ERYTHROCYTE BIOLOGY AND ITS IMPACT ON PLASMODIUM VIVAX INVASION

By

EMILY SCHEETZ

Submitted in Partial Fulfillment of the Requirements for the degree of Master of Science

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

August, 2008

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of

Emily Scheetz

candidate for the Master’s degree *.

(signed) Mark Smith (chair of the committee)

Chris King

Clive Hamlin

Peter Zimmerman

(date) 7-8-2008

*We also certify that written approval has been obtained for any proprietary material contained therein.

1 TABLE OF CONTENTS

LIST OF FIGURES………………………………………… 3

Abstract……………………………………………………... 4

Introduction…………………………………………………. 5

Chapter 1- Interaction of Parasites with human erythrocytes………………………………………………… 5

Erythrocyte Structural ……………………. 5

Assembly of the Erythrocyte………………………. 8

Duffy Expression…………………………………… 10

Genetic Abnormalities of Erythrocytes…………… 16

Chapter 2- Erythrocyte Maturation……………………… 19

Chapter 3- Experimental Support……………………….. 22

Methods……………………………………………... 25

Results………………………………………………. 28

Discussion…………………………………………... 34

Future Directions…………………………………… 36

References………………………………………….. 39

2

LIST OF FIGURES

1. Figure 1: Erythrocyte Schematic……….. 10

2. Figure 2: Map of Duffy (-) Expression in ……….. 13

3. Table 1: Invasion Pathways of 14

4. Figure 3: Map of Overlap of Malaria with Abnormalities 15

5. Figure 4: Erythrocyte Abnormalities……………………. 16

6. Figure 5: Erythrocyte Expression……………… 20

7. Figure 6: Enrichment of Cord ……………………. 29

8. Figure 7: Images of Enriched Cord Blood……………... 29

9. Figure 8: Culturing of Parasites…………………………. 31

10. Figure 9: CD71 Expression Through Time…………… 32

11. Figure 10: A Expression on Erythrocytes 33

12. Figure 11: Fy6 Expression on Erythrocytes………….. 33

13. Figure 12: Western Blot of Bound Protein……………. 34

3

Erythrocyte Biology and its Impact on Plasmodium vivax Invasion

Abstract

by

EMILY SCHEETZ

Malaria infection caused by Plasmodium species parasites is a world problem and the number of infections and deaths are staggering; 500 million and 1 million per year respectively. Although research on malaria has been extensive, a vaccine remains elusive. Characterization of the human erythrocyte proteins and their expression, and how Plasmodium species parasites exploit these proteins to gain entry into erythrocytes is an aspect of research that cannot be overlooked. In this paper, knowledge of erythrocyte biology, protein expression of erythrocytes, and genetic abnormalities of erythrocytes is combined to better understand the invasion biology of Plasmodium vivax malaria. This research may lay the groundwork for future insight into an in vitro culturing system for P. vivax as well as offer insight into vaccine development.

4

Introduction:

The burden of malaria infection has impacted humans throughout the

world for hundreds of years. Approximately 3.2 billion people are at risk for

malaria [1] and vaccine development efforts have been unsuccessful thus far [2,

3]. Understanding the interaction between Plasmodium parasite species and

erythrocytes is one of the most important aspects of vaccine development. The

first step in this understanding is the functional role of the cytoskeleton and

plasma membrane proteins of the erythrocyte. Expression of particular proteins,

mainly the Duffy for chemokines (DARC or Duffy), may

determine the invasion capacity of erythrocytes to Plasmodium vivax. Although

factors affecting Duffy expression most certainly play a role in the variability of

Duffy expression, very little is actually known. Genetic abnormalities of erythrocytes may be factors that affect Duffy expression. The localization of Duffy on the red surface may affect invasion of the parasite. Some preliminary data on Duffy expression has been assembled but a much deeper understanding must be reached. Compiling some well understood physical interactions of proteins within the human erythrocyte along with abnormalities and developing data on Duffy expression and parasite invasion, we seek to better understand the invasion pathways utilized by Plasmodium vivax by developing an in vitro culturing model and potentially be more informed about the development of a

5 vaccine against Plasmodium malaria by studying the expression of Duffy on the

erythrocyte surface.

Chapter 1- Biology and Abnormalities

The interaction between the cytoskeleton and the plasma membrane of

the erythrocyte is vital for the movement of erythrocytes into and out of the

capillaries carrying oxygen and removing carbon dioxide from the bloodstream.

This movement is also critical for the development of severe malaria and cerebral

malaria. Each protein, and the interactions between proteins, contributes to the

function or dysfunction of the erythrocyte. Basic knowledge about the

constituents involved in erythrocyte function and parasite invasion of erythrocytes

lays the ground work for filling in the gaps of scientific understanding.

CYTOSKELETAL PROTEINS

As in most cells, the cytoskeleton of erythrocytes is quite complex. The

cytoskeleton is the network of proteins underlying and interacting with the cell

membrane [4]. Multiple protein interactions must take place to keep the network

intact and give the cell shape. The cytoskeleton determines the shape, integrity,

and elasticity of erythrocytes [5-8]. Of all the cytoskeletal proteins, is the

most abundant making up 75% of the erythrocyte cytoskeleton and is present at

100,000 copies per cell [9].

