EFFECT OF BED NETS ON ACQUIRED HUMORAL IMMUNITY TO PLASMODIUM FALCIPARUM ANTIGENS IN CHILDREN FROM MUGIL, MADANG PROVINCE, PAPUA NEW GUINEA.

Transcription

1 EFFECT OF BED NETS ON ACQUIRED HUMORAL IMMUNITY TO PLASMODIUM FALCIPARUM ANTIGENS IN CHILDREN FROM MUGIL, MADANG PROVINCE, PAPUA NEW GUINEA. by VALENTINE SIBA Submitted in partial fulfillment of the requirement for the degree of Master of Science Dissertation Advisor: Professor James Kazura (MD) CASE WESTERN RESERVE UNIVERSITY January 2017 i

2 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of VALENTINE SIBA... candidate for the Master of Science degree*. (signed) Michael Benard (PhD)... (Chair of the committee) James Kazura (MD)... Emmitt Jolly (PhD)... Ryan Martin (PhD)... (Date) 31 st August 2016 * We also certify that the written approval has been obtained for any proprietary material contained therein. i

3 Dedication To my parents Peter and Yalum, and to the loving memory of Quintilla and Siwak ii

4 Table of Contents LIST OF TABLES... VII LIST OF FIGURES... VIII ACKNOWLEDGEMENT... X LIST OF ABBREVIATIONS... XI ABSTRACT... XIII 1 INTRODUCTION MALARIA: GLOBAL BURDEN AND DISTRIBUTION Global progress in controlling and eliminating malaria MALARIA IN PAPUA NEW GUINEA Malaria control efforts in Papua New Guinea PLASMODIUM FALCIPARUM Life cycle CHARACTERISTICS OF THE DISEASE PATHOGENESIS Sequestration and cytoadhesion Variant surface Antigens (VSA) Rosetting Phenotypic and antigenic variation PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN var genes and PfEMP IMMUNITY TO P. FALCIPARUM MALARIA Acquired Immunity Antibody responses to P. falciparum antigens P. falciparum antigens iii

5 1.8. BED NETS AND IMMUNITY THESIS HYPOTHESIS AND OBJECTIVES MATERIALS AND METHODS STUDY DESIGN RECOMBINANT ANTIGENS COVALENT COUPLING OF ANTIGENS TO MICROSPHERES Bead activation Protein coupling SAMPLE DILUTIONS AND PREPARATION PLATE PREPARATION AND ANALYSIS RESULTS PFEMP1 VARIANT SURFACE ANTIGENS: DBL AND CIDR CIDR α CIDR α DBL α DBL β DBL g MEROZOITE ANTIGENS: MSP AND EBA Merozoite surface proteins MSP142 FUP MSP142 FVO MSp142 3D MSP2 FC PfMSP MSP6 GST MSP7 GST iv

6 MSP-348 FL + inhibitors MSP-DBL 355 FL + inhibitors Erythrocyte Binding Antigen (EBA) EBA-140 R D7 EBA-175 R W2Mef EBA-175 R EBA-181 R CIRCUMSPOROZOITE PROTEIN (CSP) Pp CSP M3/ AGE MATCHED COMPARISON DISCUSSION ANTIBODY RESPONSES Low antibody responses High antibody responses ANTIGENICITY AND PHENOTYPIC VARIATIONS EFFECT OF BED NETS ON IMMUNITY TO PLASMODIUM FALCIPARUM ANTIGENS CONCLUSION APPENDICES APPENDIX 1: ANTIBODY COUPLING APPENDIX 2A: ANTIGEN CONCENTRATIONS APPENDIX 2B: CALCULATION APPENDIX 3A: 96-WELL MICRO FILTER PLATE LAY OUT APPENDIX 3B: SERIAL DILUTIONS APPENDIX 4: BEAD MASTER MIX CALCULATION APPENDIX 5: SECONDARY ANTIBODY (Α-F(AB)2) CALCULATION v

7 REFERENCES vi

8 List of Tables Table 1: PfEMP1 variant surface antigens Table 2: Merozoite Antigen - Merozoite surface proteins (MSP) and Erythrocyte binding antigens (EBA) Table 3: Circumsporozoite protein Table 4: Appendix 2a - Protein concentration and coupling volumes Table 5: Appendix 3b - Serial dilutions Table 6: Appendix 4 - Calculation table for calculating bead master mix Table 7: Appendix 5 - Calculation table for calculating secondary antibody vii

9 List of Figures Figure 1: Percentage of world population at risk for malaria in Figure 2: Altitude zones in Papua New Guinea (15) Figure 3: Plasmodium falciparum life cycle (20)... 8 Figure 4: Surface expression in irbc and host cell showing various host cell ligands that are able to adhere to PfEMP1 variants (31) Figure 5: Expression of PfEMP1 by Plasmodium falciparum on knobs formed on the surface of irbcs. CIDR and DBL mediate adhesion by binding to several endothelial receptors such as CD36, ICAM1 and CSA. (PV-parasitophorous vacuole; MC-maurer's cleft) (38) Figure 6: Domain architecture example of two PfEMP1 molecules. DBL, CIDR with NTS make up the semi conserved head structure of Exon Figure 7: Organization and protein structure of var genes. (A) Chromosomal organization. Groups A and B are transcribed in opposite directions and are located in subtelomeric region whereas group C are located at the central chromosome cluster. (B) Recombination between subdomains 2 and 3 in the DBL domain (29) Figure 8: Relationship between age and severity of malaria in populations exposed to high malaria transmission. Changes over time of various indices of malaria in a population in an area with high transmission of P. falciparum. Asymptomatic infection (pink), mild disease (febrile episodes blue) and sever malaria (green) (58) Figure 9: Map of Madang Province, Papua New Guinea viii

10 Figure 10: General protein coupling workflow Figure 11: Overview of workflow for 96-well plate preparation Figure 12: Antibody responses to PfEMP1 antigens Figure 13: Antibody responses to Merozoite surface proteins Figure 14: Antibody responses to Erythrocyte binding antigens Figure 15: Antibody responses to Circumsporozoite proteins Figure 16:Antibody responses in children aged 5 to 9 years from Mugil 1 (non-itn users) and Mugil 2 (ITN users) Figure 17: Antibody responses in children aged 10 to 14 years from Mugil 1 (non- ITN users) and Mugil 1 (ITN users) Figure 18: Appendix 1 - Antibody coupling for proteins Figure 19: Appendix 3a - Layout of 96-well micro filter plate ix

11 Acknowledgement First of all, I would like to thank and acknowledge my advisor, Dr. James Kazura for his supervision, advise, guidance and assistance throughout the duration of my program and towards the completion of my research. I would also like to thank the King Lab; Dr. Christopher King, Sarah, Rich and Gabby for assisting me with the samples and help in data analysis. I would also like to thank everyone in the Kazura/ Dent Lab; Dr. Kazura, Dr. Dent, Paula, Grace, Kate, Katelyn, Anil, Anna and Victoria for the friendship and for helping in one way or another. I greatly appreciate everything that you all have done to help me get through my studies and my research. I would also like to thank the PNGIMR and the Center for Global Health and Diseases through which the Global Infectious Disease Research Training Program, John E Fogarty International, National Institute of Health Scholarship has enabled me to undertake this program. I would also like to acknowledge my thesis committee members; Dr. Michael Benard, Dr. Ryan Martin and Dr. Emmitt Jolly. I would like to thank all the friends I have made both here in Case and outside of Case and finally to my family for their everlasting love, support and prayers. x

