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UNIVERSITY OF GHANA 
COLLEGE OF BASIC AND APPLIED SCIENCES 
SCHOOL OF BIOLOGICAL SCIENCES 
 
 
INTERACTION OF PLASMODIUM FALCIPARUM ANTIGENS WITH BLOOD 
GROUP DETERMINANTS: PREFERENCES AND LINK TO DISEASE 
SEVERITY 
 
 
BY 
WILLIAM VAN DER PUIJE 
(10010232) 
 
 
THIS THESIS IS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, 
CELL AND MOLECULAR BIOLOGY, LEGON IN PARTIAL FULFILMENT OF 
THE REQUIREMENT FOR THE AWARD OF DOCTOR OF PHILOSOPHY IN 
MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE 
 
 
JULY 2019
 
 
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DECLARATION 
I hereby declare that this thesis, except for the references to other persons’ 
investigations which I have duly acknowledged, is the result of my own original 
research and that it has neither in whole nor in part been submitted for another degree 
elsewhere. This work was done under the supervision of the persons mentioned below. 
 
………………………………. 
William  van der Puije 
(Candidate) 
 
………………………………. 
Michael F. Ofori (Supervisor) 
Department of Immunology, Noguchi Memorial Institute for Medical Research, 
University of Ghana, Legon 
 
………………………………. 
Gordon A. Awandare (Supervisor) 
West African Centre of Cell Biology of Infectious Pathogens (WACCBIP)  
Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon 
 
………………………………. 
Trine Staalsø (Supervisor) 
Centre for Medical Parasitology (CMP), University of Copenhagen, Denmark 
 
………………………………. 
Jørgen Kurtzhals (Supervisor) 
Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark 
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ABSTRACT 
The blood type of malaria patients may determine the outcome of disease. Blood group 
O associates with protection, while non-O blood groups (A, B or AB) are associated 
with severe malaria. Rosetting of red blood cells (RBC), which is mediated by blood 
group A and other red blood cell surface molecules, is also associated with severe 
disease in the two most virulent species of Plasmodium that infect humans. The 
interaction of parasite ligands with blood group carbohydrates on RBC and endothelial 
receptors leads to infected RBC sequestration and persistence of the parasite in the 
vasculature. The objectives of this work were to study and measure the interactions 
between parasite-expressed factors on the red cell surface and blood group molecules 
using standard and novel methods, in both laboratory strains and field isolates of 
Plasmodium falciparum. Expression levels of var and rif transcripts in laboratory 
isolates selected for binding to blood group antigens were determined, while antibody 
reactivity to surface antigens were investigated in these isolates. Venous blood samples 
were collected from 113 children aged 12 years and below diagnosed with malaria and 
resident in Hohoe, a high malaria transmission zone in the Volta Region of Ghana. The 
blood type was determined for all patients, while plasma and RBCs were processed and 
stored for subsequent work. 
Long term blood group A and B binding variants of laboratory strains of P. falciparum 
3D7, FMG and FUP parasites were successfully produced by regular panning on 
immobilized blood group oligosaccharides, though the production of binding variants in 
other strains (FCR3 and HB3) were not as successful. Selected parasites were found to 
bind to both A and B blood group carbohydrates irrespective of the blood group antigen 
used in selection. The binding isolates also showed a marked adhesion to aorta and 
dermal endothelial cell (EC) lines. A novel microtiter-plate based quantitative assay to 
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specifically measure binding of parasite infected RBC to plate-bound blood group 
antigens was developed and used to determine the interaction of both laboratory and 
field strains to the blood group A and B antigens. All blood group antigen-selected 
isolates showed a strong propensity to form rosettes in the presence of RBC from 
donors of blood type A, AB or B, in contrast to unselected isogenic isolates which did 
not form rosettes under the same conditions. 
The transcription levels of 58 var genes of ring stage parasites of 3D7 and FMG 
selected on blood group antigens and detected by quantitative real-time polymerase 
chain reaction (QPCR) showed no consistent pattern of expression between selected and 
unselected parasites, though the adhesion linked genes Pf13_0003 and IT4var32b of 
3D7 and FMG, respectively, had elevated levels in some selected isolates. 
In conclusion, a novel plate-based assay to directly measure infected erythrocyte 
adhesion to blood group antigens has been developed. Selection for long term blood 
group specific parasite binding isolates was also achieved, but with no clear changes in 
variant surface antigen transcript levels. Notwithstanding, an increased rosette 
formation and adhesion to non-O blood antigens of selected parasites which suggest a 
link to severe disease was observed. It is hoped that future investigations using field 
isolates with clearer categorization of severe cases and a larger sample size would 
further validate these findings. Whole genome transcriptional studies and sequencing 
studies in these parasites may also specifically identify the genes and molecules 
involved in adhesion. Improved knowledge in all these areas will accelerate the design 
and development of targeted syndrome specific vaccines which will improve the 
management of this disease. 
  
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DEDICATION 
 
 
To 
 
Naa Aku, Nii Ankrah and Efua 
 
for your love and support through this journey 
 
 
 
  
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ACKNOWLEDGEMENTS 
During the past few years I have been blessed to have had the pleasure of meeting quite 
a few people; those I already knew, and those I met during this study who were 
involved in various aspects of the work, both professionally and on a personal level. It 
has been a long and challenging journey, and you have made it possible for me to get 
where I am today. A thousand thanks to you all, especially the brave, young and 
promising children of Hohoe and its environs who took part in this study, along with 
their parents, guardians and families. This would not have been possible without you! 
My acknowledgement goes to the West African Centre for Cell Biology of Infectious 
Pathogens (WACCBIP) at the Department of Biochemistry, Cell and Molecular Biology 
(BCMB), University of Ghana and the Centre for Medical Parasitology (CMP), 
Department of Immunology and Microbiology, ISIM, University of Copenhagen (KU), 
Denmark as well as the Department of Immunology at the Noguchi Memorial Institute 
for Medical Research (NMIMR). These institutions and their staff provided the right 
environment to meet the challenging demands of a PhD in science. Special thanks to the 
Danish International Development Agency (DANIDA), Ministry of Foreign Affairs of 
Denmark for funding my PhD on the Malaria Vaccine Research and Capacity Building 
in Africa (MAVARECA) project. 
My profound appreciation goes to my supervisors in Ghana: Professor Gordon A. 
Awandare, Director of WACCBIP and Dr Michael F. Ofori, Department of 
Immunology, NMIMR for their support and direction throughout my PhD. Thank you 
so much for your contributions and understanding, on both academic and personal 
levels. I am indebted to my other supervisors Professor Jørgen Anders Lindholm 
Kurtzhals of CMP and Clinical Microbiology Department at Rigshospitalet, and 
Associate Professor Trine Staalsø of CMP/ISIM at KU, Denmark for their invaluable 
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direction and teaching during my stay in Copenhagen. I truly appreciate the many 
Skype meetings on my return and during my write-up, for fitting me in during your 
visits to Ghana to make sure I was on track. Thank you Jørgen for being a great group 
leader; always there, always understanding and ready to make time to listen and guide. 
Your knowledge both in the lab and field setting was invaluable to this work. Trine, 
thanks for the knowledge, the many laughs, a great companion in the office and lab, and 
for showing me a broader perspective in the subject and for being a great example of 
how passionate a true scientist can be when they love what they do. 
Special thanks to Lars Hviid our project coordinator, a great scientist full of wit and 
charm. Thank you for your frank advice and keen perception in everything, not only 
during this project but from almost two decades ago when we first met. A big thanks to 
Casper Hempel, always ready to teach and help in the lab with a smile. To Kirsten (now 
at Herlev Hospital), Jens, Mette, Pilar, Mary, Regis, Anja B, Anja J, Rebecca, Yvonne 
and all at CMP, thanks! Christian Wang thanks for your guidance. Maiken Visti, big 
thanks for your support in the lab, and of course for being great company for a coffee or 
lunch on those hectic days. 
To the staff of other collaborating institutions; Dr Nick Opoku, Shelter, Joshua, Rukaya, 
nurses Dina and Vivian, project clinicians Drs Edem Wilson and Rafiq Okine all at the 
Hohoe Municipal Hospital field site, your efforts are appreciated. Our Danish project 
clinicians Filip Castberg and Jonathan Glenthøj, great job! The Danish team: Rebecca, 
Yvonne, Peter, Mette, Maiken, Kirsten and Mads who joined us at different times to 
support or troubleshoot various field challenges, thank you! 
To my MAVARECA colleagues, from the sunny, hot Hohoe to a snow storm on our 
first Monday in Copenhagen, it’s been both challenging and wonderful, in Ghana and in 
Denmark. Thank you for the experience; sample processing at 2 a.m. in Hohoe, to new 
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year parties at Øresund. Betty, Dedo and Gerty, ayekoo! Alex Danso, our able 
supporter, thank you so much for the help and sacrifices. 
Many thanks to my lovely classmates the pioneering MCBI class of WACCBIP. You 
have been an inspiration. Sena, Reuben, Steven, Dorotheah, Tina, Hetty, Ethel, 
Nicholas, Tagoe, Aboagye, Rufai and Helena of blessed memory. 
I appreciate the efforts of friends and colleagues, past and present, of the Department of 
Immunology of the NMIMR during my PhD. Special thanks to Kakra Dickson and Eric 
Kyei-Baafour for your immense help with logistics in both the laboratory and in the 
field, especially during my six-week stretch in the field. Dorothy Anum, thank you for 
your interest in my progress through all the years. To Ben, Jasmine, Belinda, thank you 
too for your help. 
Yvonne Ashong, Joseph Quartey, Joseph Okyere, Dickson Osabutey, Doctors Irene 
Ayi, Anita Ghansah, Abena Amoah, Elias Asuming-Brempong, Irene Larbi, Charles 
Quaye, Irene Owusu Donkor, Lydia Mosi, Dziedzom deSouza, and Profs Mike Wilson, 
Kwadwo Koram, Daniel Boakye and all other ‘Parasitologists’ from my pre-PhD era, 
thank you! 
Mrs Roselyn Mills, your regular transatlantic calls were so supportive during those 
trying times; I truly appreciate your genuine concern and friendship. Dr Kodjo Ayertey, 
Dr John Arko-Mensah, Kofi Adjei-Henaku and Charles Andoh, great friends after all 
these years, thank you for the encouragement! 
To my family, I am eternally grateful to you for being there for me during this study, 
especially my wife Efua who I cannot thank enough for going all out to make sure our 
family was okay considering the demands of this endeavour. I thank my parents and 
siblings for their encouragement and belief in me; I am blessed to have you. 
  
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TABLE OF CONTENTS 
DECLARATION ............................................................................................................... i 
ABSTRACT ...................................................................................................................... ii 
DEDICATION ................................................................................................................. iv 
ACKNOWLEDGEMENTS .............................................................................................. v 
TABLE OF CONTENTS ............................................................................................... viii 
LIST OF FIGURES ......................................................................................................... xi 
LIST OF TABLES ......................................................................................................... xiii 
LIST OF ABBREVIATIONS ........................................................................................ xiv 
CHAPTER ONE ............................................................................................................... 1 
1.0   INTRODUCTION .................................................................................................... 1 
1.1 Background ........................................................................................................ 1 
1.2 Justification and study objectives ....................................................................... 6 
1.2.1 Justification ................................................................................................. 6 
1.2.2 Aims of the study ........................................................................................ 7 
1.2.3 Specific objectives ...................................................................................... 8 
CHAPTER TWO .............................................................................................................. 9 
2.0   LITERATURE REVIEW ......................................................................................... 9 
2.1 The burden of malaria ........................................................................................ 9 
2.2 Malaria: the disease .......................................................................................... 11 
2.3 Malaria: the parasite ......................................................................................... 14 
2.3.1 The Life Cycle of Plasmodium Species .................................................... 15 
2.3.2 Multigene families of P. falciparum ......................................................... 17 
2.3.2.1 The var genes .................................................................................... 19 
2.3.2.2 The rif genes ...................................................................................... 21 
2.3.2.3 The stevor genes ................................................................................ 22 
2.4 The ABO blood group system.......................................................................... 23 
2.5 Immunity to malaria ......................................................................................... 25 
2.5.2 Sequestration of P. falciparum ................................................................. 27 
2.5.3 Red blood cell rosetting in malaria ........................................................... 28 
2.6 Malaria vaccines ............................................................................................... 31 
CHAPTER THREE ........................................................................................................ 34 
3.0   METHODS ............................................................................................................. 34 
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3.1 Study location, subjects and recruitment ......................................................... 34 
3.2 Blood sampling and processing ....................................................................... 36 
3.2.1 ABO blood grouping and Rhesus status ................................................... 38 
3.3 Parasite cultures ............................................................................................... 38 
3.3.1 Freezing and thawing of parasites ............................................................ 40 
3.3.1.1 Freezing P. falciparum-containing blood samples ............................ 40 
3.3.1.2 Thawing P. falciparum-containing blood samples ............................ 40 
3.4 Detection of rosetting rates in selected parasites and field isolates ................. 41 
3.5 Blood group polysaccharide constructs ........................................................... 42 
3.6 Selection for binding to blood group constructs .............................................. 42 
3.7 Blood group antigen and EC binding assays.................................................... 44 
3.7.1 ABO polysaccharide antigen binding assay ............................................. 44 
3.7.2 Endothelial cell binding assays ................................................................. 45 
3.7.3 Blocking assay using anti-A and anti-B monoclonal antibodies .............. 46 
3.8 MACS and Flow Cytometry ............................................................................ 47 
3.8.1 Harvesting late stage P. falciparum by Magnetic-activated cell sorting .. 47 
3.8.2 Measurement of antibody reactivity to selected parasite VSA by Flow 
Cytometry ................................................................................................................ 48 
3.9 Molecular methods ........................................................................................... 49 
3.9.1 Sample processing and storage ................................................................. 49 
3.9.2 RNA extraction from Trizol-stored samples ............................................ 49 
3.9.3 DNase treatment and cDNA synthesis ...................................................... 50 
3.9.4 Quantitative Polymerase Chain Reaction to detect VSA transcripts of var 
and rif genes ............................................................................................................ 51 
3.9.5 Statistical Analysis .................................................................................... 52 
CHAPTER FOUR ........................................................................................................... 53 
4.0   RESULTS ............................................................................................................... 53 
4.1 Rosetting in parasites selected on blood group antigens .................................. 56 
4.2 Adhesion of selected parasites to blood group oligosaccharides ..................... 57 
4.2.1 Characteristics of binding isolates ............................................................ 61 
4.2.2 Binding of selected parasites to primary human ECs ............................... 66 
4.2.3 Comparison of adhesion and rosetting in selected parasites ..................... 67 
4.3 PfEMP1 and RIFIN expression patterns in selected P. falciparum laboratory 
parasite strains ............................................................................................................. 69 
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4.3.1 QPCR var and rif transcripts in 3D7 selected and unselected parasites ... 69 
4.3.2 QPCR var transcripts in FMG selected and unselected parasites ............. 81 
4.4 Fieldwork: Background and Demographics ..................................................... 84 
4.5 Plate-based ABO haem-release binding assays in clinical isolates ................. 85 
4.5.1 Relative adhesion of selected 3D7 parasites during experiments ............. 85 
4.5.2 Relative adhesion of field isolates and blood type of donor ..................... 87 
4.5.3 Summary of binding characteristics of field isolates ................................ 88 
4.6 Rosetting in field isolates ................................................................................. 89 
4.6.1 Rosetting and blood groups ...................................................................... 89 
4.6.2 Rosetting and Age ..................................................................................... 91 
4.6.3 Rosetting and Parasitaemia ....................................................................... 93 
4.7 Antibody recognition profiles of patient sera to selected parasites.................. 96 
CHAPTER 5 ................................................................................................................... 99 
5.0   DISCUSSION ......................................................................................................... 99 
5.1 Conclusions and recommendations ................................................................... 113 
5.1.1 Conclusions ............................................................................................. 113 
5.1.2 Recommendations ................................................................................... 114 
REFERENCES ............................................................................................................. 115 
 
 
  
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LIST OF FIGURES 
Figure 2.1 Actual and predicted parasite rates based on interventions ....................... 10 
Figure 2.2 Pathogenesis of severe malaria .................................................................. 13 
Figure 2.3 The life cycle of P. falciparum in the human and Anopheles mosquito 
vector ......................................................................................................... 17 
Figure 2.4 The domain structure of PfEMP1. ............................................................. 21 
Figure 3.1 The map of Ghana showing the field study site. ....................................... 35 
Figure 4.1a Dose dependent binding of blood group O specific Ulex europaeus 
agglutinin (UEA) to blood group H antigen construct .............................. 55 
Figure 4.1b Detection of immobilized A-BSA construct with Dolichos biflorus 
(DBA) lectin .............................................................................................. 55 
Figure 4.2 A large rosette containing IEs surrounded by several RBC ...................... 56 
Figure 4.3 Rosetting rates of selected parasites in RBC of blood group O, A and 
AB .............................................................................................................. 57 
Figure 4.4 Optical density readings of bound RBC in plate-based assay. .................. 58 
Figure 4.5 Reactivity of mouse monoclonal anti blood group A and B antibody 
with A-BSA and B-BSA constructs by ELISA ......................................... 59 
Figure 4.6 Inhibition of blood group B selected parasite adhesion to constructs by 
specific anti A- and B antibody ................................................................. 60 
Figure 4.7 Weak binding to the H-BSA (blood group O) construct by all parasites .. 62 
Figure 4.8 A-BSA and B-BSA selected parasites of 3D7 and FMG bind both the 3- 
and 6-spacer arm constructs of blood group B. Selected parasites ........... 63 
Figure 4.9 Percentage adhesion of selected and unselected 3D7, HB3, FCR3, FMG 
and FUP parasites. ..................................................................................... 64 
Figure 4.10 Phenotype stability over a 16-week period................................................ 65 
Figure 4.11 Adhesion of A and B selected parasites to human Aorta and Dermal 
endothelial cells (ECs) ............................................................................... 66 
Figure 4.12 Adhesion and rosetting characteristics of selected and unselected 3D7, 
FMG and FUP parasite strains................................................................... 68 
Figure 4.13 Transcription levels of var genes in blood group antigen-selected and 
unselected 3D7 parasites. .......................................................................... 71 
Figure 4.14 The rif transcripts of selected and unselected parasites. ............................ 76 
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Figure 4.15 Fold change of the top 10 var transcripts in the FMG A- and B-BSA 
selected parasites ....................................................................................... 83 
Figure 4.16 Relative adhesion of 3D7 parasites to blood group A antigen in plate 
based assays ............................................................................................... 86 
Figure 4.17 Relative adhesion of non-group O and group O patient parasite isolates 
to blood group A antigen ........................................................................... 87 
Figure 4.18 Binding of field isolates to the blood group A, B and O (H) constructs 
in 39 children 10 years and below ............................................................. 88 
Figure 4.19 Rosetting rates in blood group O versus non-group O malaria patients .... 91 
Figure 4.20 Correlation between rosetting and age in blood groups. ........................... 92 
Figure 4.21 Association between parasitaemia and rosetting isolates in 0-5, and 5 -
12 year groups ........................................................................................... 94 
Figure 4.22 Rosetting rates in two age groups of 0-5 and 5-12 years........................... 95 
Figure 4.23 Mean Fluorescence Intensity of bound antibody from donor plasma ....... 97 
Figure 4.24 Difference in mean fluorescence intensity between selected and 
unselected 3D7, FMG and HB3 parasites ................................................. 98 
  
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LIST OF TABLES 
Table 4.1 Lectins and corresponding specific antigen/carbohydrates ...................... 54 
Table 4.2 Transcription levels of 58 var genes of 3D7, in 3D7 cultures .................. 72 
Table 4.3 Transcription of the 154 rif genes of 3D7, in five 3D7 cultures ............... 77 
Table 4.4 Transcription of 56 var genes from the IT4/FCR3/FMG genome ............ 82 
Table 4.5 Characteristics of patients recruited for the study .................................... 84 
Table 4.6 Characteristics of field isolates in the different blood groups .................. 90 
 
 
  
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LIST OF ABBREVIATIONS 
ARDS  Acute respiratory distress syndrome 
ATS  Acidic terminal segment 
BD   Becton Dickinson  
BSA   Bovine serum albumin  
CD   Cluster of differentiation 
cDNA  Complementary deoxyribonucleic acid 
CIDR  Cysteine-rich interdomain region 
CM  Cerebral malaria 
CSA  Chondroitin sulphate A 
DBL  Duffy-binding like  
DC  Domain cassette 
DEPC  Diethylpyrocarbonate 
EC  Endothelial cell 
EDTA  Ethylenediaminetetracetic acid 
ELISA  Enzyme-linked immunosorbent assay 
EPCR  Endothelial protein C receptor 
FACS  Fluorescence –activated cell sorting 
FCS  Fetal calf serum 
FITC  Fluorescein isothiocyanate 
H2SO4  Sulphuric acid 
Hb  Haemoglobin 
HRP  Horseradish peroxidase 
ICAM-1  Inter cellular adhesion molecule 1 
IE  Infected erythrocyte 
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Ig  Immunoglobulin 
IgG  Immunoglobulin G 
IgM  Immunoglobulin M 
MACS  Magnetic-activated cell sorting 
MFI  Mean fluorescence intensity 
NGP  Neoglycoprotein 
OD  Optical density 
PBS  Phosphate buffered saline 
PBS-2  Phosphate buffered saline with 2% FCS 
PCR  Polymerase chain reaction 
PECAM-1  Platelet endothelial cell adhesion molecule 1 
PfEMP1  Plasmodium falciparum erythrocyte membrane protein 1 
QPCR  Quantitative real-time polymerase chain reaction 
RBC  Red blood cell 
rif/RIFIN  Repetitive interspersed family 
RNA  Ribonucleic acid 
SCD  Sickle cell disease 
SMA  Severe malarial anaemia 
STEVOR  Subtelomeric variant open reading frame 
TMB  Tetramethylbenzidine 
VSA  Variant surface antigen 
vWF  von Willebrand Factor 
WHO  World Health Organization 
 
 
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CHAPTER ONE 
1.0   INTRODUCTION 
1.1 Background 
Malaria is a major cause of morbidity and mortality, especially in Sub-Saharan Africa. 
Figures from the World Health Organization’s 2017 World Malaria Report estimates 
that in 2016, there were 216 million cases worldwide, up 5 million from 2015. This 
increase is in contrast to the continuous decline in deaths and cases observed over the 
last decade-and-a-half or so (WHO, 2017). 
The disease manifests in several disease forms, and severe forms are defined by clinical 
or laboratory evidence of vital organ dysfunction. These manifestations can occur singly 
or, more commonly, in combination in the same patient (WHO, 2012). Clinical features 
range from impaired consciousness, prostration, convulsions and respiratory distress to 
acute kidney injury, metabolic acidosis, hypoglycaemia and renal impairment among 
others (WHO, 2012). 
Cerebral malaria (CM), the deadliest form of severe malaria, together with severe 
malarial anaemia (SMA) account for most of the deaths due to the disease (Murphy and 
Breman, 2001). Unfortunately, as many as 60% of deaths due to CM are either 
misdiagnosed or get a delayed diagnosis (Maro and McLarty, 1986). Natural immunity 
to malaria and protection from severe disease is based on the production of protective 
antibodies to parasite antigens expressed on the surface of IE during the erythrocytic 
stage of infection (Chan et al.et al., 2014, Chan et al., 2012, Marsh et al., 1989). These 
variant surface antigens (VSAs) mediate adhesion to the endothelial membranes, 
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leading to sequestration of the parasites and avoidance of splenic clearance. In the brain, 
this contributes to the pathogenesis of CM (Yusuf et al., 2017, Storm and Craig, 2014). 
Another of the several life threatening complications of falciparum malaria is SMA, the 
most common syndrome of severe disease, and a major clinical issue in malaria 
endemic settings (Lamikanra et al., 2007, Weatherall et al., 2002). Defined as 
haemoglobin concentrations of less than 5g/dL, a haematocrit less than 15% and 
parasitaemia greater than 10,000/µL, severe anaemia contributes up to 46% of child 
fatalities in some developing nations (English et al., 2004). Infants and pregnant women 
are most at risk for severe anaemia due to malaria, with the risk in infants peaking at 1-2 
years of age, depending on the transmission intensity in the area. Prevalence rates of up 
to 91% in children and 60% to 80% in pregnant women have been reported 
(Schellenberg et al., 2003, Menendez et al., 2000). SMA is marked by the significant 
loss of predominantly uninfected RBCs through relatively little understood processes, 
but known to be influenced by the formation of rosettes, as well as by increased 
phagocytic clearance (Uyoga et al., 2012). There is also increased haemolysis of both 
infected and non-infected RBCs, and an abnormal development of erythroid precursors 
(Abdalla et al., 1980, Casals-Pascual et al., 2006). 
Blood groups are complex, inherited chemical systems on blood cell surfaces (and on 
other cells and body tissues) that determine blood type based on the presence or absence 
of specific antigens (Kanchan and Krishan, 2016). Currently, there are 35 blood group 
systems made up of almost 350 of these antigens (Storry et al., 2014, Lane et al., 2016), 
which may be proteins, carbohydrates, glycoproteins, or glycolipids. 
The A and B antigens are both derived from the H determinant precursor to which N-
acetylgalactosamine and D-galactose, respectively, are added by glycosyltransferases, 
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encoded by A and B alleles (Franchini and Lippi, 2015). Type O individuals encode an 
inactive enzyme that makes no modification to the H determinant, which is their blood 
group substance. Individuals with different blood types of the ABO group have been 
known to differ in their general resistance and susceptibility to some diseases (Franchini 
and Bonfanti, 2015).  
In malaria, the relationship between ABO blood group and disease was first noted in 
1967 (Athreya and Coriell, 1967), and subsequently there have been arguments to 
support the idea that the disease has over the millennia, influenced the distribution of 
these blood groups across the globe (Cserti and Dzik, 2007, Goel et al., 2015, Achur et 
al., 2008). This distribution indicates a survival advantage for group O compared to 
others, as the percentage of this group is higher in all malaria endemic areas (up to 
90%), which is consistent with selective pressure from the disease (Cserti and Dzik, 
2007). 
Several clinical studies of severe malaria over the years reflect this advantage of group 
O over other blood types, with significantly more severe outcomes in the non-O blood 
groups (Fischer and Boone, 1998, Lell et al., 1999, Pathirana et al., 2005, Tadesse and 
Tadesse, 2013, Rout et al., 2012, Panda et al., 2011, Tekeste and Petros, 2010). 
Notably, a meta-analysis of ABO phenotypes and malaria risk in non-pregnant subjects 
however showed no conclusive evidence associating the blood group phenotypes with 
the risk of uncomplicated malaria (Loscertales et al., 2007). 
The var genes of P. falciparum encode a group of VSAs referred to as P. falciparum 
erythrocyte membrane protein-1 (PfEMP1) antigens. These are expressed on the RBC 
surface and were identified as mediators of rosetting (Chen et al., 1998a, Rowe et al., 
1997). It is also known that blood group phenotypes modulate P. falciparum rosetting, 
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and subsequently severe malaria (Vigan-Womas et al., 2012). However, the removal or 
absence of PfEMP1 from IE affected (reduced) rosetting only in blood group O, but not 
in group A, eliminating PfEMP1 as the only molecule responsible for RBC binding and 
rosette formation (Goel et al., 2015). 
Recent research work has identified the repetitive interspersed family of proteins 
(RIFINs) as the main molecules involved in non-group O (or PfEMP1 independent) 
rosetting (Goel et al., 2015). RIFINs are encoded by some 150-200 repetitive 
interspersed family (rif) genes of P. falciparum, making them the largest known family 
of variable IE surface-expressed proteins. They are naturally immunogenic (Abdel-Latif 
et al., 2002), and a subgroup (A- RIFINs) are exported to the IE surface (Petter et al., 
2007). 
Only one study so far (Goel et al., 2015) indicates that RIFINs have a preference for 
binding blood group A antigens on RBC and endothelial cells (ECs), and have been 
implicated in the pathogenesis of severe P. falciparum malaria via rosetting and 
adhesion to the vascular endothelium. 
Initially described as ‘rosettins’ several years ago (Helmby et al., 1993), the functions 
of RIFINs were still unknown, though they are resistant to enzymatic degradation and 
known to be up regulated in rosetting parasites (Fernandez et al., 1999, Kyes et al., 
1999). Rosette formation has been linked to severe disease, including SMA (Doumbo et 
al., 2009, Rowe et al., 1995). Being the most commonly observed syndrome of disease 
severity (English et al., 2004), the identification of RIFINs as mediators in rosetting is a 
positive step towards better understanding of SMA. 
In early studies, a strong association between rosette formation and the ABO blood 
group was established; rosetting was found to be highest in group A or AB, intermediate 
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in B and least in group O RBCs (Carlson and Wahlgren, 1992, Udomsangpetch et al., 
1993, Rowe et al., 2007). Subsequently, all clinical forms of severe disease were found 
to have significantly higher rosetting rates (Doumbo et al., 2009), indicating the 
possibility of targeting the disruption of rosettes as interventions for severe disease. 
In P. vivax infections, rosetting is frequent and may be linked to the development of 
anaemia, as the amount of rosetting was higher in anemic individuals (Marin-Menendez 
et al., 2013). 
This study sought to determine, in patients in a malaria endemic setting, differences in 
the interaction of parasite adhesion factors on IEs in the ABO blood groups, and their 
link to severe disease. The molecular basis of this was investigated by analysis of real 
time PCR data from parasites selected for preferences to these blood group antigens. 
The study in addition, aimed to develop an assay to reliably detect and measure binding 
between parasites in IE and immobilized blood group antigens in vitro. 
  