Because spectrin is the main component of the cytoskeleton it interacts

with multiple proteins to form linkages [4]. Spectrin tetramerizes to form filaments

which then interact with , 4.1, adducin, and 4.9 [10]. Other proteins such as

C and D and anion-exchanger 1 (band 3) associate with spectrin to

6 form interactions between the plasma membrane and the cytoskeleton [10].

Adaptor proteins such as ankyrin, 4.1, 4.2, and p55 also contribute to binding spectrin and creating the meshwork of the cytoskeleton [10]. Mutations in the spectrin lead to hemolytic anemias such as spherocytosis and elliptocytosis

[11, 12] and knockouts of spectrin are lethal in nematodes, flies, and mice [13-

15].

RED BLOOD CELL SURFACE PROTEINS

Red blood cell surface proteins play various roles in erythrocyte biology.

The first important protein is band 3, also known as the anion exchanger. Named for its migration pattern when lysed erythrocytes were run on a gel, band 3 is one of the two most abundant integral proteins of the erythrocyte [16]. With one million copies per cell, band 3 makes up 30% of the total amount of membrane protein [9]. Band 3 is confined to cells of erythroid lineage and cells, and is expressed on mature erythrocytes [17]. Band 3 is involved in membrane anion transport with its C-terminal domain mediating the exchange of chloride and bicarbonate anions, which increases the erythrocyte’s ability to carry CO2 from the tissues [16]. Another of its functions is to help with membrane stability by interacting with the cytoskeleton via Ankyrin-1 [18]. The cytoplasmic domain anchors the sub-membrane protein skeleton with the bilayer by interacting with ankyrin, 4.1, and 4.2 [16].

A second surface protein expressed on erythrocytes is the , also known as CD71. CD71 is expressed very early in the erythroid lineage maturation [19, 20] The transferrin receptor transports iron bound to

7 transferrin from the extracellular space inside the cell to form hemoglobin [21].

Without CD71, patients experience severe anemia [22]. There is variable

expression on the surface of cells because of the varying needs for iron and

hemoglobin synthesis [22]. Cells undergoing replication greatly up-regulate the

expression of CD71 on their surface [22] and mature erythrocytes express very little CD71 [23]. CD71 is of interest because of its expression on reticulocytes and young cells. This fact is important when providing target cells to in vitro parasite cultures. Counting the number of young and old reticulocytes is done by flow cytometry detecting RNA and CD71 positivity respectively.

The glycophorin family of proteins is another group expressed on the

surface of erythrocytes. Glycophorins A-C play multiple roles on the surface of

erythrocytes. They are the major expressed on the surface

[16, 19]. Sialic acids may be ligands for bacteria and viruses as well as parasites.

Because the glycophorins give the erythrocyte its negative charge, they may prevent red cell aggregation in circulation and minimize cell-cell intereactions [16,

19]. Glycophorins also contribute to the glycocalyx which plays a role in

protection from mechanical damage and microbial attack [16, 19]. Each of the

previous proteins, as well as a multitude not described in detail here, interact to

create the relationship between the cytoskeleton and plasma membrane.

Cytoskeleton and Plasma Membrane Association

Spectrin tetramers form a two-dimensional lattice by binding actin and

other associated proteins forming a junctional complex. Dissociation and

reassociation of the spectrin tetramers allows for the deformabilty of the

8 erythrocyte as it moves through the microvasculature [24]. The interaction

between spectrin and the cytoplasmic surface of the plasma membrane controls the erythrocyte shape and elasticity. Spectrin interacts with ankyrin which subsequently binds to band 3 [25]. Protein 4.2 also associates with ankyrin and band 3. The ends of spectrin tetramers also interact with protein 4.1 which recognizes membrane sites of band 3 and [25]. Other protein interactions for spectrin-actin stability include complexes with dematin, adducin, tropomyosin, tropomodulin, and p55 [25]. (Figure 1) There is some evidence for the formation of membrane domains within the plasma membrane of different cell types, but this has not been substantiated for erythrocytes [25]. Membrane domains could potentially explain the use of Duffy by P. vivax, how the expression of Duffy may be altered by protein deformities, and the localization of

Duffy expression on the erythrocyte surface.

These complex interactions allow flexibility for the function of the human

erythrocyte. However, genetic alterations can result in deformed red cells as well

as dysfunctional behavior of the red cells. Some of these mutations may offer

protection from severe malaria, and understanding the relationship between the

deformed erythrocytes and protection from severe disease is quite important.

9

Figure 1- Schematic of the erythrocyte cytoskeleton. The interaction between spectrin and associated proteins is called a junctional complex. Junctional complexes are pictured just below the plasma membrane interacting with glycophorin C and band 3 by the accessory proteins 4.1, 4.2, and ankyrin. Figure by [25].

Duffy Expression

One erythrocyte protein is cornerstone of invasion by P. vivax. DARC

(Duffy antigen receptor for chemokines) does not have a structural or cytoskeletal role in erythrocyte function, but rather is a receptor. There is a large

gap in the understanding of Duffy expression on erythrocytes and its role in P.

vivax invasion, but a large body of knowledge exists about Duffy’s normal

function on erythrocytes and other cells.

10 DARC is a protein expressed on the erythrocyte surface. (DARC will be

referred to as Duffy throughout this paper.) Duffy shares to

the IL-8 family of receptors and is able to bind both C-C and C-X-C families of

chemokines [26-28] Duffy is a scavenger-like receptor for chemokines [20] but

lacks the G-protein binding activation and therefore has not been shown to

activate any signaling cascade [29]. This lack of signaling properties is supported

by the fact that Duffy lacks the canonical DRY motif common to Family A GPCRs

[30] Without this signaling, Duffy acts to control local levels of chemokines by

internalization of the chemokines [31, 32].