12 List of Abbreviations ACT ADCI ARDS ATS AQ CD CIDR CSA CQ CR CSP DBL DC DDT EBA EDC Artemisinin-based combined therapy Antibody-dependent cellular inhibition Acute respiratory distress syndrome Acidic terminal segment Amodiaquine Cluster of differentiation Cysteine-rich interdomain region Chondroitin - sulfate A Chloroquine Complement receptor Circumsporozoite protein Duffy binding-like Domain cassette Dichlorodiphenyltrichloroethane Erythrocyte binding antigen 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ICAM-1 Intracellular adhesion molecule 1 IgG INT irbc IRS KAHRP Immunoglobulin G Insecticide treated nets infected red blood cells Indoor residual spraying Knob-associated histidine rich protein xi

13 LLIN MBP MFI MQ MSP NAI NTS PBS Long lasting insecticide treated nets Maltose binding protein Median fluorescence intensity Mefloquine Merozoite surface protein Naturally acquired immunity N-terminal segment Phosphate buffered saline PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 PfMC2TM PNG RBC RIFIN SP STEVOR SURFIN TM TNF TSP Ups VSA WHO Plasmodium falciparum Maurer s cleft two trans membrane Papua New Guinea Red blood cells Repetitive interspersed family Sulfadoxine-prymethamine Subtelomeric variable open reading frame Surface-associated interspersed gene family Trans membrane Tumor necrosis factor Thrombospondin Upstream sequences Variant surface antigen World Health Organization xii

14 Effect of Bed Nets on Acquired Humoral Immunity to Plasmodium falciparum Antigens in Children from Mugil, Madang Province, Papua New Guinea. Abstract by VALENTINE SIBA Papua New Guinea (PNG) is endemic to P. falciparum and has the highest burden of malaria outside of Africa with an estimated 1.36 million cases each year. Malaria control and elimination efforts have proven to reduce the burden of the disease in PNG through the distribution and use of insecticide treated nets (INTs) especially in highly endemic areas. Evidence suggests that antibodies mediate protection against infection, and immunity against malaria is associated with repeated exposure to the disease. This study is focused on comparing antibody levels between children who did not use ITNs and children who used ITNs in a malaria endemic region of PNG. The overall results showed higher levels of antibody responses to P. falciparum in the group of children who did not use ITNs and low antibody responses to P. falciparum in the group of children who used ITNs. This suggests that the use of ITNs may have an effect on immunity to malaria in children. xiii

15 1 Introduction 1.1. Malaria: Global burden and distribution Malaria remains a major public health problem worldwide affecting humans especially throughout the tropical and sub-tropical regions (Figure 1). Malaria is a blood infection caused by mosquito borne apicomplexan parasites of the genus Plasmodium, and remains a leading cause of morbidity and mortality. The World Health Organization (WHO) reported an estimated 3.3 billion people in 97 countries being at risk of infection from malaria with 198 million cases reported in 2013, leading to deaths (1,2). In malaria endemic regions, pregnant women and children suffer more from malaria. It was also reported in 2013 that 78% of global malaria deaths occurred in children less than 5 years of age (2). Malaria can be asymptomatic, or a spectrum of clinical diseases ranging from mild to severe and can also result in death for those with poor immunity (3). 1

16 Figure 1: Percentage of world population at risk for malaria in Human malaria is caused by five major species of protozoan parasites belonging to the genus Plasmodium: P. vivax, P. falciparum, P. ovale, P. malariae and P. knowlesi (1,5). P. falciparum is the deadliest form of the disease accounting for more than 90% of the global malaria mortality (6). This thesis will be based on P. falciparum and the antibody responses to its various antigens Global progress in controlling and eliminating malaria From the 2015 World Malaria Report, there has been an increase in the number of countries moving towards malaria elimination. Before the year 2000, there were 13 countries that were estimated to have less than 1000 malaria cases each year and by 2015, an additional 33 countries have been estimated to reach this milestone (7). Global efforts made towards the control and elimination of malaria is mainly through the following three strategies: 2

17 (i) Reducing the contact between the human host and mosquito vector Most malaria endemic countries have implemented policies and programs to promote the universal access to insecticide treated bed nets (INTs)(2). One such program is the Roll Back Malaria Program by the World Health Organization (WHO) which had set an ambitious target of halving the burden of malaria by 2010 (8). The distribution and use of ITNs and long-lasting insecticide treated nets (LLINs) have shown to reduce human-vector contact. Studies conducted in malaria endemic countries like Papua New Guinea using ITNs and LLINs have reported declines in mosquito biting rates (9,10), which have contributed to the reduction in the incidence and mortality of malaria. (ii) Vector control Various methods are used to control and eliminate the mosquito vector. These measures include indoor residual spraying (IRS) of insecticide, using larvicides to eliminate mosquito larvae and the identification and clearing/reduction of mosquito breeding sites (2). IRS has proven to be an effective vector control measure (3,11). (iii) Diagnosis and treatment The early diagnosis and treatment of malaria greatly reduces the disease and may prevent death. WHO recommendations state that suspected cases of malaria be confirmed using parasite-based testing (microscopy or rapid diagnostic test) before treatment is administered (12). Resistance to antimalarials 3

18 has been a recurring problem with the resistance of Plasmodium falciparum to chloroquine and sulfadoxine-prymethamine during the 1970 s and 1980 s. However, the best available treatment, especially for Plasmodium falciparum malaria is artemisinin-based combination therapy (ACT). This treatment contains the drug artemisinin and a partner drug such as amodiaquine (AQ), lumefantrine, mefloquine (MQ) or sulfadoxine-prymethamine (SP) (2,3,12,13) Malaria in Papua New Guinea Papua New Guinea (PNG) is reported to have the highest burden of malaria in the Pacific region and outside of Africa (14). With a population of more than 7 million people, and a small landmass of km 2, PNG is characterized by its variability and complexity in its culture, ecology and its geography. This vast complexity is also reflected in the malaria situation throughout the country (15). Plasmodium species found in PNG are: P. vivax, P. falciparum, P. ovale, and P. malariae. Malaria infection is the second highest leading cause of hospital admission in PNG and the most common outpatient diagnosis with a reported estimate of 1.36 million cases per year with an estimated 800 malaria related deaths in 2011 of which most were children under five years of age (2). More than 60% of PNGs population live in malaria endemic areas (9). The transmission of malaria in the country is highly influenced by altitude where the disease is prevalent in the coastal lowlands, islands and at altitudes up to

19 and 1600 meters above sea level. However, malaria is absent at altitudes above 1700 meters (Figure 2) (9,15). P. falciparum and P. vivax are encountered frequently and are associated with severe malaria in children with P. falciparum being predominant (9,15). Figure 2: Altitude zones in Papua New Guinea (15) Malaria control efforts in Papua New Guinea Malaria vector control programs in PNG have influenced the epidemiology of malaria since the 1950s with residual spraying of dichlorodiphenyltrichloroethane (DDT) and dieldrin along with mass drug distribution of chloroquine (CQ) throughout endemic areas of the country (9,15,16). Operational challenges ceased the control programs in the 1970s but DDT spraying remained as the major control method until the mid 1980s (9). Before vector control programs, P. vivax was the predominant species but after the program was stopped, P. 5

