A way forward for culturing Plasmodium vivax

Abstract

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish P. vivax in continuous culture have failed. Unlike P. falciparum that can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth In vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is the protection from oxidative stress by glucose-6-phosphate dehydrogenase. Here we also suggest supplements to the media and procedures that may reduce oxidative stress and as a result establish a system for continuous culture of P. vivax.

A need for Plasmodium vivax continuous culture system

Malaria is a severe disease affecting more than 228 million people in developing and underdeveloped countries (WHO 2019 report) [1]. Five species of Plasmodium cause malaria in humans, of which Plasmodium falciparum and P. vivax are the most prevalent. High incidence of P. vivax is observed in Asia and South America. Africa, until recently, was thought not to have P. vivax, because Africans are predominantly Duffy blood group negative [2]. We now know that P. vivax is prevalent in Africa in Duffy-negative people, but the infection is at a low level and sometimes difficult to identify without the P. vivax polymerase chain reaction (PCR) [3]. In areas of both Duffy-positive and negative people living side-by-side (Madagascar, Ethiopia and Sudan), P. vivax is more severe in Duffy-positive Africans [4–6]. Compared to P. falciparum, the parasite density in P. vivax infection is low, and the primary disorder from P. vivax is anemia, although respiratory distress and coma may also occur although the incidence is rare [7]. It is important to develop a continuous In vitro (see Glossary) culture system for P. vivax to understand parasite biology in order to develop suitable interventions to fight the parasite.

A favorable reticulocyte intracellular environment for P. vivax

One thing that is common to all blood stage infections is the specific interaction that happens between the parasite and the host erythrocytes during invasion. In addition, the unique environment within the host cell may also aid in parasite propagation. In the case of P. vivax, preferential invasion of reticulocytes is mediated by reticulocyte-specific receptors for invasion and a possible requirement for high intracellular levels of glucose-6-phosphate dehydrogenase (G6PD) [8, 9] which declines as the erythrocyte ages. The G6PD deficiency is strongly positively selected in populations due to its ability to protect against P. vivax [10] and may reflect the parasites’ need for the antioxidant environment. G6PD is the enzyme that reduces nicotinamide adenine dinucleotide (NADP) to NADPH. NADPH then takes oxidized glutathione (GSSG) to reduced glutathione (GSH). GSH is critical for preventing oxidative stress (reactive oxygen species, ROS) in erythrocytes, including P. vivax infected erythrocytes.

A glance at the P. vivax culture system

In 1941, Trager aimed at identifying specific reagents and conditions essential for Plasmodium survival In vitro [11]. Trager selected P. lophurae which infects duck erythrocytes to test for parasite survival under In vitro conditions as the susceptible host is readily available [11]. Trager determined that the parasites survived for 6 days In vitro with parasite survival dropping from 40% on day 3 to 0.5% on day 6 [11]. In 1976, almost four decades after initial work on In vitro culture, Trager and Jensen made a breakthrough in discovering the right combination of components required for continuous In vitro culture for P. falciparum [12]. The report demonstrated that P. falciparum can be cultured continuously In vitro at 38°C in RPMI medium with human serum in 7% carbon dioxide and 1 or 5 % oxygen [12]. Ever since the establishment of a continuous culture system for P. falciparum [12], similar conditions and medium were tried in subsequent studies for P. vivax culture [13]. However, these conditions were not suitable for P. vivax continuous growth and invasion.

One notable report described a short-term culture of P. vivax Chesson strain (Papua New Guinea from monkeys) [14]. This study used purified human reticulocytes from hemochromatosis patients and a new medium called McCoy’s 5A medium. The parasites were grown in a candle jar in static conditions until the schizont stage. Subsequently, fresh reticulocytes were added, and at the time of merozoite release from infected erythrocytes, the culture was incubated in a shaker for 10-12 hrs. The shaker may be important because, under static conditions when merozoites are released from infected erythrocytes, multiple merozoites may infect one reticulocyte, reducing their survival. In a shaker, if there are limited reticulocytes, merozoites can bounce off multiple erythrocytes until they hit a reticulocyte with receptors to which they can bind and invade. Parasitemia was reported to increase 2-fold every cycle. The parasites obtained from the sixth cycle were subsequently used to infect an Aotus monkey [14]. To date only this study has reported increased parasitemia after every cycle. Using these methods, one study failed to grow P. vivax perhaps due to differences in P. vivax isolates, reticulocyte preparation or the karyotype of the Aotus monkey used [15]. Ring and trophozoite stage parasites isolated from patient samples or monkeys (Aotus or Saimiri) were reported to develop into healthy schizonts under In vitro conditions [16, 17]; nevertheless, the parasite density in subsequent cycles declined [16, 18].