The function of Duffy on erythrocytes versus vascular endothelial cells, the

other cell type where Duffy is expressed, seems to be quite different. An

intriguing role for Duffy has been examined by its expression on endothelial cells.

Hetero-oligomerization with CCR5 reduces cell chemotaxis and calcium flux

through CCR5 [33] but this interaction has not been observed for erythrocytes.

Currently, there is no knowledge of Duffy interacting with other receptors on the

erythrocyte surface. The function of Duffy on endothelial cells is the transcytosis

or neutralization of chemokines at barriers of [30]. Duffy function on

erythrocytes on the other hand may acts as a chemokine sink or as was more recently proposed, to maintain plasma concentrations of certain chemokines [32].

The Duffy antigen delays the disappearance of chemokines from the plasma which may help maintain plasma chemokine levels [34]. Duffy may control

leukocyte sensitivity to pro-inflammatory chemokines by internalizing excess

chemokines into the cell [34]. WT mice were able to remove angiogenic

11 chemokines produced by prostate cancer cells [35]. Duffy then may normally

negatively regulate angiogenic chemokine levels produced by prostate cancer

[32].

Multiple invasion pathways have been implicated in P. falciparum infection and one necessary pathway has been elucidated for P. vivax. These pathways are dependent on surface proteins on the red cell, discussed previously, and proteins produced by the parasite. Studying the expression, abundance, and biology of these red blood cell surface proteins is important for moving forward in the development of a successful vaccine against P. falciparum and P. vivax.

Interaction between P. vivax and the Duffy antigen on the surface of red

blood cells is necessary for subsequent invasion of the red cell [36-39] P. vivax accounts for at least half of all malaria cases in Latin America, Oceania, and Asia

[40]. There are 70 to 80 million clinical cases of P. vivax annually [40], which results in increased mortality in children, particularly those less than five years of age. Additionally, P. vivax infection causes debilitating symptoms in patients who

have previously been unexposed to the parasite. It has been observed that only

people that express the Duffy blood group antigen on the surface of their erythrocytes become infected with blood-stage P. knowlesi or P. vivax [37].

Duffy expression in Africa is limited, (Figure 2) compared to the rest of the

world. This is supported by the fact that there are many fewer P. vivax infections there compared to Asia, South America, and Oceania [41].

12

Figure 2- Map showing Duffy expression in Africa. Darkest yellow represents highest percentage of Duffy(-) individuals. [41].

Additionally, it has been observed that people who are heterozygous for

the Duffy antigen are half as likely to become infected by P. vivax. Using a Duffy

binding protein (DBP,) invading malaria parasites exploit this receptor to facilitate parasite invasion of erythrocytes. Several current vaccine strategies are focused on the interruption of the interaction of the Duffy antigen and the parasite DBP

[42]. While this strategy is promising, little is known about the expression of the

Duffy receptor and how effective such a strategy would be. Previous studies

have suggested that Duffy antigen expression may vary in Duffy positive

populations as well [43, 44]. This difference in expression is partly affected by the

two different FyA and FyB [36].

13 Plasmodium falciparum on the other hand utilizes multiple different

invasion pathways and multiple surface proteins on the erythrocyte. Many of the

surface receptors used for invasion are not known, and therefore the invasion

pathways are categorized by the dependency on sialic acid and sensitivity to [45] A summary of P. falciparum invasion pathways is in Table 1.

Because of the differences in invasion behaviors and pathways, developing vaccines for falciparum and vivax may require very different strategies.

Table 1. Table summarizing the invasion pathways utilized by Plasmodium falciparum. The erythrocyte receptors are listed in the far right column and parasite ligands are listed in the center column. On the left are the pathways as determined based on scialic acid dependency and trypsin sensitivity.

14 Similar to the overlap between Duffy negativity and the lack of P. vivax blood stage infections, Haldane first theorized that there was a wide variety of blood disorders that were maintained in the human population because of the selective pressure of malaria. Figure 3 is a small representation of the overlap of hereditary blood disorders with malaria endemicity. Some of these may have even been driven to fixation [46] Malaria has led to the selection of hundreds or more genetic variations that give different levels of protection against malaria

[47]. Multiple disorders alter the structure of the erythrocyte surface. Three of those disorders are in Figure 4.

Figure 3 Maps of Overlap between malaria and two erythrocyte abnormalities. Maps showing the endemicity of malaria in Africa (left map) and the frequency of sickle cell (center map) and α-thalassemia (right map.) [48]

15

Normocytes Sickle Cells Ovalocytes Thalassemia major Figure 4- Genetic Conditions of Erythrocytes. Multiple genetic abnormalities exist but only three are pictured here. Normal blood cells are in the left panel, sickle cell anemia is second from left [49] third from the left is mild Southeast Asian ovalocytosis [50], and thalassemia major is the right panel [50]. Figure compiled by Grimberg 2008.

Erythrocyte Abnormalities

Southeast Asian Ovalocytosis (SAO) is one of those disorders described.

SAO is caused by a 27 deletion of a portion of the band 3 gene [18].