20 falciparum superseded P. vivax due to the emergence of CQ resistant P. falciparum (16). Major malaria control programs began again in 2004 to 2008 with a country wide free distribution of INTs and LLINs through a grant from the Global Fund to Fight AIDS, Tuberculosis and Malaria (9,17). This has seen a decrease in the prevalence of malaria (9). Further grants have supported the continued distribution of ITNs and LLINs since 2009 which will enable further assessment of intensive control data on prevalence of Plasmodium species across PNG (9) Plasmodium falciparum Plasmodium falciparum is the most virulent form of malaria in humans and is a leading cause of death among children under 5 years of age world wide (18). The complex life cycle of Plasmodium falciparum involves a vertebrate host (human) and the insect vector (female Anopheles mosquito) Life cycle Plasmodium falciparum parasites spend most of their time inside the human host within the host s erythrocytes during their life cycle. Interaction between the host and the vector takes place when an infected anopheles mosquito bites the human host and takes a blood meal (Figure 3). During a blood meal, salivacontaining sporozoites are inoculated into the subcutaneous tissue of the host and penetrates the capillaries and, within 30 minutes, reach the liver where they begin the liver stage infection. 6

21 Sporozoites undergo pre-erythrocytic development in the liver where they invade hepatocytes and, within 10 days, multiply asexually into thousands of merozoites (1,19). Over a period of 7 to 10 days, infected liver cells rupture and release merozoites that enter the blood stream where they infect red blood cells (RBC) to begin the blood stage of the parasite lifecycle. Each merozoite that invades a RBC multiplies asexually to form16 to 32 new daughter merozoites (1). This takes place within 48 hours during which several different parasite stages develop in the following order: ring, trophozoite, schizont containing daughter merozoites. The lifecycle continues when the infected RBC (irbc) ruptures and releases merozoites that invade new RBCs. During the blood stage of the parasite s life cycle, some of the merozoites develop to form to sexual forms of the parasite, known as gametocytes, which are essential to continue transmission from the human host to the mosquito vector. Gametocytes circulate in the blood stream until they are ingested by a female Anopheles mosquito (1,19). After ingestion into the midgut of the mosquito, differentiation into the remaining sexual stages of the parasite occur. Inside the mosquito s midgut, an individual gametocyte forms one female macrogamete or up to eight male microgametes (1,19). Fusion of gametes produce zygotes, which develop into motile ookinetes that penetrate the midgut wall fto form oocysts. Oocysts enlarge over time and burst to release sporozoites which migrate to the salivary gland of the mosquito where they are ready to be transmitted to the next human host when the mosquito takes a blood meal (19). 7

22 The on-going asexual blood stage of the parasite s life cycle leads to the clinical symptoms of malaria (18). Figure 3: Plasmodium falciparum life cycle (20) Characteristics of the disease Various symptoms are associated with malaria from asymptomatic, mild to severe symptoms. Malaria is usually categorized as either uncomplicated or severe. Malaria paroxysm is the sudden onset of clinical symptoms with flu like manifestations occurring 1-2 weeks after infection accompanied by a combination of shivering and cold feelings for about an hour despite having an elevated body temperature. This is commonly referred to as the cold stage that 8

23 an infected person experiences. The hot stage usually lasts 2-6 hours where an infected person would experience headaches, fever, vomiting, nausea and seizures (in children). This is followed by the sweating stage where there is a decline in body temperature and malaise. Paroxysm is caused when parasites reach the erythrocytic stage in their life cycle and depending on the parasite species, febrile attacks would periodically occur every 24, 48 or 72 hours. Fever associated substances are released from ruptured irbc during the parasites blood stage. The released substances include toxic factors that stimulate macrophages and dendritic cells resulting in the production of proinflamatory cytokines. These release high levels of tumor necrosis factor α (TNF α) which have an effect on the progression of the disease and is mostly associated with severe malaria (21 24). Severe malaria progresses from uncomplicated malaria and results in severe anaemia, renal failure, cerebral malaria, hemolysis pulmonary edema, convulsions, acute respiratory distress syndrome (ARDS), circulatory collapse, hypoglycemia, abnormal bleeding, acidosis, hyperlactemia and hyperparasitemia (25,26) Pathogenesis Pathogenesis of Plasmodium falciparum infection is complex as it involves parasite-induced erythrocyte alterations and micro-circulatory abnormalities which are accompanied by local and systemic immune reactions resulting in multiple clinical outcomes (27). P. falciparum uses several techniques in order to 9

24 evade the host immune system and cause severe disease, as it needs to proliferate and survive without being destroyed. These include sequestration and cytoadherence of irbc, rosetting and antigenic / phenotypic variation Sequestration and cytoadhesion Sequestration is the adherence of infected erythrocytes that contain late developmental stages of the parasite (trophozites and schizonts) to the endothelium of capillaries and venules. It is one of the characteristics of P. falciparum that plays a major role in malaria infections. It favors parasitic development by protecting it from splenic clearance and is responsible for severe forms of cerebral malaria (28). In order to avoid clearance by the spleen, the mature irbc sequester in various organs. A vital feature of irbc that enables sequestration in Plasmodium falciparum is the expression of knob structures on the irbc membrane called knob-associated histidine-rich proteins (KAHRP) (Figure 4) (18). Knobs present the major virulence factor known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) where it mediates cytoadhesion to the host endothelium. KAHRPs are found on the cytoplasmic side of erythrocytes and interact with spectrin, actin and ankyrin and anchor PfEMP1 to the cytoskeleton of RBC. Sequestration can be either adherence to endothelial cells lining of blood vessels (cytoadhesion) or by binding to uninfected erythrocytes (rosetting). Cytoadhesion is the ability of RBC to adhere to vascular endothelium and has been seen in organs such as the brain, intestines, liver, lung, skin and in cell 10

25 linings of the placenta (29,30). Mediation of cytoadhesion is through specific interactions between members of the PfEMP1 family encoded by var genes and receptors on the surface of endothelial cells (Figure 4). Various cell types bind to P. falciparum such as cluster of differentiation 36 (CD36), chondroitin-sulfate A (CSA), intracellular adhesion molecule-1 (ICAM-1), thrombospondin (TPS), E- selectin and vascular cell adhesion molecule 1 (VCAM-1). Severe malaria is associated with specific PfEMP1 variants that contain a combination of adhesion domains referred to as domain cassettes (DC) 8 and 13. Severe malaria does not occur in the majority of Plasmodium falciparum infections. This suggests that irbc sequestration is adapted to limit death and favor parasite transmission (29). Figure 4: Surface expression in irbc and host cell showing various host cell ligands that are able to adhere to PfEMP1 variants (31). 11

26 Variant surface Antigens (VSA) VSAs are thought to be responsible for the cytoadherence of P. falciparum. Apart from PfEMP1, there are several VSA families that exist such as repetitive interspersed family (RIFIN) proteins, subtelomeric variable open reading frame (STEVOR) proteins, surface-associated interspersed gene family (SURFIN) proteins and Plasmodium falciparum Maurer s cleft two transmembrane (PfMC- 2TM) proteins which are important because of their significance in targets of natural acquired immunity (NAI) (18). The most studied VSA is PfEMP1, which is an important target for naturally acquired immunity Rosetting Rosetting is the clustering of mature irbc to uninfected RBCs. This has been thought to contribute to microvasculature obstruction and has been associated with severe disease (18,32). However, studies have observed that rosetting phenotypes are not always associated with severe disease (32 34). Rosetting isolates have been reported to be associated to A or B blood groups forming larger and stronger rosettes whereas the rosettes in blood group O are much smaller and weaker (35) Phenotypic and antigenic variation Phenotypic variation is a vital survival strategy mediated by the differential control of members of the PfEMP1 family. Encoded with ~60 var genes, each parasite 12