Several groups have reported long term continuous culture. Interestingly, in a recent study, the longest In vitro culture was established using P. vivax isolates from western Thailand cultured in McCoy’s 5A medium at a 5% hematocrit and at 5% CO2 and 5% O2. Although the cultures were maintained for 26 months, the parasite densities were very low and detected only by PCR [19]. In addition, in another recent study, long term P. vivax culture was demonstrated using isolates from Madagascar cultured in Saimiri erythrocytes [18]. AIM V media was used with 4 % hematocrit maintained at 37 C in 10% CO2. P. vivax was continuously propagated for 233 days; however, the parasitemia was very low [18]. These studies fail to offer a path forward for continuous culture. Several improvements in P. vivax short term culture have been made by altering the source of susceptible host cells (reticulocytes) and altering media components and serum [16, 20–22]. Despite this there are no established conditions for continuous culture of P. vivax.

Schuffner’s dots in P. vivax and P. cynomolgi

P. vivax and P. cynomolgi are two highly related parasites that contain speckled red dots on Giemsa-stained infected erythrocytes, reflecting an unusual ultrastructure consisting of caveola-vesicle complexes [23] (Figure 1). The function of these structures is unknown. These structures are lost in culture, even in the most developed culture system [14], and may relate to the pathologic effects of the culture conditions.

Figure 1: Schuffner’s dots in P. vivax and P. cynomolgi infected erythrocytes.

A. Illustration shows the P. vivax infected reticulocytes in a ring form with Schuffner’s dots in red as in Geimsa-stained films. Electron microscopy images (B and C) represent P. vivax and P. cynomolgi infected erythrocytes, respectively, showing caveola-vesical complexes (indicated by arrows) which are Schuffner’s dots specific to these two species. Figures in panel B and C are adapted from Aikawa et al. [23] with permission.

Plasmodium vivax invasion restricted to reticulocytes

Maturation of reticulocytes is defined based upon the degree of CD71 expression (CD71^high, CD71^medium, CD71^low and CD71^negative) where CD71^high are young reticulocytes, and CD71^negatlve are mature erythrocytes [24, 25]. The three P. vivax parasite ligands that bind reticulocytes are Duffy Binding Protein 1 (DBP1), Erythrocyte Binding Protein (EBP) or DBP2, and Reticulocyte Binding Protein (RBP2b).

P. vivax requires Duffy blood group antigen for optimal invasion but can invade Duffy-negative erythrocytes in Africa possibly due to some Duffy-negative individuals may still express low level (leaky expression) of Duffy blood group antigen on their erythrocytes [2, 3, 26]. Rosettes of erythrocytes around COS7 cells expressing DBP-RII (erythrocyte binding domain of DBP1) bind approximately 30% better to reticulocytes than mature erythrocytes in which the reticulocytes were removed [25]. Thus, DBP-RII binding is not exclusive to reticulocytes. Again, the higher anti Duffy blood group antigen (Fya and Fyb) binding on Duffy-positive reticulocytes [27] does not interfere with blood typing of erythrocytes with no requirement for purification of reticulocytes in blood banks. In addition, studies indicate that binding of recombinant DBP-RII to Duffy-positive mature erythrocytes is much lower [27–29] compared to binding of COS7 cells expressing DBP-RII to Duffy-positive erythrocytes [30]. This difference is due to multimeric interactions in the COS7 expression system.