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1.2 Justification and study objectives 
1.2.1 Justification 
The interactions between parasite IE surface molecules and the ABO blood group 
antigens have far-reaching implications for therapy and vaccine development in malaria. 
Though advances continue to be made in drug interventions and vector control that have 
led to significant reduction in the incidence and mortality rates of malaria over the past 
two decades, additional tools for accessing these interactions, and ultimately a blood 
group specific RBC rosette blocking therapy or vaccine (targeting/reducing severe 
disease) would significantly buttress current efforts to combat malaria where it is most 
needed. There have been warnings against complacency (Gulland, 2015) following a 
UNICEF/WHO publication of the reduction (by 50%) in malaria deaths since 2000, and 
reinforced by the fact that a subsequent report indicated an end to the downward trend 
of global malaria cases (WHO, 2017). Additionally, there is still a limit to our 
knowledge of the molecular basis and interactions between parasite phenotypes, their 
proteins that interact with blood group antigens and how all this affects the clinical 
outcome of an infection. There is therefore the need, based on previously reported 
studies, to attempt to further understand some of these interactions and outcomes. The 
interaction of the infected erythrocyte with host receptors and tissues has over the years 
focused on the variant surface antigens of the parasite, particularly PfEMP1. More 
recently, and with the publication of the full parasite genome sequence of P. falciparum, 
the role and significance of some other groups of these antigens have been elucidated, 
offering a better understanding of host-parasite dynamics and the relationship to disease 
states and approaches to therapy. 
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1.2.2 Aims of the study 
This study was aimed at going a step further in studying the interaction of parasite 
infected RBCs with the ABO blood group antigens in particular, by developing a 
system that is able to specifically measure these interactions, both in the laboratory 
setting and for use in the field. In addressing these challenges, the study also sought to 
utilize and adapt existing laboratory isolates for use in this system and to further 
determine if these changes in phenotype are reflected in transcription of VSA genes 
previously implicated in blood group specific rosetting. 
This would hopefully address hitherto unanswered questions relating to human blood 
group antigen interactions with this complicated parasite, while providing an alternative 
way to study particular aspects of the disease. It will also provide, in the long term, 
further insights to improved therapy and vaccine development approaches that take into 
account blood group specific differences in the target population. The achievement of 
all these was based on meeting the objectives of the study listed below. 
 
  
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1.2.3 Specific objectives 
 To develop a reliable, quantitative in vitro plate-based assay that directly 
measures the ability of P. falciparum-infected erythrocytes to bind human 
blood group A and B determinants 
 
 To produce and phenotypically characterize in vitro adapted and genetically 
characterized strains of P. falciparum with high binding affinity for blood 
group A and blood group B antigens 
Hypothesis: based on interaction with different host cell surface molecules 
including blood group antigens, it should be possible to select laboratory 
strains of P. falciparum for binding to blood group antigens in vitro. 
 
 To investigate the expression of 2 major VSA gene families (var and rif) 
implicated in the virulence-linked rosetting phenotype through binding to 
blood group A and B antigens 
Hypothesis: preferential binding by IE to non-O blood type, which is linked 
to severity of disease, will mirror differences in the VSA gene families that 
mediate adhesion. 
 
 To investigate the direct binding of field isolates to ABO antigens and test 
antibody reactivity of patient sera to VSA in parasites selected on blood group 
antigens 
Hypothesis: There will be field isolates that bind particular blood group 
antigens, while sera from infected/exposed individuals will differentially 
recognize parasites selected for binding to different blood group antigens. 
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CHAPTER TWO 
2.0   LITERATURE REVIEW 
2.1 The burden of malaria 
The WHO’s 2017 World Malaria Report indicates that there were 216 million cases of 
the disease in 2016, up from the 211 million cases reported in 2015 (WHO, 2017), and 
ending the decade and a half long trend of declining cases since 2000. The 2000-2015 
decline of 60% represented averting (preventing) the deaths of approximately 6.2 
million lives that would have been lost to malaria during the period (WHO/UNICEF, 
2015a), in addition to an estimated 663 million averted clinical cases (Bhatt et al., 
2015). The success chalked in this era was due to the massive expansion in the delivery 
of core malaria interventions: the distribution of a billion insecticide treated nets (ITNs) 
in Africa, the introduction of rapid diagnostic tests (RDTs) and the use of artemisinin-
based combination therapies (ACT) in treatment (WHO/UNICEF, 2015a). The 
depiction in Figure 2.1 shows how ITNs represented the most widespread intervention, 
as well as the largest contributor (68%) of averted cases (Bhatt et al., 2015). 
Currently, there are 91 malaria endemic countries, with the top 15 carrying 80% of the 
global burden, and with a vast majority of cases (90%) in the WHO Africa Region, 
which also carries 91% of malaria deaths (WHO, 2017). In the African sub region, 
malaria due to P. falciparum accounted for 99% of reported cases in 2016, and was 
responsible for 91% of the 445,000 malaria deaths recorded globally (WHO, 2017). 
In the 2000 to 2015 period, falciparum malaria prevalence in endemic Africa was 
halved, while clinical disease incidence was reduced by 40% (Bhatt et al., 2015). Total 
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estimated deaths due to malaria were similar for the most recent years, with 446,000 
and 445,000 for 2015 and 2016 respectively (WHO, 2017). 
 
 
Figure 2.1 Actual and predicted parasite rates based on interventions. Geostatistical 
model-derived predictions (time series of population-weighted mean) of P. 
falciparum parasite rate (PfPR) in endemic Africa. The actual (red line) and 
predicted (black line) scenario with no ITNs, ACTs or IRS intervention. Coloured 
areas show relative contribution of each intervention in reducing parasite rate. 
From Bhatt et al. (2015) 
 
Since 2000, over a 15-year period, the children under 5-years global malaria death rate 
has also fallen by 65 per cent (WHO/UNICEF, 2015a). Though these deaths were 
almost halved in the 2010 to 2016 period, malaria continues to be a major killer for this 
age group in high transmission locations, with a child succumbing to malaria every two 
minutes (WHO, 2018a).  
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With declining investments in malaria control since 2014, raising the US$ 6.5 billion 
that will be needed per year by 2020 in order to meet WHO global malaria strategy 
targets by 2030 will present its own challenges. This figure is far more than what is 
currently available, and more than twice the US$ 2.7 billion spent in 2016 (WHO, 
2017). 
These challenges remain, but on the positive side more countries are headed towards 
malaria elimination. In 2010, 37 countries reported less than 10,000 cases of malaria, 
the number increasing to 44 countries in 2016. Additionally, the WHO has in 2016 
identified 21 countries that could potentially eliminate malaria by the year 2020 (WHO, 
2017). 
 
2.2 Malaria: the disease 
 
Malaria in humans is a disease caused by five species of the protozoan parasite of the 
genus Plasmodium and transmitted by the female Anopheles mosquito. Plasmodium 
falciparum and P. vivax are responsible for the majority of cases and severe forms of 
the disease, while P. ovale and P. malariae are less virulent and infect fewer people 
(White et al., 2014, Antinori et al., 2012). More recently a fifth species of simian origin, 
P. knowlesi, has become a significant cause of severe human malaria in South East Asia 
(White, 2008, Cox-Singh et al., 2008). 
Most infections with malaria take a mild course, mainly with flu-like symptoms 
including fever, chills, headache, fatigue, nausea, vomiting and muscle pain. However, 
a significant proportion of patients, mainly children under the age of five years and 
pregnant women, end up with complications leading to severe disease and fatal 
outcomes (WHO, 2000a). The observed clinical manifestation of infection with 
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Plasmodium is the result of responses to the replicating erythrocytic stages of the 
parasites. 
In uncomplicated malaria, the main presentation is fever and mild anaemia, sometimes 
with an enlarged liver in children, or mild jaundice in adults. Severe falciparum malaria 
on the other hand may present with additional symptoms such as coma resulting from 
CM and acidosis irrespective of age, though SMA and hypoglycaemia are more 
common in children. Figure 2.2 sums up the various facets of pathogenesis of severe 
disease. In adults, the severe disease symptoms more commonly observed are 
pulmonary oedema, acute kidney injury and jaundice (White et al., 2014). The majority 
of deaths from severe disease are attributed to CM and SMA (Murphy and Breman, 
2001). Late stages of the parasite (trophozoites or schizonts) are known to sequester in 
cerebral vessels of CM patients, adhering via surface knobs on IEs (MacPherson et al., 
1985). 
  
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Figure 2.2 Pathogenesis of severe malaria. Various factors including host response to 
infection as well as parasite phenotypes like rosetting and cytoadherence lead to 
microvascular obstruction, metabolic acidosis and severe anaemia that culminate 
in CM and other forms of severe disease. ARDS, acute respiratory distress 
syndrome. From Fairhurst and Wellems (2015) 
In Ghana, though some progress has been made over the years in controlling the 
disease, malaria is still a leading cause of morbidity and mortality, and is also to blame 
for poverty and low productivity in many areas. In the 2005 to 2015 period, outpatient 
cases due to malaria in public health facilities reduced by 57%, along with a significant 
65% fall in deaths due to malaria. In particular, the number of deaths in children under 5 
years of age decreased by 70% in the same period (Aregawi et al., 2017). In line with 
global objectives, Ghana is targeting 100% of households owning at least one ITN by 
2020, and 85% of children under 5 years and pregnant women sleeping under bed nets 
(https://www.unicef.org/ghana). At the current rate however, the target of 40% 
reduction in malaria cases and death rates by 2030 will be a challenge to achieve, 
though interventions such as the intermittent preventive treatment in pregnant women 
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with sulfadoxine-pyrimethamine, IPTp-SP, have reduced the infection in this group by 
up to 57% over a 6-year period in parts of the country (Hommerich et al., 2007).  
The main cause of malaria in Ghana is P. falciparum, and to a much lower extent P. 
malariae and P. ovale (Owusu et al., 2017, Kweku et al., 2008). The mosquito vectors 
known to transmit the disease are Anopheles gambiae (sensu stricto), which transmits 
malaria across all the three ecological zones of the country, whereas transmission by A. 
melas, A. arabiensis and A. funestus is limited to some ecological zones (Duah et al., 
2016) 
 
2.3 Malaria: the parasite 
 
Plasmodium parasites are the causative agents of malaria. Transmitted through the bite 
of female Anopheles mosquitoes, this parasite has evolved with humans for millennia, 
predating the development of modern humans (Cserti and Dzik, 2007). These 
protozoans belong to the phylum Apicomplexa, which includes several obligate 
intracellular parasites. There are more than 100 species of Plasmodium, infecting a 
variety of animals including mammals, birds and reptiles (CDC, 2018). Within the 
highly diverse and successful Plasmodium genus, there are 5 species that can infect 
humans, namely, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium 
malariae, Plasmodium ovale and Plasmodium vivax (Keeling and Rayner, 2015). 
P. falciparum is the most virulent of these, causing the most severe forms of the disease 
and a majority of related deaths, quickly turning fatal if not promptly recognized. P. 
vivax and P. knowlesi can also cause severe disease (Cox-Singh et al., 2008, Kantele 
and Jokiranta, 2011), though these are not as common as in P. falciparum. P. knowlesi 
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has however emerged as an important cause of human malaria (Singh et al., 2004), and 
in parts of Southeast Asia is now the most common cause of the disease (William et al., 
2013, Yusof et al., 2014). More recently there are calls to acknowledge the importance 
of knowlesi malaria in the WHO’s World Malaria Report so as to increase identification 
and improve monitoring of the parasite (Barber et al., 2017). 
 
2.3.1 The Life Cycle of Plasmodium Species 
 
The life cycle of Plasmodium species is complicated, consisting of an exogenous sexual 
phase (sporogony) and an endogenous asexual phase (schizogony) that occur 
respectively in the female anopheles mosquito and vertebrate hosts. In humans (Figure 
2.3), infection begins with the injection of sporozoites, along with the saliva of an 
infected mosquito during its blood meal, into the dermis of the skin. Sporozoites enter 
the circulation and are quickly transported to the liver where they migrate through 
several hepatocytes, finally settling into one, within the parasitophorous vacuole of the 
parasite (Mota et al., 2002). Here, it differentiates and multiplies several times 
mitotically, up to 40,000 merozoites per hepatocyte (Cowman et al., 2016), which are 
released into the blood stream via vesicles (merosomes) that bud from the hepatocyte 
(Sturm et al., 2006). The length of this pre-erythrocytic stage is species dependent, 
ranging from between 5-7 days in P. falciparum up to 14-16 days in P. malariae 
(Antinori et al., 2012). The release of merozoites from hepatocytes marks the end of the 
pre-erythrocytic stage and the beginning of the asexual erythrocytic phase (blood stage) 
of the life cycle. 
Free merozoites very quickly invade red blood cells, the process taking only 1-2 
minutes to complete (Paul et al., 2015). Successful merozoite invasion of RBC is 
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dependent on the recognition, attachment and entry into the erythrocyte. Initial contact 
may occur anywhere on the merozoite surface, followed by re-orientation of the parasite 
to enable alignment of the apical end to the entry point on the RBC membrane to form a 
tight junction (Cowman and Crabb, 2006). This tight junction, powered by the actin-
myosin motor of the parasite, moves from the apical to the posterior end of the 
merozoite, pushing the parasite into the RBC cytoplasm. At the same time, this action 
forms a parasitophorous vacuole around the merozoite (Cowman and Crabb, 2006, 
Keeley and Soldati, 2004). 
Within the RBCs, the early ring stages mature through trophozoites into late schizont 
forms over a 48-hour period. RBCs with these late stages eventually rupture to release 
between 16 and 36 merozoites per RBC, producing a new wave of the asexual blood 
stage cycle (Cowman et al., 2016, Antinori et al., 2012). All clinical symptoms 
attributed to malaria manifest during this stage of the disease. 
During the erythrocytic cycle, some merozoites develop into male and female 
gametocytes, which are sexual forms of the parasite capable of being picked up by the 
vector during feeding. Gametocytes sequester and develop in the bone marrow, 
reappearing in the peripheral circulation when mature (Cowman et al., 2016). 
In the mosquito gut, gametocytes develop into gametes and fuse to form zygotes. The 
zygote develops into an ookinete and then an oocyst in the mosquito gut. The mature 
oocyst then bursts to release sporozoites that migrate to the salivary glands of the 
mosquito to complete the cycle during the next blood meal. 
 
 
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Figure 2.3 The life cycle of P. falciparum in the human and Anopheles mosquito vector. 
Sporozoites in saliva are injected into the skin at bite site (A), migrate to the liver 
(B), and invade hepatocytes. Here, they multiply and burst out of the liver cells 
and invade RBCs, marking the beginning of the asexual cycle (C) of merozoite 
growth and maturation to trophozoites and schizonts. Some parasites form 
gametocytes (sexual forms) that are taken up by the vector during a blood meal 
(D). In the mosquito gut they mature into gametes and reproduce sexually 
resulting in an ookinete, eventually resulting in an oocyst full of sporozoites, 
which burst out and migrate to the salivary glands. White boxes show 
approximate number of parasites for that stage. From White et al. (2014). 
 
 
2.3.2 Multigene families of P. falciparum 
During the asexual stages of malaria, P. falciparum modifies the infected erythrocyte by 
inserting highly polymorphic variant surface antigens (VSAs) that undergo clonal 
antigenic variation (Roberts et al., 1992, Brannan et al., 1994) in the membrane. The 
parasite has 14 chromosomes with a haploid nuclear genome of about 23 Mb. The genes 
that code for the variant antigens associated with the IE membrane are mostly located in 
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the subtelomeric regions of the chromosomes (Gardner et al., 2002), with the 3 main 
variant gene families; the var family of genes which encode the P. falciparum 
erythrocyte membrane protein 1 (PfEMP1), the repetitive interspersed family, rif, that 
code for RIFINs, and the subtelomeric variant open reading frame (stevor) genes that 
code for STEVOR proteins, all located in subtelomeric block 4 of the five unique 
subtelomeric blocks. This region contains all the stevor genes, 36 of the 59 var genes 
and 149 of the 159 rif genes (Rasti et al., 2004). Apart from the three mentioned before, 
there are other lesser studied VSAs such as the SURFIN protein family, which are 
encoded by a group of 10 surface-associated interspersed genes (surf genes) and have 
structural and sequence similarities with other exported surface antigens (Winter et al., 
2005). 
These VSAs serve an important role by facilitating parasite cytoadhesion to vascular 
ECs of the host, thereby avoiding clearance in the spleen and contributing to the 
pathogenesis of malaria (Berendt et al., 1994). They also serve as an immune evasion 
strategy due to the high turnover in protein sequence, making it difficult for the host 
immune system to keep up with the changing antigens presented. The immune response 
to the blood stage is primarily targeted at VSAs (Staalsoe et al., 2003), and antibody 
reactivity to these has been consistently linked to disease severity and conditions, and 
also to protection against clinical malaria (Chan et al., 2014, Chan et al., 2012, Marsh et 
al., 1989). For example, VSA antibody levels were found to correlate positively with 
the age of healthy children, but negatively with that of malaria patients (Ofori et al., 
2002). Similarly, fewer VSAs were recognized by sera from children with SMA 
compared with sera from children with CM, while sera from those with severe disease 
were generally less reactive to several of the RIFIN and STEVOR VSA families 
(Travassos et al., 2018). 
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2.3.2.1 The var genes 
The var family of genes, constituting a family of about 60 copies per genome, encode a 
group of VSAs termed P. falciparum erythrocyte membrane protein-1 (PfEMP1). These 
large molecules (250-350kDa), exported to and expressed on knobs on the surface of IE 
(Oh et al., 2000), are the best characterized member of the VSA, and known to mediate 
cytoadhesion to endothelial receptors of the host (Baruch et al., 1995, Gardner et al., 
2002, Su et al., 1995, Treutiger et al., 1997, Newbold et al., 1997b). As well as 
mediating sequestration, clonal antigenic variation within this family of genes enables 
the parasite evade host antibody responses (Kyes et al., 2001). The parasite also exhibits 
allelic exclusion, which means that usually only one of these var genes is expressed at a 
time (Chen et al., 1998b, Scherf et al., 1998), enhancing its host immune evasion 
capabilities. The subtelomeric location means some var genes are more vulnerable to 
recombination, while centrally located var genes are relatively conserved, though 
recombination can affect their stability (Gardner et al., 2002).  
Structurally, PfEMP1 molecules are composed of several distinct domain structures. 
The extracellular component is made up of an N-terminal segment (NTS) which is 
variable, followed by several Duffy-binding-like (DBL) domains and cysteine-rich 
inter-domain regions (CIDRs). The more conserved intracellular acidic terminal 
segment (ATS) is the C-terminal component that may play a role in anchoring the 
molecule to erythrocyte skeletal proteins (Oh et al., 2000), and is linked to the 
extracellular component via a transmembrane domain (Rasti et al., 2004), shown in 
Figure 2.4. There are 4 main groups (A, B, C, and E) of PfEMP1 based on the var gene 
sequence that encodes the proteins (Smith, 2014).  There are variants of PfEMP1 
classified based on the domain cassette (DC) system consisting of certain DBL and 
CIDR domain structure combinations. Parasites with specific DCs (DC8, DC13) are 
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associated with binding to the vascular endothelium of the brain and thus to severe 
malaria in children (Claessens et al., 2012, Avril et al., 2012, Lavstsen et al., 2012). 
As the primary parasite protein mediating cytoadhesion (Pasternak and Dzikowski, 
2009), PfEMP1 binds to a variety of host endothelial receptors including cluster of 
differentiation 36 (CD36), chondroitin sulphate A (CSA), PECAM-1, inter cellular 
adhesion molecule 1 (ICAM-1) and endothelial protein C receptor (EPCR), the latter 
two of which are expressed on brain microvascular ECs. Figure 2.4 shows a schematic 
depiction of the domain structure of PfEMP1 showing the group structure and some of 
the corresponding host receptors mentioned. 
EPCR is a host receptor important in the cytoprotective protein C pathway for 
regulating blood clotting and for endothelial barrier integrity (Bouwens et al., 2013). 
The binding of PfEMP1 may interfere with this pathway, resulting in blood-brain 
barrier dysfunction and pathological damage of the endothelium (Moxon et al., 2013). 
However, the ultimate outcome of disease could well depend on the specific domain 
compositions of the PfEMP1 variant in question (Smith, 2014). 
 
 
  
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Figure 2.4 The domain structure of PfEMP1. Structurally, PfEMP1 consists of a 
hypervariable, extracellular N‐terminal head structure and 2 to 6 subsequent 
domains of Duffy binding like (DBL) or cysteine-rich interdomain region 
(CIDR), a transmembrane domain (TM) and an intracellular acidic terminal 
segment (ATS). Receptors are indicated in bold. Figure reproduced from 
Storm et al. (2019) 
 
 
2.3.2.2 The rif genes 
With 160 copies per haploid genome (Zhou et al., 2019), RIFINs are the largest and 
most diverse multigene family in P. falciparum, but have not been as well studied or 
characterized as the var genes, having only recently generated much research interest 
(Ch'ng et al., 2017). First described by Kyes et al. (1999), RIFINs consist of two sub-
groups, most (70%) belonging to the group possessing a 25 amino acid insertion-
deletion (indel) region (subgroup A, or A-RIFINs ), and subgroup B (B-RIFINs ) that 
lack an indel (Joannin et al., 2008). These subgroups also differ in their localisation, 
with A-RIFINs shown to be exported to the host cell membrane while B-RIFINs remain 
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within the parasite (Haeggstrom et al., 2007, Bachmann et al., 2012, Petter et al., 2007, 
Joannin et al., 2008). Importantly, and unlike the var genes, several rif genes are 
simultaneously transcribed in the parasite, with multiple proteins expressed at the 
Maurer’s clefts (MCs) and on the IE surface (Kyes et al., 1999, Fernandez et al., 1999, 
Khattab and Klinkert, 2006). These 27-45kDa membrane proteins seem to be expressed 
in all stages of the parasite in the human host, though their functions at these stages are 
not fully known (Petter et al., 2007, Wang et al., 2009, Mwakalinga et al., 2012). Along 
with other VSA genes like the var and stevor, rif genes cluster mainly in the 
subtelomeric regions of all 14 chromosomes of the parasite (Gardner et al., 2002). 
RIFINs may serve as potential vaccine candidates, mediating blood group A rosetting 
and believed to influence blood group distribution, all of which has implications in 
disease control and management (Goel et al., 2015, Tan et al., 2016). A study by Abdel-
Latif et al. (2003) in a comparison of antibody responses to RIFINs between immune 
adults and semi-immune children, reported much higher recognition by the immune 
adults, indicating that naturally acquired antibodies target these RIFINs. In addition, the 
fact that removal of recombinant RIFIN-adsorbed antibodies reduced reactivity to VSAs 
in the plasma of immune individuals point to the contribution of naturally acquired anti-
RIFIN antibodies to VSA antibody responses (Abdel-Latif et al., 2004). 
 