27 expresses only a single var gene at a time. This leads to the irbc adhereing within the microvasculature, resulting in sever disease (36). This variation gives rise to functionally different PfEMP1 adhesion ligands while changing the antigenic makeup of a molecule in order to decrease or evade recognition by existing antibody responses. It has been reported that this functional diversity may be a key factor in the pathogenesis of the disease (37) Plasmodium falciparum erythrocyte membrane protein 1 PfEMP1 is a var gene product and is one of the most extensively characterized proteins expressed on the surface of irbc. The virulence of P. falciparum is caused by the parasites ability to adhere to erythrocytes and to evade the host immune attack. PfEMP1s are the major antigenic ligands responsible for the sequestration of irbc in different host organs, contributing to the manifestation of the disease (38). PfEMP1s range in size from kDa and contain in their main structure: an N-terminal segment (NTS); variable numbers of Duffy Binding Like domains (α-ε); one or two CIDR (α-γ); a TM domain; a C2 domain; and the conserved intra-cellular acidic terminal segment (ATS) (38,39). PfEMP1 has been reported to be a major molecule responsible for the cytoadhesive properties of irbcs to uninfected erythrocytes, the formation of rosettes and the binding to endothelial receptors. This enables the parasite to complete its life cycle, multiply and re-invade without being removed by the spleen (38). Extracellular domains of PfEMP1 (DBL and CIDR) can bind to a variety of host receptors such as cluster determinant 36 (CD36), inter-cellular 13

28 adhesion molecule 1 (ICAM1), thrombospondin (TSP), complement receptor 1(CR1) and chondroitin sulfate A (CSA) (38,39). PfEMP1 is the major antigen that is exposed by Plasmodium falciparum parasites on the surface of irbcs (Figure 4). Its antigenic diversity and regulation in switching between antigenic variants enables the parasite to maintain long-lasting chronic infections (38). Figure 5: Expression of PfEMP1 by Plasmodium falciparum on knobs formed on the surface of irbcs. CIDR and DBL mediate adhesion by binding to several endothelial receptors such as CD36, ICAM1 and CSA. (PV-parasitophorous vacuole; MC-maurer's cleft) (38) var genes and PfEMP1 PfEMP1 is encoded by the highly polymorphic var multigene family which are capable of undergoing clonal antigenic variation (18,38). With ~60 var genes, only a single PfEMP1 variant is expressed at any given time (18), this makes it harder for antibodies to recognize the new protein. As the parasite divides inside the human blood cells, the var genes continuously transfer genetic information between one and other through recombination. Through this process, it is able to make new var genes by combining pieces of other existing var genes together. 14

29 Var genes are encoded in Exons 1 and 2 (Figure 6). Exon 1 is the largest of the two in which most sequence variability occurs and it encodes an NTS along with segments of DBL domains (α, β, γ, δ and ξ subtypes) and CIDR (α, β and γ). Exon 1 is separated from Exon 2 by the trans-membrane (TM) sequence to which Exon 2 encodes the ATS (40). Figure 6: Domain architecture example of two PfEMP1 molecules. DBL, CIDR with NTS make up the semi conserved head structure of Exon 1. The majority of var genes are classified into three main groups based on their upstream sequences (UpsA, UpsB and UpsC), and their chromosome location and direction of transcription (Figure 7). The var gene repertoire also contains three strain-transcendent variants (var1, var2csa and type3 var) which are present in all parasite genotypes (29). 15

30 Figure 7: Organization and protein structure of var genes. (A) Chromosomal organization. Groups A and B are transcribed in opposite directions and are located in subtelomeric region whereas group C are located at the central chromosome cluster. (B) Recombination between subdomains 2 and 3 in the DBL domain (29) Immunity to P. falciparum malaria Immune response to malaria is activated when parasite molecules are inoculated in the blood stream. This is regulated by the innate and the adaptive (humoral) immune system through a process involving protection against extracellular (sporozoites and merozoites), and intracellular liver and blood stages (41 43). The level of malaria immunity varies amongst individuals. Non-immune individuals who are infected with the parasite for the first time, even with low levels of parasitaemia, tend to develop acute clinical symptoms that can lead to severe malaria and death (44,45). But for individuals who have had several infections, they develop anti-immunity towards malaria, which serves as a defense mechanism against clinical symptoms and therefore decreasing the risk of severe malaria, even if they have high levels of parasitaemia. Repeated infections of malaria leads to anti parasite immunity which protects individuals 16

31 against both low and high levels of parasitaemia and does not often cause clinical symptoms (44,45). Passive immunity against malaria protects a child during the first six months of life and is due to the transfer of maternal antibodies from a malaria immune mother to her child. However, naturally acquired immunity takes time, and as mentioned earlier, requires repeated infections and parasite exposure. Acquired immunity develops and strengthens human immune responses to the parasite and decreases the risk of mild and severe malaria. Innate protection and acquired immunity (humoral and cell-mediated responses) are the defense mechanisms that reduce parasitaemia (44,46,47) Acquired Immunity Plasmodium falciparum is susceptible to the hosts natural acquired immunity (NAI) which increases over time (48). Studies conducted in PNG and in other malaria endemic countries have reported that protective immunity to P. falciparum is acquired after repeated disease episodes during childhood (8,16,49 53). As children get older and are exposed to repeated P. falciparum infections, they acquire protective immunity over time which depends on the intensity of transmission (53 55). Immunity to severe disease and death is acquired within the first five years of life while immunity to mild disease is acquired later in life. This development of protective immunity is slow and it requires the exposure to multiple antigenic variants of the parasite (45,56). After reaching adulthood, individuals in malaria endemic areas will have naturally 17

32 acquired a more protective immunity to the disease (Figure 3) (57,58). Even though acquired immunity to malaria decreases the risk of an individual to mild and severe malaria, it does not prevent malaria infection. Figure 8: Relationship between age and severity of malaria in populations exposed to high malaria transmission. Changes over time of various indices of malaria in a population in an area with high transmission of P. falciparum. Asymptomatic infection (pink), mild disease (febrile episodes blue) and sever malaria (green) (58). Protection against P. falciparum infection is mediated by antibodies and the acquisition of immunity is associated with acquiring a broad repertoire of antibodies to parasite encoded variant surface antigens (VSA) (53). Protective antibodies are effective through either binding of irbc surface antigens (59), blocking of irbc invasion or by antibody-dependent cellular inhibition (ADCI) which is when monocytes produce cytokines that mediate the killing of intracellular parasites, thus leading to parasite clearance (60,61). Throughout the development of immunity to P. falciparum malaria, the disease becomes less severe with a decrease in the number of parasites circulating 18

33 through the blood stream. Protective immunity appears to be mediated through a repertoire of immunoglobulin G (IgG) antibodies against target surface antigens (59,62) Antibody responses to P. falciparum antigens The mechanism by which antibodies mediate protective immunity is not fully understood. Antibodies against P. falciparum are directed against the different stages of the parasites life cycle. Antibody functions include; inhibition of merozoite invasion/intracellular growth, antibody-dependent cellular killing with opsonization and clearance of irbc by antibody binding to host cell surfaces and inhibition of sequestration in the microvasculature (60). Antibodies targeting VSA are thought to interfere with irbc sequestration, rosetting and inhibit adhesion which are features that contribute significantly to the pathogenesis of Plasmodium falciparum (18) P. falciparum antigens Most of the studies conducted have relied on recombinant purified PfEMP1 domains to study antibody responses to PfEMP1 in humans (18). P. falciparum antigens that are targets of antibodies include blood stage Merozoite antigens such as Merozoite surface proteins (MSP), Erythrocyte-binding antigens (EBA); pre-erythrocytic Circumsporozoite protein (CSP) and PfEMP1 variant surface antigens; Duffy binding like (DBL) and Cysteine-rich interdomain region (CIDR). 19