Two other P. vivax ligands, EBP (or DBP2) and RBP2b preferentially bind reticulocytes for invasion. Binding studies confirm that EBP binds strongly to CD71^high reticulocytes [25]. EBP is structurally similar to DBP, but it does not bind to Duffy blood group antigen [30], and its reticulocyte receptor is unknown. The copy number of EBP in Madagascar was between 1 and 5 copies, suggesting selection for EBP [31]. Another parasite ligand found to only bind reticulocytes was RBP2b, and its receptor was shown to be CD71 [32]. As CD71 is critical in the uptake of transferrin for hemoglobin development, CD71 could not be knocked out. Instead, CD71 mutations that did not affect transferrin uptake but did affect its ability as a receptor led to the breakthrough in receptor identification [32].

The proportion of reticulocytes in peripheral blood is between 0.5-1.6% [33]; hence purifying reticulocytes from blood for culturing P. vivax In vitro is challenging. To improve on reticulocyte yield, several attempts were made to purify reticulocytes from hemochromatosis patients who have a higher percentage of circulating reticulocytes [14, 15, 33], depending on when they started treatment (repeated removal of a unit of blood). Other attempts were made to culture the P. vivax in reticulocytes obtained from cord blood [16, 20, 21]. The main reason to derive reticulocytes from cord blood is due to its increased proportion of reticulocytes in the range of 2-8 % [21, 34]. While In vitro invasion assays were achievable to some extent, obtaining a continuous source of reticulocytes remains a problem. In order to obtain more reticulocytes, CD34+ hematopoietic stem cells from the cord blood were purified and differentiated In vitro with appropriate growth factors such as stem cell factor and erythropoietin [35–37]. The cells differentiated through hematopoiesis developed to young reticulocytes by day 15 and by five days later became mature erythrocytes. The reticulocytes from day 15 were suitable for P. vivax merozoite invasion [38]. Although there was successful P. vivax merozoite invasion in the first cycle, the invasion efficiency dropped even after continuous supplementation with fresh reticulocytes [15, 16, 38].

Attempts have been made to develop an immortalized hematopoietic progenitor cell line for genetic manipulations and to have a continuous supply of reticulocytes [39, 40]. Reticulocytes derived from immortalized cell lines need to be tested for P. vivax continuous cultivation In vitro.

Oxidative stress in culturing P. vivax

The missing factor/condition required for successful growth and invasion of P. vivax remains a puzzle. Most mammalian cells grown In vitro require a certain proportion of oxygen (O₂). The required percentage of oxygen varies based on the nature of the cell type and its growth In vivo. The concentration of oxygen differs between the brain, kidney, heart, liver, intestine, skin and bone marrow [41, 42] and varies from low oxygen to room air conditions. P. vivax has been shown to grow in reticulocytes in bone marrow where the oxygen concentration is low [24, 41,43, 44].

An important factor that may have been overlooked and is not yet known for reticulocytes or P. vivax is how they are affected by oxidative stress from exposure to atmospheric O₂. Performing usual culture maintenances like media changes in the hood under ambient air might trigger oxidative stress in parasites or reticulocytes (Figure 2A) adapted to hypoxic conditions. Consequently, this oxidative stress has the potential to negatively impact growth, especially when the percentage of oxygen in bone marrow where reticulocytes are made is between 1.3 to 4.2 % [41].

Figure 2: Hypoxia conditions to combat oxidative stress in P. vivax and reticulocytes.

Panel A shows the P. vivax culture maintenance (medium change) in the hood where P. vivax and reticulocytes are exposed to ambient air. The parasites develop from rings, trophozoites to schizonts but fail to invade uninfected fresh reticulocytes and the cycle stops. Panel B shows a hypothetical experiment in which P. vivax culture maintenance (medium change) and growth occurs in a hypoxia chamber at 37°C and low oxygen concentration between 1 to 5 % throughout. The parasites undergo healthy development from rings, trophozoites to schizonts then invade the healthy reticulocytes and the cycle continues.

There is precedent for this hypothesis; a study on the transplantation of hematopoietic stems cell (HSCs) has identified that HSCs isolated in low oxygen/hypoxic conditions from cord blood or bone marrow have better stem cell potency when compared to those that are briefly exposed to ambient conditions [45]. The toxicity to HSCs was due to extra physiologic oxygen shock/stress (EPHOSS) damage in HSCs after 30 minutes of exposure to atmospheric air [45]. The drastic decrease in the recovery of the HSCs was linked to ROS production and mitochondrial permeability transition pore formation [45]. A similar phenomenon was also reported in cardiac stem cells. Cardiac stem cells cultured at 5% O₂ showed increased cell production, reduced ROS levels, less cell senescence and increased resistance to oxidative stress when compared to those cultured at 20% O₂ [46]. Another recent study also showed that growing these heart derived stem cells continuously at physiological conditions in an hypoxia chamber (cell culture processing and maintenance at 5% O₂ at 37°C) resulted in enhanced proliferation and regenerative potency when compared to conditions wherein cells were exposed to ambient air [47].