2.3.2.3 The stevor genes 
Another major gene family of P. falciparum are the stevors, with some 30 copies 
encoding antigenically variant STEVORs of 30-40kDa (Zhou et al., 2019). Initially 
thought to be contained within the MC (Kaviratne et al., 2002), it was eventually 
demonstrated that STEVOR moves out of the MC and is expressed on the IE surface 
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(Niang et al., 2009), thus playing a role in the antigenic variation of late stage parasites. 
STEVORs, along with RIFINs, are involved in the pathogenesis of malaria and immune 
evasion, though the knowledge on the protection offered by antibodies to these two 
VSA families is not extensive (Yam et al., 2017, Wahlgren et al., 2017). In contrast to 
patient isolates however, STEVOR expression in culture adapted parasites is very low, 
an indication that this protein is not necessary for survival in culture. STEVORs also 
mediate rosetting (Niang et al., 2014), which is presumed to protect against invasion-
blocking antibodies that would otherwise limit merozoite attachment and erythrocyte 
invasion. 
 
 
2.4 The ABO blood group system 
The human ABO blood group system in red cells was discovered over a century ago by 
Karl Landsteiner. These carbohydrate antigens are widely distributed throughout the 
body. They can be found in saliva, gut, endothelia of blood vessels as well as on the 
epithelial cells of some tissues, particularly those in direct contact with the environment 
such as in the trachea, nose and in the urinary, respiratory and reproductive tracts (Ravn 
and Dabelsteen, 2000, Eastlund, 1998, Szulman, 1960). Though found in the tissues of 
many animal species, the presence of the ABO blood group antigens on the RBC is 
unique and limited to humans and some apes (Oriol et al., 1986).  
Blood group antigens, which are terminal structures of glycan chains, are synthesized 
by the action of glycosyltransferases encoded by the ABO gene that sequentially add 
monosaccharide units to a variety of disaccharide precursors. To produce the H antigen, 
an α1,2 fucosyltransferase catalyzes the addition of a fucose in α1,2 linkage to the 
precursors. In humans, two α1,2 fucosyltransferases encoded by the FUT1 and FUT2 
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genes are involved. Mutations in these genes lead to the Bombay (absence of ABH 
antigens on RBCs) and non-secretor phenotype (absence of ABH antigens in saliva and 
epithelia) respectively (Marionneau et al., 2001). Following biosynthesis of the H 
antigen, the subsequent addition of an N-acetylgalactosamine in α1,3 linkage will yield 
blood group A antigen, while the addition of D-galactose at the same position yields 
blood group B antigen. 
Two of the three main alleles of the blood group system, A and B, are co-dominant 
while the O allele is recessive, the combinations of these resulting in the four major 
phenotypes (A, B, AB and O) observed in humans (Yamamoto et al., 1990).  
The biological significance, and therefore the driving force for the evolution of the 
ABO system in humans is not fully elucidated, though pathogens have been suggested 
as the cause due to several associations (Seymour et al., 2004, Frattali Eder and 
Spitalnik, 1997). Some studies suggest the modulation of cellular interactions on the 
RBC surface by ABH antigens to stabilize glycan molecules as an alternate explanation 
for this evolution (Cohen et al., 2009). Blood groups have been associated with 
susceptibility to several diseases including tropical infections caused by parasitic 
helminths like schistosomiasis (Kassim and Ejezie, 1982) and onchocerciasis (Opera, 
2007), as well as viral infections like HIV (Abdulazeez et al., 2008). However, in some 
of these instances, there has been no clear-cut or conclusive proof of such associations 
(Anstee, 2010). Malaria is no exception, and associations between the disease and blood 
groups are well known (Fry et al., 2008, Rowe et al., 2007, Miller et al., 2002). In a 
Ghanaian study, it was observed that patients with complicated cases of malaria were 
twice as likely to be of non O blood groups, and for blood group O patients with 
complicated disease, parasitaemia was low (Afoakwah et al., 2016). 
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There are also studies that link blood groups to genetic blood disorders such as 
haemoblobinopathies and thalassemias. In sub Saharan Africa, sickle cell disease 
(SCD), recognized as a problem of major public health significance (WHO, 1994), is 
linked to decreased susceptibility to malaria (Awotua-Efebo et al., 2004, Aidoo et al., 
2002), also for the heterozygous (AS) individuals (Williams et al., 2005, Amodu et al., 
2012). In a study in Nigeria, blood group O was the blood type most commonly 
associated with the SS genotype (Alagwu et al., 2016). Interestingly, in another 
Nigerian study, SCD patients with non-group O blood types presented with almost 
double the relative risk and significantly higher vaso-occlusive crisis episodes compared 
to blood group O SCD patients (Ahmed et al., 2014). 
Some studies indicate that the most common blood group observed in β-thalassemia is 
group O+, and though this group also shows the most complications of the disease, they 
occur much later in life and are milder in severity compared to other blood types (Sinha 
et al., 2017, Marbut et al., 2018). 
 
 
2.5 Immunity to malaria 
Immune response to the Plasmodium species, though studied for many decades, is still 
unclear and remains challenging in its complexity. The importance of antibodies in 
protection has been investigated in several species (Brown, 1969, Miller et al., 1975, 
Mitchell et al., 1976). In humans, the importance of humoral immunity was 
demonstrated in a classical study where passive transfer of purified IgG antibodies from 
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immune individuals to patients with malaria resulted in parasite clearance and recovery 
(Cohen et al., 1961).  
Clinical immunity to malaria develops slowly and only after repeated episodes over 
time following recurrent infections with P. falciparum in holoendemic areas, with adults 
eventually becoming protected from clinical disease (Bull et al., 1998, Marsh et al., 
1989) as antibodies are acquired from successive exposure to the repertoire of surface 
antigens (Giha et al., 2000, Contamin et al., 1996). This immunity is acquired in a 
systematic manner, with protection first acquired from the most severe forms of the 
disease in the children, followed by protection from less severe, uncomplicated malaria 
as they grow (Hviid, 2005, Gupta et al., 1999). Generally, asyptomatic P. falciparum 
infections associate with decreased incidence of clinical disease, but offer no protection 
in new infections (Buchwald et al., 2019). Immunity however is not directly linked to 
age, since adults with no prior exposure to the disease will develop severe disease 
(Barcus et al., 2003). Studies suggest that protective antibody responses in falciparum 
malaria are directed primarily to the parasite encoded exported PfEMP1 variant antigens 
on the IE surface (Bull et al., 1998, Giha et al., 2000, Dodoo et al., 2001). 
Studies have shown associations between specific IgG antibody classes and subclasses 
that are associated with protection and those that are not (Bouharoun-Tayoun and 
Druilhe, 1992, Domingos et al., 2018). Studies have also indicated the protective role 
IgM may have in malaria, with lower levels in nonimmune individuals and a negative 
correlation with parasite densities observed (Boudin et al., 1993, Wahlgren et al., 1986). 
Anti-malarial IgE on the other hand, which increase during P. falciparum infection, has 
been linked both to reduced risk from clinical disease (Bereczky et al., 2004, Duarte et 
al., 2007) and to severe disease (Perlmann et al., 1994, Calissano et al., 2003). 
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The VSAs consisting of exported parasite proteins mediate the virulence-linked 
mechanisms of cytoadhesion/sequestration and rosetting through products of the 3 main 
protein multigene families represented by PfEMP1 (Smith et al., 2013), RIFIN (Goel et 
al., 2015) and STEVOR (Niang et al., 2014) proteins, which are attributed to the cause 
of severe malaria (Ringwald et al., 1993). 
 
2.5.2 Sequestration of P. falciparum 
Sequestration is described as the adhesion of late-stage P. falciparum-infected 
erythrocytes to ECs (Udeinya et al., 1981), primarily within the venules. This ability is 
critical to the organism as it enables the parasite to avoid clearance by the spleen. In 
addition, the hypoxic venular environment with reduced oxygen tension is ideal for the 
development of the parasite (Scheibel et al., 1979). The brain, heart, lung, liver, kidney, 
subcutaneous tissues and placenta are all sites where P. falciparum-infected 
erythrocytes have been found to be sequestered (Miller, 1969, Yamada et al., 1989). 
Sequestration of IE in the brain microvasculature is the histopathological indicator of 
CM, which can lead to obstruction of these vessels with fatal consequences (Milner et 
al., 2015). Apart from the mechanical obstruction caused  by rosetting and cytoadherent 
RBCs (Chen et al., 2000a), sequestration also induces pathology due to hyper 
inflammatory host responses leading to blood-brain barrier dysfunction and cerebral 
edema (Brown et al., 1999). Other complications including acidosis, respiratory distress 
and SMA may be indicated (Rowe et al., 2009). 
Host receptors for cytoadhesion are numerous and varied, and includes the intercellular 
adhesion molecule 1 (ICAM-1). Expressed on the endothelium and several immune 
cells, this receptor is one of the most well described receptors for PfEMP1. ICAM-1 is 
upregulated following adhesion of IE and in response to pro-inflammatory cytokines 
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(Berendt et al., 1989, Chakravorty and Craig, 2005). In CM cases of severe malaria, this 
sequestration occurring via ICAM-1 mediated adhesion results in pathology in vivo, 
with histologically demonstrable co-localization of ICAM-1 and IE in the brain ECs of 
patients who succumbed to the disease (Turner et al., 1994). In addition, other studies 
show higher adhesion to ICAM-1 in parasites from severe disease, than in those from 
uncomplicated disease or controls (Ochola et al., 2011, Newbold et al., 1997a). 
The common membrane protein CD36 which is expressed on the vascular endothelium 
and several other cells such as monocytes, macrophages, dendritic cells and platelets 
(Silverstein and Febbraio, 2009) is also a known receptor for P. falciparum 
cytoadhesion. CD36 binding is fairly common in most P. falciparum strains (Turner et 
al., 1994, Rowe et al., 2009, Newbold et al., 1997a), and is also linked to the 
phagocytosis of IE (McGilvray et al., 2000). There is however no clear role for CD36 in 
the pathogenesis of malaria, as there has been no clear-cut association to disease 
severity (Rogerson et al., 1999, Newbold et al., 1997a). 
 
2.5.3 Red blood cell rosetting in malaria 
Rosetting was first described as a spleen-related property by David and colleagues in 
1988, defined as the adhesion of uninfected RBC(s) to an infected one, or ‘the 
agglutination of uninfected erythrocytes around parasitized erythrocytes’ (David et al., 
1988). This phenomenon involves mature or late stage parasites of certain strains of P. 
falciparum (Handunnetti et al., 1989), and has not only been described in all species 
known to infect humans (Lowe et al., 1998) such as P. vivax (Udomsangpetch et al., 
1995, Chotivanich et al., 1998) and P. ovale (Angus et al., 1996), but also in some 
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simian (Udomsangpetch et al., 1991) and murine (Mackinnon et al., 2002) malaria 
parasites. 
The ability of IEs to adhere to uninfected ones and to the endothelium is thought to be 
crucial to the pathophysiology observed in CM, SMA and other syndromes of severe 
malaria (Sherman et al., 2003, Miller et al., 2002, Carlson et al., 1990). Rowe and 
colleagues for example reported significantly lower rosetting rates in milder forms of 
disease, with increasing rates as severity increased (Rowe et al., 1995). In this same 
study, rosetting frequency of isolates from donors of blood group O was less than 
rosetting observed in non-group O (A and AB) isolates, while in another study 
significantly high levels of rosetting was observed in all clinical forms of severe disease 
including coma, SMA, neurological impairment and seizures (Doumbo et al., 2009). 
Other studies have demonstrated that more pronounced rosetting is observed in isolates 
from severe malaria patients, particularly those from cases with CM (Treutiger et al., 
1992, Rowe et al., 1995, Ho et al., 1991). In addition, rosetting has been shown to 
enhance vaso-occlusion (Kaul et al., 1991), but the relation between rosetting and 
virulence remains unclear, since rosetting is also observed in other, less virulent, 
malaria species (Rowe et al., 2009). 
The changes occurring on the surface of IEs stem from extensive modifications made by 
the parasite, which exports proteins to the erythrocyte surface, making sequestration 
possible by binding to host endothelial receptors. The biological role or functional 
significance of rosetting per se is not yet clear, though there may be pathogenic 
implications such as occluding capillaries, enhancing reinvasion due to proximity of 
uninfected erythrocytes, physically blocking immune attack on IE and even a host 
defense mechanism to discourage cytoadhesion (Wang and Hviid, 2015). Conversely, 
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rosetting may also reflect a bystander effect to EC adhesion in which RBC happen to 
share common receptors with the ECs in question. 
PfEMP1, RIFIN and STEVOR proteins all mediate the rosetting, previously thought to 
be mediated by PfEMP1 alone. P. falciparum RIFINs were shown to preferentially bind 
to blood group A (and perhaps B) antigens on the erythrocyte surface, making them the 
parasite ligand primarily responsible for rosetting and microvascular binding in non-
group O blood types. This interaction seems to result in larger rosettes and increased 
binding of IEs in the microvasculature of individuals with blood group A (Carlson et 
al., 1994), rosettes that are also more resistant to heparin-induced rosette disruption 
(Ch'ng et al., 2016). These properties of non-group O (specifically group A) rosettes, in 
addition to the fact that they shield IE from antibody binding (Moll et al., 2015), 
ultimately implicate them in disease severity. STEVOR on the other hand was found to 
mediate rosetting independent of PfEMP1 through Glycophorin C (GPC) on the RBC 
surface (Niang et al., 2014). 
In a 2005 study, it was reported that a novel group of parasite derived polymorphic 
surface proteins, the surface-associated interspersed gene family (SURFIN) proteins 
(Winter et al., 2005), may also have a role in rosetting, either directly or as an 
accessory molecule (Quintana et al., 2018). 
IgM was thought to mediate rosetting through Fc receptor binding to IE and therefore 
affect disease severity (Rowe et al., 2002, Ghumra et al., 2008), but subsequent studies 
indicate that this IgM binding does not directly mediate the formation of rosettes, 
though it may, along with α2- macroglobulin, be important for some forms of the 
phenomenon (Stevenson et al., 2015a, Stevenson et al., 2015b, Akhouri et al., 2016). 
Also, complement receptor 1 is considered a malaria resistance gene, as polymorphisms 
that reduce CR1 on the RBC reduce rosetting and protect from severe disease 
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(Cockburn et al., 2004). Understanding the phenomenon of adhesion and rosetting, 
along with the ligands and receptors involved in the development of severe disease, has 
huge implications in disease management and possible drug and vaccine targets. 
 
2.6 Malaria vaccines 
There have been significant declines in malaria morbidity and mortality in the past 
decade or so, but the global burden of malaria remains unacceptably high. Of concern as 
well is the fact that for the parasite, there are reports of development of resistance to the 
first line drug Artemisinin (Noedl et al., 2008), as well as the observation that 
mosquitoes may be changing feeding behavior to adapt to pressures from large scale 
ITN coverage (Russell et al., 2011). A highly effective malaria vaccine is very much 
needed to support the efforts in worldwide malaria control, and would be critical for 
total elimination of the disease in the most affected areas (Laurens, 2018). This could 
provide at least some level of immunity to subjects (at best, sterilizing immunity), while 
buttressing current successes chalked in ITN schemes, vector management and ACTs 
(WHO, 2018b). 
There is currently just one malaria vaccine, RTS,S/AS01 (also known as Mosquirix™), 
approved in 2015 by the European Medicines Agency for use in children in malaria 
endemic countries (Gosling and von Seidlein, 2016). Unfortunately efficacy is limited, 
and protection is also short-lived for this vaccine, especially in infants (White et al., 
2015). There are however suggestions that this vaccine may additionally prove to be 
beneficial in transmission interruption efforts in areas of low-endemicity (Gosling and 
von Seidlein, 2016). 
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Placental malaria, which results from P. falciparum infection in pregnant women, is 
characterized by the sequestration of IE in the intervillous spaces of the placenta where 
they bind to CSA through a PfEMP1 variant VAR2CSA (Duffy et al., 2006, Clausen et 
al., 2012). This results in inflammation and other negative outcomes including perinatal 
mortality, low birth weight and premature births (Rogerson et al., 2007, Fried and 
Duffy, 2017) Immunity to these placental parasites, like other forms of malaria, is 
achieved over successive pregnancies (Ricke et al., 2000, Doritchamou et al., 2019b), 
which has made VAR2CSA a potential vaccine candidate for malaria in pregnancy. 
Clinical trials of two VAR2CSA vaccine candidates designed to block IE binding to 
CSA; PAMVAC (Mordmuller et al., 2019) and PRIMALVAC (Sirima et al., 2020), are 
underway, and results of some studies underscore the need for targeted vaccines based 
on parasite, individual or geographical differences (Doritchamou et al., 
2019a)Antibodies have long been known as important for protective immunity against 
malaria, as shown in classical passive transfer studies (Cohen et al., 1961). 
Unfortunately, the generation of these protective antibodies by vaccination has been 
challenging, as definite correlates of immunity have been difficult to ascertain (Fowkes 
et al., 2010). There have been several different subunit malaria vaccines developed to 
clinical testing stages, but results from these have not been encouraging (Thera and 
Plowe, 2011, Chuang et al., 2013, Ogutu et al., 2009, Hodgson et al., 2014, Genton et 
al., 2002). However, recent trends point to a growing interest in whole parasite vaccines 
targeting the liver stage to block infection (Laurens, 2018) 
With reports of developmental advances in several areas of malaria vaccine research, 
from infection-blocking pre-erythrocytic vaccines, to infection- and disease-limiting 
vaccines, and transmission blocking vaccines that halt the spread of infection (Coelho et 
al., 2017), there is an upside to the situation. Improvement and advances in post-
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genomic technologies have facilitated the identification of numerous vaccine 
candidates, elucidating the gap in development of robust correlates of protection to 
adequately validate these candidates (Tuju et al., 2017). Recent work could lead to the 
identification of a subset of VSAs for development of a severe disease prevention 
vaccine or a test to predict children at risk of severe disease children (Travassos et al., 
2018). Other available data could also make it possible to target antigens related to 
phenotypes such as rosetting (Vigan-Womas et al., 2011) or to target parasites that 
prefer, and are linked to more severe disease syndromes (Storm et al., 2019). Vaccines 
targeted at these antigens would prevent the specific severe disease manifestation of 
cytoadhesion and/or sequestration in malaria. More recently, studies comparing 
transcriptomes of parasites from severe and uncomplicated disease revealed both known 
and novel PfEMP1 linked to severe disease, and could greatly enhance the identification 
of severe disease vaccine targets (Tonkin-Hill et al., 2018). 
  
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CHAPTER THREE 
3.0   METHODS 
3.1 Study location, subjects and recruitment 
The study was conducted at the Hohoe Municipal Hospital in the Volta Region of 
Ghana, which is situated in the town of Hohoe (7.1519°N, 0.4738°E), with a population 
of about 63,000 inhabitants. The municipality is in the eastern central zone of Ghana, 
east of the Volta river and near the border to the Republic of Togo (Figure 3.1). The 
Hohoe district is located in the Wet Semi-equatorial climatic zone of the region, with 
perennial transmission of P. falciparum peaking seasonally in June-July during the 
April to November wet season. There is therefore an intense and prolonged malaria 
transmission season for the municipality. There are occasional reports of P. ovale and P. 
malariae infection, but by far the most dominant malaria species in the zone is P. 
falciparum (Kweku et al., 2008). Annual precipitation values range between 1016 and 
1210 mm, with a 4-5-month dry season between November and April. (Ghana 
Statistical Service, 2014). The 2010 national census puts the population of the 
municipality at 262,046, with 96,504 under 15 years-of-age (Ghana Statistical Service, 
2012). 
 
  
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Figure 3.1 The map of Ghana showing the field study site. Hohoe is the district 
capital, located in the Hohoe Municipality of the Volta Region in eastern 
Ghana, about 221 km from the capital Accra. Map of the municipality 
adapted from the Hohoe District Analytical Report, Ghana Statistical Service 
(2014) 
 
Permanently resident children aged 12 years and below from Hohoe and surrounding 
settlements who presented with malaria at the Hohoe Municipal Hospital were included 
in the project following written consent from their parents or guardians and satisfactory 
study requirements. Details of the study were described to each parent or guardian, 
including the option to exit the study after consent. Translation to the local language 
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during the consent seeking process was done where necessary. In addition, parents or 
guardians who did not sign the consent form, and children who did not meet the study 
requirements (due to diagnosis with a major medical condition such as cardiovascular 
disease), or those with SS sickling genotypes, were excluded. 
Appropriate protocols and permits for the study were reviewed and granted by the 
Noguchi Memorial Institute for Medical Research Institutional Review Board (Study 
number 026/13-14), University of Ghana, and the Ghana Health Service Ethical Review 
Committee (GHS-ERC 08/05/14), Ministry of Health, Ghana. 
The World Health Organization standard definitions and criteria for severe malaria and 
complications were employed in the collection and categorization of clinical and 
research blood samples (WHO, 2000b). Diagnosis involved a positive result from a P. 
falciparum-specific rapid test kit (First Response HRP2, India) and confirmation by 
microscopy of Giemsa-stained blood film. Patients with asexual P. falciparum 
parasitaemia of 2,500 IEs/µl of blood or more and temperature greater than 37.5°C, 
without an alternative cause of febrile illness were eligible for inclusion in the study. 
Recruited malaria patients were clinically examined by designated project clinicians and 
categorized into uncomplicated or severe cases following WHO guidelines on 
uncomplicated and severe malaria. 
 
3.2 Blood sampling and processing 
Rapid diagnostic testing for malaria was usually done as initial screening in the waiting 
area using finger-prick capillary blood, and the results confirmed by microscopy. For 
rapid test and/or microscopy positive children, venous blood samples were subsequently 
drawn by project staff for parasite counts (microscopy), in vitro cultures, 
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cryopreservation of parasite isolates, ABO blood grouping, blotting onto filter paper for 
subsequent parasite typing as well as for the tests described below. Fully automated 
blood chemistry and 18-parameter haematology indices were also performed. All 
patients were screened for HbS and G6PD deficiency. 
Trained phlebotomists collected venous blood aseptically from the upper arm with a 
single use, sterile butterfly needle kit into evacuated 6ml EDTA and Heparin blood 
collection tubes (Becton Dickinson, USA) for molecular and cellular assays 
respectively. The vacuum tubes were centrifuged at 800 × g for 5 minutes to obtain the 
blood pellet and plasma, which was collected and stored in aliquots at -40°C. 
For each patient, thick and thin blood smears were prepared on a microscope slide for 
estimation of parasitaemia according to WHO protocols (WHO/UNICEF, 2015b, WHO, 
2010). Briefly, a small aliquot of blood was spotted near one edge of the slide and 
spread evenly but thickly in a circular motion to about 1.5cm in diameter. A second 
blood spot was then placed near the first and spread evenly along the length of the slide 
using the edge of a second slide to form a thin film. The slides were air dried, after 
which the thin smear was fixed by briefly flooding with, or dipping in 100% methanol. 
Once dry, the slides were stained for 15 minutes in a freshly prepared 10% Giemsa stain 
solution using Giemsa/staining buffer with a pH of 7.2. The stain was washed off by 
rinsing the slide under a gentle flow of water, air dried and examined under oil 
immersion with a light microscope (Olympus CX) at 1000× magnification. Parasitaemia 
was calculated from counts of P. falciparum infected RBC per 400 RBC when >1%, or 
else from counts of parasites per 200 WBC in the thick smear and adjusted to parasites 
per µl of blood using the measured RBC or WBC counts. 
 
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3.2.1 ABO blood grouping and Rhesus status  
Blood grouping tests were carried out on all samples collected from patients using a 
commercially available ABO- and Rhesus-grouping kit (Fortress Diagnostics, UK) 
based on agglutination of RBC upon addition of anti-A, anti-B or anti-Rhesus reagents 
from the test kit. All reagents and samples were brought to room temperature for the 
test. 
A drop of anti-A, anti-B and anti-Rh serum was placed on a clean, hard but smooth 
white surface, and to each of which a drop of blood from a patient donor was added. 
Each was mixed well with a small wooden stick and observed for agglutinating RBCs. 
A donor’s blood type was categorized as blood type A if agglutination was observed 
with the anti-A serum only, or blood type B if the agglutination was observed with the 
anti-B serum only. Agglutination of RBC with both anti-A and anti-B test sera indicates 
the AB blood type, while no agglutination in both test sera is indicative of a donor of 
blood group O. 
Similarly, an Rh-positive test of agglutinating RBCs following the addition of anti-Rh 
serum was used to determine if patients were Rhesus positive or negative. 
 
3.3 Parasite cultures 
P. falciparum field isolates obtained from patient blood draws were either cultured for 
use in rosetting assays or cryopreserved for later work. Blood stage culture procedures 
were all performed in a biosafety level II cabinet using standard methods (Cranmer et 
al., 1997), with slight modification. 
After collecting the plasma from the EDTA and heparin tubes as described in the 
previous section, the remaining RBC pellets were washed twice by centrifugation in 
excess, sterile, incomplete medium (RPMI 1640 medium with 25 mM Hepes, Sigma-
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Aldrich, Germany, supplemented with 5µg/mL gentamicin sulphate) to dilute out any 
traces of anticoagulant. After washing, the medium was pipetted off and a 200 µl 
aliquot of the remaining pellet containing the IEs was added to a sterile 25 cm2 culture 
flask (Thermofisher Scientific, Denmark) containing 5mL of complete parasite medium 
(HEPES buffered RPMI 1640 medium supplemented with Albumax II (Life 
Technologies, Paisley UK), gentamycin and L-glutamine at 5, 0.05 and 0.18 mg/mL 
respectively and for field isolates, additional supplementation with 2% human serum) to 
obtain a final haematocrit of 4%. The flask was gassed with a mixture of 2.0% O2, 5.5% 
CO2 and 92.5% N2 for 30 seconds, capped and incubated at 37°C, with regular media 
changes every 24 to 48 h, along with an estimation of parasitaemia from Giemsa-stained 
smears as described in the previous section to monitor the health and growth of P. 
falciparum in the cultures. 
Maintenance of laboratory strains of P. falciparum used in this study, including 3D7, 
FMG and FUP followed the same procedure, but without supplementation of the 
complete media with human serum. Blood group O+ erythrocytes obtained from the 
blood bank (Rigshospitalet, Copenhagen, Denmark) were used for culturing of 
laboratory strains, after washing at least twice in incomplete medium to remove plasma 
and leucocytes. The washed RBC was re-suspended at 50% haematocrit, stored at 4°C 
and used for a maximum of three weeks. Genotyping of parasites in culture and tests for 
Mycoplasma contamination was carried out regularly every few weeks as described 
(Bengtsson et al., 2013). 
 
