ABSTRACT
The use of reverse vaccinology in vaccine candidate antigen discovery has led to identification of many Plasmodium falciparum novel antigens. Some of these antigens are essential for parasite survival and could be evaluated as targets of protective immunity to clinical malaria that is acquired naturally. Targeting essential antigens identified through functional genomic studies involving loss-of-function, could aid in the development of an effective next generation sub-unit vaccine. For these studies to be successful, the antigens must be expressed in their correctly folded structure in a heterologous expression system. This study aimed at expressing PF3D7_1146100, one of the novel, essential P. falciparum antigens, and assess the protective role of antibodies against it in clinical malaria using sera from a prospective cohort of children (N=343) from Junju, Kilifi, Kenya. PF3D7_1146100 was sub-cloned into pTT28, an expression vector, and transfected into Expi 293F cells for recombinant protein expression. The antigen was purified and characterized using SDS PAGE and Western blotting. Immunogenicity of the recombinant protein and the correlation between its immune responses and protection against clinical malaria was determined using indirect ELISA. In a univariate logistic regression analysis, anti-PF3D7_1146100 antibodies were correlated with protection against clinical malaria at an odds ratio of 0.63(0.42- 0.95) at 95% C.I and p value=0.027. After adjusting for age and exposure, the odds ratio was 0.73(0.44-1.16) at 95% C.I with a p-value=0.176. The essentiality and immunogenicity of this antigen indicates that it is a potential vaccine candidate targeting the blood stage of the parasite. The successful expression of PF3D7_1146100 and its analysis as a target of the immune system, provides a basis for studying other novel essential and conserved antigens. This also provides an opportunity to study its function, a knowledge which could contribute to vaccine and drug development not only against P. falciparum but also other Apicomplexa organisms
TABLE OF CONTENTS
DECLARATION i
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES x
LIST OF ABBREVIATIONS xi
DEFINITION OF TERMS xiii
ABSTRACT xiv
CHAPTER ONE: INTRODUCTION
1.1 Background 1
1.2 Problem Statement 3
1.3 Justification of the Study 3
1.4 General Objective 4
1.4.1 Specific Objectives 4
CHAPTER TWO: LITERATURE REVIEW
2.1 Malaria vaccine development 5
2.2 Current malaria vaccine status 6
2.3 Life cycle complexity and malaria vaccine development 7
2.4 The Plasmodium falciparum life cycle 8
2.5 Pathogenesis of P. falciparum occurs in the blood stage cycle 10
2.6 Naturally acquired immunity (NAI) to clinical malaria 11
2.7 Reverse vaccinology and antibody targets discovery 13
2.8 Functional genomics and discovery of essential vaccine candidate antigens 14
2.9 Recombinant expression of P. falciparum antigens 14
CHAPTER THREE: MATERIALS AND METHODS
3.1 Study design 17
3.2 Ethical Approval 18
3.3 Methods 18
3.3.1 Essential Gene Selection 18
3.3.2 Sequence retrieval and construct design 19
3.3.3 Transformation and Plasmid Midiprep 20
3.3.4 Restriction digest 21
3.3.5 PF3D7_1146100 ligation to the expression vector 21
3.3.6 Transfection of Expi 293 cells 23
3.3.5 Protein purification 24
3.3.6 SDS PAGE 24
3.3.7 Western Blot 25
3.3.8 Indirect ELISA 26
3.3.9 Statistical analysis 27
CHAPTER FOUR: RESULTS
4.1 Construct design 30
4.2 Successful sub-cloning, expression and characterization of PF3D7_1146100 antigen 30
4.3 Concentration of recombinant PF37_1146100 required for an optimal reaction with immune sera 32
4.4 The epitopes present in recombinant PF3D7_1146100 antigen 33
4.5 Sero-epidemiological information on the Junju 2014 cohort of children 34
