Non-human primate model systems have been instrumental in malaria research for decades whether for furthering basic understanding of Plasmodium biology, malaria pathogenesis, or preclinical investigations pertinent to developing new interventions (Coatney et al., 1971; Collins, 1974; Galinski and Barnwell, 2012; Beignon et al., 2014). Notably, they were critical for the discovery of dormant forms of the parasite in the liver, known as hypnozoites. These forms were first discovered in rhesus macaques (Macaca mulatta) experimentally infected with P. cynomolgi (Krotoski et al., 1982b), and then in chimpanzees infected with P. vivax (Krotoski et al., 1982a). Research from the field has confirmed that hypnozoites can stay dormant for weeks, months, or years after a primary infection and then activate and result in relapses, with new cycles of blood-stage parasitemias and illness (White and Imwong, 2012). Recently, NHPs were critical for the development of an in vitro, primary hepatocyte culture system that supported the cultivation of P. cynomolgi LSFs for approximately 40 days and provided the first tangible evidence that hypnozoites existed and were capable of activating and multiplying to generate merozoites (Barnwell and Galinski, 2014; Dembele et al., 2014).

Various NHP-simian and human malaria parasite combinations can be used to study Plasmodium biology. Many strains of the parasites that infect NHPs, including four validated relapsing species, are available and can be used to address scientific questions relating to LSF biology (Table 1). Different strains of P. cynomolgi and P. vivax that possess distinctive relapse patterns can be utilized to study the consequences of frequent versus infrequent relapses on the host immune system. A suitable mouse model is not currently available to study such phenomena. Indeed, humanized mice containing human hepatocytes have been demonstrated to support P. falciparum liver-stage growth (Vaughan et al., 2012; Kaushansky et al., 2014). These models also appear to have some utility for P. vivax because they appear to support the development of hypnozoites (Mikolajczak et al., 2013). However, these mice lack intact immune systems and, thus, are deficient when addressing immunobiological questions, whether for P. falciparum or other primate malaria species (Kaushansky et al., 2014).

Systems biology approaches have been recently pioneered to study infectious diseases and vaccine efficacy (Li et al., 2014; Petrizzo et al., 2014; Zak et al., 2014). Systems biology foregoes traditional reductionist approaches and focuses on a biological system in its entirety. Typically, multiple ‘omics technologies are employed to generate large datasets and methods are developed to integrate those datasets. Advanced mathematics and statistics are required to generate computational models of specific biological processes, such as hematopoiesis, immune responses, and infectious disease pathogenesis. Indeed, these strategies are being utilized by the Malaria Host–Pathogen Interaction Center (MaHPIC) using Old World and New World monkey models to study malaria and bring a wealth of data and novel results to the research community via online resources. We believe that systems biology approaches will likewise have utility for studying Plasmodium LSFs and the mechanisms behind relapses.

Systems biology methods can be attempted to study LSFs using transgenic, fluorescent LSFs isolated by fluorescence-activated cell sorting from ex vivo liver tissue. We and others (Voorberg-van der Wel et al., 2013) hope to demonstrate that adequate, purified ex vivo parasite material for downstream experimentation can be attained using such strategies. Performing transcriptomic, proteomic, and metabolomic analyses on the ex vivo material holds potential for identifying biochemical and molecular pathways important in hypnozoite biology. A hypnozoite proteome may help elucidate new vaccine candidates whereas transcriptome and metabolome data could give insight into other potential biochemical and molecular pathways that could become drug targets. Indeed, headway is being made in understanding hypnozoite biology with the recent demonstration that epigenetic programming could be responsible for latency (Dembele et al., 2014). Notably, however, modifying epigenetic changes in vivo may have detrimental side effects to the host organism, and thus, this may not be a feasible treatment strategy in humans.

We are well aware of the scientific and physiological challenges, but also optimistic that highly sensitive systems biology approaches that include metabolomics can help identify biomarkers that could predict the presence of hypnozoites and serve as diagnostic tools. Metabolomics is a relatively new, yet powerful, scientific discipline that is gaining traction in the fight against malaria (Olszewski et al., 2009; Kafsack and Llinás, 2010; Lakshmanan et al., 2011; Salinas et al., 2014). It is exciting to consider how high-resolution mass spectrometry could potentially identify a metabolic biomarker(s) (predictably of host origin) in the serum, plasma, urine, or saliva that is indicative of the presence of LSFs and the need for treatment. Potential biomarkers are best sought from an in vivo infection where host–parasite interactions can be investigated in the context of the normal physiology of the parasite and the host. Experimental NHP models can be informative in this regard. Diets and other variables can be controlled, and infected samples can be collected daily, or even multiple times daily if useful, over the course of a designated infection period through multiple relapse episodes without the immediate, ethical need for treatment.

Extracellular vesicles (EVs) are a heterogeneous population of small vesicles found in virtually all bodily fluids including serum, plasma, urine, saliva, etc., and are categorized into subtypes based on their physical properties such as density, size, and shape as well as their biogenesis (Kowal et al., 2014; Robbins and Morelli, 2014). These vesicles are produced by all multi- and unicellular organisms examined to date, and different types of EVs contain specific protein and RNA cargo dependent upon the cell-type the EV originated from (Villarroya-Beltri et al., 2014).