34 A major factor in understanding the potential clinical significance of the different antibody responses is the association they have with protective immunity (54). The acquisition of antibodies to P. falciparum antigens in children has been reported to be highly structured. Antibodies to different recombinant PfEMP1 domains were sequentially acquired, with the children acquiring first, antibodies to variants encoded by var genes (18) Bed nets and Immunity The use of insecticide bed nets has proven to be an effective malaria control strategy in reducing malaria exposure. Studies have reported a reduction in malaria related morbidity and mortality in young children in areas of high malaria transmission through the use of bed nets (8,64). However, there have been concerns about the usage of ITNs and their effect on the acquisition of immunity, as bed nets are seen as a prerequisite for the development and the maintenance of acquired immunity. When used widely in a community, ITNs reduce the population of sporozoite-positive mosquitoes which is reflected in reduced plasma antibody levels to circumsporozoite proteins (8). Malaria immunity relies on the acquisition of a repertoire of agglutinating antibodies that recognize a broad spectrum of VSAs that are expressed on irbc such as PfEMP1. Studies investigating the effects of ITNs usage on plasma levels and repertoires of VSA antibodies, have reported reduced levels of VSA antibodies and 20

35 narrowing the repertoire of recognized VSA which could make ITNs users more vulnerable to parasites that may express rare VSA antigens (8). 21

36 1.9. Thesis hypothesis and objectives This study hypothesizes that: i) Children who do not use ITNs have higher antibody responses towards P. falciparum irbc antigens than children who use ITNs. ii) The use of ITNs alters the natural acquisition of immunity to P. falciparum infection. This study aims to: i) Determine if the use of ITNs have an influence on antibody levels to irbc surface antigens and overall immunity to malaria. 22

37 2 Materials and Methods 2.1 Study design This age matched cohort study is designed to determine whether antibody responses to malaria proteins have changed since the introduction of vector control and national program to control malaria in PNG by using ITNs. Plasma samples collected from two different cohorts (2004 and 2013) of children aged 5-14 from the Mugil area of Madang Province (Figure 9). A total of 636 samples were used in this study. The first cohort (Mugil 1) consisted of 206 plasma samples collected in 2004 (65 69). These samples were collected prior to the distribution and use of INTs. The second cohort (Mugil 2) consisted of 430 plasma samples collected between 2012 and These samples were collected after the introduction of vector control measures and the national malaria control program of the distribution and use of INTs. 23

38 Figure 9: Map of Madang Province, Papua New Guinea. 2.2 Recombinant Antigens Three groups of antigens consisting of a total of nineteen recombinant protein antigens specific for Plasmodium falciparum were selected and covalently coupled to nineteen different beads/microspheres. The first group of antigens consisted of five PfEMP1 variant surface antigens (Table 1), the second group of consisted of 13 merozoite antigens (Table 2) and the third group only consisted of a circumsporozoite antigen (table 3). The recombinant protein antigens were from several specific P. falciparum antigenic regions: i) PfEMP1 variant surface antigens Duffy binding like domains and Cysteine rich inter domains (DBL and CIDR); 24

39 ii) Merozoite antigens - Merozoite surface proteins (MSP); Erythrocyte binding antigens (EBA); iii) Circumsporozoite proteins (CSP) A Maltose binding protein (MBP) was also coupled to be used as a control antigen for the PfEMP1 variant surface antigens. Each antigen was coupled with a microsphere bead that was color-coded specifically based on the ration of two flourochromes that is detected by the BioPlex system (BioPlex System; Bio-Rad. USA). Each bead contains about 10 8 COOH-groups on its surface and during coupling, 10 6 COOH-groups will randomly couple with the added antigens. Antigens Microspheres / Beads 1 IT4var19 CIDR α1.1 #57 2 IT4var07 CIDR α1.4 #68 3 IT4var19 DBL α2 #57 4 IT4var19 DBL β12 #68 5 IT4var19 DBL γ6 #69 Table 1: PfEMP1 variant surface antigens. Antigens Microspheres / Beads 1 Ec MSP1 42 FUP #38 25

40 2 Ec MSP1 42 FVO #42 3 Ec MSP1 42 3D7 #54 4 MSP2 FC27 #86 5 Ec PfMSP3 #78 6 PfMSP6 GST #87 7 PfMSP7 GST #85 8 MSP DBL 348 FL + inhibitors #52 9 MSP DBL 355 FL + inhibitors #53 10 EBA-140 R3-5 # D7 EBA-175 R3-5 #79 12 W2Mef EBA-175 R3-5 #89 13 EBA-181 R3-5 #44 Table 2: Merozoite Antigen - Merozoite surface proteins (MSP) and Erythrocyte binding antigens (EBA). Antigens Microspheres / Beads 1 PpCSP - M3/4 #80 Table 3: Circumsporozoite protein. 2.3 Covalent coupling of Antigens to microspheres Coupling of proteins is a two-step carbodiimide procedure during which microspheres are first activated with 1-Ethyl-3-[3-dimethylaminopropyl] 26

41 carbodiimide hydrochloride (EDC) in the presence of N-hydrosulfosuccinimide (Sulfo-NHS) to form a sulfo-nhs-ester intermediate, which is then replaced by the reacting with the target molecule (antibody) to form a covalent amide bond (Appendix 1). Figure 10 shows the general workflow in coupling proteins, which will be explained in the following paragraphs. Figure 10: General protein coupling workflow. 27

42 Bead activation A total volume of 50μl (containing 1.25 x 10 7 beads) was prepared to be used for this study. Coupling of the antigens to the beads/microspheres involved the following steps. First, each of the twenty (including the MBP) stock microspheres (xmap, Luminex, USA) was resuspended by vortexing and sonication for 10 seconds. A total of 50μl was pipetted into single 2ml micro centrifuge tubes for each of the COOH beads. The microspheres were pelleted by micro centrifugation at rpm for 2 minutes after which the supernatant was removed. The pellet was resuspended in 100μl of sterile filtered water by vortexing and sonication for 10 seconds. The microspheres were then pelleted by micro centrifugation at rpm for 2 minutes. The supernatant was carefully removed and 1mL of 100mM activation buffer; monobasic sodium phosphate (Nah 2 PO 4 ), with a ph of 6.2 was pipetted to the pellet and resuspended by vortex and sonication for 10 seconds. The microspheres were then pelleted by micro centrifugation at rpm for 2 minutes. After micro centrifugation, 920μl of the supernatant was carefully removed. EDC and Sulfo_NHS were reconstituted in separate 2μl micro centrifuge tubes. The remaining 80μl was resuspended by vortexing and sonication for 10 seconds and 10μl of 50mg/mL Sulfo-NHS (in sterile filtered water) was added to the microspheres and mixed gently by vortex. After that, 10μl of 50gm/mL EDC (in sterile filtered water). After gently mixing by vortex, the microspheres were incubated on a rotator in the dark for 20 minutes at room temperature. 28

43 Protein coupling After incubation, the microspheres were pelleted by micro centrifugation at rpm for 2 minutes and the supernatant aspirated. To each pelleted microsphere, 250μl of 1x PBS (ph7.4) was added and resuspended by vortexing and sonication for 10 seconds. This step was repeated one more time to wash the microspheres before adding the proteins. Before adding the proteins to the coupled microspheres, the concentrations of each protein antigen were calculated. Appendix 2(a) shows the concentration of each of the twenty protein antigens, the concentrated volume to be coupled and the final diluted volume to use. After micro centrifuging, the activated washed microspheres were resuspended in 1xPBS (ph7.4) by vortexing and sonication for 10 seconds and the appropriate volume of protein antigen were added to bring the final volume to 250μl (Appendix 2(b)). The mixture was gently mixed by vortexing and incubate at 4 C overnight on a rotator. After incubation, each activated microspheres were pelleted by micro centrifuging at rpm for 2 minutes and the supernatant was aspirated. 500μl of PBS- TBN blocking/ storage buffer was added and resuspended by vortexing and sonication for 10 seconds. This was repeated for a total of three washes. After the final aspiration, the microspheres were resuspended in 112.5μl of PBS-TBN by vortexing for 10 seconds and stored at 4 C in the dark. 29