Band 3 protein is still expressed on erythrocytes despite the nine

deletion, however, the protein is misfolded and will not bind stilbene disulfonates

or transport ions and the erythroctes become very rigid [51]. The erythrocytes

also have altered morphology and leak cations when cooled [52] which may have

implications for cold storage of SAO patient samples or their use in culturing in

vitro. Only heterozygotes are viable and they show protection against P.

falciparum malaria [18]. SAO also confers protection against severe forms of

malaria; mainly cerebral malaria [48]. Two possible mechanisms have been

proposed. Cortes et al found that SAO showed resistance to invasion by many

parasite isolates in vitro. Secondly, they found an increase of binding to CD36,

an endothelial receptor, by parasite infected cells. This binding may act as a

16 decoy because it is not expressed on the vascular endothelium of the brain [48] which may protect from the development of cerebral malaria. Without the adherence of infected erythrocytes in the brain, cerebral malaria complications are thwarted. The relationship between SAO and Duffy expression has not been explored, however, it can be hypothesized that erythrocyte disorders may affect

Duffy expression levels based on the localization of Duffy and some mutated surface protein. Altering the protein structure of one protein (band 3 for example) may change the ability of a parasite to interact with Duffy or change the folded conformation of Duffy on the surface of the altered erythrocyte.

Another group of hereditary erythrocyte disorders are the hemoglobinopathies. These are disorders of the structure or function of hemoglobin in the erythrocyte [48]. Hemoglobin S produces a variant structural form of hemoglobin which forms long, multi-stranded fibers [53]. Instead of the normal at position β6, there is valine, which causes the sickling of the hemoglobin due to decreased solubility [54]. This mutation causes severe sequelae (sickle cell anemia) including anemia by the breakdown of abnormal erythrocytes and the obstruction of blood vessels leading to severe pain and ischemic injury in multiple organs [1]. However, it also confers greater than 90% protection from severe and lethal malaria in carriers [48]. More mild structural modifications of hemoglobin can be seen with hemoglobin C, which shows difficulty for the growth and multiplication of P. falcpiarum or the inability of the parasites to adhere to the erythrocyte [48]. Hemoglobin C homozygotes may also express lower levels of Plasmodium falciparum erythrocyte membrane protein-1

17 (PfEMP-1), a major adhesion protein used by the parasite [48]. Hemoglobin E heterozygotes also show more mild structural differences in hemoglobin and incidence and severity of malaria infection is markedly less compared to patients with normal hemoglobin [48]. More mild modifications include different amino acid substitutions at β6 that only slightly reduce the solubility of hemoglobin compared to hemoglobin S where solubility is greatly reduced.

Decreased production of the alpha and beta subunits of hemoglobin characterizes another set of erythrocyte disorders that offer protection from severe malaria. α and β-thalassemia seem to confer protection from severe malaria-related anemia [48]. These cell surface changes overlap with populations where malaria is endemic [46]. Expression of Duffy on the erythrocyte surface, however, has not been determined for these disorders. A reduction in hemoglobin subunit production may also cause changes in protein expression.

Because hemoglobin interacts with spectrin of the cytoskeleton, altered cell shape or expression may occur. Investigating further how Duffy expression may be altered would be interesting for future experiments.

Hereditary elliptocytosis has a higher incidence in Sub-Saharan Africa than other places in the world [18]. The frequency of mutations in the spectrin gene is higher here and it is postulated that these mutations arose because of a form of protection against malaria [18]. These erythrocytes are reduced in their ability to deform as determined by osmotic gradient ektacytometry [18]

Mechanical weakness and erythrocyte membrane skeleton fragility are the two most common defects due to mutated alpha or beta spectrin [24].

18 Glucose-6-phosphate deficiency (G6PD) has a high prevalence and

diverse genetic background in malaria endemic regions [55]. It is the most

common enzyme defect in humans [56]. Its normal function is to provide cells

with NADPH and subsequently counterbalance oxidative stress [56]. Because

erythrocytes lack mitochondria, the pentose phosphate pathway (in which G6PD

is utilized) is the erythrocyte’s only defense against oxidative stress [56].

Mutations in the G6PD gene alter the enzymatic activity and may lead to brought on by exogenous agents [56]. Multiple effects including early of malaria infected cells, more efficient lysis of infected cells, and or accelerated oxidative membrane damage of the erythrocyte

are observed in mutated G6PD individuals [57, 58].

Complement receptor 1 (CR-1) is a major receptor for endothelial

adherence in P. falciparum malaria and is highly polymorphic in malaria endemic

regions [48]. A lower expression in results in less severe

malaria infection [59]. Considering such a diverse group of erythrocyte

abnormalities, there is quite a bit left to learn about how these abnormalities may

affect other proteins’ expression and the ability of parasites to invade altered

erythrocytes.

Chapter 2- Erythroid Lineage and Reticulocyte Maturation

Cells of erythroid lineage progress through different stages of maturation

in relatively regular fashion. Scicchitano et al developed a stem cell liquid culture system as an in vitro model to study erythroid . Hemoglobin A expression starts very low on day 1 of in vitro culture. It increases up to 61% on

19 day 4, a 60-fold increase from day 1, and decreases slowly as the cell matures through days 7 and 15 [19]. expression follows a somewhat similar pattern of bell-shaped expression but levels start, peak, and finish higher from day 1 to 15 [19]. CD71 expression however reaches a higher percentage

(98%) by day 4 and quickly decreases through day 15 [19]. Decreased expression of the transferrin receptor makes sense because the cell has finished producing necessary hemoglobin levels and bringing in more iron would poison the cell. Figure 5 by Okumura et al is representative of protein expression in different stages of erythrocytes.