Trager et al demonstrated that P. falciparum grows better continuously at 1% oxygen concentration compared to 5% [12]. However, the ideal oxygen concentration for P. vivax cultures has not yet been determined, and exposure to higher oxygen concentrations may be deleterious to the growth of P. vivax In vitro. Growth for P. vivax from rings to schizonts (for one cycle) was achieved by culturing in Iscove’s Modified Dulbecco’s Medium (IMDM) medium at 1 % oxygen [22]. However, reinvasion and continuous culturing needs to be tested under this condition.

The growth and proliferation of HSCs and cardiac stems cells are suppressed when exposed to oxidative stress [45–47]. Reticulocytes differentiate, develop and mature in the bone marrow and the bone marrow harbors P. vivax parasites. As a result, reticulocytes and P. vivax might prefer a low oxygen niche similar to HSC’s or cardiac stem cells. Such environmental control can be obtained in a hypoxia chamber (Figure 2A). Since Oxidative stress (ROS) has not been measured in P. vivax and remains to be determined under all conditions.

Supplementing culture medium with molecules to reduce oxidative stress

In addition to experimenting with P. vivax cultures in an environmentally controlled hypoxia chamber to overcome oxidative stress, addition of supplements may further improve the culture conditions. Traditional media formulations used for P. vivax culture contain molecules with antioxidant properties such as glutathione, selenium, and pyruvate [48–51]. Altering the concentration of these antioxidant compounds may help reduce oxidative stress and improve culture outcomes. Recent attempts to further supplement IMDM with various antioxidant compounds including glutathione, selenium, pyruvate, ascorbic acid, and an antioxidant supplement yielded no appreciable improvements to P. vivax growth over a single cycle [22]. However, these molecules may prove more useful when used in conjunction with other culture conditions.

Permeability of the reticulocyte membrane to antioxidant compounds is an important consideration in an antioxidant’s ability to reduce intracellular oxidative stress. Previous studies have indicated that glutathione may not permeate erythrocyte membranes as efficiently as other thiol compounds [52]. Insufficient membrane transmissibility may explain why previous studies have seen minimal benefit from the addition of glutathione to P. vivax culture [22] (Table 1). Alternative thiol compounds that have not been thoroughly investigated in P. vivax culture but demonstrate the ability to improve intracellular glutathione levels during oxidative stress in erythrocytes include N-acetylcysteine (NAC) and N-acetylcysteine amide (AD4) [52, 53] (Table 1). Substituting traditional antioxidant compounds with more membrane permeable alternatives may help alleviate intracellular oxidative stress.

Table 1:

Supplements and procedure to support P. vivax continuous culture system

Supplements and Procedure	Mechanism of Oxidative Stress Reduction	Previously Tested in P. vivax Culture?	Sources
Ascorbic Acid	• Neutralizes free radicals and functions as a chain breaking antioxidant in lipid peroxidation • May act as a pro-oxidant in the presence of redox active ions	Yes	[19, 22, 49, 51]
Glutathione	Neutralizes free radicals and peroxides, acts as a substrate of seleno-proteins, and reduces other thiols and antioxidants	Yes	[19, 22, 48]
Lipoic Acid	• Essential to mitochondrial function in P. falciparum • Reduces oxidized forms of other antioxidants	Alternative media formulations containing lipoic acid have been tested	[22, 54–57]
N-acetylcysteine (NAC)	• Restores intracellular levels of reduced glutathione • Scavenges hydroxyl radicals and hydrogen peroxide	No	[52]
N-acetylcysteine amide (AD4)	Same mechanism of NAC with increased membrane permeability	No	[53]
Pyruvate	Scavenges hydrogen peroxide generated in culture media	Yes	[22, 51]
Salen-Manganese Complexes	Mimic the function of SOD and catalase	No	[58, 59]
Selenium	Component of seleno-proteins including glutathione peroxidases and thioredoxin reductases	Yes	[22, 50]
Thioredoxin Mimetics	Mimic thioredoxin by catalyzing the reduction of other proteins	No	[60]
Hypoxia incubator Chamber	Provides a controlled low oxygen environment and temperature mimicking the In vivo environment of the cell of interest.	No	[45, 47]