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3.3.1 Freezing and thawing of parasites 
3.3.1.1 Freezing P. falciparum-containing blood samples 
P. falciparum-IE obtained from patients in the field during blood collection or from 
continuing cultures in the laboratory were prepared for long-term cold storage using the 
Glycerolyte method. In short, cultures with at least 2% (ideally 5%) ring forms were 
chosen for freezing. The cultures were initially transferred into sterile 12 mL tubes and 
centrifuged at 2000rpm (769 × g) for 5 minutes to pellet the RBC. The 
media/supernatant was removed and the pellet re-suspended and its volume estimated. 
The total volume of glycerolyte to be used was determined from the pellet volume, V, 
by multiplying V by a factor of 5/3. Next, one-fifth of V (glycerolyte) was added 
dropwise to the pellet using a 2 mL syringe and an appropriate sized needle. The 
mixture was incubated for 5 minutes at room temperature followed by the addition of 
the rest of the glycerolyte (the remaining 4/5 × V), also dropwise, while gently shaking 
the tube. The mixture was then transferred into cryotubes at a volume of 800 µl each, 
placed into a ‘Mr Frosty’ freezing container (Thermo Scientific) and stored at -80°C 
overnight before they were transferred to an ultra-low -150°C freezer or into liquid 
nitrogen. 
3.3.1.2 Thawing P. falciparum-containing blood samples 
Malaria parasites were thawed from -150°C or liquid nitrogen storage using two sterile 
NaCl solutions (12% and 1.6%) at room temperature. In brief, the vial to be thawed was 
removed from cold storage, quickly thawed in a water bath at 37°C and transferred 
aseptically into a 50 mL centrifuge tube. The volume of thawed suspension, V, was 
noted, following which 0.1 × V of the 12% NaCl solution was added while gently 
shaking the tube. The tube was left for 5 minutes, after which 10 × V of 1.6% NaCl was 
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also added dropwise as before. The tube was centrifuged at 20°C for 5 minutes at 500 × 
g, after which the supernatant was discarded and the pellet was resuspended in 10 × V 
of culture medium and centrifuged at 500 × g under the same conditions. Following this 
step, the pellet was re-suspended in the appropriate amount of culture medium, 
transferred to a culture flask, gassed and placed in a 37°C incubator. 
 
3.4 Detection of rosetting rates in selected parasites and field isolates 
Rosetting assays were used to measure the rosetting rates in blood group antigen 
selected P. falciparum laboratory strains, and also in parasite isolates from patients. In 
the field, rosetting assays were performed following parasite culture in the donor’s own 
RBC for 24-36 hours when the parasites were at late trophozoite/early schizont stages. 
For laboratory strains (3D7, FMG, HB3 and FUP), the selected parasites were cultured 
in blood type O RBC using standard protocols as described in Section 3.3. Mature 
parasites to be used for rosetting assays were concentrated, as described in detail in 
Section 3.8.1, by magnetic-activated cell sorting (MACS) and mixed with uninfected 
RBC of blood type O, A or AB for 1 h prior to counting the rosettes. 
For the rosetting assay ethidium bromide was added (at a final concentration of 
approximately 7 µg/mL ) to a 100 µL aliquot taken from a thoroughly but gently mixed 
late stage culture, incubated for at least 2 minutes at 37°C after which the samples were 
ready for observation. Two to 5 µL of the mixture was then transferred by micropipette 
to a well of an 8-well 6 mm diagnostic microscope slide (ThermoFisher Scientific, 
USA) and gently covered with a coverslip to avoid trapping bubbles underneath. The 
RBCs and rosettes were observed using a fluorescence microscope at 400× 
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magnification. The rosetting rates were assessed by detection and counting the number 
of rosettes observed per 200 IEs and expressed as a percentage. 
 
3.5 Blood group polysaccharide constructs 
Four bovine serum albumin (BSA)-coupled blood group neoglycoprotein (NGP) 
constructs were purchased as lyophilized powders from Dextra Laboratories Limited, 
Reading, UK (https://dextrauk.com/) for use in the parasite selection and binding assay 
experiments. These NGPs, namely Blood Group A-BSA (6-atom spacer), Blood Group 
B-BSA (6-atom spacer), Blood Group B-BSA (3-atom spacer) and Blood Group H 
disaccharide-BSA (3-atom spacer) (product codes NGP6305, NGP6323, NGP0323 and 
NGP0205 respectively) were reconstituted to a stock concentration of 250 µg/mL with 
sterile PBS. They were further diluted to a final concentration of 10 µg/mL for the 
experiments. 
 
3.6 Selection for binding to blood group constructs 
The 3D7, FMG, HB3 and FUP parasite strains were selected on blood group 
polysaccharides and used for subsequent assays. All procedures were performed under 
sterile conditions, and all media pre-warmed to 25-37°C. 
A day before selection, one well of a 24-well plate (Nunc, Thermo scientific) was 
coated overnight at 4°C with 400 µL of 10 µg/mL oligosaccharide-BSA in PBS. On the 
day of selection, the buffer was aspirated and the well was blocked for 2 hours at room 
temperature with 800 µl of 2% BSA in PBS. The blocked well was then washed 3 times 
with 1 mL of culture medium each time.  
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Late trophozoites and schizont stages of the culture to be used for selection were 
purified by MACS as per the manufacturer’s instructions (Section 3.8.1) under sterile 
conditions, using PBS with 2% FCS, RBC-wash or culture medium. 
The IEs were re-suspended in a final volume of 400 µl in culture medium, transferred to 
the coated well and incubated with gentle shaking for an hour at room temperature. 
Following incubation, repeated gentle washing to remove unbound IEs was done by 
first adding, and then aspirating 1 mL of culture medium (the well was not emptied 
before first wash). The washing procedure was repeated 3 to 7 times depending on how 
well parasites bound. An inverted microscope was used to check bound cells after every 
2-3 washes. 
After washing, between 200 and 400 µl of culture medium and 50 µl of packed RBC 
were added to the excess medium in the wells to ensure a minimum volume of 600 µl 
per well. The plate was incubated in an exicator under micro-aerobic conditions at 37°C 
overnight. The following day, 500 µl of culture medium was carefully removed from 
the top, leaving the RBCs at the bottom. A thin smear was prepared from this to observe 
selected parasites that were now at ring stages, before the remaining RBC were 
transferred to a culture flask, adjusting for the appropriate haematocrit for the volume 
used. 
  
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3.7 Blood group antigen and EC binding assays 
3.7.1 ABO polysaccharide antigen binding assay 
Parasites selected on blood group A or B polysaccharide constructs, along with field 
isolates cryopreserved from patients from the field site were tested for binding to the 
blood group antigens in a modification of the method described by Hempel and 
colleagues for immobilized cells (Hempel et al., 2015).  
The wells of flat-bottomed optical 96-well polystyrene plates (Nunc Polysorb, Thermo 
Fisher Scientific) were coated overnight at 4°C with 50 µL of blood group A-, B- or H-
BSA constructs diluted to a concentration of 10 µg/mL in 0.05 M Carbonate buffer, pH 
9. Following the coating step, the excess constructs were flipped out and the wells were 
blocked with 100 µL/well of 20 mg/mL (2%) BSA in PBS for a minimum of 2 hours at 
room temperature. The wells were washed twice with 100 µL complete parasite 
medium, followed by the addition of 100 µL of the late stages of control (unselected) 
7
and IE of selected and field isolates to the wells at a concentration of 1.25 × 10  
6
cells/mL (or 1 × 10  cells/100 µL). The plate was incubated with gentle shaking at room 
temperature for 45-60 minutes. A gravity wash step to remove unbound IE was done by 
immersion and inversion of the plate in 2% FCS in PBS for 60 to 90 minutes, after 
which it was taken out and blotted to remove the excess wash buffer. Standards (known 
concentration/quantity of RBC or IE) were added to the plate and incubated for 15 
minutes with 100 µL of 0.1% Triton X-100 (Sigma-Aldrich) diluted in PBS to 
permeabilize the bound erythrocytes in the wells. This disruption of IE enables the 
release of intra-erythrocytic haem which acts as a quantifiable pseudoperoxidase 
(Hempel et al., 2015). Tetramethylbenzidine (TMB, R&D Systems, Oxon UK) was 
added to the wells at 100 µL/well, and after incubation in the dark for about 5 minutes 
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while monitoring colour development, the reaction was stopped by the addition 50 µL 
of 1M H2SO4 to the wells. Optical densities of the wells were read at a wavelength of 
450 nm with a 540 reference on a plate reader (Multiskan EX, Thermo Labsystems, 
MA, USA). 
 
The proportion of adherent IEs were determined by the formula: 
(sampleOD –  backgroundOD)
 
(totalOD –  backgroundOD)
where backgroundOD = OD of wells with no added IE, and totalOD = OD of well with 
6
total number (100%) of IE (1 × 10  RBC or IE). 
 
3.7.2 Endothelial cell binding assays 
Primary human endothelial cells of the aorta and foreskin dermis were cultured in vitro 
and used in cell binding assays to assess the binding characteristics of blood group A- 
and B-binding 3D7, HB3 and FMG parasites. The assays were performed using 96-well 
flat-bottomed tissue culture plates (NUNC, Denmark) containing monolayers of the 
relevant cell lines. Briefly, and generally as described in the method by Hempel et al. 
5
(2015), ECs were seeded in the wells at approximately 1.5 × 10  cells/ml and grown to 
confluence 48 hours prior to the experiment. On the day of the experiment, MACS-
purified late stages of the parasites (Section 3.8.1) were added in triplicate to the EC-
6 7
containing wells at a concentration of 1 × 10  IE in 100 µL per well (about 1.25 × 10  
cells/mL). Subsequent steps were essentially as described for the blood group binding 
assay described in the previous section. Thus, the plate was incubated with gentle 
shaking for about 45- minutes to 1 hour at room temperature and then gravity-washed in 
2% FCS in PBS for 60 to 90 minutes to remove unbound erythrocytes. After blotting on 
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tissue paper to remove excess wash buffer, the permeabilization step followed with the 
addition of 100 µL of PBS supplemented with 0.1% Triton X-100 to the wells. After 15 
minutes, TMB was added to the wells at 100 µL/well, and colour development was 
allowed to proceed in the dark. The reaction was stopped with 50 µL of 1M H2SO4/well 
and ODs read at 450nm in an ELISA plate reader. Similarly lysed IE of known 
concentration were included on the plate, as well as wells with EC but with no added IE 
to serve as standards and background references respectively. The percentage of 
adherent cells were determined using the formula 
(sampleOD –  backgroundOD)
 
(totalOD –  backgroundOD)
as described in the preceding section (3.7.1). 
 
3.7.3 Blocking assay using anti-A and anti-B monoclonal antibodies 
A variation of the binding assays described previously was conducted using 
commercially procured monoclonal antibodies to blood group A and B antigens. The 
antibodies were used to confirm the uniqueness and difference between the blood group 
constructs by specifically blocking adhesion of IE of selected parasites to the respective 
polysaccharide constructs A-BSA and B-BSA used in this study. As described earlier in 
Section 3.7.1, 96-well microtiter plates were coated overnight with the blood group A-, 
B- and H-BSA constructs. The plates were blocked as before for 2 hours with 2% BSA 
in PBS and washed four times with 100 µL complete parasite medium. After the 
washing step, mouse Monoclonal Anti-Blood Group A and Monoclonal Anti-Blood 
Group B antibody (SAB4700677 and SAB4700676 respectively, Sigma Aldrich) were 
diluted in PBS and incubated at 100 µL per well for 1 hour. The washing step was 
repeated after the incubation period, followed by the addition of horseradish peroxidase 
46 
 
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(HRP)-conjugated Goat anti-mouse IgM secondary antibody (Jackson ImmunoResearch 
115-035-020 from TriChem, Denmark) diluted 1:5000 in PBS, 100 µL/well for an hour. 
After another washing step, TMB substrate was added and the colour allowed to 
develop prior to reading the plate in an ELISA plate reader to obtain the ODs as before. 
All steps except for the coating were performed at room temperature. 
 
3.8 MACS and Flow Cytometry 
3.8.1 Harvesting late stage P. falciparum by Magnetic-activated cell sorting 
Late trophozoite stages of P. falciparum were harvested from culture by MACS 
(Miltenyi Biotec, Germany) following the manufacturer’s instructions. Briefly, the 
column and 3-way stopcock were assembled in the magnet and prepared by attaching a 
20 mL syringe filled with PBS containing 2% FCS (PBS-2) to the left opening of the 
stopcock. The tap was set to a position to open the way between the syringe and column 
but close the way out. The plunger was moved back and forth until the column was well 
rinsed and all bubbles were dislodged from the column. A blunt needle (20G) was 
attached to the lower outlet of the 3-way stopcock, which was opened between the 
column and the lower outlet to allow the PBS-2 to run through to a waste receptacle 
below. A further 20 mL of PBS-2 was added gradually to the top of the column and 
allowed to run through, making sure the column did not run dry so as to avoid air 
pockets. The culture to be separated was then re-suspended with 5 mL of PBS-2 and 
added to the column, followed by 40-50 mL more PBS-2 to rinse out excess, unattached 
ring-stage IEs and uninfected RBC. A plunger attachment was inserted at the top of the 
column, to which a syringe filled with 20 mL of PBS-2 was fitted. The column with the 
attached syringe was twisted to unscrew it from the 3-way stopcock, detached from the 
magnet and inserted into a 50mL tube. The IE containing the late stage parasites trapped 
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in the column were removed by pushing the PBS-2 in the attached syringe through the 
column into the 50 mL tube. The late stage parasite concentration in the resulting 
suspension was estimated by counting the IE using an Improved Neubauer 
haemocytometer. 
 
3.8.2 Measurement of antibody reactivity to selected parasite VSA by Flow 
Cytometry 
Antibody reactivity to variant surface antigens of parasites selected on blood group 
antigens was investigated by flow cytometry. Plasma samples from children enrolled in 
the study and stored at minus 40°C were thawed and added to 96-well polystyrene 
round-bottom microtiter plates (Nunc, Thermo Fisher Scientific) at 5µL per well. IEs 
containing late stage parasites obtained from MACS separation as described in Section 
6
3.8.1 were then adjusted to a concentration of 2x10  cells/mL in 2% FCS in PBS and 
added to the well at 100 µL per well. The plate was then incubated for 30 minutes at 
4°C, after which the cells were washed twice in PBS-2 by centrifugation at 500 × g for 
4 minutes each time. The wash buffer was removed after each wash by flicking the plate 
once, leaving the IE pellets at the bottom of the wells. Next, 100 µL of FITC conjugated 
Rabbit anti-Human IgG antibody (Jackson ImmunoResearch) was diluted 1:150 in PBS-
2 with 20 µL/mL of 0.1 mg/mL Ethidium Bromide solution (2 µg/mL) and incubated 
again for 30 minutes at 4°C. The cells were then washed twice as before, followed by 
the addition of 100 µL of PBS-2, after which the plate was read with a flow cytometer 
(Cytomics FC500, Beckman Coulter, USA). WinList version 6.0 (Verity Software 
House Inc.) was used to analyze the data generated. Uninfected and IE were gated 
according to ethidium bromide fluorescence. 
 
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3.9 Molecular methods 
3.9.1 Sample processing and storage 
Laboratory isolates, both unselected and selected for binding to blood group 
oligosaccharides (Section 3.6), were processed and stored in TRIzol from cultures at 
ring stages (at least 2%) by transferring the contents of the culture flasks to centrifuge 
tubes and pelleting IE by centrifugation at 500 × g for 5 minutes. Following 
centrifugation, and removal of the supernatant/medium, 200 µL of the RBC pellet 
containing the parasites was pipetted into a cryo-storage tube, to which 1.6mL of 
TRIzol (ThermoFisher Scientific InVitrogen, USA) at room temperature was added. 
This was thoroughly mixed by pipetting several times to remove clumps and maintain 
parasite RNA integrity. After a brief shake in the vortex, the tubes were transferred to a 
-80°C storage facility until use. 
 
3.9.2 RNA extraction from Trizol-stored samples 
Samples stored in TRIzol and retrieved from -80°C storage were thawed to room 
temperature, 500 µL of which was transferred into an Eppendorf tube after pipetting up 
and down a few times for a uniform mixture. To this tube 200 µL of chloroform 
(Sigma-Aldrich, Germany) was added and shaken vigorously for about 15 seconds to 
achieve a good mix and then centrifuged at 12,000 × g for 15 minutes at 4°C. The 
supernatant was collected and transferred to a new Eppendorf tube, after which 500 µL 
of 2-Propanol (Sigma-Aldrich, Germany) and 5 µL of Glycogen (ThermoFisher 
Scientific, USA) were added. The mixture was shaken and then incubated for 10 
minutes at room temperature, before spinning at 12,000 × g for 10 minutes at 4°C in the 
centrifuge. The supernatant was removed, and the remaining pellet was washed by 
centrifugation (5 minutes at 7,500 × g at 4°C) by adding 1mL of 75% Ethanol in 25% 
49 
 
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DEPC (diethylpyrocarbonate) water (deionized) to the pellet. The supernatant was then 
carefully pipetted off and the pellet air dried for a few minutes, before adding 10 µL of 
DEPC water and placing the tube in a heat block at 65°C for 5 minutes to dissolve the 
pellet. The extract was then transferred onto ice or stored in the freezer for the next step. 
All steps described in this extraction procedure were performed in a fume hood, and 
using RNase-free tips and consumables to minimize contamination. 
 
3.9.3 DNase treatment and cDNA synthesis  
Following RNA extraction, samples were treated with DNase to digest possible 
genomic DNA contaminants. Briefly, 10 µL of the extracted RNA was transferred to a 
PCR tube and incubated for 20 minutes at 37°C with 1.4µL of 10× DNase reaction 
buffer (Sigma-Aldrich, Germany) and 2µL of DNase I (Sigma-Aldrich, Germany). At 
the end of the incubation period, 2µL of stop solution (Sigma-Aldrich, Germany) was 
added to the mixture and kept for 10 minutes at 70°C to stop the reaction. 
To confirm presence of extracted RNA and the effectiveness of DNase treatment of the 
sample, an aliquot of the DNase treated RNA sample was tested by QPCR using a 
primer set for the p90 seryl-tRNA synthetase housekeeping gene (Salanti et al., 2003).  
In brief, 0.5 µL of the treated RNA sample was added to 10 µL of SYBR Green PCR 
Master Mix (Qiagen, UK), 7.5 µL DEPC water and 2 µM forward and reverse primer 
pair of the p90 housekeeping gene. The QPCR was performed on a Rotorgene RG-3000 
thermal cycler (Corbett Research, Australia). 
Complementary DNA (cDNA) synthesis from the DNA-free RNA was achieved by 
reverse transcription from random hexamers using Superscript II (InVitrogen, USA), as 
described in the manufacturer’s protocol. In brief, to a 200 µL PCR tube was added 15 
µL of the RNA sample, 0.75 µL of random primers (3 µg/µL in RNase-free 
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water)(ThermoFisher Scientific, USA) and 1.5 µL of 1.74 µM Deoxynucleotide 
triphosphate (dNTP) mix (InVitrogen, USA). The tube was then vortexed, spun briefly, 
incubated for 10 minutes at 65°C, and then transferred directly onto ice. Six microliters 
of First Strand Buffer (ThermoFisher Scientific, USA), 3 µL of dithiothreitol (DTT) 
(ThermoFisher Scientific, USA), and 1 µL each of RNaseOut and Superscript II 
(ThermoFisher Scientific, USA) were added to the reaction tube, which was then 
vortexed to mix the contents. Following a brief spin, the tube was placed in a PCR 
machine with the following settings for cDNA synthesis: 1 cycle each of 25°C for 10 
minutes, 42°C for 50 minutes, 70°C for 15 minutes, and then 8°C. 
cDNA samples were again tested for gDNA contamination by QPCR with p90 primers 
as described earlier in this section. 
 
3.9.4 Quantitative Polymerase Chain Reaction to detect VSA transcripts of 
var and rif genes 
Complementary DNA samples from blood group selected parasites processed as 
described in section 3.9.1, and free from gDNA contaminants, were prepared for reverse 
transcriptase (RT)-PCR on a thermal cycler (Rotorgene RG-3000, Corbett Research, 
Australia). The reactions were carried out in a total reaction volume of 20 µL, in 0.1 mL 
clear polypropylene tube and cap strips containing 10 µL QuantiTect SYBR Green PCR 
master mix (Qiagen, UK), 2 µM forward and reverse gene primer pairs, and 0.2 µL 
cDNA sample. Primer pairs of 154 rif, 60 var, and primers to the endogenous control 
genes seryl-tRNA synthetase (p90) and fructose-bisphosphate aldolase (p61), were used 
(Wang et al., 2010, Dahlback et al., 2007). 
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Optimised cycling conditions for cDNA of P falciparum were as described previously 
(Salanti et al., 2003) as follows: 95°C for 15 minutes, followed by 40 cycles of 95°C for 
30 s, 54°C for 40 s and 68°C for 50 s. 
 
3.9.5 Statistical Analysis 
Statistical analysis of the data generated in this study were carried out using Sigmaplot 
12 (Systat Software, Inc., San Jose, CA) and GraphPad Prism (Version 6, GraphPad 
Software, San Diego, CA). Characteristics of study participants were recorded as 
median with minimum and maximum values. As most of the data were not normally 
distributed, the non-parametric Mann-Whitney and Kruskal Wallis tests were used to 
determine differences between two groups, or in comparing more two groups, 
respectively. Spearman’s Rank-Order coefficients were also calculated to assess 
correlations, and in cases where post hoc tests were required following multiple 
comparisons, the Dunn’s Multiple Comparison test was employed. P values ≤ 0.05 were 
considered statistically significant. 
  
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CHAPTER FOUR 
4.0   RESULTS 
The initial aspect of this study was to establish blood group A and B-binding variants of 
well-characterized P. falciparum laboratory strains and to demonstrate the resulting 
phenotypic changes. P. falciparum 3D7, FMG and FUP isolates were continuously 
panned on blood group oligosaccharides immobilized on polystyrene plates to yield 
blood group A and B binding phenotypes. The effect of this selection of parasites on 
rosetting was then investigated by conducting rosetting assays of IEs containing 
selected parasites and RBCs from donors of blood types A, AB and O. 
A plate-based quantitative read-out specifically measuring selected parasite-IE binding 
to immobilized A- and B oligosaccharide binding was therefore developed to measure 
this interaction. Initial development of the assay involved confirmation of binding of the 
BSA-conjugated oligosaccharide constructs to the polystyrene plates. This was achieved 
by detection of the bound constructs at different concentrations using blood group A, B 
and O specific biotinylated lectins as indicated in Table 4.1. 
Ulex europaeus lectin (UEA) binding to the H-BSA construct was measured as the 
optical density (OD) and was concentration dependent. Additionally the Galanthus 
nivalis lectin, or GNL, used here as a negative control lectin, showed no indication of 
binding to the targets of interest (Figure 4.1a). Similarly, Dolichos biflorus agglutinin 
(DBA), which specifically binds the blood group A oligosaccharide, showed specific 
and concentration dependent binding to the immobilised blood group A construct 
(Figure 4.1b). 
 
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Table 4.1 Lectins and corresponding specific antigen/carbohydrates 
Lectin Blood Group Specificity 
Dolichos biflorus agglutinin (DBA) Anti-A1 
Griffonia (or Bandeiraea) simplicifolia lectin Anti-B 
(GSL-I or BSL-I) 
Ulex europaeus agglutinin (UEA-I) Anti-H (O) 
Galanthus nivalis lectin (GNL) Anti-rabbit RBC (does not bind 
A,B or H) 
 
The DBA, GSL-I and UEA-I lectins bind specifically to blood group A, blood group B and 
blood group O antigens respectively. GNL on the other hand, is known to agglutinate rabbit, but 
not human erythrocytes 
 
  
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3
UEA
2.5 GNL
2
1.5
1
0.5
0
10ug/ml 5ug/ml 2.5ug/ml 1.25ug/ml
Lectin Concentration 
 
Figure 4.1a Dose dependent binding of blood group O specific Ulex europaeus 
agglutinin (UEA) to blood group H antigen construct. The H-BSA 
construct was coated at 10 µg/ml. Each data point is the mean of triplicate 
readings in an experiment. No OD signal was observed with the non-specific 
Galanthus nivalis lectin (GNL) at any concentration. 
 
1.4
DBA 10
1.2
DBA 5
1 DBA 2.5
0.8
0.6
0.4
0.2
0
5ug 2.5ug 1.25ug
A-BSA coating  concentration (µg/ml) 
 
Figure 4.1b Detection of immobilized A-BSA construct with Dolichos biflorus (DBA) 
lectin. ODs show concentration dependent binding of the DBA lectin to the 
blood group A oligosaccharide construct coated at different concentrations. 
Each data point indicates mean of triplicate readings in an experiment 
55 
 
Optical density (O.D) 
Optical density (OD) 
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4.1 Rosetting in parasites selected on blood group antigens 
To investigate the effect of selecting blood group binding phenotypes on rosette 
formation, parasites (3D7, FMG and FUP) selected by regular panning on BSA-coupled 
blood group oligosaccharides, along with their corresponding isogenic unselected 
cultures, were tested for rosetting using blood type A, AB and O RBCs from donors 
without malaria. Rosetting rates are the number of rosettes counted in ethidium bromide 
stained parasites per 200 IEs under a fluorescent microscope (Figure 4.2).  
 
 
Figure 4.2 A large rosette containing IEs surrounded by several RBC. Ethidium 
bromide-stained late stage P. falciparum parasites can be seen in the rosette 
and in surrounding IEs (arrowed) 
 
All parasites selected on blood group sugars clearly formed rosettes, while none were 
observed in the unselected 3D7, FMG and FUP parasites (Figure 4.3). It was observed 
particularly that FMG B-selected IEs in group A and AB RBC formed numerous giant 
rosettes. In addition, the rosetting rates observed were generally higher in group A 
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RBCs than rates observed in blood group AB erythrocytes. Irrespective of the parasite 
isolate or whether selected or unselected, no rosetting was observed in blood type O 
erythrocytes (Figure 4.3). 
 
 
100
90 grp O
80 grp A
70 grp AB
60
50
40
30
20
10
0
 
Figure 4.3 Rosetting rates of selected parasites in RBC of blood group O, A and 
AB. Unselected parasites (3D7, FUP and FMG) do not form rosettes in RBC 
of blood group O, A or B. However, A- and B-selected parasites (A3, A8 
and B3, B8) all show increased rosetting rates in blood group A and group 
AB, but not in group O RBCs. Rosetting rates are the average of counts from 
duplicate wells for each isolate.  
 