44 2.4. Sample dilutions and preparation All samples and controls were tested in duplicates. Each of the 636 plasma samples were tested in duplicates and each duplicate was diluted to a 1:100 serial dilution by adding 2μl of the plasma sample to 198μl of PBS-TBN (Apendix 3a). The negative controls used were from North American individuals never exposed to malaria. Each plasma samples and duplicates were diluted to 1:100 (2μl of plasma in 198μl of PBS-TBN). Positive controls were from a pool of Papua New Guinean adults. A 10 serial 2- fold dilution for positive controls were done for each duplicate for every plate. The positive PNG controls were diluted from 1:20 to 1:12800 (Appendix 3b). Apart from the Positive controls and negative controls, blanks were also run in duplicates. Blank wells contained only PBS-TBN and microspheres, which allowed determination of the fluorescence background in the assay Plate Preparation and Analysis Figure 11 illustrates an overview of the steps taken in preparing the plates, which will be explained further in the following paragraphs. 30

45 Figure 11: Overview of workflow for 96-well plate preparation. Using a MultiScreen 96-well filtration plate (Merck KGaA, Germany), 100μl of PBS-TBN was pipetted into each well to pre wet the wells and then vacuumed out. The bottom of the plate was sealed with a Titer Top plate sealer and into 31

46 each well, 50μl of diluted plasma samples and controls were added. Duplicates for each were added alongside each sample. The bead master mix was calculated and prepared prior to setting up the plates (Appendix 4). The master mix was vortexed and sonicated for 30 seconds and poured into a multi-channel pipettor trough. Using a multi-channel pipette, 50μl of the bead master mix was added into each well and resuspended by pipetting up and down 5-6 times. After resuspending, the plate was incubated on a plate shaker for 20 minutes in the dark. During incubation, the BioPlex machine was warmed up and calibrated. After incubation, the sealer was removed and the wells were vacuumed. The plate was washed twice with 100μl PBS-TBN, vacuuming after each wash. The bottom of the plate was sealed and 100μl of diluted anti human IgG secondary antibody (Appendix 5) was resuspended into each well. The plate was then incubated on a plate shaker for 15 minutes in the dark. After incubation, the sealer was removed and the plate was washed three times with 100μl PBS-TBN, vacuuming after each wash. The bottom of the plate was sealed with a new Titer Top plate sealer and each well was resuspended with 100μl of PBS-TBN and the plate was placed in the BioPlex to be analyzed. The analysis of the data collected from the multiplex assay was done using the Bio-Plex Manager Software Version 6.1 (Bio-Rad, USA). Statistical analysis (Mann-Whitney unpaired t test) was conducted using Prism GraphPad software version 6 with p values less than 0.05 regarded as statistically significant. 32

47 3 Results The antibody responses to each of the antigens for children in Mugil 1 and Mugil 2 cohorts were analyzed as the accumulated fold-over Median Fluorescence Intensity (MFI) according to the type of P. falciparum antigenic region they belonged to. Antibody responses between the two cohorts were reported as significantly different if P<0.05. Comparing P. falciparum surface antigens across multiple samples in the two groups enabled a clearer observation of the differences in the antibody responses to each of the antigens PfEMP1 variant surface antigens: DBL and CIDR In analyzing PfEMP1 variant surface antigens (CIDR α1.1, CIDR α1.4, DBL α2, DBL β12, and DBL g6), the MFI of the maltose binding protein (MBP-His6 control) was subtracted from the MFI values of each PfEMP1 variant surface antigens after which the fold-over value was determined. All samples with an MFI > 0 after subtracting MBP MFI from antigen MFI were recorded as 0. Figure 12 shows the results of all PfEMP1 variant surface antigens. 33

48 Fold over - MFI (SD, Mean) 70 All PfEMP1 antigens p= p< p< p= p< CIDR α1.1 CIDR α1.1 CIDR α1.4 CIDR α1.4 DBL α2 DBL α2 DBL β12 DBL β12 DBL g6 DBL g6 Mugil 1 vs Mugil 2 Figure 12: Antibody responses to PfEMP1 antigens CIDR α1.1 There was a significant difference in antibody response to CIDR α1.1 for Mugil 1 (Mdn = 0) and Mugil 2 (Mdn = 0), p> This indicated that antibody responses to DBL α2 were higher for Mugil 1 than for Mugil CIDR α1.4 For CIDR α1.4, there were no significant differences in the level of antibody responses. The Mann-Whitney test indicated that antibody responses for Mugil 1 (Mdn = 0.1) was lower than Mugil 2 (Mdn = 0.2), p=

49 DBL α2 There was a significant difference in antibody response to DBL α2 for Mugil 1 and Mugil 2. The Mann-Whitney test indicated that antibody responses to DBL α2 was higher for Mugil 1 (Mdn = 0.7) than for Mugil 2 (Mdn = 0.07), p> DBL β12 Significant differences were observed in antibody response levels to DBL β12 for Mugil 1 (Mdn = 0.9) and Mugil 2 (Mdn = 0.3), p= This indicated that children in the Mugil 1 cohort had higher antibody responses to DBL β12 than children in the Mugil 2 cohort DBL g6 Differences observed in antibody response levels were significant. The Mann- Whitney test indicated that Mugil 1 (Mdn = 0.5) had higher antibody responses than Mugil 2 (Mdn = 0), p>0.0001, indicating higher antibody responses from children in Mugil 1 than Mugil Merozoite antigens: MSP and EBA Merozoite Surface Protein antigens (MSP1 42 FUP, MSP1 42 FVO, MSP1 42 3D7, MSP2 FC 27, PfMSP3, MSP6 GST, MSP7 GST, MSP-DBL 348 FL + inhibitors and MSP-DBL 355 FL + inhibitors, EBA-140 R3-5, 3D7 EBA-175 R3-5, W2Mef 35

50 Fold over - MFI (SD, Mean) EBA-175R3-5 and EBA181 R3-5) were analyzed without subtracting the MFI of the maltose binding protein (MBP-His6 control) Merozoite surface proteins Each antigen showed significant differences between the two groups with Mugil 1 samples showing higher levels of antibody levels that the Mugil 2 samples (Figure 13). 120 p< p< All MSP p< p< p< p< p< p< p< MSP1 42 FUP MSP1 42 FVO MSP1 42 3D7 MSP2 FcC27 PfMSP3 MSP6 GST Mugil 1 vs Mugil 2 MSP7 GST 348 FL 355 FL Figure 13: Antibody responses to Merozoite surface proteins 36

51 MSP1 42 FUP The Mann-Whitney test indicated a significant difference in antibody responses to MSP1 42 FUP with Mugil 1 (Mdn = 58) having higher antibody responses than Mugil 2 (Mdn = 5), p< MSP1 42 FVO Differences were seen in the level of MSP1 42 FVO for Mugil 1 (Mdn = 55) and Mugil 2 (Mdn = 7, p< This indicated a significant difference with antibody responses to MSP1 42 FVO higher for children in Mugil 1 than children in Mugil MSp1 42 3D7 There was a significant difference in antibody response levels to MSP1 42 3D7, with Mugil 1 (Mdn = 68) having a higher level than Mugil 2 (Mdn = 5), p< MSP2 FC27 There was a significant difference in antibody levels to MSP2 Fc27. Mugil 1 (Mdn = 87) had higher response levels than and Mugil 2 (Mdn = 28), p< PfMSP3 There was a significant difference in the antibody response level of PfMSP3 with children from Mugil 1 (Mdn = 3) having higher antibody responses than Mugil 2 (Mdn = 0.3), p<