Another possible antioxidant supplement is lipoic acid. Lipoate is a coenzyme for several enzymes involved in oxidative metabolism. P. falciparum mitochondrial lipoylation (covalent bonding of lipoate) relies specifically on scavenged lipoate. Lipoate analogues are capable of arresting P. falciparum growth In vitro. However, addition of lipoic acid restores growth, indicating that scavenged lipoate is essential for P. falciparum intraerythrocytic survival [54]. Three lipoylated proteins have been localized to the mitochondria: branched chain dehydrogenase (BCDH), alpha-ketoglutarate dehydrogenase (KDH), and the H-protein [55]. The redox state of lipoate is essential to these enzymes as lipoylation of BCDH and KDH requires fully reduced lipoate while lipoylation of H protein does not [55]. These proteins are activated through distinct pathways involving two enzymes (PfLipL1 and PfLipL2). PfLipL1 recognizes the redox state of lipoate and functions as a molecular switch that determines downstream lipoylation [56]. Addition of lipoic acid to P. vivax cultures may help ensure proper mitochondrial function. Lipoate also protects against oxidative damage by quenching ROS and reducing oxidized forms of other antioxidants such as ascorbic acid and glutathione [57] (Table 1). During previous investigations to determine the appropriate media formulation for P. vivax growth, several media containing lipoic acid including DMEM/F12 and Ham’s Nutrient Mixture F12 and F10 have been used and were found to be inferior to IMDM. However, IMDM and alternative media formulations differed in composition by many elements, not just lipoic acid, and additional supplementation of IMDM with lipoic acid was not reported [22]. As result, the possible benefits of lipoic acid in P. vivax culture remains to be tested.

Adding mimetics of natural proteins involved in decreasing oxidative stress could also improve P. vivax media formulations. Salen-manganese (salen Mn) complexes are synthetic complexes that mimic the function of superoxide dismutase (SOD) and catalase by scavenging both superoxide and hydrogen peroxide. Salen Mn complexes have been utilized to reduce oxidative stress in rat ischemic brain models and to mitigate radiation injury [58, 59]. Salen Mn complexes may help to reduce extracellular ROS in P. vivax culture (Table 1). Thioredoxin mimetics are another possible culture supplement. These peptides possess the catalytic site of thioredoxin and are blocked at both the N- and C-termini to increase their membrane permeability. Thioredoxin mimetic peptides were significantly more effective at preventing oxidative damage in thioredoxin disrupted bovine chromaffin cells than conventional thiol-containing antioxidants and ascorbic acid [60] (Table 1). Addition of salen Mn complexes or thioredoxin mimetics may provide one possible avenue to reduce oxidative stress in P. vivax culture.

Concluding remarks

Unlike P. falciparum, P. vivax parasite density in symptomatic patients is low, in part due to the fact that P. vivax is limited to reticulocytes. Although sufficient reticulocytes were provided to maintain P. vivax culture In vitro, continuous culture has not yet been established (Figure 2A). Several questions remain - could this potentially be due to the difference in the environment in which P. vivax parasites develop and grow In vivo compared to the environment In vitro? (Figure 2B), Will isolating reticulocytes and P. vivax under hypoxic conditions improve culture? (Figure 2B), Do reticulocytes or P. vivax undergo EPFIOSS when isolated from bone marrow or cord blood at atmospheric conditions? (See Outstanding questions). In addition, modulation of media components may support the reticulocyte or parasite’s ability to cope with oxidative stress and may play an essential role in obtaining a suitable In vitro culture system for P. vivax. Possible supplementation avenues include addition of media components with antioxidant properties, lipoic acid and antioxidant enzyme mimetics.

Outstanding questions.