 
4.2 Adhesion of selected parasites to blood group oligosaccharides 
With the culture of blood group polysaccharide-binding phenotypes, the plate-based 
assay described earlier at the start of this chapter was developed further and used to 
directly measure adhesion of IEs to immobilized blood group antigens. 
57 
 
Rosettes/200 IE 
3D7
3D7-B8
3D7-A8
FUP
FUP-B3
FUP-A3
FMG
FMG-B3
FMG-A3
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Following three rounds of selection by panning on blood group A and B constructs, 3D7 
parasites selected by this method consistently bound to A- and B-BSA constructs at 
much higher levels compared to the levels observed for the unselected parent culture. 
Binding to the group O antigen construct (H-BSA) by the selected parasites was low, 
and comparable to binding of the selected parasites (Figure 4.4). 
 
 
1
0.9 A-BSA
0.8 B-BSA
0.7 H-BSA
0.6
0.5
0.4
0.3
0.2
0.1
0
3D7 A2 B2 Medium
 
Figure 4.4 Optical density readings of bound RBC in plate-based assay. Typical 
binding profile of selected (A2, B2) and unselected 3D7 parasites to blood 
group constructs (A-BSA, B-BSA and H-BSA) following 3 rounds of 
selection by panning. Error bars denote standard deviation (SD) of duplicate 
readings from the same experiment. 
 
 
To confirm the presence and availability of epitopes and to test the specificity of the 
blood group constructs used in the adhesion assays, monoclonal antibodies against 
blood group A and B polysaccharides were used. Panels A and B of Figure 4.5 show 
58 
 
Optical Density (OD) @ 450nm 
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clear, specific binding of the respective specific antibody to the A-BSA and B-BSA 
constructs. On the other hand, the monoclonal antibodies against blood group A do not 
recognize or bind B-BSA and vice versa. 
 
 
A B
1.4 1.4
Anti A on A-BSA Anti A on B-BSA
Anti B on A-BSA Anti B on B-BSA
1.2 1.2
1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
10µl 5µl 2,5µl 1,25µl 10µl 5µl 2,5µl 1,25µl
Antibody added/well
 
Figure 4.5 Reactivity of mouse monoclonal anti blood group A and B antibody with 
A-BSA and B-BSA constructs by ELISA. Panel A shows ODs of reactivity 
with blood group A-BSA construct, panel B shows reactivity with the B-
BSA construct. The grey bars show OD of secondary antibody alone. Error 
bars are SD of duplicate readings 
 
 
Additionally, it was demonstrated that these monoclonal antibodies to the blood group 
constructs could inhibit the specific binding of IE of selected parasites to the constructs 
(Figure 4.6). With no inhibiting antibodies added, the adhesion to both A-BSA and B-
BSA constructs remained high at 60% and 58.5% respectively (White bars). This 
59 
 
Optical density (OD)
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dropped to 1% with the addition of anti A antibody and to 15.6% when anti B antibody 
was added. The addition of anti B antibody appeared to have no effect on the binding of 
the parasite isolate (B-selected) to A-BSA (Grey bar), while the anti A antibody reduced 
the binding to the B-BSA to 43.8%. Adhesion to the H-BSA construct with no added 
antibody was 6%. 
 
 
70
no antibody added
5uL anti A added
60 5uL anti B added
50
40
30
20
10
0
H-BSA A-BSA B-BSA
construct  
Figure 4.6 Inhibition of blood group B selected parasite adhesion to constructs by 
specific anti A- and B antibody. White bars represent adhesion to 
constructs with no antibody added, Black bars: adhesion with 5µL anti A 
antibody added, Grey bars: adhesion with 5µL of anti B antibody added. 
Error bars show SD of multiple readings 
 
 
60 
 
Percent adhesion
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4.2.1 Characteristics of binding isolates 
Binding of parasites to the H antigen was no different between 3D7, A-BSA selected or 
B-BSA selected parasites, and as expected, the unselected 3D7 parasites also showed no 
preference for binding to any of the 3 blood group antigens. 
A-BSA selected parasites bound to blood group A polysaccharides 7 to 8 times more 
than unselected 3D7 parasites. The binding of parasites selected on B-BSA to the group 
B construct was usually slightly less, at 2 to 4 times more than the binding observed in 
the unselected 3D7 (Figure 4.7). 
It was noted that selection of P. falciparum laboratory strains on either A or B blood 
group antigens always resulted in the selected parasites binding to both A and B 
constructs (Figure 4.4 and 4.7). In addition, the selected isolates bound better to the 
blood group A antigen than to the B antigen, with the binding of selected parasites to 
the blood group A polysaccharide usually about 1.5 times more than the binding to the 
blood group B polysaccharide. 
Binding of all parasite isolates to the H antigen was observed to be quite weak, and 
attempts to select for binding to this construct following two rounds of selection yielded 
no clear result (Figure 4.7). 
The H antigen construct was supplied with a 3 atom spacer arm, while the A and B 
constructs were linked with a 6 atom spacer to BSA (Section 3.5). To determine 
whether the lack of binding to the H antigen was due to the shorter spacer arm of this 
construct, binding to a B construct with the same shorter arm was tested. As Figure 4.8 
shows, A and B selected parasites of 3D7 and FMG bound to both constructs (B3 and 
B6), albeit with consistently lower binding to the B-construct with the shorter spacer 
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(32-68%). Interestingly, selected FMG parasites on the other hand bound both B 
constructs equally well (100-105%). These results indicate that the shorter spacer arm of 
the H construct does not explain the lack of binding seen. 
 
 
80
BSA 
BSA-A 
BSA-B 
BSA-H 
60
40
20
0
RBC 3D7 u 3D7 BGH2 3D7 BGA2 3D7 BGB2
RBC added  
Figure 4.7 Weak binding to the H-BSA (blood group O) construct by all parasites. 
Percentage binding of parasites selected after 2 rounds of panning on H-BSA, A-
BSA, and B-BSA constructs (BGH2, BGA2 and BGB2 respectively). Low level 
binding of IE to H-BSA was observed for all selected parasites (yellow bars). 
rd
BGH2 parasites (3  group of columns), showed no preference for binding to any 
of the constructs, even for H-BSA (less than 15%). Error bars denote standard 
deviation of duplicate measurements. 
 
 
  
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Binding (% of total)
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0.7
BSA
0.6 A6
B6
0.5 B3
H3
0.4
0.3
0.2
0.1
0
3D7 3D7 A 3D7 B FMG A FMG B
Parasite strain  
Figure 4.8 A-BSA and B-BSA selected parasites of 3D7 and FMG bind both the 3- and 
6-spacer arm constructs of blood group B. Selected parasites 3D7 A and 3D7 
B show better binding to the 6-spacer arm construct of B-BSA, B6 (green bars) 
than to the 3-spacer arm construct B3 (purple bars). Error bars represent standard 
deviation of duplicate readings 
 
 
Subsequently, to investigate if phenotypic changes caused by selection on A or B 
antigen are a general phenomenon or limited to some strains, the experiments were 
repeated using other established laboratory strains of P. falciparum: FCR3, FMG, FUP 
and HB3. As observed with the 3D7 strain, selection of the FMG and FUP strains on the 
blood type sugars was successful and resulted in binding that was several times higher 
than for the corresponding unselected parental parasites. In contrast, FCR3 and HB3 
parasites, even after 3 and up to 6 panning cycles respectively, did not show marked 
differences from the unselected stock (Figure 4.9). 
 
63 
 
Optical Density (OD) @ 450nm 
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100
BSA 
BSA-A 
BSA-B 
80 BSA-H 
60
40
20
0
3D7u A4 B4 Hb3u A6 B6 FCR3uA3 B3 FMGu A5 B5 FUPu A4 B4
Parasite  
Figure 4.9 Percentage adhesion of selected and unselected 3D7, HB3, FCR3, FMG 
and FUP parasites. Following a minimum of 3 rounds of selection, blood 
group A and B selected parasites of 3D7 (A4, B4), FMG (A5, B5) and FUP 
(A4, B4 last 2 cluster of columns) show binding several fold higher than 
unselected parasites 3D7u, FMGu and FUPu respectively. Error bars 
represent standard deviation of triplicate readings. 
 
 
Over time, the phenotype of selected parasites may diminish gradually. To investigate 
this phenomenon in the current study, the selected laboratory strains were monitored 
using this new assay method and re-selected by panning periodically to restore an 
optimum binding phenotype in the selected parasites. Older cultures maintained over 12 
weeks without subsequent selection were tested in comparison to the more recently 
selected parasites. 
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Binding (% of total)
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A comparison of the binding capacity of selected parasites over a period of 4 months 
(16 weeks) without further reselection indicated that these selected parasites 
consistently bound to the A- and B-BSA constructs over the period, thus maintaining 
the acquired binding phenotype. This was in contrast to binding to BSA and the blood 
group O antigen construct H-BSA which was, as expected, recorded at low levels (less 
than 15%) over the same period (Figure 4.10). 
 
 
Figure 4.10 Phenotype stability over a 16-week period. Binding of A-BSA selected 
parasites of 3D7 to BSA and H-BSA (open diamond and black circles 
respectively) remain at low levels over the period, while binding to the A- 
and B-BSA constructs (open triangles and black squares respectively) are 
maintained at between 50-90%. 
 
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4.2.2 Binding of selected parasites to primary human ECs 
Selected parasites (3D7, HB3 and FMG) were tested on ECs to investigate binding 
characteristics of IE containing these parasites on human aorta and dermal ECs. 
All three parasite strains showed some binding to both aorta and dermal EC, with 3D7 
and FMG selected parasites binding at a higher rate than their unselected isogenic lines. 
The HB3 strain however showed no difference between selected and unselected 
parasites in binding to the EC (Figure 4.11) 
 
medium 
Aorta EC 
50 Dermal EC 
25
0
3D7' 3D7-A4 3D7-B4 Hb3 Hb3-A5 Hb3-B5 FMG FMG-A5 FMG-B5
 
Figure 4.11 Adhesion of A and B selected parasites to human Aorta and Dermal 
endothelial cells (ECs). Parasites selected on A and B carbohydrates 
adhered better (3D7-A4 & B4, FMG-A5 & B5) to the ECs than unselected 
lines, except for HB3, where selection (Hb3-A5, Hb3-B5) does not seem to 
affect binding characteristics. Error bars indicate standard deviation of 
duplicate measurements 
 
 
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Adhesion (%)
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4.2.3 Comparison of adhesion and rosetting in selected parasites 
Blood group A and B selected 3D7, FMG and FUP parasites previously tested for 
binding to BSA-coupled blood group oligosaccharides were also tested for rosetting 
using RBC from donors of blood type O, A and AB. Blood groups A and B selected 
parasites bound significantly better than the corresponding unselected 3D7, FMG and 
FUP parasites, with unselected parasites binding at 20% or below (Figure 4.12). Results 
from the rosetting assays also showed rosetting only in selected parasites and in non-O 
RBCs (blood groups A and AB RBC). Rosetting was not observed in unselected 3D7, 
FMG or FUP parasites (Figure 4.12). 
 
  
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Figure 4.12 Adhesion and rosetting characteristics of selected and unselected 3D7, 
FMG and FUP parasite strains. Blood group A and B-selected parasites of 
all strains had much higher adhesion and formed rosettes, while the 
corresponding unselected parasites showed much lower adhesion and formed 
no rosettes in donor RBC from blood type A, AB and O. The error bars on 
the adhesion panel show the standard deviation of duplicate readings. 
 
  
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4.3 PfEMP1 and RIFIN expression patterns in selected P. falciparum 
laboratory parasite strains 
The var and rif expression profiles of ring stage parasites selected on blood group 
antigens (Section 3.6) were investigated in 3D7 and FMG following storage in TRIzol 
and subsequent RNA extraction, cDNA generation and QPCR experiments and 
analysis. 
 
4.3.1 QPCR var and rif transcripts in 3D7 selected and unselected parasites 
To determine if any of the 58 var or 154 rif genes of 3D7 were expressed at a higher 
level in the blood group A and B adhesive parasites compared to the unselected control, 
QPCR experiments including primer sets designed to detect all the known var and rif 
sequences of 3D7 were performed. The experiments included cDNA prepared from 
both the A-BSA and B-BSA selected 3D7 following 3 rounds of panning, as well as 
from the unselected parental 3D7 that had been cultured in parallel. The results show 
that there was no single var gene that was clearly dominant before or after selection 
(Figure 4.13) 
One var gene, namely PF13_0003, was transcribed at a slightly higher rate in both 
selected lines compared to the control, with fold increases of 1.3 and 1.7 respectively in 
transcription for the A-BSA and the B-BSA selected lines (Table 4.2). With the 
exception of PFE1640w/3D7var1 that showed a minor increase in transcription in the 
A-BSA selected 3D7 (but not in the B-BSA selected), transcription of all other var 
genes was decreased in the selected parasites. Therefore, the transcription of PF13_0003 
was more prominent relative to the other var genes in the selected parasites. 
th
Consequently, in unselected 3D7 this transcript was the 10  most highly transcribed var 
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gene, constituting 3.5% of all var transcripts, whereas in the A and B selected lines it 
was the most dominant or second most dominant var gene constituting 18.4% and 
18.2% of var transcripts respectively (Figure 4.13 and Table 4.2). 
 
  
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60
PFL0030c 
PF13_0003 
PFC0005w 
50 PFD0020c 
PFD0625c 
PFD1235w 
MAL6P1.4 
MAL6P1.1 
PFD0995c 
PF08_0140 
40
30
20
10
0
unselected A3 B3 unselected A9
Parasite line  
Figure 4.13 Transcription levels of var genes in blood group antigen-selected and 
unselected 3D7 parasites. Transcripts of var genes relative to housekeeping 
genes as measured by QPCR are shown, with the 10 highest average 
transcripts indicated in colour (also indicated by the same colour scheme in 
Table 4.2) 
 
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transcripts relative to housekeeping
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Table 4.2 Transcription levels of 58 var genes of 3D7, in 3D7 cultures 
Table 4.2   3D7 (exp 1) 3D7 BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
PFE1640w A/var1 2324 0.03 0.05 3466 0.04 0.29 1.55 854 0.01 0.07 0.54 7264 0.01 0.02 5100 0.01 0.02 0.82 
PFL0030c var2 172825 1.97 3.43 34066 0.40 2.82 0.20 66002 1.10 5.75 0.56 1709906 2.33 4.96 257683 0.41 1.21 0.18 
PF11_0008 A/DC5 70931 0.81 1.41 18407 0.22 1.52 0.27 12840 0.21 1.12 0.27 1019305 1.39 2.95 233575 0.37 1.10 0.27 
PF13_0003 A/DC5 176254 2.01 3.50 221654 2.61 18.35 1.30 208968 3.49 18.19 1.74 301536 0.41 0.87 2669053 4.24 12.57 10.34 
MAL6P1.314 A 74746 0.85 1.48 24394 0.29 2.02 0.34 29689 0.50 2.58 0.58 1395762 1.90 4.05 153609 0.24 0.72 0.13 
PFI1820w A 33403 0.38 0.66 7030 0.08 0.58 0.22 12840 0.21 1.12 0.56 324057 0.44 0.94 150621 0.24 0.71 0.54 
PFA0015c A 25875 0.29 0.51 7654 0.09 0.63 0.31 7506 0.13 0.65 0.43 1032742 1.41 2.99 160814 0.26 0.76 0.18 
PFD1235w A 200917 2.29 3.99 22699 0.27 1.88 0.12 19912 0.33 1.73 0.15 967282 1.32 2.80 711033 1.13 3.35 0.86 
PFD0020c A 309538 3.52 6.15 98408 1.16 8.15 0.33 35664 0.60 3.11 0.17 5485119 7.47 15.90 505833 0.80 2.38 0.11 
PF08_0140 DC8 193176 2.20 3.84 21682 0.26 1.79 0.12 12591 0.21 1.10 0.10 986472 1.34 2.86 588053 0.94 2.77 0.70 
MAL6P1.316   119770 1.36 2.38 21260 0.25 1.76 0.18 15224 0.25 1.33 0.19 540065 0.74 1.57 423859 0.67 2.00 0.92 
PF11_0521   179751 2.05 3.57 21682 0.26 1.79 0.13 14830 0.25 1.29 0.12 754197 1.03 2.19 282423 0.45 1.33 0.44 
PF08_0141   227537 2.59 4.52 34066 0.40 2.82 0.16 9439 0.16 0.82 0.06 657299 0.89 1.91 443741 0.71 2.09 0.79 
PFL0020w   8783 0.10 0.17 2371 0.03 0.20 0.28 2149 0.04 0.19 0.36 70931 0.10 0.21 11563 0.02 0.05 0.19 
MAL6P1.4   244531 2.78 4.86 38077 0.45 3.15 0.16 34066 0.57 2.97 0.20 832038 1.13 2.41 1117169 1.78 5.26 1.57 
PFA0005w   52828 0.60 1.05 42007 0.50 3.48 0.82 29689 0.50 2.58 0.83 164005 0.22 0.48 837505 1.33 3.95 5.97 
PF10_0001   34515 0.39 0.69 3823 0.05 0.32 0.11 4808 0.08 0.42 0.20 967282 1.32 2.80 62633 0.10 0.30 0.08 
PF10_0406   31907 0.36 0.63 9195 0.11 0.76 0.30 9689 0.16 0.84 0.45 81923 0.11 0.24 76229 0.12 0.36 1.09 
PF11_0007   73292 0.83 1.46 16148 0.19 1.34 0.23 20710 0.35 1.80 0.42 389270 0.53 1.13 865380 1.38 4.08 2.60 
PFL0935c   7858 0.09 0.16 3676 0.04 0.30 0.49 1973 0.03 0.17 0.37 32116 0.04 0.09 16795 0.03 0.08 0.61 
PFL2665c   30278 0.34 0.60 10012 0.12 0.83 0.34 8119 0.14 0.71 0.39 230536 0.31 0.67 159765 0.25 0.75 0.81 
PFL0005w   20983 0.24 0.42 4904 0.06 0.41 0.24 3225 0.05 0.28 0.23 84097 0.11 0.24 44267 0.07 0.21 0.61 
PF13_0364   34515 0.39 0.69 8500 0.10 0.70 0.26 8389 0.14 0.73 0.36 79284 0.11 0.23 60616 0.10 0.29 0.89 
PFB1055c   37337 0.42 0.74 9563 0.11 0.79 0.27 6938 0.12 0.60 0.27 157686 0.21 0.46 326186 0.52 1.54 2.42 
PFB0010w   9255 0.11 0.18 4715 0.06 0.39 0.53 3310 0.06 0.29 0.53 3225 0.00 0.01 47885 0.08 0.23 17.35 
PFC1120c   72814 0.83 1.45 16043 0.19 1.33 0.23 11413 0.19 0.99 0.23 217342 0.30 0.63 131270 0.21 0.62 0.71 
PFC0005w   155634 1.77 3.09 19145 0.23 1.58 0.13 20175 0.34 1.76 0.19 2356802 3.21 6.83 305511 0.49 1.44 0.15 
PFD0005w   99706 1.13 1.98 14830 0.17 1.23 0.15 10550 0.18 0.92 0.16 271542 0.37 0.79 145769 0.23 0.69 0.63 
PFD1245c   5852 0.07 0.12 2294 0.03 0.19 0.41 1146 0.02 0.10 0.29 15628 0.02 0.05 13890 0.02 0.07 1.04 
PFE0005w   16905 0.19 0.34 3849 0.05 0.32 0.24 1935 0.03 0.17 0.17 56035 0.08 0.16 40125 0.06 0.19 0.84 
PF08_0142   22257 0.25 0.44 5410 0.06 0.45 0.25 4808 0.08 0.42 0.32 105759 0.14 0.31 52140 0.08 0.25 0.58 
PF07_0139   84649 0.96 1.68 25875 0.31 2.14 0.32 21967 0.37 1.91 0.38 483170 0.66 1.40 223110 0.35 1.05 0.54 
PFI1830c   52483 0.60 1.04 13620 0.16 1.13 0.27 9626 0.16 0.84 0.27 164005 0.22 0.48 141074 0.22 0.66 1.00 
PF13_0001   23454 0.27 0.47 6047 0.07 0.50 0.27 4593 0.08 0.40 0.29 102352 0.14 0.30 69551 0.11 0.33 0.79 
MAL6P1.1   464554 5.29 9.22 123757 1.46 10.24 0.28 232051 3.88 20.20 0.73 2419350 3.29 7.01 2280887 3.63 10.75 1.10 
PFI0005w   31286 0.36 0.62 9882 0.12 0.82 0.33 7264 0.12 0.63 0.34 84649 0.12 0.25 74258 0.12 0.35 1.02 
PFA0765c   42841 0.49 0.85 13268 0.16 1.10 0.32 8119 0.14 0.71 0.28 52140 0.07 0.15 28174 0.04 0.13 0.63 
PFL1950w   55306 0.63 1.10 11488 0.14 0.95 0.22 7216 0.12 0.63 0.19 54945 0.07 0.16 83002 0.13 0.39 1.76 
PFL1955w   97128 1.11 1.93 13095 0.15 1.08 0.14 7408 0.12 0.64 0.11 213114 0.29 0.62 71397 0.11 0.34 0.39 
PFD0635c   14259 0.16 0.28 1675 0.02 0.14 0.12 2685 0.04 0.23 0.28 22257 0.03 0.06 18168 0.03 0.09 0.95 
PF07_0050   230536 2.62 4.58 4358 0.05 0.36 0.02 6759 0.11 0.59 0.04 278748 0.38 0.81 127041 0.20 0.60 0.53 
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Table 4.2continued 3D7 (exp 1) 3D7BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
MAL7P1.55   28359 0.32 0.56 6414 0.08 0.53 0.23 9075 0.15 0.79 0.47 95864 0.13 0.28 51462 0.08 0.24 0.63 
PFD1005c   40654 0.46 0.81 4872 0.06 0.40 0.12 3977 0.07 0.35 0.14 62224 0.08 0.18 57900 0.09 0.27 1.09 
PF08_0106   15526 0.18 0.31 5445 0.06 0.45 0.36 3398 0.06 0.30 0.32 74258 0.10 0.22 140153 0.22 0.66 2.20 
PF08_0103   48517 0.55 0.96 13799 0.16 1.14 0.29 13095 0.22 1.14 0.40 246138 0.34 0.71 2040598 3.24 9.61 9.69 
MAL7P1.50   100033 1.14 1.99 10311 0.12 0.85 0.11 8093 0.14 0.70 0.12 331570 0.45 0.96 1761042 2.80 8.30 6.20 
PFL1960w   24716 0.28 0.49 4416 0.05 0.37 0.19 2279 0.04 0.20 0.14 176254 0.24 0.51 64295 0.10 0.30 0.43 
PFD0615c   33623 0.38 0.67 18049 0.21 1.49 0.56 13531 0.23 1.18 0.59 223110 0.30 0.65 83548 0.13 0.39 0.44 
PFD0625c   505833 5.75 10.04 122949 1.45 10.18 0.25 115912 1.94 10.09 0.34 2403559 3.27 6.97 2251209 3.58 10.61 1.09 
PFD1000c   77234 0.88 1.53 8172 0.10 0.68 0.11 9439 0.16 0.82 0.18 369403 0.50 1.07 100361 0.16 0.47 0.32 
PFD1015c   39088 0.44 0.78 4003 0.05 0.33 0.11 5305 0.09 0.46 0.20 76730 0.10 0.22 66002 0.10 0.31 1.00 
MAL6P1.252   11870 0.14 0.24 2309 0.03 0.19 0.20 1686 0.03 0.15 0.21 55669 0.08 0.16 30477 0.05 0.14 0.64 
PF07_0048   29112 0.33 0.58 4623 0.05 0.38 0.16 5776 0.10 0.50 0.29 246138 0.34 0.71 101684 0.16 0.48 0.48 
PF07_0049   57522 0.65 1.14 9377 0.11 0.78 0.17 5481 0.09 0.48 0.14 588053 0.80 1.70 147691 0.23 0.70 0.29 
MAL7P1.56   29495 0.34 0.59 6207 0.07 0.51 0.22 6499 0.11 0.57 0.32 141074 0.19 0.41 131270 0.21 0.62 1.09 
PF07_0051   48200 0.55 0.96 6127 0.07 0.51 0.13 3183 0.05 0.28 0.10 140153 0.19 0.41 53524 0.09 0.25 0.45 
PF08_0107   12508 0.14 0.25 810 0.01 0.07 0.07 584 0.01 0.05 0.07 116674 0.16 0.34 14830 0.02 0.07 0.15 
PFD0995c   91569 1.04 1.82 6007 0.07 0.50 0.07 4082 0.07 0.36 0.07 3979560 5.42 11.54 114404 0.18 0.54 0.03 
Total 
  5036654 57.30 100.00 1207986 14.25 100.00 0.25 1148536 19.20 100.00 0.34 34497669 46.95 100.00 21227210 33.75 100.00 0.72 
var 
House-keeping   87897     84788       59827       734699     628878       
 
Classification as UPS-A var genes and Domain Cassette (DC) group according to Rask et al. (2010). For all cultures tested the absolute number of detected 
transcripts (Abs #), the number of transcripts relative to the average transcript number of two house-keeping genes (relative) and the percentage of all var 
transcripts detected (%) are given. For the selected parasites, fold change in transcription relative to the unselected control parasite is given (fold). The 10 most 
highly transcribed genes are highlighted in colors corresponding to the color-coding of Figure 4.13. 
 