52 MSP6 GST There was a significant difference in antibody response to MSP6 GST with higher response levels for Mugil 1 (Mdn = 3) than Mugil 2 (Mdn = 0.5), p< MSP7 GST There was significant differences in the level of antibody responses to MSP7 GST with Mugil 1 (Mdn = 0.8) having higher antibody response levels than Mugil 2 (Mdn = 0.4), p> MSP-348 FL + inhibitors Significant differences were observed in the antibody response levels of MSP- DBL 348 FL. Children from Mugil 1 (Mdn = 58) had higher antibody responses than children from Mugil 2 (Mdn = 3), p< MSP-DBL 355 FL + inhibitors There was significant differences in the antibody response levels to MSP-DBL 355 FL with higher antibody responses for Mugil 1 (Mdn = 4) than Mugil 2 (Mdn = 0.5), p< Erythrocyte Binding Antigen (EBA) Erythrocyte Binding Antigens were analyzed without subtracting the MFI of the maltose binding protein control (MBP-His6 control). Each antigen showed 38

53 Fold over - MFI (SD, Mean) significant differences between the two groups with children from Mugil 1 showing higher antibody responses than children from Mugil All EBA p< p< p< p< EBA 140 R3-5 EBA 140 R3-5 W2Mef EBA 175 R3-5 W2Mef EBA 175 R3-5 3D7 EBA 175 R3-5 Mugil 1 vs Mugil 2 3D7 EBA 175 R3-5 EBA 181 R3-5 EBA 181 R3-5 Figure 14: Antibody responses to Erythrocyte binding antigens EBA-140 R3-5 There was a significant difference in the level of antibody reactivity to EBA-140 R3-5. There were higher antibody responses in children from Mugil 1 (Mdn = 94) than Mugil 2 (Mdn = 8), p<

54 D7 EBA-175 R3-5 There was a significant difference in the antibody responses to 3D7 EBA-175 R3-5 with greater reactivity in children from Mugil 1 (Mdn = 21) than Mugil 2 (Mdn = 0.9), p< W2Mef EBA-175 R3-5 Significant differences were seen in the reactivity levels to W2Mef EBA-175 R3-5, with a greater reactivity levels in children from Mugil 1 (Mdn = 30) than Mugil 2 (Mdn = 1), p< EBA-181 R3-5 There were significant differences in the antibody responses to EBA-181 R3-5 with higher antibody responses in children from Mugil 1 (Mdn = 2) than children from Mugil 2 (Mdn = 0.2), p< Circumsporozoite Protein (CSP) CSPM3-4 was analyzed without subtracting the MFI of the maltose binding protein control (MBP-His6 control) with Mugil 1 samples having higher antibody responses than the Mugil 2 samples (Figure 15). 40

55 Fold over - MFI (SD, Mean) CSPM p< Mugil 1 vs Mugil 2 Figure 15: Antibody responses to Circumsporozoite proteins Pp CSP M3/4 There was a significant difference in the antibody response levels to Pp CSP M3/4 with higher responses observed in children from Mugil 1 (Mdn = 6) than the children from Mugil 2 (Mdn = 1), p< Age matched comparison An important focus in this study was also looking at age matched antibody responses between certain age groups in both ITN users and non-itn users. By 41

56 grouping the children according to age groups; 5 to 9 years and 10 to 14 years, it was easier to observe the levels of antibody responses. Children from 2004 aged 5 to 9 years of age were seen to have a significantly higher antibody response across all Plasmodium falciparum antigens compared to children of the same age group from The Mann Whitney test was used to determine the p value between the two cohorts, which saw evidence that repeated exposure to infection by children from 2004 has increased their antibody levels and are therefore more responsive to Plasmodium falciparum antigens than that of children from 2013 who slept under ITN (Figure 16). 42

57 F o ld o v e r - M F I (S D, M e a n ) p < p < p < p < p < p < p < p = p = p < p < p < p < C ID R 1.1 C ID R 1.1 p = p < C ID R 1.4 C ID R 1.4 D B L 2 D B L 2 D B L 1 2 D B L 1 2 D B L g 6 D B L g 6 M S P F U P M S P F U P p < M S P F V O M S P F V O M S P D 7 M S P D 7 M S P 2 F C 2 7 M S P 2 F C 2 7 P fm S P 3 P fm S P 3 M S P 6 G S T M S P 6 G S T M S P 7 G S T M S P 7 G S T M S P -D B L F L p < p < p < M S P -D B L F L M S P -D B L F L M S P -D B L F L E B A R 3-5 E B A R 3-5 W 2 M e f E B A R 3-5 W 2 M e f E B A R D 7 E B A R D 7 E B A R 3-5 E B A R 3-5 E B A R 3-5 C S P M 3-4 C S P M 3-4 Figure 16: Antibody responses in children aged 5 to 9 years from Mugil 1 (non-itn users) and Mugil 2 (ITN users) Similar observations were also noted for children aged 10 to 14 years from both cohorts (Figure 17). Repeated exposure to infections in children from 2004 saw significant differences with higher antibody levels and responsiveness to Merozoite antigens and the pre-erythrocytic Circumsporozoite protein when compared to children of the same age group in 2013 who used ITNs. However, there were no significant differences seen through all PfEMP1 variant surface antigens. 43

58 F o ld o v e r - M F I (S D, M e a n ) p < p < p < p < p < p = p < p =1203 p < p =0027 p < p = C ID R 1.1 C ID R 1.1 p =1013 p =3053 C ID R 1.4 C ID R 1.4 D B L 2 D B L 2 D B L 1 2 p =2489 D B L 1 2 D B L g 6 D B L g 6 M S P F U P M S P F U P M S P F V O M S P F V O M S P D 7 M S P D 7 M S P 2 F C 2 7 M S P 2 F C 2 7 P fm S P 3 p < P fm S P 3 M S P 6 G S T M S P 6 G S T M S P 7 G S T M S P 7 G S T M S P -D B L F L M S P -D B L F L M S P -D B L F L M S P -D B L F L E B A R 3-5 E B A R 3-5 W 2 M e f E B A R 3-5 p < p < p < W 2 M e f E B A R D 7 E B A R D 7 E B A R 3-5 E B A R 3-5 E B A R 3-5 C S P M 3-4 C S P M 3-4 Figure 17: Antibody responses in children aged 10 to 14 years from Mugil 1 (non-itn users) and Mugil 1 (ITN users) 44

59 1 Discussion There still remains a need for in depth understanding of the functional background for malaria immunity, however, studies have indicated that immunity to malaria relies strongly on the acquisition of a repertoire of antibodies that would be able to recognize a broad spectrum of antigens expressed on irbcs (8,70 73). The acquisition of natural immunity is directed against various merozoite antigens. The main focus of this study was to compare the two groups of children (Mugil 1 and Mugil 2) and observe the antibody levels to Plasmodium falciparum recombinant surface antigens between the two groups to determine if the use of bed nets could have had an impact on the natural acquisition of antibodies Antibody responses Comparisons between the levels of antibodies against recombinant antigens between children who did not use bed nets (Mugil 1) and children who used bed nets (Mugil 2) saw significant differences (p< for most antigens). The high antibody levels observed in children who did not use bed nets was reflective of several other studies that reported the significance of bed nets and their influence on immunity to malaria (8,64,74). This could be interpreted in the Mugil 1 group who would have experienced repeated exposure to the disease than the Mugil 2 group, resulting in higher antibody recognition to PfEMP1 surface 45