Why does P. vivax demonstrate restricted tropism for reticulocytes? Clearly, restricted tropism is not limited only to reticulocyte receptor compatibility.

What combination of factors in addition to ligand-receptor interactions may be required for restricted tropism?

Does P. vivax require oxygen for its survival in reticulocytes? If oxygen is not required for parasite invasion and development in reticulocytes, is the presence of oxygen in the culture environment deleterious to P. vivax growth? Could susceptibility to oxidative stress be a contributing factor for P. vivax restricted tropism to reticulocytes? How can the effects of oxidative stress be reduced?

Apart from the parasite perspective, what is the ideal oxygen concentration for reticulocyte maintenance in in vitro culture? Are reticulocytes too susceptible to oxygen shock? How does oxygen concentration alter the process of reticulocyte maturation? Are reticulocytes grown in hypoxic conditions better able to support P. vivax maturation in vitro?

Unlike P. falciparum, do P. vivax and reticulocytes require maintenance in only hypoxic environment (hypoxia incubator chamber) to support proper maturation of reticulocytes and the efficient invasion and healthy development of P. vivax in in vitro culture?

Could supplementing culture medium with elements that reduce oxidative stress improve P. vivax in vitro growth?

Highlights.

• P. vivax demonstrates a restricted tropism for reticulocytes which poses a problem for in vitro culture due to their low levels in peripheral blood.

• Reticulocyte development occurs in hypoxic conditions in the bone marrow in vivo, therefore they may experience oxygen shock/stress at higher oxygen concentrations possibly altering their ability to support P. vivax development and subsequent invasion.

• Immortalized erythroid progenitor cells are a promising source for obtaining a continuous supply of reticulocytes to be used in vitro culture.

• The link between G6PD deficiency and protection against P. vivax may indicate that the parasite is susceptible to oxidative stress.

• The optimal media formulation for P. vivax culture is not known. Future culture attempts may benefit by focusing on media components that reduce oxidative stress for both the parasite and reticulocyte.

Glossary

Antioxidant

A compound that decreases oxidative damage through direct reaction with free radicals, inhibiting the activity of enzymes that generate free radicals, or increasing the expression of antioxidant enzymes

Extra physiologic oxygen shock/stress (EPHOSS)

A state of oxygen shock/stress experienced by cells typically found in hypoxic conditions such as stem cells when exposed even briefly to ambient air conditions

Glucose-6-phosphate dehydrogenase (G6PD)

An essential enzyme in the pentose phosphate pathway which provides reducing energy via the production of NADPH. NADPH is then utilized by glutathione reductase to reduce glutathione. G6PD deficiency is a genetic disorder which predisposes affected individuals to hemolytic anemia. G6PD deficiency provides protection against P. vivax

Glutathione

An antioxidant tripeptide composed of glutamate, cysteine, and glycine capable of neutralizing reactive oxygen species including peroxides and free radicals

Hematopoietic stem cell (HSC)

Stem cells found in the bone marrow that give rise to mature blood cells via hematopoiesis. HSC differentiation into erythrocytes is termed erythropoiesis

Hemochromatosis

A hereditary disorder characterized by excess dietary iron absorption eventually leading to organ damage. Treatment for hemochromatosis involves routine bleeding a unit of blood that leads to reticulocytosis

Immortalized hematopoietic progenitor cell line

Erythroid progenitor cells capable of unlimited proliferation in the progenitor state via a Tet-inducible HPV16-E6/E7 expression system. The progenitor cells then follow normal erythropoiesis and enucleation into functional reticulocytes and erythrocytes

In vitro

Culturing parasites or host cells outside the living organism in a culture dish or flask in an incubator at a certain atmospheric environment

Oxidative stress

An imbalance between reactive oxygen species and cellular antioxidant defenses. Oxidative stress leads to damage of cellular proteins, lipids, and DNA

Reactive oxygen species (ROS)

Chemically reactive species of radicals of oxygen and non-radical derivatives including peroxides, superoxide, hydroxyl radicals, singlet oxygen, and hydrogen peroxide

Reticulocyte

Immature erythrocytes that lack a cell nucleus but retain ribosomal RNA. Their maturation process is characterized by a progressive loss of Transferrin Receptor 1 (CD71)