 
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In the first experiment, the total transcription of rif genes were approximately two-fold 
lower in the selected parasites than in the control (Figure 4.14). For the A-BSA selected 
parasite there was no obviously dominant rif transcript, whereas one gene, PF13_0004, 
was dominant in the B-BSA selected 3D7 (constituting 17.7% of all rif transcripts and 
1.3 fold upregulated relative to the control parasite (Table 4.3)). In all three 3D7 
parasite lines, approximately 2/3 of the rif transcripts were of group A. The PFA0020w 
group A RIFIN reported by Goel et al. (2015) to be expressed in blood group A-
preferring rosetting 3D7, was transcribed at a very low level in all the 3 sublines of 
3D7. 
To confirm the results obtained in the first QPCR experiment, a second experiment 
including only unselected and A-BSA selected 3D7 was done. Seen in isolation, the 
positive fold change observed for PF13_0003 in the first experiment appeared to be 
much more prominent with a 10.3-fold higher transcription of the gene in A-BSA 
selected versus unselected 3D7 (Table 4.2). However, comparing the level of 
transcription with the first experiment, it was obvious that transcription had decreased in 
the unselected 3D7 but had been maintained at roughly the same level in the A-BSA 
selected line (Figure 4.13, yellow stacks, and Table 4.2). Consistent with the first 
experiment, no single dominant var transcript was detected. The overall decrease in 
transcription in the selected versus unselected line was much less apparent than in the 
first experiment. For the rif genes it now appeared that the PF13_0004 gene was now 
very dominant in the A-BSA selected parasite, responsible for more than 1/3 of all rif 
transcripts, whereas it was not prominent in the first experiment (Table 4.3). 
Two genes, not very prominently transcribed in the first experiment, were now clearly 
dominant in the unselected 3D7: PF11_0009 and PFD0025w, representing 19.5% and 
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14.5% of the total rif transcription respectively. No decrease in total transcription of rif 
genes was observed in the second experiment. 
 
  
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2.5
2.0
1.5
1.0
0.5
0.0
3D7u 3D7-A3 3D7-B3 3D7u 3D7-A9
parasite  
Figure 4.14 The rif transcripts of selected and unselected parasites. Relative to the 
housekeeping gene, total rif gene transcription was less in selected parasites 
(3D7-A3 and 3D7-B3) than in the unselected (3D7u)  
 
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Table 4.3 Transcription of the 154 rif genes of 3D7, in five 3D7 cultures 
Table 4.3   3D7 (exp 1) 3D7 BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
MAL13P1.2 A 776 0.0153 0.66 1714 0.0219 2.16 1.44 1319 0.0205 1.81 1.34 6649 0.0121 0.94 16307 0.0287 2.02 2.39 
MAL13P1.4 A 178 0.0035 0.15 206 0.0026 0.26 0.75 130 0.0020 0.18 0.58 687 0.0012 0.10 1313 0.0023 0.16 1.86 
MAL13P1.500 A 122 0.0024 0.10 51 0.0007 0.06 0.27 43 0.0007 0.06 0.28 184 0.0003 0.03 363 0.0006 0.04 1.92 
MAL13P1.515 A 9 0.0002 0.01 6 0.0001 0.01 0.45 4 0.0001 0.01 0.33 95 0.0002 0.01 252 0.0004 0.03 2.56 
MAL13P1.520 A 2 0.0000 0.00 3 0.0000 0.00 0.77 1 0.0000 0.00 0.35 284 0.0005 0.04 193 0.0003 0.02 0.66 
MAL13P1.535 A 5487 0.1079 4.64 2436 0.0312 3.07 0.29 3853 0.0598 5.29 0.55 50516 0.0916 7.13 42608 0.0751 5.27 0.82 
MAL7P1.184 A 199 0.0039 0.17 130 0.0017 0.16 0.42 11 0.0002 0.02 0.04 704 0.0013 0.10 265 0.0005 0.03 0.37 
MAL7P1.185 A 214 0.0042 0.18 189 0.0024 0.24 0.58 95 0.0015 0.13 0.35 1313 0.0024 0.19 836 0.0015 0.10 0.62 
MAL7P1.213 A 678 0.0133 0.57 674 0.0086 0.85 0.65 235 0.0036 0.32 0.27 1357 0.0025 0.19 2399 0.0042 0.30 1.72 
MAL7P1.216 A 343 0.0067 0.29 198 0.0025 0.25 0.38 75 0.0012 0.10 0.17 434 0.0008 0.06 783 0.0014 0.10 1.75 
MAL7P1.217 A 62 0.0012 0.05 69 0.0009 0.09 0.72 36 0.0006 0.05 0.46 248 0.0004 0.03 367 0.0006 0.05 1.44 
MAL7P1.222 A 113 0.0022 0.10 100 0.0013 0.13 0.58 95 0.0015 0.13 0.66 895 0.0016 0.13 487 0.0009 0.06 0.53 
MAL7P1.57 A 740 0.0145 0.63 397 0.0051 0.50 0.35 209 0.0032 0.29 0.22 1551 0.0028 0.22 1901 0.0034 0.24 1.19 
MAL8P1.208 A 1244 0.0244 1.05 1056 0.0135 1.33 0.55 1009 0.0157 1.39 0.64 7883 0.0143 1.11 7288 0.0128 0.90 0.90 
MAL8P1.218 A 750 0.0147 0.63 540 0.0069 0.68 0.47 424 0.0066 0.58 0.45 2015 0.0037 0.28 1887 0.0033 0.23 0.91 
MAL8P1.219 A 311 0.0061 0.26 336 0.0043 0.42 0.70 251 0.0039 0.34 0.64 1775 0.0032 0.25 2189 0.0039 0.27 1.20 
PF07_0003 A 312 0.0061 0.26 78 0.0010 0.10 0.16 153 0.0024 0.21 0.39 976 0.0018 0.14 398 0.0007 0.05 0.40 
PF07_0132 A 130 0.0025 0.11 113 0.0014 0.14 0.57 86 0.0013 0.12 0.53 413 0.0007 0.06 316 0.0006 0.04 0.74 
PF07_0134 A 220 0.0043 0.19 109 0.0014 0.14 0.32 65 0.0010 0.09 0.23 688 0.0012 0.10 306 0.0005 0.04 0.43 
PF07_0138 A 71 0.0014 0.06 63 0.0008 0.08 0.58 68 0.0011 0.09 0.76 405 0.0007 0.06 405 0.0007 0.05 0.97 
PF08_0104 A 165 0.0032 0.14 145 0.0019 0.18 0.57 114 0.0018 0.16 0.55 434 0.0008 0.06 3372 0.0059 0.42 7.55 
PF08_0138 A 75 0.0015 0.06 56 0.0007 0.07 0.49 28 0.0004 0.04 0.30 424 0.0008 0.06 294 0.0005 0.04 0.67 
PF08_0139 A 3656 0.0719 3.09 2907 0.0372 3.67 0.52 1510 0.0235 2.07 0.33 8075 0.0146 1.14 20598 0.0363 2.55 2.48 
PF10_0004 A 141 0.0028 0.12 107 0.0014 0.13 0.49 55 0.0009 0.08 0.31 406 0.0007 0.06 225 0.0004 0.03 0.54 
PF10_0393 A 129 0.0025 0.11 54 0.0007 0.07 0.27 35 0.0005 0.05 0.21 288 0.0005 0.04 401 0.0007 0.05 1.36 
PF10_0396 A 789 0.0155 0.67 472 0.0060 0.59 0.39 115 0.0018 0.16 0.12 1142 0.0021 0.16 1084 0.0019 0.13 0.92 
PF10_0398 A 455 0.0089 0.38 1024 0.0131 1.29 1.47 297 0.0046 0.41 0.52 1384 0.0025 0.20 4499 0.0079 0.56 3.16 
PF10_0400 A 60 0.0012 0.05 53 0.0007 0.07 0.58 22 0.0003 0.03 0.29 91 0.0002 0.01 163 0.0003 0.02 1.74 
PF10_0403 A 227 0.0045 0.19 194 0.0025 0.24 0.56 88 0.0014 0.12 0.31 77 0.0001 0.01 314 0.0006 0.04 3.95 
PF10_0405 A 1783 0.0350 1.51 2624 0.0336 3.31 0.96 1554 0.0241 2.13 0.69 3616 0.0066 0.51 6187 0.0109 0.77 1.66 
PF11_0009 A 3786 0.0744 3.20 488 0.0062 0.61 0.08 1574 0.0244 2.16 0.33 137877 0.2499 19.46 34178 0.0603 4.23 0.24 
PF11_0010 A 184 0.0036 0.16 107 0.0014 0.13 0.38 195 0.0030 0.27 0.84 2901 0.0053 0.41 763 0.0013 0.09 0.26 
PF11_0021 A 411 0.0081 0.35 398 0.0051 0.50 0.63 358 0.0056 0.49 0.69 1042 0.0019 0.15 1311 0.0023 0.16 1.22 
PF11_0520 A 2109 0.0415 1.78 189 0.0024 0.24 0.06 405 0.0063 0.56 0.15 20598 0.0373 2.91 10289 0.0181 1.27 0.49 
PF11_0529 A 743 0.0146 0.63 319 0.0041 0.40 0.28 724 0.0112 0.99 0.77 5369 0.0097 0.76 6080 0.0107 0.75 1.10 
PF13_0004 A 8022 0.1577 6.79 2851 0.0365 3.59 0.23 12854 0.1996 17.66 1.27 32789 0.0594 4.63 279053 0.4919 34.51 8.28 
PF13_0005 A 1105 0.0217 0.94 792 0.0101 1.00 0.47 856 0.0133 1.18 0.61 2838 0.0051 0.40 14879 0.0262 1.84 5.10 
PF14_0004 A 13 0.0003 0.01 8 0.0001 0.01 0.39 4 0.0001 0.01 0.22 35 0.0001 0.01 16 0.0000 0.00 0.43 
PF14_0006 A 278 0.0055 0.24 193 0.0025 0.24 0.45 315 0.0049 0.43 0.89 4931 0.0089 0.70 1834 0.0032 0.23 0.36 
PF14_0769 A 224 0.0044 0.19 163 0.0021 0.21 0.48 255 0.0040 0.35 0.90 1818 0.0033 0.26 472 0.0008 0.06 0.25 
PF14_0772 A 624 0.0123 0.53 160 0.0020 0.20 0.17 296 0.0046 0.41 0.37 7084 0.0128 1.00 1170 0.0021 0.14 0.16 
77 
 
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Table 4.3continued 3D7 (exp 1) 3D7 BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
PFA0010c A 934 0.0184 0.79 768 0.0098 0.97 0.54 687 0.0107 0.94 0.58 6449 0.0117 0.91 5264 0.0093 0.65 0.79 
PFA0020w A 398 0.0078 0.34 559 0.0072 0.71 0.92 302 0.0047 0.42 0.60 1372 0.0025 0.19 945 0.0017 0.12 0.67 
PFA0040w A 83 0.0016 0.07 51 0.0006 0.06 0.40 38 0.0006 0.05 0.36 825 0.0015 0.12 229 0.0004 0.03 0.27 
PFA0045c A 94 0.0018 0.08 57 0.0007 0.07 0.39 57 0.0009 0.08 0.48 831 0.0015 0.12 748 0.0013 0.09 0.88 
PFA0050c A 220 0.0043 0.19 176 0.0023 0.22 0.52 100 0.0016 0.14 0.36 1198 0.0022 0.17 1065 0.0019 0.13 0.86 
PFA0080c A 847 0.0166 0.72 719 0.0092 0.91 0.55 583 0.0091 0.80 0.54 4618 0.0084 0.65 3895 0.0069 0.48 0.82 
PFA0740w A 69 0.0014 0.06 14 0.0002 0.02 0.14 14 0.0002 0.02 0.16 138 0.0002 0.02 336 0.0006 0.04 2.37 
PFA0760w A 1799 0.0354 1.52 1527 0.0195 1.93 0.55 934 0.0145 1.28 0.41 6796 0.0123 0.96 7022 0.0124 0.87 1.00 
PFB0030c A 22 0.0004 0.02 97 0.0012 0.12 2.90 54 0.0008 0.07 1.94 73 0.0001 0.01 256 0.0005 0.03 3.42 
PFB0060w A 75 0.0015 0.06 165 0.0021 0.21 1.43 46 0.0007 0.06 0.48 111 0.0002 0.02 206 0.0004 0.03 1.81 
PFB1010w A 18 0.0004 0.02 27 0.0003 0.03 0.97 12 0.0002 0.02 0.53 79 0.0001 0.01 51 0.0001 0.01 0.64 
PFB1050w A 2109 0.0415 1.78 2237 0.0286 2.82 0.69 1360 0.0211 1.87 0.51 6506 0.0118 0.92 16924 0.0298 2.09 2.53 
PFC0010c A 1524 0.0299 1.29 722 0.0092 0.91 0.31 926 0.0144 1.27 0.48 5463 0.0099 0.77 8986 0.0158 1.11 1.60 
PFC0040w A 206 0.0041 0.17 264 0.0034 0.33 0.84 79 0.0012 0.11 0.30 254 0.0005 0.04 550 0.0010 0.07 2.11 
PFC1095w A 74 0.0015 0.06 45 0.0006 0.06 0.40 30 0.0005 0.04 0.32 120 0.0002 0.02 262 0.0005 0.03 2.12 
PFC1115w A 1338 0.0263 1.13 1129 0.0144 1.42 0.55 728 0.0113 1.00 0.43 4643 0.0084 0.66 10255 0.0181 1.27 2.15 
PFD0025w A 3001 0.0590 2.54 653 0.0084 0.82 0.14 1088 0.0169 1.49 0.29 102352 0.1855 14.45 17468 0.0308 2.16 0.17 
PFD0030c A 2708 0.0532 2.29 731 0.0094 0.92 0.18 368 0.0057 0.50 0.11 22898 0.0415 3.23 3985 0.0070 0.49 0.17 
PFD0040c A 89 0.0017 0.07 152 0.0019 0.19 1.11 54 0.0008 0.07 0.48 418 0.0008 0.06 249 0.0004 0.03 0.58 
PFD0045c A 205 0.0040 0.17 107 0.0014 0.14 0.34 51 0.0008 0.07 0.20 306 0.0006 0.04 306 0.0005 0.04 0.97 
PFD0050w A 38 0.0007 0.03 19 0.0002 0.02 0.33 6 0.0001 0.01 0.13 10 0.0000 0.00 6 0.0000 0.00 0.56 
PFD0055w A 155 0.0030 0.13 61 0.0008 0.08 0.26 27 0.0004 0.04 0.14 101 0.0002 0.01 109 0.0002 0.01 1.05 
PFD0060w A 283 0.0056 0.24 329 0.0042 0.41 0.76 80 0.0012 0.11 0.22 363 0.0007 0.05 520 0.0009 0.06 1.39 
PFD0640c A 34 0.0007 0.03 19 0.0002 0.02 0.35 10 0.0002 0.01 0.24 32 0.0001 0.00 55 0.0001 0.01 1.70 
PFD0645w A 1701 0.0334 1.44 731 0.0094 0.92 0.28 403 0.0063 0.55 0.19 14012 0.0254 1.98 5602 0.0099 0.69 0.39 
PFD1230c A 52 0.0010 0.04 3 0.0000 0.00 0.04 6 0.0001 0.01 0.10 127 0.0002 0.02 141 0.0002 0.02 1.08 
PFD1240w A 3660 0.0719 3.10 1542 0.0197 1.94 0.27 1275 0.0198 1.75 0.28 6180 0.0112 0.87 8463 0.0149 1.05 1.33 
PFE0020c A 104 0.0020 0.09 37 0.0005 0.05 0.23 19 0.0003 0.03 0.14 150 0.0003 0.02 91 0.0002 0.01 0.59 
PFF0015c A 870 0.0171 0.74 1359 0.0174 1.71 1.02 558 0.0087 0.77 0.51 4623 0.0084 0.65 3289 0.0058 0.41 0.69 
PFF0025w A 679 0.0133 0.57 399 0.0051 0.50 0.38 424 0.0066 0.58 0.49 7506 0.0136 1.06 2053 0.0036 0.25 0.27 
PFF0035c A 160 0.0031 0.14 164 0.0021 0.21 0.67 31 0.0005 0.04 0.15 550 0.0010 0.08 266 0.0005 0.03 0.47 
PFF0855c A 180 0.0035 0.15 124 0.0016 0.16 0.45 61 0.0009 0.08 0.27 747 0.0014 0.11 733 0.0013 0.09 0.95 
PFF1555w A 183 0.0036 0.15 295 0.0038 0.37 1.05 145 0.0022 0.20 0.62 277 0.0005 0.04 384 0.0007 0.05 1.35 
PFF1560c A 102 0.0020 0.09 70 0.0009 0.09 0.45 26 0.0004 0.04 0.20 109 0.0002 0.02 149 0.0003 0.02 1.32 
PFF1565c A 66 0.0013 0.06 43 0.0005 0.05 0.42 21 0.0003 0.03 0.25 52 0.0001 0.01 178 0.0003 0.02 3.33 
PFF1575w A 1066 0.0210 0.90 1470 0.0188 1.85 0.90 644 0.0100 0.88 0.48 1922 0.0035 0.27 3977 0.0070 0.49 2.01 
PFF1590w A 1881 0.0370 1.59 1844 0.0236 2.33 0.64 1262 0.0196 1.73 0.53 10188 0.0185 1.44 8208 0.0145 1.02 0.78 
PFI0010c A 3951 0.0777 3.34 1731 0.0222 2.18 0.29 1377 0.0214 1.89 0.28 6803 0.0123 0.96 17583 0.0310 2.17 2.51 
PFI0020w A 1318 0.0259 1.11 246 0.0032 0.31 0.12 430 0.0067 0.59 0.26 12292 0.0223 1.74 10573 0.0186 1.31 0.84 
PFI0030c A 271 0.0053 0.23 366 0.0047 0.46 0.88 95 0.0015 0.13 0.28 1030 0.0019 0.15 1016 0.0018 0.13 0.96 
PFI0035c A 47 0.0009 0.04 42 0.0005 0.05 0.59 19 0.0003 0.03 0.32 94 0.0002 0.01 87 0.0002 0.01 0.90 
PFI0050c A 76 0.0015 0.06 95 0.0012 0.12 0.82 36 0.0006 0.05 0.37 158 0.0003 0.02 346 0.0006 0.04 2.13 
PFI0065w A 80 0.0016 0.07 69 0.0009 0.09 0.56 53 0.0008 0.07 0.52 144 0.0003 0.02 268 0.0005 0.03 1.81 
PFI0070w A 58 0.0011 0.05 72 0.0009 0.09 0.81 71 0.0011 0.10 0.97 119 0.0002 0.02 217 0.0004 0.03 1.78 
PFI0075w A 218 0.0043 0.18 406 0.0052 0.51 1.21 155 0.0024 0.21 0.56 439 0.0008 0.06 751 0.0013 0.09 1.66 
78 
 
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Table 4.3continued 3D7 (exp 1) 3D7 BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
PFI1815c A 1483 0.0291 1.25 1134 0.0145 1.43 0.50 861 0.0134 1.18 0.46 4172 0.0076 0.59 3782 0.0067 0.47 0.88 
PFL0010c A 1735 0.0341 1.47 680 0.0087 0.86 0.26 956 0.0148 1.31 0.44 12618 0.0229 1.78 9968 0.0176 1.23 0.77 
PFL0025c A 5034 0.0989 4.26 3605 0.0461 4.54 0.47 2340 0.0363 3.21 0.37 27626 0.0501 3.90 9195 0.0162 1.14 0.32 
PFL2615w A 51 0.0010 0.04 76 0.0010 0.10 0.98 41 0.0006 0.06 0.64 141 0.0003 0.02 274 0.0005 0.03 1.88 
PFL2625w A 159 0.0031 0.13 75 0.0010 0.09 0.31 105 0.0016 0.14 0.52 954 0.0017 0.13 256 0.0005 0.03 0.26 
PFL2640c A 133 0.0026 0.11 189 0.0024 0.24 0.93 97 0.0015 0.13 0.58 184 0.0003 0.03 435 0.0008 0.05 2.29 
PFL2645c A 189 0.0037 0.16 83 0.0011 0.11 0.29 120 0.0019 0.17 0.50 575 0.0010 0.08 803 0.0014 0.10 1.36 
PFL2660w A 79 0.0016 0.07 233 0.0030 0.29 1.92 95 0.0015 0.13 0.95 320 0.0006 0.05 960 0.0017 0.12 2.92 
PFB0040c A 55 0.0011 0.05 74 0.0010 0.09 0.87 56 0.0009 0.08 0.80 74 0.0001 0.01 335 0.0006 0.04 4.39 
PF10_0003 B1 358 0.0070 0.30 241 0.0031 0.30 0.44 223 0.0035 0.31 0.49 3186 0.0058 0.45 980 0.0017 0.12 0.30 
PF10_0397 B1 43 0.0009 0.04 51 0.0007 0.06 0.77 16 0.0003 0.02 0.30 87 0.0002 0.01 146 0.0003 0.02 1.64 
PF10_0404 B1 1592 0.0313 1.35 1602 0.0205 2.02 0.66 1602 0.0249 2.20 0.80 2635 0.0048 0.37 16060 0.0283 1.99 5.93 
PF13_0006 B1 5184 0.1019 4.39 4150 0.0531 5.23 0.52 4260 0.0661 5.85 0.65 3938 0.0071 0.56 20374 0.0359 2.52 5.03 
PF14_0003 B1 3585 0.0705 3.03 1026 0.0131 1.29 0.19 1551 0.0241 2.13 0.34 19041 0.0345 2.69 34327 0.0605 4.25 1.75 
PF14_0770 B1 122 0.0024 0.10 103 0.0013 0.13 0.55 55 0.0009 0.08 0.36 326 0.0006 0.05 122 0.0002 0.02 0.36 
PFA0095c B1 429 0.0084 0.36 144 0.0018 0.18 0.22 482 0.0075 0.66 0.89 5128 0.0093 0.72 2384 0.0042 0.29 0.45 
PFA0745w B1 225 0.0044 0.19 150 0.0019 0.19 0.43 67 0.0010 0.09 0.24 708 0.0013 0.10 786 0.0014 0.10 1.08 
PFB1015w B1 86 0.0017 0.07 107 0.0014 0.13 0.81 48 0.0007 0.07 0.44 255 0.0005 0.04 190 0.0003 0.02 0.72 
PFB1040w B1 140 0.0027 0.12 147 0.0019 0.19 0.69 59 0.0009 0.08 0.33 124 0.0002 0.02 271 0.0005 0.03 2.12 
PFD1010w B1 1715 0.0337 1.45 138 0.0018 0.17 0.05 276 0.0043 0.38 0.13 36 0.0001 0.01 18 0.0000 0.00 0.47 
PFI0025c B1 8518 0.1674 7.21 8353 0.1069 10.53 0.64 2570 0.0399 3.53 0.24 5981 0.0108 0.84 16504 0.0291 2.04 2.68 
PFL0015c B1 258 0.0051 0.22 72 0.0009 0.09 0.18 79 0.0012 0.11 0.24 1636 0.0030 0.23 216 0.0004 0.03 0.13 
PFL2605w B1 58 0.0011 0.05 56 0.0007 0.07 0.63 16 0.0003 0.02 0.22 49 0.0001 0.01 55 0.0001 0.01 1.08 
PFL2655w B1 14 0.0003 0.01 12 0.0001 0.01 0.54 3 0.0001 0.00 0.19               
MAL13P1.530 B2 237 0.0047 0.20 73 0.0009 0.09 0.20 55 0.0008 0.08 0.18 955 0.0017 0.13 298 0.0005 0.04 0.30 
MAL7P1.215 B2 178 0.0035 0.15 141 0.0018 0.18 0.51 55 0.0009 0.08 0.25 409 0.0007 0.06 406 0.0007 0.05 0.97 
PF11_0020 B2 340 0.0067 0.29 463 0.0059 0.58 0.89 128 0.0020 0.18 0.30 945 0.0017 0.13 1196 0.0021 0.15 1.23 
PF14_0766 B2 132 0.0026 0.11 84 0.0011 0.11 0.41 48 0.0007 0.07 0.28 920 0.0017 0.13 807 0.0014 0.10 0.85 
PFA0030c B2 1204 0.0237 1.02 908 0.0116 1.15 0.49 351 0.0055 0.48 0.23 2156 0.0039 0.30 3387 0.0060 0.42 1.53 
PFC0030c B2 75 0.0015 0.06 64 0.0008 0.08 0.55 22 0.0003 0.03 0.23 224 0.0004 0.03 226 0.0004 0.03 0.98 
PFF1570w B2 1371 0.0269 1.16 2314 0.0296 2.92 1.10 537 0.0083 0.74 0.31 1148 0.0021 0.16 2471 0.0044 0.31 2.09 
PFI0015c B2 111 0.0022 0.09 100 0.0013 0.13 0.59 103 0.0016 0.14 0.74 271 0.0005 0.04 457 0.0008 0.06 1.64 
PFI1810w B2 153 0.0030 0.13 388 0.0050 0.49 1.66 190 0.0029 0.26 0.98 410 0.0007 0.06 496 0.0009 0.06 1.18 
MAL7P1.219 B3 157 0.0031 0.13 179 0.0023 0.23 0.74 88 0.0014 0.12 0.44 371 0.0007 0.05 498 0.0009 0.06 1.31 
PF10_0394 B3 908 0.0179 0.77 258 0.0033 0.33 0.19 211 0.0033 0.29 0.18 1084 0.0020 0.15 976 0.0017 0.12 0.88 
PF11_0515 B3 1147 0.0225 0.97 418 0.0054 0.53 0.24 641 0.0099 0.88 0.44 7563 0.0137 1.07 4877 0.0086 0.60 0.63 
PF14_0005 B3 211 0.0041 0.18 158 0.0020 0.20 0.49 132 0.0021 0.18 0.50 2490 0.0045 0.35 1063 0.0019 0.13 0.42 
PF14_0008 B3 271 0.0053 0.23 164 0.0021 0.21 0.39 177 0.0027 0.24 0.52 6040 0.0109 0.85 2218 0.0039 0.27 0.36 
PFB0015c B3 271 0.0053 0.23 371 0.0048 0.47 0.89 181 0.0028 0.25 0.53 434 0.0008 0.06 3770 0.0066 0.47 8.44 
PFB0055c B3 20 0.0004 0.02 30 0.0004 0.04 1.00 14 0.0002 0.02 0.55 24 0.0000 0.00 86 0.0002 0.01 3.46 
PFB1005w B3 89 0.0018 0.08 61 0.0008 0.08 0.45 14 0.0002 0.02 0.12 90 0.0002 0.01 245 0.0004 0.03 2.65 
PFC1100w B3 15 0.0003 0.01 24 0.0003 0.03 1.00 13 0.0002 0.02 0.66 16 0.0000 0.00 35 0.0001 0.00 2.08 
PFE1630w B3 352 0.0069 0.30 254 0.0032 0.32 0.47 170 0.0026 0.23 0.38 521 0.0009 0.07 1099 0.0019 0.14 2.05 
PF10_0402 B3 170 0.0033 0.14 106 0.0014 0.13 0.41 32 0.0005 0.04 0.15 131 0.0002 0.02 210 0.0004 0.03 1.56 
79 
 