Figure 5- Schematic representation of the expression of multiple proteins by the human erythrocyte. Of particular interest are CD71 (the transferrin receptor) and Glycophorin A. CD71 can be used as a marker for young reticulocytes and Glycophorin A is utilized by Plasmodium falciparum to invade erythrocytes. Both interesting to observe for in vitro parasite culture. Dotted lines are previously reported data not from Okumura et al 1992.

Erythropoiesis is the lineage commitment and proliferation of hematopoietic stem cells which is then followed by terminal differentiation of

20 erythroblasts into mature erythrocytes. Following the last cell division, loss of the

nucleus marks the complete cessation of erythroid [60]. Some RNA

is retained for a few days as the cell enters the circulation and these cells are

called reticulocytes [60].

Terminal differentiation is characterized by many specialized processes

including protection from oxidant damage, accumulation of hemoglobin,

cytoskeleton formation, and autophagocytosis of organelles. After the final cell

division and enucleation, reticulocytes complete the phenotypic specialization

required for transporting oxygen and surviving in the absence of new gene

activity. Due to their unique position at the final stage of the erythroid

developmental pathway, reticulocytes are one of the most functionally specialized cellular populations in humans [60]. They are also extremely important in the lifecycle of and invasion of P. vivax and further characterization of surface protein expression is underway.

Another important distinction also needs to be made between “young”

reticulocytes obtained from cord blood and “older” reticulocytes obtained from

adult blood. There is noticeably different messenger RNA expression between

cord blood reticuloctyes and adult reticulocytes [60]. Plasmodium vivax is known

to favor invasion of reticulocytes and young cells [61-63] so variability in protein

expression, mainly Duffy, is a missing link in the understanding of P. vivax

invasion. Whether this variable RNA expression matches with protein expression is not yet elucidated [60]. These differences may indicate developmental changes in erythroid gene expression or RNA stability [60]. Differential RNA

21 expression may be most indicative of an immature erythrocyte’s function. Iron uptake is a major function of immature red cells and expression of for hemoglobin synthesis may account for differences between younger reticulocytes, producing more hemoglobin, and older reticulocytes nearing maturity and no longer synthesizing hemoglobin. Mutations in expression of the erythrocyte proteome can cause defects in shape, function, and cellular content, as mentioned previously. The role reticulocytes play in the malaria parasite invasion picture has been examined in some detail and supplementing this understanding with new techniques may further our in vitro culturing abilities as well as infection prevention.

Chapter 3- Reticulocyte Enrichment and Culturing

Currently the techniques for culturing Plasmodium vivax (P. vivax) are quite limited. The most common method is to remove PBMCs from infected blood and allow the parasites to grow in the remaining blood [64]. There is limited reinvasion with this method and parasites only grow and mature in the red cells they start in [65]. For P. vivax culturing to be successful, a fresh supply of reticulocytes must be given regularly. Another method being developed is a stem

cell preparation that yields a high reticulocytemia [64]. This method, however, is

very costly and labor intensive. A third method for P. vivax culturing involves using blood from patients with hemochromatosis who have 3-10 times more reticulocytes than normal adults [66]. Spinning this blood down using an ultracentrifuge enriches for reticulocytes further by up to 20%.

22 Described here is a method using umbilical cord blood (2-8% reticulocytes), a readily available resource for culturing P. vivax. Expectant mothers who are willing to donate can be found in any world setting. After obtaining the cord blood, we further enrich it for reticulocytes. Blood was exposed to a hypotonic lysing solution of NaCl. Because of osmotic pressure differences, this solution preferentially lyses all cells that do not contain RNA or DNA. We observe a consistent enrichment of 10-fold higher reticulocytes up to 30% of the total cell number. Malaria cultures were then able to receive a higher number of target cells for invasion and the hematocrit does not get diluted from the normal

5%. This enrichment method shows the viability of enriched reticulocytes for invasion by P. falciparum. This data may support the potential for P. vivax in vitro culturing in enriched blood which will lead to new insights into P. vivax biology and its invasion of red cells.

Examining why P. vivax prefers reticulocytes over mature red blood cells is an important aspect of the biology of P. vivax. Reticulocytes are thought to express more Duffy on their surface but a substantial pool of data on this area of research is not available [43, 44]. Expressing more molecules of Duffy may cause reticulocytes to be more susceptible to P. vivax invasion by sheer percentages. Once in the blood stream, a parasite has less than one minute to bump into and invade an erythrocyte before being cleared by the human immune system [67]. Because P. vivax must have Duffy to invade, the more molecules of

Duffy expressed on an erythrocyte, the more likely the parasite is to find a Duffy molecule to attach to and enter the red cell.

23 P. vivax parasites require interaction of their Duffy binding protein (DBP)

with the Duffy blood group antigen on the surface of erythrocytes in order to

attach and invade [38, 39]. The invasion forms of P. vivax (merozoites) express

DBP which facilitates parasite attachment and entry into erythrocytes [38, 39, 68-

71]. Duffy heterozygous humans show a markedly reduced number of infections

by P. vivax compared to homozygous positive humans [39].