4.6 Presence of naturally acquired antibodies against recombinant PF3D7_1146100 antigen 35
4.7 Naturally acquired anti-PF3D7_1146100 antibodies’ variation with age 36
4.8 Anti-PF3D7_1146100 antibody responses and malaria outcome 37
4.9 Malaria outcome and levels of anti-schizont antibodies 38
4.10 Anti-PF3D7_1146100 antibodies’ association with protection against clinical malaria 39
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion 43
5.2 Conclusion 50
5.3 Recommendations 50
REFERENCES 52
APPENDICES 64
LIST OF TABLES
Table1: Characteristics of the Junju 2014 cohort as obtained from their epidemiological data. 35
Table 2: A Logistic regression analysis summary 42
LIST OF FIGURES
Figure 1.1 : The global prevalence of malaria in 2017 (WHO, 2018) 2
Figure 2.1: Malaria vaccine candidates in clinical development that target the life cycle stages of P. falciparum (Adapted from Draper et al. (2018)) 8
Figure 2.2: The P. falciparum life cycle depends on transmission cycle between humans and Anopheles mosquito (Adapted from Cowman et al. (2017)) 10
Figure 3.1: An illustration of the longitudinal Junju cohort study set up. 18
Figure 3.2: The PF3D7_1146100 gene cassette 19
Figure 3.3: A plasmid map of the PF3D7_1146100 construct 20
Figure 3.4: pTT28 expression vector used for cloning and expression of PF3D7_1146100 23
Figure 4.1: A confirmatory digest of the successfully cloned PF3D7_1146100 gene (indicated by the arrow) in a pTT28 expression vector 31
Figure 4.2: Characterization of the purified recombinant PF3D7_1146100(≈ 𝟐𝟐𝟐𝟐. 𝟑𝟑𝑘𝐷𝑎) protein as indicated on SDS PAGE (A) and western blot (B) images 32
Figure 4.3: A titration curve of PF3D7_1146100 protein 33
Figure 4.4: Antibody levels against native (magenta bars) and heat-treated (orange bars) PF3D7_1146100 antigen 34
Figure 4.5: Interaction of antibodies from the children cohort sera with the recombinant PF3D7_1146100 antigen 36
Figure 4.6: Antibodies against the recombinant PF3D7_1146100 antigen increased with age. 37
Figure 4.7: Variation in anti-PF3D7_1146100 antibody levels between malaria negative individuals (0) and malaria positive individuals (1) 38
Figure 4.8: Anti-schizont antibody among malaria positive and disease free individuals.
Figure 4.9: Classification of participants into the low, medium and high tertile based on their anti-PF3D7_1146100 antibody levels 40
Figure 4.10: Influence of tertile classification on the count of malaria clinical episodes an individual had throughout the follow up. 41
LIST OF APPENDICES
Appendix 1: The genomic sequence of PF3D7_1146100 64
Appendix 2: The cDNA sequence of PF3D7_1146100 65
Appendix 3: The predicted protein sequence of PF3D7_1146100 65
Appendix 4: The R code used for generation of the titration curve 65
Appendix 5: R code used for analysis of recombinant PF3D7_1146100 epitope data 67
Appendix 6: R code used for analysis of the antibody responses for the Junju 2014 cohort of children 68
Appendix 7: Logistic regression model 76
Appendix 8: Anti-schizont antibodies increase with age 78
Appendix 9: Anti-disease immunity increases with age 79
Appendix 10: Distribution of malaria episode levels in each age category 80
Appendix 11: Ethical approval document 81
LIST OF ABBREVIATIONS
ACT : Artemisinin based Combination Therapy ADCC : Antibody dependent cellular cytotoxicity AMA1 : Apical membrane antigen 1
APS : Ammonium persulfate cDNA : Complementary DNA CDS : Coding sequence
CHMI : Controlled Human Malaria Infection CMV : Cytomegalovirus
CSP : Cicumsporozoite antigen DALYs : Disability-adjusted-life years DNA : Deoxy ribonucleic acid
DEG : Database of Essential Genes EBA 140 : Erythrocyte binding antigen 140 EBA 175 : Erythrocyte binding antigen 175 ELISA : Enzyme Linked Immunosorbent GOI : Gene of interest