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Table 4.3continued 3D7 (exp 1) 3D7 BGA3 (exp 1) 3D7 BGB3 (exp1) 3D7 (exp2) 3D7 BGA9 (exp2) 
Gene name Type Abs. # relative % Abs. # relative % Fold. Abs. # relative % fold Abs. # relative % Abs. # relative % fold 
MAL13P1.495   518 0.0102 0.44 330 0.0042 0.42 0.41 345 0.0054 0.47 0.53 627 0.0011 0.09 1191 0.0021 0.15 1.85 
MAL7P1.200   127 0.0025 0.11 63 0.0008 0.08 0.32 19 0.0003 0.03 0.12 431 0.0008 0.06 154 0.0003 0.02 0.35 
PF08_0105   202 0.0040 0.17 220 0.0028 0.28 0.71 93 0.0014 0.13 0.36 591 0.0011 0.08 3138 0.0055 0.39 5.17 
PF10_0002   188 0.0037 0.16 139 0.0018 0.18 0.48 133 0.0021 0.18 0.56 898 0.0016 0.13 309 0.0005 0.04 0.33 
PF10_0005   267 0.0052 0.23 746 0.0096 0.94 1.82 153 0.0024 0.21 0.45 1954 0.0035 0.28 908 0.0016 0.11 0.45 
PF10_0006   510 0.0100 0.43 388 0.0050 0.49 0.49 469 0.0073 0.64 0.73 3737 0.0068 0.53 2128 0.0038 0.26 0.55 
PF10_0399   42 0.0008 0.04 35 0.0004 0.04 0.53 7 0.0001 0.01 0.14 44 0.0001 0.01 130 0.0002 0.02 2.90 
PF10_0401   242 0.0048 0.20 134 0.0017 0.17 0.36 62 0.0010 0.09 0.20 419 0.0008 0.06 308 0.0005 0.04 0.71 
PF11_0517   207 0.0041 0.17 91 0.0012 0.11 0.29 44 0.0007 0.06 0.17 879 0.0016 0.12 393 0.0007 0.05 0.43 
PF11_0519   408 0.0080 0.34 153 0.0020 0.19 0.24 66 0.0010 0.09 0.13 1274 0.0023 0.18 658 0.0012 0.08 0.50 
PF14_0002   1415 0.0278 1.20 1140 0.0146 1.44 0.52 588 0.0091 0.81 0.33 2618 0.0047 0.37 2420 0.0043 0.30 0.90 
PF14_0768   117 0.0023 0.10 58 0.0007 0.07 0.32 28 0.0004 0.04 0.19 196 0.0004 0.03 284 0.0005 0.04 1.41 
PFA0710c   67 0.0013 0.06 112 0.0014 0.14 1.09 28 0.0004 0.04 0.33 141 0.0003 0.02 345 0.0006 0.04 2.39 
PFB0035c   23 0.0004 0.02 79 0.0010 0.10 2.26 42 0.0007 0.06 1.46 30 0.0001 0.00 202 0.0004 0.03 6.58 
PFB1035w   195 0.0038 0.16 275 0.0035 0.35 0.92 158 0.0024 0.22 0.64 328 0.0006 0.05 421 0.0007 0.05 1.25 
PFC0035w   121 0.0024 0.10 118 0.0015 0.15 0.64 66 0.0010 0.09 0.44 402 0.0007 0.06 382 0.0007 0.05 0.92 
PFD0070c   3421 0.0672 2.89 1643 0.0210 2.07 0.31 6248 0.0970 8.58 1.44 25371 0.0460 3.58 35200 0.0621 4.35 1.35 
PFD1020c   422 0.0083 0.36 204 0.0026 0.26 0.31 218 0.0034 0.30 0.41 2314 0.0042 0.33 1805 0.0032 0.22 0.76 
PFE0025c   234 0.0046 0.20 260 0.0033 0.33 0.72 93 0.0014 0.13 0.31 937 0.0017 0.13 597 0.0011 0.07 0.62 
PFF1545w   442 0.0087 0.37 204 0.0026 0.26 0.30 91 0.0014 0.13 0.16 381 0.0007 0.05 278 0.0005 0.03 0.71 
PFI0055c   71 0.0014 0.06 57 0.0007 0.07 0.52 15 0.0002 0.02 0.16 118 0.0002 0.02 231 0.0004 0.03 1.91 
PFI1805w   163 0.0032 0.14 190 0.0024 0.24 0.76 54 0.0008 0.07 0.26 919 0.0017 0.13 537 0.0009 0.07 0.57 
PFL2585c   65 0.0013 0.06 38 0.0005 0.05 0.38 38 0.0006 0.05 0.46 215 0.0004 0.03 155 0.0003 0.02 0.70 
PFL2630w   103 0.0020 0.09 76 0.0010 0.10 0.48 36 0.0006 0.05 0.28 1848 0.0034 0.26 986 0.0017 0.12 0.52 
Total 
  118215 23236 100.00 79323 10153 100.00 0.44 64401 11304 100.00 0.49 708443 12842 100.00 808531 14253 100.00 1.11 
Rif 
House-keeping   50876     78124       72797       551682     567251       
 
Classification of A or B RIFINs according to Joannin et al. (2008). For all cultures tested the absolute number of detected transcripts (Abs #), the number of 
transcripts relative to the average transcript number of two house-keeping genes (relative) and the percentage of all rif-transcripts detected (%) are given. For 
the selected parasites, fold change in transcription relative to the unselected control parasite is given (fold). The 10 most highly transcribed genes are 
highlighted in color.  
 
80 
 
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4.3.2 QPCR var transcripts in FMG selected and unselected parasites 
From the results obtained from the 3D7 QPCR, PF13_0003 appeared to be the most 
likely candidate among the var genes as being the mediator of blood group A and B 
adhesion in this parasite. It would be expected that the blood group A and B binding 
FMG parasites express one or more var genes with similar structural motifs as those 
proposed to mediate binding between PF13_0003 and blood group A and B. To test 
this, a similar QPCR analysis was performed for the FMG parasites using primers 
generated to detect 56 var genes of the IT4 genotype to which the FMG strain belongs. 
In contrast to what was observed for PF13_0003, the two SD3-loop containing UPS-A 
var genes of IT4 previously proposed to mediate blood group A dependent rosetting, 
IT4var9 and IT4var60, were transcribed at a very low level (<0.2% of all var transcripts 
in all FMG sublines), and were down-regulated in the selected relative to the unselected 
parasites (Table 4.4). The one DC5 type var gene of the IT4 genome, IT4var2, was 
transcribed at less than 1% in all sublines. 
IT4var21, which has also been previously associated with rosetting and binding to blood 
group A, was transcribed at a slightly higher level but was down regulated in the 
selected parasites (Figure 4.15). As in the first 3D7 experiment, only one var gene was 
upregulated in both selected sublines, namely IT4var32b (Figure 4.15), with a 6.7- and 
1.3- fold increased transcription in A and B-BSA selected parasites, respectively. 
One var gene (IT4var19), which like IT4var32b belongs to the DC8 group of var genes, 
was erroneously omitted from the analysis, so a prominent expression of this gene 
cannot be ruled out in the selected parasites. The overall trend however, as for the 3D7 
experiment, was a general decrease in var expression in the A and B-BSA selected 
parasites. 
81 
 
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Table 4.4 Transcription of 56 var genes from the IT4/FCR3/FMG genome 
Table 4.4   FMG FMG BGA5 FMG BGB5 
Gene name Type Abs. # relative %* Abs. # relative %* Fold. Abs. # relative %* fold 
IT4var35 A/var1 16272 0.16 0.57 17718 0.16 3.55 0.98 11081 0.14 3.02 0.88 
IT4var04 var2 3585 0.04 0.13 3077 0.03 0.62 0.77 2495 0.03 0.68 0.89 
IT4var02 A/DC5 2838 0.03 0.10 3712 0.03 0.74 1.18 2184 0.03 0.60 0.99 
A/Rosetting/SD3 
IT4var09 1652 0.02 0.06 374 0.00 0.07 0.20 232 0.00 0.06 0.18 
loop 
A/Rosetting/SD3 
IT4var60 3624 0.04 0.13 335 0.00 0.07 0.08 159 0.00 0.04 0.06 
loop 
IT4var03 A 818 0.01 0.03 665 0.01 0.13 0.73 292 0.00 0.08 0.46 
IT4var07 A 79892 0.81 2.82 1285 0.01 0.26 0.01 628 0.01 0.17 0.01 
IT4var08 A 3350 0.03 0.12 1313 0.01 0.26 0.35 862 0.01 0.24 0.33 
IT4var18 A 17411 0.18 0.61 1791 0.02 0.36 0.09 1322 0.02 0.36 0.10 
IT4var22 A 807 0.01 0.03 345 0.00 0.07 0.39 181 0.00 0.05 0.29 
IT4var64 A 2468 0.02 0.09 900 0.01 0.18 0.33 480 0.01 0.13 0.25 
IT4var06 DC8 3329 0.03 0.12 3562 0.03 0.71 0.97 1912 0.02 0.52 0.74 
IT4var20 DC8 6067 0.06 0.21 1345 0.01 0.27 0.20 735 0.01 0.20 0.16 
IT4var32b DC8 22184 0.22 0.78 164543 1.50 32.95 6.69 21517 0.28 5.87 1.25 
IT4var21 Rosetting 35161 0.36 1.24 12522 0.11 2.51 0.32 9045 0.12 2.47 0.33 
IT4var01   13905 0.14 0.49 2175 0.02 0.44 0.14 1544 0.02 0.42 0.14 
IT4var05   9026 0.09 0.32 2410 0.02 0.48 0.24 1405 0.02 0.38 0.20 
IT4var10   8697 0.09 0.31 3554 0.03 0.71 0.37 1733 0.02 0.47 0.26 
IT4var11   12305 0.12 0.43 4232 0.04 0.85 0.31 2653 0.03 0.72 0.28 
IT4var12   21283 0.22 0.75 3307 0.03 0.66 0.14 2506 0.03 0.68 0.15 
IT4var13   5846 0.06 0.21 1337 0.01 0.27 0.21 648 0.01 0.18 0.14 
IT4var14   65499 0.66 2.31 39302 0.36 7.87 0.54 5264 0.07 1.44 0.10 
IT4var15   23429 0.24 0.83 5017 0.05 1.00 0.19 2578 0.03 0.70 0.14 
IT4var16   18589 0.19 0.66 2933 0.03 0.59 0.14 1446 0.02 0.39 0.10 
IT4var17   7730 0.08 0.27 2845 0.03 0.57 0.33 1430 0.02 0.39 0.24 
IT4var23   13966 0.14 0.49 3432 0.03 0.69 0.22 2795 0.04 0.76 0.26 
IT4var24   6992 0.07 0.25 1891 0.02 0.38 0.24 1217 0.02 0.33 0.22 
IT4var25   10493 0.11 0.37 3179 0.03 0.64 0.27 2312 0.03 0.63 0.28 
IT4var26   12332 0.12 0.44 3972 0.04 0.80 0.29 3350 0.04 0.91 0.35 
IT4var27   1685817 17.07 59.47 37623 0.34 7.53 0.02 174151 2.27 47.54 0.13 
IT4var28   20643 0.21 0.73 4793 0.04 0.96 0.21 3432 0.04 0.94 0.21 
IT4var29   11203 0.11 0.40 4123 0.04 0.83 0.33 2332 0.03 0.64 0.27 
IT4var30   47625 0.48 1.68 7068 0.06 1.42 0.13 4450 0.06 1.21 0.12 
IT4var31   49210 0.50 1.74 9511 0.09 1.90 0.17 4963 0.06 1.35 0.13 
IT4var32a   229783 2.33 8.11 9892 0.09 1.98 0.04 8967 0.12 2.45 0.05 
IT4var33   10795 0.11 0.38 2506 0.02 0.50 0.21 1494 0.02 0.41 0.18 
IT4var34   21098 0.21 0.74 10795 0.10 2.16 0.46 4866 0.06 1.33 0.30 
IT4var36   26941 0.27 0.95 6976 0.06 1.40 0.23 5050 0.07 1.38 0.24 
IT4var39   8325 0.08 0.29 2723 0.02 0.55 0.30 1729 0.02 0.47 0.27 
IT4var40   7099 0.07 0.25 2358 0.02 0.47 0.30 1363 0.02 0.37 0.25 
IT4var41   30711 0.31 1.08 4909 0.04 0.98 0.14 2175 0.03 0.59 0.09 
IT4var44   26765 0.27 0.94 27960 0.26 5.60 0.94 18069 0.24 4.93 0.87 
IT4var45   17335 0.18 0.61 4689 0.04 0.94 0.24 2567 0.03 0.70 0.19 
IT4var46   15611 0.16 0.55 4996 0.05 1.00 0.29 2926 0.04 0.80 0.24 
IT4var47   10379 0.11 0.37 3972 0.04 0.80 0.35 2119 0.03 0.58 0.26 
IT4var51   8947 0.09 0.32 2901 0.03 0.58 0.29 1814 0.02 0.50 0.26 
IT4var54   12092 0.12 0.43 1082 0.01 0.22 0.08 568 0.01 0.16 0.06 
IT4var58   42701 0.43 1.51 13575 0.12 2.72 0.29 9165 0.12 2.50 0.28 
IT4var59   29399 0.30 1.04 9785 0.09 1.96 0.30 7514 0.10 2.05 0.33 
IT4var61   9408 0.10 0.33 3454 0.03 0.69 0.33 2595 0.03 0.71 0.35 
IT4var62   24208 0.25 0.85 7193 0.07 1.44 0.27 3887 0.05 1.06 0.21 
IT4var63   5584 0.06 0.20 2838 0.03 0.57 0.46 1446 0.02 0.39 0.33 
IT4var65   25847 0.26 0.91 12413 0.11 2.49 0.43 7935 0.10 2.17 0.39 
IT4var66   13997 0.14 0.49 3609 0.03 0.72 0.23 3037 0.04 0.83 0.28 
IT4var67   11614 0.12 0.41 3660 0.03 0.73 0.28 2955 0.04 0.81 0.33 
IT4var68   1993 0.02 0.07 869 0.01 0.17 0.39 764 0.01 0.21 0.49 
Total var*   2834680 28.70 100.00 499346 4.56 100.00 0.16 366342 4.77 100.00 0.17 
House-keeping  98785   109517    76856    
Var gene transcripts in unselected, A- and B- selected FMG cultures. For all cultures tested the 
absolute number of detected transcripts (Abs #), the number of transcripts relative to the average 
transcript number of two house-keeping genes (relative) and the percentage of all var transcripts 
detected (%) are given. For the selected parasites, fold change in transcription relative to the 
unselected control parasite is given (fold). The 10 most highly transcribed genes are highlighted 
in colors corresponding to the color-coding of Figure 4.15. *The DC8 var gene IT4var19 was 
by error not included in the analysis. 
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IT4var27 FMG BGA5 FMG BGB5
IT4var32a
IT4var32b
IT4var14
IT4var7
IT4var44
IT4var58
IT4var31
IT4var30
IT4var21
-100 -10 0 10 100 -100 -10 0 10 100
Fold change Fold change  
Figure 4.15 Fold change of the top 10 var transcripts in the FMG A- and B-BSA 
selected parasites. As shown, IT4var32b (dark green bar) was the only 
transcript upregulated in both selected parasites. 
 
  
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4.4 Fieldwork: Background and Demographics 
A total of 113 patients, aged 5 months to 12 years, were recruited in 2016 after the 
inclusion criteria were met for this aspect of the study. There were 60 male and 53 
females, representing 53% and 47% of the cohort respectively. The categories of 
disease included 53 (46.9%) cases of severe malaria (SM) and 52 (46.02%) cases of 
uncomplicated malaria (UM). Six (6) patients, or 5.31% of the cohort, were not 
categorized. Table 4.5 summarizes characteristics of recruited patients. 
 
 
Table 4.5 Characteristics of patients recruited for the study 
 
Number recruited 113 
Age range  5 months to 12yrs  
Gender:  
          Male 60 
          Female 53  
Parasite positive (Microscopy) 90 of 113 
Parasitaemia Range (per µl) 0 to 433,525 parasites  
Clinical category:  
          Severe Malaria 53 
          Uncomplicated Malaria 52  
          Not categorized & febrile cases 8 
*Blood group:   
          A 24 
          B 27 
          AB 4  
          O 57 
*Sickling positive 6 of 112  
*no blood group and sickling record for 1 patient  
 
 
 
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Tests to determine the blood type were done for each patient, and all blood types were 
observed among the patients recruited for the study. Blood group AB was the least 
common, while blood group O as expected, was the most common blood type recorded 
in the study (Table 4.5). The sickling positive patients were excluded from the analysis. 
 
4.5 Plate-based ABO haem-release binding assays in clinical isolates 
Following studies of the rosetting and binding characteristics of parasite lines selected 
on blood group carbohydrates, further investigations into the binding characteristics of 
clinical parasite isolates from patients were conducted. These field isolates, stored in 
liquid nitrogen during field work, were transported to the laboratory where they were 
subsequently thawed, cultured and used in in vitro experiments to study their binding 
profiles to immobilized blood group oligosaccharides on polystyrene plates. 
In all, 39 field isolates were tested for their ability to bind the BSA-conjugated blood 
group oligosaccharides immobilized on 96 well Polysorb plates. Each plate included at 
least the unselected 3D7 laboratory strain and the blood group A ‘control’ parasites. 
 
4.5.1 Relative adhesion of selected 3D7 parasites during experiments 
The laboratory selected strains used on each plate as a reference during the assays 
showed consistent binding over the course of the experiments. The relative adhesion to 
blood group A polysaccharides for 3D7, 3D7-A and 3D7-B selected parasites for all 
assays combined show consistently higher rates of adhesion during the experiments. 
Relative adhesion of both 3D7-A and 3D7-B were comparable and significantly higher 
than for the unselected 3D7 parasites (Figure 4.16). 
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Figure 4.16 Relative adhesion of 3D7 parasites to blood group A antigen in plate 
based assays. Combined relative adhesion of 3D7 parasites over the course 
of the experiments indicate a significantly higher relative adhesion to A-
BSA by the 3D7 BGA (blue box; n = 20) and 3D7 BGB (green box; n = 14) 
selected parasites (p < 0.0001, Kruskal-Wallis test and Dunn’s post hoc test) 
over the unselected isogenic 3D7 line (grey box; n = 18). 
  
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4.5.2 Relative adhesion of field isolates and blood type of donor 
Comparing relative adhesion of field isolates from patients based on grouping into 
blood type O and non-O indicates that parasite binding to blood group A is independent 
of the blood type of the patient donor. Thus, the binding of patient isolates to the blood 
group A construct, A-BSA, shows no significant difference (P = 0.4619, Mann Whitney 
test) between blood type (O versus non-O) and capability to bind blood group A antigen 
(Figure 4.17). 
 
1 5
1 0
5
0
 O  O
rp rp
 g g
n
o
N
B lo o d  g ro u p  o f p a tie n t
 
Figure 4.17 Relative adhesion of non-group O and group O patient parasite isolates 
to blood group A antigen. Boxplots of relative adhesion (median values) to 
blood group A antigen by field isolates from patients with blood group A 
and AB (Non grp O, brown box; n = 13) and from patients with blood group 
th
O (grp O, blue box; n = 26). Upper and lower limits of boxes are the 25  and 
th
75  percentile respectively, while whiskers show the minimum and 
maximum values. Outliers are shown as data points beyond these values. 
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4.5.3 Summary of binding characteristics of field isolates 
For most isolates, the binding to the A and B constructs were similar, and binding of 
clinical isolates to the blood group H construct was only observed in one isolate. 
Of the 39 field isolates tested, 8 bound to at least one blood group oligosaccharide. 
Interestingly, a lone isolate bound to the blood group H antigen, though this was only 
slightly above the cut-off value. This isolate also happened to bind both the A and B 
construct above cut-off values. All 8 of the binding field isolates were observed in 
children 5 years and below. Figure 4.18 summarizes the binding characteristics of field 
isolates to the A- and B-BSA constructs. Five of the 8 field isolates that bound to ABO 
polysaccharides were from donors with blood type O, two were of blood type B, and the 
remaining binding isolate was a donor of blood type A. 
 
 
 
Age (years) 
 
Figure 4.18 Binding of field isolates to the blood group A, B and O (H) constructs in 
39 children 10 years and below. Black squares denote positive binding to 
blood group antigens (mean + 2SD of unselected 3D7), and was observed 
only in children 5 years and below. One isolate from a 2-year old patient 
bound to all three constructs. 
 
 
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4.6 Rosetting in field isolates 
During field work, malaria parasites from vein-puncture blood samples of recruited 
patients were processed and cultured in the donor’s RBC and monitored until they were 
at late trophozoite stages and then used for rosetting assays. 
A total of 70 field isolates from the cohort were successfully assayed. At least a rosette 
was observed in 50 of the parasite cultures, representing 71% of the total assayed 
cohort. 
4.6.1 Rosetting and blood groups 
Table 4.6 shows the characteristics and distribution of the various blood groups in the 
sampled cohort. Using the Kruskal Wallis test, the differences observed in rosetting 
rates between the different blood groups were found to be statistically insignificant (p = 
0.3533), though the mean rosetting rate in blood group O was about half of those 
observed for both blood groups A and B. 
  
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Table 4.6 Characteristics of field isolates in the different blood groups 
 
Blood Total Rosetting Range  Mean # of KW test 
Median 
group isolates isolates  (rosettes/200 IE) rosettes p value 
A 13 9 0-105 19.9 7 
B 18 15 0-127 21 12 
P > 0.05 
AB 2 2 4-13 8.5 
8.5 
O 37 24 0-86 7 10.9 
The total number of rosetting isolates, as well as rosetting rates for each patient blood type in 
the field isolates are shown for the various blood groups. The Kruskal-Wallis (KW) test for the 
difference in medians for these groups showed no significant difference between the blood 
types. 
 
 
A comparison of mean rosetting rates between patients of blood group O and non-group 
O categories showed no significant difference (p>0.05) (Figure 4.19). In other tests, 
results of a comparison between parasite density (number of parasites per microlitre) in 
rosetting and non-rosetting isolates showed there was no significant difference in 
parasite load (as detected by microscopy) between these groups (Mann-Whitney, p = 
0.112). 
 
 
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6 0
M a n n -W h itn e y , p  =  0 .0 8 9 2
4 0
2 0
0
 O  O
p n
u o
ro N
G
B lo o d  ty p e  g r o u p in g
 
Figure 4.19 Rosetting rates in blood group O versus non-group O malaria patients. 
Mean values of the number of rosettes counted per 200 IE in blood group O 
(grey bar, n = 37) and non-O (black bar, n = 33) patients were statistically 
insignificant (p ˃ 0.05). Error bars are SD of mean number of rosettes 
 
 
4.6.2 Rosetting and Age 
In general, rosetting rates were found to decline with age (n=70, ρ= -0.322, p=0.00657, 
Spearman Rank order). This observed effect was due to non-O blood types, since no 
significant change in rosetting rates were observed for blood type O when patients were 
grouped according to blood type (Figure 4.20, A). On the other hand, in patients with 
blood types other than group O, the rosetting rates declined significantly with increasing 
age (Figure 4.20, B). 
 
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Figure 4.20 Correlation between rosetting and age in blood groups. Panel A shows 
the correlation between rosetting rates and age in patients of blood group O. 
In Panel B, this correlation is more evident and significant in patients with 
non-O blood types. 
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4.6.3 Rosetting and Parasitaemia 
To determine whether the level of parasitaemia observed in patients had any effect on, 
or association with, the rosetting rate, tests of association were conducted on these 
parameters. The tests showed no significant association between parasitaemia and 
rosetting for the cohort, even for the higher risk age group of children 5 years and 
below, as well as in patients over 5 years of age (Figure 4.21, A and B respectively). 
There is a gradual increase in rosetting rate as the parasite density increases in the 0-5 
year group, though this association was not significant (P = 0.235). This association was 
much less obvious in the 5years+ group (P= 0.83: Figure 4.21, B) 
  
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1 5 0
A : u p  to  5  y e a rs
n =  4 2 , P  =  0 .2 3 5
2
R =  0 .0 3 5
1 0 0
5 0
0
0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0
P .f  d e n s ity  (p e r  µ L )
 
 
 
1 5 0
B : 5  -  1 2  y e a rs
n =  2 8 , P =  0 .8 3
2
R =  0 .0 0 2
1 0 0
5 0
0
0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 5 0 0 0 0 0
P .f  d e n s ity  (p e r  µ L )
 
 
 
Figure 4.21 Association between parasitaemia and rosetting isolates in 0-5, and 5 -12 
year groups. Rosetting rates versus parasite density showing the line of best 
fit for the 0 - 5 year group (Graph A), and the 5 - 12 year group (Graph B) 
Each point represents an individual patient record. 
 
 
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However, when analyzed  solely on the basis of the age category of 0-5 and 5 plus 
years, the rosetting rates in field isolates from children 5 years and below were 
significantly higher (p<0.05) than rosetting rates recorded in isolates from children over 
5 years of age (Figure 4.22). 
 
 
1 5 0
P <  0 .0 5
1 0 0
5 0
0
u p  to  5 y rs 5 y rs  +
A g e  g ro u p
 
Figure 4.22 Rosetting rates in two age groups of 0-5 and 5-12 years. Rosetting rates 
observed for the 0-5 year lower age group (n= 42) were significantly higher 
(p = 0.038, Mann-Whitney test) than rates observed in the older 5 years plus 
age group (n= 28) group. Median values depicted by the long black 
horizontal bars, along with shorter bars representing the interquartile ranges 
 
 
 
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4.7 Antibody recognition profiles of patient sera to selected parasites 
After studying the wild type clinical parasite isolates, investigations into the immune 
reactivity of patient sera to the well-characterized A- and B-binding parasites were 
done. Flow cytometric analysis of patient sera profiles to RBC infected with parasites 
selected on blood group antigens indicated increased recognition of IE of the selected 
parasite lines (Figure 4.23). As shown, generally higher mean fluorescence intensities 
(MFI) were observed for selected parasites BGA7 and BGB7 than for the isogenic, 
unselected 3D7 strain. 
 