Examining the surface Duffy expression of reticulocytes and red blood

cells over time is important to determine the levels of expression as the cells

reside in culture. Should the lifespan of the receptor on the cells in culture be short, supplementing cultures with blood more often would be very important. By examining the expression of Duffy on normal red cells, cord blood cells, and abnormal red cells we will gain insight into the expected sensitivities to P. vivax

invasion of different types of red cells. Staining for the Duffy antigen by flow has

proven an effective method for observing cells positive for Duffy expression [43,

44] .

Previous studies of a limited number of samples have suggested that

there is variation in the expression of Duffy antigen on the surface of erythrocytes

between patients and between very young and very old erythrocytes [43, 44].

Examining more closely how other mutations or disorders may affect Duffy

expression has not been undertaken. Parasite access to the Duffy antigen may

be influenced by Duffy , Duffy antigen expression, or properties of the

folded conformation of Duffy.

24 A second portion of this examination is to observe the binding activity of

the Duffy Binding Protein. The strength of the interaction between the protein on

the parasite and the receptor on the cell plays a large role in determining whether the parasite can enter the cell or not. To investigate this invasion pathway in vitro, we have generated recombinant DBP and show that this protein is able to bind red cells in a classical red cell binding assay.

Methods

Cord blood was enriched for reticulocytes by Histopaque separation and

NaCl lysis.

Cord blood was washed three times with incomplete media (RPMI 1640

containing HEPES and sodium bicarbonate- iMCM) After each wash, the blood

was spun at 200 x g for 10 minutes. The supernatant was removed following

each wash. After the final wash, the pellet was diluted to 50% hematocrit with

iMCM. Histopaque 1077 (Sigma, St. Louis, MO) was put in a 15mL conical tube

and warmed to 37°C. An equal volume of the warm, diluted blood was carefully

layered on top of the warm Histopaque. (Total volumes of eight mLs or less work

most efficiently.) The conical was then spun at 200 x g for 10 minutes. The

supernatant was then removed and the pellet was washed one time as above. 10

volumes of 0.2% NaCl was then added to the pellet. After five minutes at room

with periodic mixing, 10 volumes of 1.6% NaCl were added to the 50

mL conical. The 50 mL conical was then spun as previously stated. The

supernatant was removed and again the pellet was washed one time as before in

iMCM. The red blood cell pellet was brought to 50% hematocrit with iRPMI and

25 again layered over warm Histopaque in a 15 mL conical. Spinning was done as before and the supernatant was removed. Lastly, the RBC pellet was washed two or three times with iRPMI. Final enriched pellet could be stored at 4° C for about two weeks at 50% hematocrit with complete media (iMCM plus supplemented with 5% albumax, 80 ng/mL gentamicin, and 200 ng/mL hypoxanthine- CMCM)

Infectivity of enriched and unenriched cord blood with Plasmodium falciparum

Cultures were started with approximately 1% parasitemia and 5% hematocrit of enriched or unenriched cord blood [72]. Cultures were grown at 37oC for 72 hr in an atmosphere of 5% CO2, 1 % O2, 94 % N2. Samples were taken at the initiation of the cultures and every 24 hours for three days. Three separate five

µL samples were taken of each culture and stained with 4µM Hoechst 33342 for

DNA, 0.5µg/mL α-CD71 for the transferrin receptor for 45 minutes and finally

100nM Thiazole orange for RNA for 30 minutes. Stained samples were flowed on a BD LSRII (Franklin Lakes, NJ) flow cytometer and 100,000 events were counted. Parasitemia was measured by fold increase in DNA fluorescence over time 0 infection of enriched and unenriched cord bloods and three adults.

Tracking reticulocyte maturation and red blood cells surface markers through time

Enriched and unenriched cord blood was put in 5% hematocrit cultures with complete media at 37° C for three days. Sampling was done as before but samples from each culture were stained with a different set of markers. One

26 group was stained with thiazole orange (TO) and α-CD71 using the same

concentrations previously stated. A second group was stained with TO and α-

Glycophorin A (GPA.) GPA staining was done with 10ul of 1ng/ml PE conjugated

anti-GPA (Abcam, Cambridge, Massachusetts.) A third group was stained with

TO and α-Fy6, a portion of the Duffy antigen. Anti-Fy6 antibody was used at 2ul

of 1:500 dilution of mouse ascites per one million cells. Anti-Fy6 was obtained

from John Barnwell and is specific to amino acids 22-25 of the N-terminal region

of the Duffy antigen on RBCs.

Classical red blood cell binging assay using recombinant Duffy binding

protein (P. vivax rDPBII)

Whole adult blood was washed three times in iMCM and centrifuged at 1200

rpm. Packed red cells are brought to a 33% hematocrit in iMCM. Control tubes of

330µL of 33% hematocrit blood were treated with 8mL of iRPMI and 2mL of

5mg/mL at 37°C for 75minutes rocking. These were also washed three times as before. Properly refolded P. vivax RDBPII [73] was added to a final volume of 340µL and 60µL of FBS was added. This was kept at room temperature for 15-20 minutes. Then, 300µL of the treated and untreated blood were added and incubated with the protein at RT on a rocking platform for one hour. 600µL of dibutylpthalate was put in microcentrifuge tubes and the RBC- protein mixture layered on top. The tubes were then spun for one minute at

13,000 rpm. The supernatant was carefully removed and placed in clean tubes.