GPI : Glycophosphatidyl inositol
GST-DBL 1 α: Glutathione-S-Transferase- Duffy Binding Like domain 1 α
HEK 293 : Human Embryonic Kidney cells 293 HIV : Human Immunodeficiency Virus
IPTi : Intermittent Prophylaxis Treatment for infants
IPTp : Intermittent Prophylaxis Treatment for pregnant women iRBCs : Infected Red Blood Cells
IRS : Indoor Residual Spraying ITNs : Insecticide Treated Bed Nets
KDHSS : Kilifi Demographic Health Surveillance System LB : Luria- Bertani
LMICs : Low and Middle Income Countries LSA 3 : Liver Stage Antigen 3
MSP 1 : Merozoite Surface Protein 1 MSP 7 : Merozoite Surface Protein 7 NAI : Naturally Acquire Immunity
NEB : New England Biolabs
OPA : Opsonic phagocytosis assay
PAGE : Polyacrylamide Gel Electrophoresis PBS : Phosphate Buffered Saline
PVDF : Polyvinylidene difluoride RBCs : Red Blood Cells
RH 5 : Reticulocyte binding homolog 5 SERU : Scientific Ethical Review Unit SDS : Sodium Dodecyl Sulphate
SOC : Super Optimal Broth with Catabolite Repression TBST : Tris Buffered Saline with Tween 20
TEMED : Tetramethylethylenediamine TLP : Tripartite Leader Peptide
TMHMM : Transmembrane Helices; Hidden Markov Model WGCFS : Wheat Germ Cell Free System
WHO : World Health Organization
DEFINITION OF TERMS
Antigenic polymorphisms refers to allelic or genetic variations at a given gene loci that result in protein forms with distinct antigenic properties in different parasite clones or strains.
Antigenic variation refers to alterations in the phenotype of an antigen as a result of regulated expression of diverse family of genes in a clone of parasites during the natural course of an infection.
Codon bias is the high preference and frequent use of a certain codon that codes for an amino acid in an organism.
Codon optimization is the process of changing the codons of the gene of interest to conform to the codon preference of the organism used for its recombinant expression.
Essential gene is a gene described to be important for growth and survival of an organism through results of its disruption in loss-of-function studies.
Functional genomics involves the use of genomic data to study gene function and host pathogen interaction.
Heterologous expression system is a system in which a protein of interest is produced in an organism that is different from the natural host (origin of the target gene).
Reverse vaccinology is the process of discovering antigens through in silico screening of the genomic data encoding a complete antigenic repertoire of an organism.
CHAPTER ONE
INTRODUCTION
1.1 Background
Malaria is among the world’s most common infectious diseases disproportionately affecting poverty-stricken regions within the tropics and sub-tropics. It claims the lives of more than 400,000 individuals with over 200 million cases being reported every year(WHO, 2020). The populations mostly at risk are children aged under five years, HIV infected individuals, pregnant women, travellers, refugees, displaced persons and migrant workers from malaria naïve areas (WHO, 2018). In 2018, the mortality of children between 0 to 5 years accounted for approximately 67% of all the reported deaths due to malaria, with sub Saharan Africa accounting for most of them(WHO, 2019).
The protozoan parasites of the genus Plasmodium are the agents causing malaria. Only five Plasmodium species are involved in infecting humans: Plasmodium knowlesi, Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax and Plasmodium ovale. The major parasite causing infection in sub-Saharan Africa is P.falciparum and is responsible for over 90% of all reported cases globally (Figure 1.1), hence the attention it has received in the malaria vaccine development research(WHO, 2019).
