F]FDG-PET imaging MRI	[106], [111], [112]

There is no doubt that the complexity of the interaction between pathogens and the host immune system can only be properly understood when studied in the native tissue environment [160]. Indeed the in vivo dynamic imaging techniques can reveal differences with in vitro data such as the CD8+T cell-mediated elimination of malaria liver stages [161]. Development of these new imaging tools that are applicable for NHP models will allow a better understanding of the biology of Plasmodium and of the host immune responses against parasites and parasitized cells as well as the development of novel therapeutic strategies. To conclude, in vivo imaging in NHP models of malaria could be very helpful to explore the following top four topics: the study of the early events in transmission to characterize the dynamic of skin stage and liver infection, the longitudinal characterization of hypnozoite forms and infection relapses, the time-lapse imaging of the invasion of red blood cells, and the dynamic imaging of host-parasite interactions and immunity.

Conflict of interest

The authors declare no financial or commercial conflict of interest.