Then 20µL of 1.5 M NaCl was added into the pellet while swirling and dispensing and the tube rested for one minute. The tube was then spun at 13,000 rpm for

27 one minute and the supernatant was collected that contained the eluted protein.

A Western blot was then run to probe for eluted protein.

Results

Reticulocyte enrichment using hypotonic lysis

Enrichment of cord blood ranged from a starting reticulocytemia of 1-2% up to 28% reticulocytes after enrichment. (Fig. 6) This calculates to a 4-fold enrichment on average. Cells were in good condition (Figure 7 A-B) before and after enrichment and morphology looked quite similar to unenriched cord blood and the few reticulocytes it contained. Enrichment varied by cord blood sample and also varied by volume of packed blood used for the first step of Histopaque extraction/WBC removal. If more blood was used, lysis was not as efficient as when smaller volumes were used. During the enrichment process, there was a large loss of blood volume. A starting volume of eight mls of packed blood yielded approximately two mls of enriched blood.

28 Matched CB and Enriched CB Samples 30

25

20

15

10

% Reticulocytes % 5

0 CB EN

Figure 6- Matched cord blood showing enrichment. 11 cord bloods were enriched for reticulocytes. The average starting reticulocytemia is 2.41% and the average enriched percentage is 8.5%, making the enrichment average about 4- fold.

Figure 7- Images of unenriched and enriched cord blood A) Bright field microscopy image of unenriched cord blood. One reticulocyte is shown at the arrow. B) Bright field microscopy image of enriched cord blood. Reticulocytes containing reticulin are stained blue with Retix-chex (Streck Inc, Omaha, NE).

29

Plasmodium falciparum equally infects enriched and unenriched cord

blood and adult blood

Both enriched and unenriched cultures were able to sustain parasite

growth for three days. (Figure 8). Starting parasitemias tended to fluctuate

between the samples because of the difficulty pipetting packed red cells. In some

cases a low fold-increase in parasitemia was attributed to the cultures having

high parasitemias, which resulted in the death of the culture. Since no fresh

RBCs were added, the availability of RBCs for re-invasion was too low in these

instances. However, the invasion process of parasites entering red cells did not seem to be hindered by the enriched blood. (Figure 8) The enriched culture initially showed a lower fold-increase than the unenriched culture at 24 hours. By

72 hours however, the parasitemias had almost an identical fold increase.

30 Pooled Data 3.5 CB 3.0 ** EN 2.5 2.0 1.5 n=10

Parasitemia 1.0 ** p= .002 Fold Increase in 0.5 0.0 0 10 20 30 40 50 60 70 80 Hours in Culture

Figure 8- Parasite growth in unenriched and enriched cord blood over three days in culture. A portion of ten different cord bloods were enriched for reticulocytes and parasites were grown in both enriched and unenriched blood. Increasing parasitemias were compared to the starting parasitemia of the culture.

The cultures started in adult blood also exhibited a large fold increase in

parasitemia over the initial day in culture. Adult blood seemed to facilitate a larger

increase in parasitemia compared to cord blood. However, all three preparations

of blood were viable for parasite invasion. (Data not shown.)

Reticulocyte maturation through time; variable expression of surface molecules and RNA

CD71+ cells started at 0.7% of live events and steadily decreased through

time in culture until reaching just above the level of detection at 0.1%. (Figure 9)

Thiazole orange positive cells in unenriched cord blood varied between samples,

but the range was 2-3% or less. Enriched samples reached up to 30%

reticulocytemia but a 10-fold increase was common. (Figure 6).

31 CD71+ Cells in Culture

0.7 Cord Blood

0.6 Enriched

ll 0.5

Ce0.4 s

1+ 0.3

CD7 0.2 % 0.1 0.0

-5 5 15 25 35 45 55 65 75 Hours in Culture

Figure 9- Tracking CD71 expression through 3 days in culture. A portion of ten cord bloods were enriched for reticulocytes and put in culture for 3 days. Samples were stained with anti-CD71 antibody conjugated with APC on the first day and the last day. The percentage of cells that were CD71+ decreased through time in culture.

Glycophorin A (GPA) expression was tracked over time and only slightly diminished while the cells were in culture. (Day 0 pictured in Figure 10). GPA expression over three days in culture did not noticeably decrease. Up to 99% of

cord blood cells stain positive for GPA and this percentage does not drop below

90% over the three day span. Staining for Fy6 also showed that >90% of cord

blood cells stained positively for Duffy. (Figure 11B) This percentage slowly

decreased over one week in culture as the cells increased in age.

32

Figure 10- Glycophorin A staining of erythrocytes. A) Erythrocytes alone, no PE stain B) Erythrocytes were stained with PE conjugated Glycophorin A stain. (Abcam, Cambridge, MA). Single red blood cells were gated by forward scatter and side scatter and subsequently gated by positive or negative staining for GPA.

Figure 11- Fy6 Expression on the erythrocyte surface- A) Human red blood cells stained with anti-Mouse IgG conjugated with PE (Sigma, Omaha, NE). B) Human red blood cells stained with anti-Fy6 made in mouse ascites and the same secondary as in A.

Duffy Binding Protein Binds to Red Cells

A Western blot (Fig. 12) was run using the rDBPII eluted from binding red

cells. The recombinant protein bound very well to the red cells using a range of protein concentrations (2, 4, 10, and 20 µg.) Full length bands of the Sal1 varient of rDBPII can be seen. Blood from a person with Southeast Asian Ovalocytosis

33 was also used and binding occurred comparatively to normal adult cells. Blood

without the addition of protein showed no cross reactivity by Western so the

antibody probe to the protein reacted specifically in the presence of protein.