In the quest to eradicate and eliminate malaria, a number of control strategies have been put in place. Artemisinin based combination therapy (ACTs), indoor residual spraying (IRS) and insecticide treated bed nets (ITNs) among others, have made a major contribution to the reduction in deaths and cases due to malaria (Bhatt et al., 2015). However, the resistance developed by parasites to antimalarial drugs and vectors to insecticides, pose a challenge towards achieving zero malaria. Lack of an effective vaccine has been a drawback to efforts put forward for malaria elimination. In fact, the current licensed vaccine being rolled out in Ghana, Malawi and Kenya demonstrated moderate efficacy at best in Phase III trials (Neafsey et al., 2015; Draper et al., 2018). Furthermore, only a limited number of P. falciparum antigens in the erythrocytic stage have been assessed in their protective role against clinical malaria despite the fact that >5400 antigens are encoded in its genome (Tuju et al., 2017). The few that advanced to clinical trial stages, have failed mainly due to their antigenic polymorphisms and variations. Antigenic polymorphisms induce strain specific immunity against clinical malaria which does not protect an individual from infection by other strains. There is therefore a need for discovery of novel targets of protective anti-malarial immunity that are less polymorphic and essential for parasite survival. Assessing the role of the novel recombinant antigens in naturally acquired immunity to malaria will hasten their prioritization and validation as vaccine candidates.
Figure 1.1 : The global prevalence of malaria in 2017 (WHO, 2018)
1.2 Problem Statement
The rising cost of malaria intervention and poverty resulting from malaria case management, calls for development of an effective malaria sub-unit vaccine (El-Houderi et al., 2019; Haakenstad et al., 2019; Winskill et al., 2019). Reverse vaccinology has enabled the identification of many novel antigens that have a potential of being vaccine candidates (Doolan et al., 2008; Crompton et al., 2010; Trieu et al., 2011; Davies et al., 2015; Kamuyu et al., 2018). Gene knock-out studies have also been done to identify proteins/ antigens essential for parasite survival especially in the invasion of host cells and tissues (Zhang et al., 2018). To date, the application of these two strategies in identifying novel essential P. falciparum antigens that have a potential of being vaccine candidates, and even assessing their role in the context of naturally acquired immunity, has not been explored. Targeting these antigens for vaccine development could help overcome the constraint posed by antigenic polymorphisms.
1.3 Justification of the Study
Passively transferred antibodies from individuals immune to malaria residing in the Gambia, were able to eliminate parasitemia and clinical symptoms in West and East African children as well as Thai adults suffering from malaria (Cohen et al., 1961; McGregor et al., 1963; Sabchareon et al., 1991).This indicates that these antibodies could be targeting parasite proteins that have limited polymorphisms (Bull and Marsh, 2002; Duffy et al., 2005; Doolan et al., 2009). Targeting multiple and highly conserved antigens as vaccine candidates could overcome the constraint of antigenic polymorphisms currently observed in blood stage vaccine candidates, which contribute to their failure in clinical trials (Draper et al., 2018). Reticulocyte binding homolog 5 (RH5), a recent blood stage vaccine candidate in the making, is highly conserved, essential for parasite survival and has been shown to illicit a strain transcending immunity to some P. falciparum strains (Douglas et al., 2011; Douglas et al., 2015).
Exposure to multiple antigens has been linked with protection against malaria and that antibodies against them, do work in synergy (Osier et al., 2008; Osier et al., 2014b; Bustamante et al., 2017). A vaccine made up of multiple conserved proteins will help alleviate the malaria burden majorly felt in sub-Saharan Africa and reduce the mortality of the most susceptible populations (Plowe et al., 2009; Tanner et al., 2015).
1.4 General Objective
To assess the role of recombinant PF3D7_1146100; a novel, essential P. falciparum vaccine candidate antigen in naturally acquired immunity to malaria
1.4.1 Specific Objectives
1. To optimize the recombinant expression of PF3D7_1146100 in Expi 293 F cells, a mammalian system.
2. To evaluate the association between immune responses to the recombinant antigen and protection against clinical malaria.
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