  
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3D7
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 dk men women
age (years)  
 
3D7 BGA7
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 dk men women
age (years)  
 
3D7 BGB7
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 dk men women
age (years)  
 
Figure 4.23 Mean Fluorescence Intensity of bound antibody from donor plasma. The 
three panels show MFI values for antibody reactivity to unselected 3D7, A-
BSA selected 3D7 BGA7 and B-BSA selected 3D7 BGB7 parasites. Each 
circle represents an individual donor. dk= pool of malaria naïve Danish 
plasma 
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The  recognition profile for selected and unselected parasites as measured by the MFI 
st nd
shows differences between 3D7 and FMG parasites (Figure 4.25, 1  and 2  panels), but 
rd
no real change in HB3 parasites (Figure 4.24, 3  panel). Again, the observed change in 
recognition increased in the older children. 
 
 
Figure 4.24 Difference in mean fluorescence intensity between selected and unselected 
3D7, FMG and HB3 parasites. Generally, Recognition of the IEs by IgG in a 
pool of plasma from non-exposed donors was always less than 5.5 (3D7) or less 
than 1.5 (FMG and HB3). 
 
 
 
 
  
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CHAPTER 5 
 
5.0   DISCUSSION 
Falciparum malaria pathogenesis is linked to the ability of the parasite to evade host 
immune responses. Adhesion to the endothelium by mature blood stages of the parasite 
is critical in this process, enabling the parasite to avoid clearance by the spleen. This 
may impair circulation in micro vessels and activate ECs, leading to complications and 
severe disease (Warrell et al., 1988, Moxon et al., 2013). The related rosette formation 
phenomenon, mediated by exported parasite antigens on the IE surface, is also linked to 
disease severity (Carlson et al., 1990). One among many (Yam et al., 2017) receptors 
on RBC for rosetting is blood group A or B glyco-antigens (Carlson and Wahlgren, 
1992). The well-established protection against severe falciparum malaria of blood group 
O individuals (Degarege et al., 2019) has been linked to reduced rosetting (Rowe et al., 
2007). Several members of the var gene family (Vigan-Womas et al., 2012, Chen et al., 
2000b, Angeletti et al., 2012), and most recently members of the rif gene family (Goel 
et al., 2015) have been proposed to encode blood group A (and/or B) adhesion 
molecules. The parasite strains used in these studies were originally selected for 
adhesion to whole cells, EC-like or RBC (rosette-selection) or passed through animals. 
All these procedures are likely to select for adhesion to multiple host receptors. 
This study sought to investigate the interaction of IE with ABO blood group antigens 
directly, and in the absence of other IE or EC receptors for IE adhesion.  To achieve this 
without the effects of other factors, blood group A- and B- specific binding phenotypes 
were successfully generated by repeated panning of known laboratory strains of P. 
falciparum on immobilized blood group oligosaccharide constructs. The binding 
patterns of these phenotypes were studied by developing a novel assay to specifically 
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measure the adhesion of IE to blood group antigens. For these generated phenotypes, a 
good correlation was found between IE binding to blood group antigens and rosetting in 
non-O blood group RBCs. 
The in vitro detection and measurement of host IE interactions are usually done by 
static, flow-based or cellular adhesion assays (Butler et al., 2016, Adams and Rowe, 
2013), rosetting assays or flow cytometric determination of surface markers or rosettes 
using multiplet discrimination (Ch'ng et al., 2016). The majority of these involve either 
the subjective and laborious manual counting of Giemsa or fluorescence stained RBC 
by microscopy, which is the ‘gold standard’, or the use of specialised or expensive 
equipment. More recently there have been developments in this area, where assays have 
been developed to reliably quantify bound parasites using image analysis software 
without the use of specialized equipment or radioactive labelling (Paton et al., 2011), 
some requiring just an ELISA reader (Hempel et al., 2015). 
A limitation of most of these methods for studies on the interaction between host 
receptors and the IE is that a range of cell surface receptors may impact this interaction 
(Andrews et al., 2005). In order to study the isolated effect of ABO blood group 
binding, a micro titre plate-based assay was developed using a simple protocol and a 
micro titre plate absorbance reader. The quantification method used here capitalises on 
the inherent peroxidase activity of RBC (Huy et al., 2005), which has been shown to 
have the same detection level as currently used methods (Hempel et al., 2015). 
The binding and specificity of the commercially obtained glyco-constructs was 
confirmed in a lectin-binding assay, in which lectin binding can be detected using 
ELISA-based methods that take advantage of the specific binding between particular 
lectins and corresponding carbohydrates (Shao, 1992).  
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The procedure for the blood group binding assay itself was broadly based on the method 
of Hempel and colleagues to measure cytoadhesion of P. falciparum IEs to immobilised 
ECs (Hempel et al., 2015). In the present study, the three BSA-coupled blood group 
antigens were incubated overnight in PBS to coat the antigens onto polystyrene micro 
titre plates before incubation with IE of both selected and unselected laboratory adapted 
parasites, or with field isolates. Next, a critical gravity washing step was done where the 
plate was fully submerged and inverted in wash buffer to remove unbound cells from 
the wells (Paton et al., 2011). Bound RBC were then disrupted to release haem, which 
acts as a pseudo-peroxidase for oxidation of the TMB chromogenic substrate 
(Streefkerk, 1972), and the plate subsequently read in a spectrophotometer for 
quantification using standards included on the plate. In conclusion, this assay was able 
to detect and quantitatively measure the binding of P. falciparum IEs to plate-bound, 
BSA-coupled oligosaccharides of blood group A, B and O. 
Following a number of pannings on ABO oligosaccharide coated wells, it was possible 
to select parasites with the same blood group-binding phenotype; binding that could be 
blocked by the relevant selective anti-A and -B antibodies (Figure 4.6). This indicates 
that the binding was specific for the A- and B-antigens and not due to irrelevant binding 
to e.g. the plastic surface of the wells or the BSA-anchor. Of concern was a slight 
difference in construct spacer arm length to the BSA for the O- on one hand, and the 
non-O blood groups on the other, as these had 3- and 6- spacer arms respectively. 
However, a comparison of adhesion to blood group B constructs with both 3- and 6-
spacer arm lengths showed no difference, for the FMG parasite line, and only a minor 
difference for the 3D7 line, indicating that this difference in spacer arm length had no 
significant effect on binding to the constructs.  
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The biological relevance of the test was indicated by the association between ABO-
binding and rosetting. As mentioned in the literature background (Section 2.5.3), the 
rosetting phenotype is strongly associated with non-O blood groups (Rowe et al., 1995, 
Carlson et al., 1994, Barragan et al., 2000). Thus, the 3D7, FMG and FUP parasites 
selected on blood group A or B antigens spontaneously formed rosettes in non-group O 
RBC, unlike their parental unselected lines that did not form rosettes in any blood type. 
The assay has the added advantage of being easy to execute, with higher throughput and 
limited requirement for reagents and equipment. As with other static assays however, 
this assay may be less representative of an in vivo physiological environment when 
compared to a flow-based assay. In the present study the focus was not to try to 
replicate an in vivo environment, where binding characteristics may be different or 
change altogether (Adams et al., 2000, Crabb et al., 1997). It was primarily to eliminate 
exogenous factors in investigating IE/ABO-blood group antigen interactions. The 
previously mentioned alternative methods, though fairly accurate, nonetheless require 
expensive equipment or reagents, specialist training and, especially under field 
conditions, may be impractical in some cases. 
In summary, this new assay to measure IE binding to ABO blood group antigens offers 
a fast and objective measure of the binding of IE to multiple blood group antigens 
within the same assay. This novel assay is able to discriminate between binding 
phenotypes and also correlates satisfactorily with IE preference for rosetting in blood 
group A, B and AB, making it a useful alternative to the subjective counting of rosettes 
by microscopy, particularly in instances where RBC numbers in large rosettes are 
difficult to determine. 
Panning on either blood group A or B BSA-coupled oligosaccharides adsorbed onto 
culture dishes was able to induce successful selection of A- and B-binding phenotypes 
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in some but not all the tested culture-adapted laboratory isolates of P. falciparum. This 
procedure yielded blood group A and B binding sublines for 3D7, FMG and FUP, with 
several times (up to 7 fold) higher binding than the unselected parent lines. In contrast, 
no A- or B-binding could be induced with the isolates HB3 or FCR3, despite several 
attempts of repeated panning. This may be due in part to the fact that there are 
differences in binding preference of some isolates between surface expressed proteins 
on IE and immobilized antigens in a non-physiological environment (Azasi et al., 
2018), or that the parasites in question simply lack the relevant VSA genes, or have 
them ‘switched off’.  
Interestingly, parasites selected for binding to blood group A antigens also bound blood 
group B antigens, as did the blood group B selected parasites, which bound blood group 
A sugars. Also, rosetting in non-group O RBC could be induced by panning on either 
A- or B-oligosaccharides, an indication that these parasites may share a common ligand 
that mediates blood group A or B binding to IE. This may be a reflection of the 
structural similarity between these two blood types, where the only difference is the 
addition (to the base H antigen oligosaccharide) of N-acetylgalactosamine (GalNAc) for 
blood type A or galactose (Gal) for blood type B.  
In summary, specific adhesion phenotypes of rosetting isolates that bind to blood group 
polysaccharides have been successfully established for both 3D7 and FMG strains of 
Plasmodium. These isolates, which are stable and maintain the phenotype over several 
months, would be useful for studies into, and comparison of blood group adhesion 
phenotypes and in vitro studies of adhesion-related manifestations of the disease.  
The binding characteristics of RBC infected with late stages of the 3D7 and FMG 
strains that were successfully selected for binding to blood group A and B, along with 
the HB3 strain for which selection was not possible, were tested on human aorta and 
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dermal ECs to investigate cytoadhesion in these parasites. The selected 3D7 and FMG 
strains showed higher binding rates than their unselected parental strains. 
The ABO blood group antigens are known to be expressed on both RBCs and other 
tissues, including the endothelium (Wang et al., 2013), and also on all but one vascular 
EC line (O'Donnell et al., 2000). Endothelial cell binding assays reflect the in vivo 
property of sequestration by late stage parasites to avoid splenic clearance (Udeinya et 
al., 1981, Utter et al., 2017). The differential phenotype of higher binding observed in 
selected 3D7 and FMG parasites in this study suggests a strong link between binding to 
A- and B- antigens and adhesion to ECs. It could be inferred here that circulating 
parasites with this phenotype could have a better chance of survival in vivo through 
sequestration and immune evasion (Ockenhouse et al., 1991). The rosetting and cyto-
adhesive phenotype shared by all successfully selected parasites in the current study 
strengthens the notion that these two attributes are linked, mediated by the parasite-
derived variant surface antigens PfEMP1 (Rowe et al., 2009) and/or RIFINs (Goel et 
al., 2015), and dependent on blood type. Some studies on the other hand have identified 
distinct receptor-ligand interactions for both rosetting and cytoadhesion in the same 
parasite via separate PfEMP1 domains (Adams et al., 2014), indicating that rosetting is 
distinct from cytoadhesion. 
In the case of HB3, for which selection for binding to the A and B constructs was not 
possible, a different picture also emerged with regards to EC adhesion. Unlike for 3D7 
and FMG, the unselected HB3 bound to EC before selection, indicating that this parasite 
was able to adhere to EC by an adhesin that does not bind blood group A or B presented 
on BSA constructs. Selection on blood group B did not induce any detectable changes 
in adhesive behavior. The A-selected HB3, however displayed a very high degree of 
binding to cell-culture wells without EC, probably reflecting that in this case a VSA 
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with high affinity for the culture-dishes or BSA rather than the A-oligosaccharide had 
been selected for by panning. It is noteworthy that the selection was done in 24-well 
culture plates made of the same material as the 96 well plates used for EC adhesion 
assay, whereas the direct binding to A and B oligo saccharide constructs was performed 
in 96-well ‘Polysorb’ plates. The apparent ‘accidental’ selection for a parasite with a 
high capacity for binding to empty cell culture dishes highlight the importance of 
validating the specific adhesion phenotypes carefully, particularly in studies where 
parasites are manipulated in vitro to bind to various receptors found on EC or 
syncytiotrophoblast.  
In all, the blood group A and B binding phenotypes produced by selection on blood 
group antigens show a clear preference for rosetting in RBC from non-group O donors. 
As indicated earlier (Figure 4.10), there is a good correlation between the rosetting and 
binding assay results for the selected phenotypes, and could serve as a good correlate of 
rosetting and in turn, sequestration, as rosetting is a phenotype known to be associated 
with severe malaria (Carlson, 1993, Carlson et al., 1990, Rowe et al., 1995, Doumbo et 
al., 2009). This could improve the study of malaria and blood group linked studies on 
rosetting and binding phenotypes in both laboratory and field based studies. 
To detect potential VSAs of interest that may be involved in the blood group antigen 
binding phenotypes, the 3D7 and FMG parasite strains selected on blood group A and B 
antigens were analysed for their gene expression profiles by QPCR, under the 
assumption that these phenotypes are attributable to particular var or rif genes, and that 
expression can be correlated to increased mRNA levels. 
The changes observed in the phenotypes of parasites, including their cytoadhesion 
characteristics, have often been investigated by analysing the var gene expression 
profiles of these parasites (Smith et al., 1995, Golnitz et al., 2008, Salanti et al., 2003, 
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Albrecht et al., 2011). In the current study, expression profiles of 58 var and 158 rif 
genes in selected and unselected 3D7 parasites were initially determined. From the 
analysis of this data, only one var gene previously associated with blood group A 
mediated rosetting, Pf13_0003, appeared to have slightly increased transcription levels 
as a consequence of blood group A and B selection (with fold changes of 1.3 and 1.7 
respectively). All other var genes were transcribed at a lower level in the selected 3D7 
parasites, resulting in an overall 3- fold decrease in total var transcription. Similarly, no 
transcriptional changes in rif genes previously associated with preferential rosetting in 
blood group A or a change in the balance between group A and B rif genes could be 
detected. 
Pf13_0003 is known to encode the 3D7 orthologue of adhesion domains that mediate 
rosetting (Bull et al., 2005, Gardner et al., 2002), and it would appear that Pf13_0003, 
and therefore DC5 var genes (and/or SD3-loop containing var genes) mediate the 
interaction observed in the isolates from the present study (Angeletti et al., 2012). To 
validate these findings, the analysis was repeated for unselected 3D7 and blood group A 
selected 3D7. In this second experiment, the difference in transcription of Pf13_0003 
between blood group A selected and unselected 3D7 was more pronounced (Table 4.2). 
Comparing the results of the second QPCR experiment with the first however, it 
appears the observed effect resulted from a decrease in transcription that had happened 
during the time between the two experiments in the unselected 3D7, while the 
expression of Pf13_0003 remained at approximately the same level in the blood group 
A selected substrain. The total decrease in var transcription was less pronounced in the 
second experiment with 3D7. This was mainly due to increased transcription of group B 
var genes not previously associated with rosetting. 
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Though the initial QPCR experiments with the 3D7 parasite lines did not reveal 
consistent upregulation of any var gene, it appeared that DC5 or SD3 loop containing 
var genes such as PF13_0003 would be the most likely candidates mediating blood 
group A and B adhesion among those candidates already suggested. This necessitated a 
look at the closely related var genes of the blood group selected FMG parasite line for 
similar trends in transcription. Thus, transcription of 56 var genes, including the DC5 
var gene IT4var2, the SD3 containing IT4var9 (R29var) and IT4var60 as well as 
IT4var21 that has also been implicated in rosetting (Vigan-Womas et al., 2008, Adams 
et al., 2014) were analysed by QPCR. Results from analysis of the transcript levels 
turned out not to be in agreement with that for 3D7, as both the SD3 loop containing var 
genes as well as IT4var21 and IT4var2 showed overall very low transcription in all 3 
FMG lines (less than 1% of all var transcripts in all parasites), with decreased 
transcription in selected relative to the unselected control for all but the DC5 var gene 
IT4var2 that was transcribed at the same level in all parasites (fold changes 1.2 and 1.0 
in the A and B selected FMG parasites, respectively).  The lone var transcript for the 
FMG selected parasites showing an elevated transcript level in the selected lines 
compared to the parental line was IT4var 32b (Fold changes of 6.7 and 1.3 in the A and 
B selected lines respectively (Figure 4.13). This var gene belongs to the DC8 var gene 
group, responsible for binding to EPCR, and has not previously been suggested to 
mediate rosetting via blood group A. This finding was in contrast to the profile observed 
for the 3D7 isolate, where two DC8 var genes were downregulated 3 to 10-fold in 
selected parasites. These results, due to the differences in transcript changes, may point 
to the fact that the adhesion phenotype selected for is not linked to a particular var gene 
expression, and that the observed upregulation of Pf13_0003 in 3D7 and IT4var32b in 
FMG could be explained as random events. Alternatively, the phenotype may be 
independent from DC structure and be mediated by a yet unidentified motif shared 
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between PF13_0003 of 3D7 and IT4var32b. With the limitation that not all IT4 var 
genes were included in the FMG analysis, the existing data set is consistent with an 
overall down regulation of var genes as the main trend in blood group A and B selected 
parasites. This could be an artefact appearing from an increased transcription of the 
housekeeping genes used for normalisation of var gene transcription that had arisen 
from the selection procedure. The number of housekeeping transcripts detected in each 
different parasite sub line tested in the same experiments were more similar than the 
total number of var transcripts detected, and the relative abundance of the 2 
housekeeping genes was similar, indicating that the transcription of the housekeeping 
gene was similar in selected and unselected parasites. 
In the first QPCR experiment with 3D7, transcription of rif genes were also slightly 
decreased in selected parasites relative to the unselected, and there was no prominent or 
specific transcription that was consistent in both the A and the B selected line linked to 
the phenotype under observation. In the second experiment one rif gene, Pf13_0004 
appeared to be very prominently transcribed in the blood group A selected 3D7 relative 
to unselected 3D7. This however was not seen in the first experiment where the 
phenotype in question had already been acquired. No major changes in the balance 
between group A and B RIFINs were detected, and the rif gene previously described to 
mediate blood group A dependent rosetting by Goel et al. (2015), PFA0020w, was 
transcribed at a very low level in both selected and unselected parasites. The data 
presented here are not consistent with group A RIFINs in general or certain RIFINs 
being the main blood group A and B binding adhesins. 
This may highlight the limitations of the method used here, since the transcription 
profiles of the var and rif genes of these parasites, investigated using QPCR, only offers 
a limited look at the broader aspect which can be achieved by other approaches such as 
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RNA-seq or gene arrays to identify novel transcripts as well as differentially expressed 
genes (Tarr et al., 2018). 
This study has successfully developed blood group binding isolates and answers basic 
questions on transcripts that may be linked to the observed phenotype, and paves the 
way for further molecular investigations into understanding interactions between 
parasite and host in falciparum malaria. 
The distribution of blood group A, B, AB and O frequencies across the sampled patients 
were respectively 21%, 24%, 4% and 51%, generally similar to frequencies reported in 
other studies in Ghana (Acquaye, 2004) and in the sub region (Tadesse and Tadesse, 
2013, Zerihun et al., 2011). The higher frequencies of blood group O is not unexpected, 
as this phenotype is known to protect from severe disease (Panda et al., 2011, Degarege 
et al., 2019), and is indeed highest in malaria endemic regions worldwide (Cserti and 
Dzik, 2007). 
A total of 39 clinical P. falciparum isolates were tested for their ability to bind the blood 
group antigens. Eight of these isolates showed binding to at least one blood group 
construct. Of these, 5 were from patients of blood type O, 2 from blood type B and 1 
from blood type A. 
Generally, as observed for the laboratory strains, most field isolates that did show 
binding, bound to both the A and B constructs. Binding to the H antigen construct 
(group O antigen) by field isolates was virtually absent and only noted in one borderline 
case, which interestingly also bound the A and B blood group constructs. 
Another observation made about the 8 blood group A and B binding cohort from the 
field isolates is that all the donors of the binding parasites were below the age of 5 
years, the age most at risk of severe disease. Considering that immunity to malaria 
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occurs over many years, and the fact that this immunity is first acquired to the most 
virulent strains (Gupta et al., 1999), it is understandable that isolates that bind blood 
group antigens (and therefore most likely to form rosettes or adhere to ECs, leading to 
severe disease) are found in children under 5 years who are unlikely to have a full 
repertoire of protective antibodies. 
No difference was observed in the binding capacity of IE to the blood group A antigen, 
irrespective of whether the donor was blood group O or a non-group O patient. In other 
words, the ability to bind to the A antigen was independent of donor blood type. 
Therefore, blood group A and B-binding parasites can be found in hosts with blood 
group O as well. It is therefore possible that the blood group A and B adhesin may bind 
other host receptors in addition to the blood group determinants such as PECAM1 
(CD31) or von Willebrand factor, which are known to carry ABH determinants. It is 
noteworthy that the DC5 group A var genes, which contain PF13_0003, have 
previously been implicated in PECAM1 mediated endothelial adhesion (Berger et al., 
2013).  
Rosetting assays were carried out in 70 clinical isolates, for 50 of which at least one 
rosette was observed. The distribution of rosettes observed between the various blood 
groups in this study were not statistically different, though it is worth mentioning that 
the mean rosetting rates observed for non-group O patients was about twice that 
observed for blood group O patients. Previous studies including cohorts with larger 
numbers and younger patients however showed significantly lower rosetting rates in 
blood group O than non-O patients (Rowe et al., 1995, Udomsangpetch et al., 1993).  In 
the present study, rosetting rates in children 5 years or less were significantly higher 
than their older counterparts. The elevated rosetting rates in young children could be 
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attributed to the non-group O blood groups, since there was no decline in rosetting with 
age for blood type O patients. 
To test if blood group A- and B-binding parasites were most commonly encountered by 
patients with non-O blood type, investigations into the ability of patient antibody to 
recognise parasites selected for binding to blood group antigens was determined by flow 
cytometry. Selected and unselected parasites of 3D7, FMG and HB3 strains were 
incubated with sera from the study cohort. 
Overall recognition of selected parasites by patient antibody was very similar to their 
unselected parental parasite line. This is a very different scenario from the dramatic 
change in serological recognition previously described for pregnant women following 
selection for binding to CSA (Sharling et al., 2004, Salanti et al., 2004). However, 
detailed analysis subtracting the MFI values obtained for the unselected parasite from 
the MFI value obtained for the selected parasite revealed that some individuals 
recognised the selected parasites slightly better than the unselected parasite. This 
increased recognition of selected parasites was found to increase with age, with older 
children recording higher MFI shifts compared to younger children. There was no 
difference between plasma donors of non-O and O blood types with regard to MFI shift 
in selected parasites. This is in concordance with the observation that blood group A 
and B adhesion was found in type O parasite donors as well as in non-O donors. This 
observation was made in both 3D7 and FMG isolates, though not for the HB3 isolate, 
where, as expected, no difference in recognition between selected and unselected 
parasites was observed in plasma from children. 
The overall very subtle change in recognition pattern following selection for binding to 
blood group A and B is in concordance with the finding that no major changes in the 
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composition of the major var and rif VSA families that have been shown to be the main 
targets of naturally acquired IE antibodies, were detected in the QPCR analysis. 
The findings accrued from this study could impact interventions in several ways. For 
example the binding assay provides an alternative procedure to quantify adhesion to 
blood group polysaccharides (or other ligands) without radioactive labelling of parasites 
or laborious error prone counting by microscopy. This information could be relevant in 
a clinical setting where parasite binding preference could predict or confirm severe 
disease such as CM and inform preventive treatment and management. Also, 
identification of specific parasite VSA epitopes that bind blood group antigens could 
lead to tailored anti-severe malaria vaccine candidates for vulnerable groups. 
 
 
 
 
  
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5.1 Conclusions and recommendations 
 
5.1.1 Conclusions 
 
In conclusion, the data presented here demonstrates that P. falciparum IE adhesion to 
blood group A and B antigens can be measured directly in a simple plate-based assay 
using oligosaccharides coupled to BSA. The binding measured by this assay correlates 
well with preference for rosette formation with RBC of non-O blood types, a phenotype 
that is associated with severe disease (Rowe et al., 2007). The A/B adhesive IE 
phenotype was detected by this assay in patient isolates obtained from Ghanaian 
children under 5 years of age. It was also demonstrated that the blood group A/B 
adhesion phenotype is linked with avid endothelial adhesion. Altogether these clinical 
and in vitro observations suggest and support the notion that the A/B adhesive 
phenotype is associated with parasite virulence. Surprisingly, the recognition patterns of 
plasma obtained from semi-immune Ghanaian children were very similar for isogenic 
parasites regardless of whether they would bind strongly to blood group A/B 
polysaccharides or not. This could indicate that the parasite adhesin selected for by 
panning on A/B oligosaccharides constitute only a minor part of all the VSAs expressed 
on P. falciparum IEs, or that the adhesin responsible for A/B binding might be of 
relatively low immunogenicity. The unexpected lack of any major changes in serotype, 
however, was mirrored by the lack of overall major and consistent changes in the 
composition of transcribed var and rif genes before and after selection on ABO blood 
group oligo saccharides. The most consistent change observed was an overall decrease 
in var gene transcription in the blood group A and B adhesive parasites compared to 
their unselected parental lines. In summary, the real time quantitative PCR data 
presented in this work are not in concordance with either a certain sub type of group A-
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var genes, the DC5 type, or SD loop containing, or type A rif genes being responsible 
for blood group A/B mediated rosetting. 
 
5.1.2 Recommendations  
Two major recommendations for future work have emerged from this project. Firstly, 
the study of binding to A/B oligosaccharides in patient isolates should be expanded to 
validate the association between binding to the A/B antigens, preference for rosette 
formation in non-O RBC and EC adhesion found in patient isolates. Furthermore, the 
larger group of patients should include well defined severe cases to evaluate whether the 
A/B adhesive phenotype is indeed associated with increased virulence. The other major 
recommendation is to broaden the search for the parasite adhesin binding blood group 
A/B beyond var and rif genes. As both the 3D7 parasite clone and the FMG strain of It4 
genotype have fully sequenced genomes, these parasites could be used in whole genome 
transcriptional studies as well as in proteomic studies searching for genes differentially 
expressed between the A/B selected parasites and the unselected lines. For 
transcriptional analysis RNA sequencing we propose to use mRNA sequencing, as this 
method, unlike array-based methods, does not depend on exact knowledge of gene 
sequences. Thus, this approach might also be employed in studies of field isolates with 
different degrees of blood group A/B adhesion capacity. 
 
  
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