60 antigens as the maintenance and development of host immunity through this repeated exposure would be needed (starting at an early age) in order to prevent severe disease outcomes later in life. Children from the Mugil 1 cohort were part of a 2004 treatment-reinfection study, which the children were treated with a course of 7-day artesunate monotherapy after infection of Plasmodium falciparum and were actively followed up every two weeks to assess re-infections (65 69) Low antibody responses Various surface antigens were observed in this study to have low antibody responses. These low levels of antibody responses in both groups of children could be indicative of weak immune recognition. A recent study has found that precursor regions of merozoite surface proteins; especially for MSP6 and MSP7, were less strongly recognized by immune sera as they were likely to have been degraded after being made resulting in lower immunogenicity (75). Apart from MSP6 and MSP7, there were also low antibody responses observed in other surface antigens such as; DBL α2, DBL β12, DBL g6, CIDR α1.1, CIDR a1.4, PfMSP3 and EBA-181 R3-5. This was seen especially through out the children in the Mugil 2 group of which the majority of children had low immune responses. This could be due to limited exposure to infection through the use of bed nets resulting in the inhibition of immune recognition of PfEMP1 antigens. 46

61 High antibody responses The results from this study have also shown children from both groups having high levels of antibodies; especially towards the merozoite surface protein antigens suggesting the presence of MSP antibodies in both exposed and protected children. Studies have reported that less antigenic diversity in various surface antigens make them easier to be recognized by the immune system (76,77) Antigenicity and phenotypic variations Antigenicity and phenotypic binding associated within the PfEMP1 var gene family plays an important role in P. falciparum malaria. The recognition of surface antigens by antibodies has been seen to be a major factor for combating malaria. Protection has been associated with the acquisition of antibodies against antigenic phenotypes in which acquired immunity ranges in protection from death, severe malaria and uncomplicated malaria (56,78). In this study a large number of children from both groups had low antibody levels to; DBL α2, DBL β12, DBL g6, CIDR α1.1, CIDR a1.4, PfMSP3, MSP6 GST, MSP7 GST, EBA-181 R3-5. This could be from phenotypic diversity by the parasite, which would make it difficult for host immune recognition as the switching of genes by the parasite during infection enables the parasite to go undetected by the host making it difficult for antibodies to recognize the new proteins (18). 47

62 4.4. Effect of bed nets on immunity to plasmodium falciparum antigens From the results, it was seen that antibody responses to Plasmodium falciparum antigens were significantly higher throughout children in the Mugil 1 cohort who did not use ITN than the children from in the Mugil 2 cohort who used ITNs. This supported previous studies (64,74,79,80) that looked into the effects of ITNs on antibody levels to P. falciparum antigens in children, suggesting that children who used ITNs had lower antigen antibody levels and recognized smaller proportions of antigens that were expressed by the parasite than children who did not use ITNs and were exposed to repeated infections. 48

63 5 Conclusion Antibody levels to PfEMP1 surface antigens observed between the two groups of children in this study showed significant differences in the level of antibody responses depending on the type of recombinant surface antigen. The use of bed nets not only limits the natural acquisition of antibodies but it also makes it harder for antibodies to recognize and respond to phenotypic variations by the PfEMP1 surface antigens. This could also be seen throughout the Mugil 2 group of children who had lower antibody levels than the children in the Mugil 1 group. Children who used bed nets showed weaker antibody responses to DBL α2, DBL β12, DBL g6, CIDR α1.1, CIDR a1.4, PfMSP3 and EBA-181 R3-5. The low antibody levels indicate weak immune recognition by antibodies to these surface antigens. Decreased exposure to P. falciparum infection by ITNs reduces antibody responses to antigens. These findings also suggest that high levels of surface antigens are good indicators for predicting protection from malaria. 49

64 APPENDICES Appendix 1: Antibody Coupling Antibody coupling for proteins is a simple two-step carbodiimide procedure during which microsphere carboxyl groups are first activated with EDC reagent in the presence of Sulfo-NHS to form a sulfo-nhs-ester intermediate. The reactive intermediate is then replaced by reaction with the primary amine of the target molecule (antibody, or peptide) to form a covalent amide bond. Figure 18: Appendix 1 - Antibody coupling for proteins. 50

65 Appendix 2a: Antigen concentrations Protein Protein Concentration Concentration to couple Dilution with PBS Volume for 50μl microsphere (Total volume - 250μl) 2μl protein into 4.08μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 4.48μl IT4var07 CIDR α ug/μl 0.5ug/μl 1xPBS Add 1μl to 248μl 1xPBS IT4var19 DBL α2 2.25ug/μl 0.5ug/μl 2μl protein into 7μl 1xPBS Add 1μl to 248μl 1xPBS IT4var19 DBL β ug/μl 0.5ug/μl No dilution needed Add 2μl (Protein) to 248μl 1xPBS 2μl protein into 1.88μl IT4var19 DBL g6 0.97ug/μl 0.5ug/μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 5.76μl Ec MSP1-42 FUP 0.97ug/μl 0.25ug/μl 1xPBS Add 1μl to 248μl 1xPBS Ec MSP1-42 FVO 0.32ug/μl 0.6ug/μl No dilution needed Add 2μl (Protein) to 248μl 1xPBS Ec MSP1-42 3D7 0.31ug/μl 0.5ug/μl No dilution needed Add 2μl (Protein) to 248μl 1xPBS 2μl protein into 18μl MSP2 Fc27 1.0ug/μl 0.1ug/μl 1xPBS Add 1μl to 248μl 1xPBS Ec PfMSP3 1.0ug/μl 0.5ug/μl 2μl protein into 2μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 13.3μl PfMSP6 GST 2.3ug/μl 0.3ug/μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 0.8μl PfMSP7 GST 0.7ug/μl 0.5ug/μl 1xPBS Add 1μl to 248μl 1xPBS MSP-DBL 348 FL + 2μl protein into 18μl inhibitors 1.0ug/μl 0.1ug/μl 1xPBS Add 1μl to 248μl 1xPBS MSP-DBL 355 FL + 2μl protein into 24.7μl inhibitors 1.0ug/μl 0.075ug/μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 58μl EBA 140 R ug/μl 0.1ug/μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 48μl 3D7 EBA 175 R ug/μl 0.08ug/μl 1xPBS Add 1μl to 248μl 1xPBS 2μl protein into 48μl 3D7 EBA 175 R ug/μl 0.08ug/μl 1xPBS Add 1μl to 248μl 1xPBS PfEMP1 variant surface antigens IT4var19 CIDR α ug/μl 0.5ug/μl Merozoite antigens Circumsporozoite protein PpCSP - M3/4 0.9ug/μl 0.01ug/μl 2μl protein into 178μl 1xPBS Add 1μl to 248μl 1xPBS Maltose binding protein MBP-His6 control 1.0ug/μl 0.5ug/μl No dilution needed Add 2μl (Protein) to 248μl 1xPBS Table 4: Appendix 2a - Protein concentration and coupling volumes 51

66 Appendix 2b: Calculation The proteins first needed to be diluted in PBS before they were added to the microspheres. The following calculations were used to calculate the volume of PBS that was needed for each protein to be diluted in for 50μl microsphere. The Final volume of the coupled microspheres with their added protein is 250μl. Protein Concentration x 2 = Volume of PBS (μl) Concentration to couple Volume of PBS (μl) - 2μl Protein = Final volume of PBS 2μl Protein is added to the Final volume of PBS (1μl from this is then diluted to 249μl of PBS) 52

67 Appendix 3a: 96-well micro filter plate lay out Figure 19: Appendix 3a - Layout of 96-well micro filter plate 53