The roles of liver-derived EVs on liver physiology have been reviewed elsewhere (Imani Fooladi and Mahmoodzadeh Hosseini, 2014). Notably, multiple studies have implicated liver-derived EVs in the life-cycle of liver pathogens. For example, exosomes, a specific EV subtype that originates from a multivesicular body within the cell, derived from liver non-parenchymal cells were demonstrated to transfer resistance against hepatitis B virus infection to hepatocytes via an IFN-α mediated mechanism (Li et al., 2013). Despite these implications of EVs in the liver, putative roles of EVs have not been reported with regards to Plasmodium liver-stage biology, but warrant attention. Indeed, EVs were recently shown to be released from Plasmodium infected erythrocytes and implicated in a “density-dependent sensing mechanism” that influences P. falciparum gametocytogenesis (Mantel et al., 2013; Regev-Rudzki et al., 2013). These studies were the first to raise the possibility for a natural role of EVs during the life-cycle and provide a firm rationale that such mechanisms may exist for LSFs.

Extracellular vesicles could predictably serve as a communication mechanism between LSFs and influence hypnozoite activation, and NHP, primary hepatocyte cultures can be used to assess if such mechanisms exist. Cultures can be infected with sporozoites, and EVs derived from uninfected and infected hepatocytes can be isolated from the culture medium (Momen-Heravi et al., 2013). The EVs can then be placed on LSF cultures harboring different forms of the parasites, including hypnozoites, to directly test if EVs isolated at different points of the LSF cycle (e.g., when hypnozoites are activating) result in specific biological outcomes. For example, one may predict that EVs isolated from cultures with activated hypnozoites may signal other hypnozoites to exit dormancy and multiply. These experiments are not perfect, however, and will be unable to delineate if the effect is caused by host and/or parasite-derived EVs, but it is a starting point to explore mechanisms and specific EV components that could potentially be used to force hypnozoite activation.

If such a strategy of ‘waking up’ hypnozoites is feasible, akin to the novel ideas suggested by others based on drug interventions to target putative epigenetic control mechanisms of dormancy (Dembele et al., 2014), only the blood-stages would have to be treated, making treatment more straightforward with multiple safe options compared to primaquine with its contraindications (White et al., 2014). Indeed, as members of the MaHPIC, we have been developing EV research strategies utilizing NHPs infected with simian malaria parasites. EVs are challenging to purify, but we have managed to purify them from small volumes of plasma, observe them by electron microscopy, and confirm their identity through other biochemical and physical means (unpublished data).

Hepatocyte-derived EVs in the circulation could also comprise possible biomarkers of hypnozoites similar to biochemical biomarkers identified by metabolomics (Deng et al., 2009; Vlassov et al., 2012). Hepatocytes and other tissue-resident cells in the liver release EVs that contain specific protein and RNA cargo that become altered during liver injury (Farid et al., 2012). Similarly, we hypothesize that the microRNA and protein content of liver-derived EVs will be altered when hepatocytes are infected with Plasmodium. If this proves to be the case, EVs could be potentially isolated from serum, plasma, saliva, and/or urine and specific diagnostic tests developed to use EVs as diagnostic markers of hypnozoites. Although it may seem unlikely that EVs from Plasmodium-infected hepatocytes could serve as biomarkers because of the scarcity of infected hepatocytes (a relative few in an entire liver), EVs are currently being explored to develop sensitive methods to detect cancer in bodily fluids (Rak, 2013). If EV-based diagnostic approaches are feasible and promising for cancers where few malignant cells exist, such approaches are worth exploring for developing technologies to detect hypnozoites.

The immunobiology behind relapses, recrudescences, and chronic infections (Figure 1) during malaria is poorly understood although “immune exhaustion” during chronic infections has been investigated recently (Wykes et al., 2014). This neglected area of research needs attention because each of these distinctive infection profiles could have unique effects on the host immune response; e.g., to alter the host’s memory pool.

The team of immunologists at the MaHPIC has been using NHP models to understand the effects of relapses on the host immune system. Indeed, the consortium has determined that relapses cause continuous expansion of the circulating memory B-cell pool using the P. cynomolgi-rhesus model system (unpublished data). Currently, the team is performing follow-up experiments to better understand this phenomenon and its immunological relevance using sampling strategies that are not possible in humans. Blood is being collected before, during, and after relapses to monitor the alterations in the B-cell compartment by flow cytometry, with a special interest in memory B cells. The identities of predominant B-cell clones based on immunoglobulin sequences are being examined using Ig-Seq technologies (Georgiou et al., 2014). The first goal is to determine the clonal diversity of the B-cell recall response against relapsing or challenge parasites. The second goal is to assess which B-cell clones respond during consecutive blood-stage infections. If consecutive blood-stage infections are selecting for a particular subset of B-cells or, contrastingly, inducing proliferation of different B-cells, the host’s memory B-cell pool could be significantly altered. We predict that alterations of the B-cell compartment could translate into poor recall responses because memory B-cells, critical for detecting, expanding, and producing antibodies to eliminate the parasites, might be eliminated from the memory B-cell niche by other B-cells that predominantly proliferate. If this proves to be the case, the impact must be considered in light of developing a vaccine that relies on neutralizing antibodies mediated by memory B-cells.

Non-human primate models of malaria have enabled major contributions toward understanding liver-stage biology, hypnozoites, and relapses, and will continue to provide the means to investigate this enigmatic part of the Plasmodium life-cycle. Primaquine is the only FDA-approved drug against hypnozoites despite its contraindications. Additionally, excessive use of this drug can support the rise of primaquine resistant parasites (John et al., 2012; Price, 2014). A biomarker test would help restrict treatment to only those individuals in need, and be useful in malaria elimination campaigns where it is preferable to only treat infected individuals instead of everyone to ensure elimination of the parasite reservoir. New knowledge, techniques, and possible diagnostics, vaccines, and medications that may result from studying LSFs using NHP models will inevitably be key to malaria eradication efforts.

All authors contributed to the writing, figure and table, and reviewed and approved the finalized article.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.