Figure 12- Western blot of recombinant Duffy binding protein eluted from erythrocytes. Lane 1 (labeled from left) is a size marker. Lanes 2 and 3 are 10 and 20 ug of the Sal1 varient of protein bound to normal human erythrocytes. Lane 4 is 15 ug of Sal1 bound to an SAO patient’s cells. Lanes 5-6 are normal adult red cells bound with 10 and 20 ug of the O variant rDBPII. Lanes 7-8 are cord blood cells bound with 10 and 20 ug of O rDBPII. Lane 9 are red cells that were not incubated with protein and lane 10 is the positive control of Sal 1 protein alone.

Discussion

This simple and inexpensive method of extracting recticulocytes from a

readily available source without the need for ultracentrifuges can potentially enhance P. vivax culturing techniques in the U.S. as well as PNG by offering

more targets for parasite invasion without a significant increase in the hematocrit of the culture. The enrichment method is not technically demanding and only

34 slightly more time consuming than previously used methods. Not only is there an

increase in reticulocytes, but there may also be an increase in the percentage of

Duffy positive cells in enriched cord blood which will be addressed further in

future experiments.

When put into cultures of enriched and unenriched cord blood, P. falciparum was able to invade erythrocytes and grow. Even though P. falciparum

does not prefer reticulocytes like P. vivax, it was important to find out if the blood

was viable for invasion at all. The variability in fold increase in parasitemia may

be attributed to the starting parasitemias of each culture. A higher starting

parasitemia may inhibit the culture from increasing invasion and parasitemia.

Because the volume of uninfected blood added to each culture was ultimately

very similar, the percentage of cells able to be invaded would be lower if there

were more parasites to start, inhibiting large fold increases in parasitemia without

the supplementation of fresh blood. Variances in invasion may also have

occurred from different enrichment percentages. There was quite a lot of

variability in the amount of enrichment obtained for each of ten cord bloods

tested. Depending on the starting number of reticulocytes, the amount of parasite invasion could have been affected, although this is unlikely. The culturing period was short and more blood should be added to supplement the cultures for long term in vitro propagation. However, to determine viability of erythrocytes manipulated in the enrichment protocol was very important. By increasing the number of viable reticulocytes, the likelihood of P. vivax invading in vitro also increases.

35 Tracking erythrocyte and reticulocyte markers through time is important for

the development of an in vitro culturing system as well as to gain insight into

surface protein expression over time. Glycophorin A receptors on the cell surface

of erythrocytes stain at very high levels up to 90% after three days in culture.

GPA is a highly utilized invasion pathway for Plasmodium falciparum and may be an example for similar expression levels of other erythrocyte surface proteins such as Duffy. Duffy expression on enriched erythrocytes did not diminish rapidly in culture. The sustained expression of Duffy both in culture and while cells are being stored in the refrigerator is invaluable when optimizing culturing conditions for P. vivax.

Examining the binding of recombinant DBP to red cells was an important

experiment to examine an in vitro model for parasite invasion. Recombinant DBP

had been shown to bind to the N-terminal portion of the Duffy receptor by ELISA

and the red cell binding assay was the first step in using the Duffy receptor for

binding in its native conformation. Correctly folded rDBP was able to bind the red

cells with quite high affinity as show by Western blot. Even at low concentrations

of protein, when probed, the Western still showed that a large quantity of protein

bound the erythrocytes. The rDBPII seems to bind with strong affinity to normal

and SAO adult erythrocytes.

Future Directions

Flow cytometry experiments are currently underway to test the binding of

rDBPII to red cells as well as testing Duffy expression on erythrocytes in a

quantitative and more physiological system. Using intact, live erythrocytes with

36 the native conformation of Duffy on their surface is a more biologically sound

experiment. Testing of one adult sample showed 70% of erythrocytes bound

rDBPII. Future experiments include optimizing rDBP binding to erythrocytes as

well as examining a competition assay between anti-Fy6 and rDBP. Blocking

rDBP from binding Duffy could support the use of anti-Duffy antibodies as a

viable vaccine candidate. Other experiments include characterizing the Duffy

expression on many adult blood samples including those with genetic

abnormalities, heterozygotes, multiple cord blood samples, and Duffy negative

individuals.

Despite malaria affecting millions of people every year and the amount of

time researchers have spent designing drugs and vaccine candidates, there are

many experiments and more research to be done. Developing an in vitro system

for P. vivax culturing would provide a direct system to study parasite biology and

perform viable vaccine candidate experiments.

Characterization of erythrocyte surface receptors that are required for

malaria invasion is important for understanding parasite biology and the design of vaccine strategies. The previous results lead to investigating the interactions of a

common erythrocyte surface receptor and its corresponding parasite .

These observations will inform strategies that may be considered for later clinical

or field development and could lead to the further development of malaria vaccine candidates.

With the potential for developing agents that block malaria invasion of

erythrocytes, it is critical to perform further studies on the influence of red blood

37 cell age and of Duffy antigen/receptor expression/conformation on parasite binding and invasion. Understanding the variability of Duffy expression and the subsequent binding of rDBPII may offer insight into the invasion variability of P. vivax and insight into Vivax malaria prevention.

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42