DECLARATION I, ORUNI AMBROSE, hereby do declare that to the best of my knowledge, this is my original research report and has

not been submitted to any University or institution for the award of any degree or certificate in the same or related field. SignedDate. This research report has been submitted for examination with the approval of my supervisors:

Ass. Prof. Enock Matovu Principal Investigator, Molecular Biology Laboratory II Department of Veterinary Parasitology and Microbiology, College of Veterinary Medicine, Animal resource development and Biosecurity, Makerere University, P.O. Box 7062, Kampala, Uganda. Signed..Date Ms. Monica Namayanja (MSc) Senior Researcher, Molecular Biology Laboratory I, Department of Veterinary Parasitology and Microbiology, College of Veterinary Medicine, Animal resource development and Biosecurity, Makerere University, P.O. Box 7062, Kampala, Uganda. Signed..Date

DEDICATION I dedicate this report to my parents; Mr. and Mrs. Okee and to my brothers and sisters; Ojok, Acellam, Kolo, Ajok, Adong, Aryemo for always supporting me in all that I do.

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ACKNOWLEDGEMENT I would like to send my sincere gratitude to Ms. Monica Namayanja for the effort she put in from the time I began my research to the finish, no words can estimate how thankful I am. I would also like to greatly thank Ass. Prof. Enock Matovu for supervising and funding my research project, without which I wouldnt have done any work. A special thank you also goes to my parents, brothers and sisters for supporting me throughout my research period. And last but not least Prof. G. W. Lubega, members of Molecular Biology Laboratory I and II, my fellow students; Wachiuri Kelvin, Cuu Gloria and my dear friends; Allan, Henry, Steven, Rebecca, Austin and Wilson, who all contributed in one way or the other for the success of this project.

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TABLE OF CONTENTS DECLARATION................................................................................................................................. i DEDICATION....................................................................................................................................ii ACKNOWLEDGEMENT ................................................................................................................iii TABLE OF CONTENTS ................................................................................................................. iv LIST OF FIGURES AND TABLES................................................................................................ ix ABBREVIATIONS AND ACRONYMS .......................................................................................... x ABSTRACT ......................................................................................................................................xii CHAPTER ONE ................................................................................................................................ 1 1.0 INTRODUCTION ........................................................................................................................ 1 1.1Background ..................................................................................................................................... 1 1.2 Statement of the problem ............................................................................................................... 2 1.3 Objectives ...................................................................................................................................... 3 1.3.1 General objective ........................................................................................................................ 3 1.3.2 Specific objective ........................................................................................................................ 3 1.4 Justification and significance ......................................................................................................... 3 1.5 Research Question.......................................................................................................................... 4 CHAPTER TWO ............................................................................................................................... 4 2.0 LITERATURE REVIEW ........................................................................................................... 4 2.1 African Trypanosomiasis ............................................................................................................... 4 2.2 Trypanosoma brucei ...................................................................................................................... 5 2.2.1 Life cycle of Trypanosoma brucei .............................................................................................. 6 2.3 Management of Human African Trypanosomiasis ........................................................................ 8 2.3.1 Diagnosis of Human African Trypanosomiasis .......................................................................... 8 2.3.1.1 Serological techniques ............................................................................................................. 9 2.3.1.1.1 The Card Agglutination Test for Trypanosomiasis (CATT) ................................................ 9 2.3.1.1.2 Antibody detection .............................................................................................................. 10 2.3.1.1.3 The LATEX agglutination test for T. b. gambiense............................................................ 10 2.3.1.1.4 Immunofluorescence Assays .............................................................................................. 11 iv

2.3.1.2 Parasitological techniques ...................................................................................................... 11 2.3.1.2.1 Chancre aspirate .................................................................................................................. 11 2.3.1.2.2 Lymph node aspirate ........................................................................................................... 12 2.3.1.2.3 Wet and thick blood films ................................................................................................... 12 2.3.1.2.4 Microhematocrit centrifugation technique .......................................................................... 12 2.3.1.2.5 Quantitative buffy coat ....................................................................................................... 13 2.3.1.2.6 Mini-anion-exchange centrifugation technique .................................................................. 14 2.3.1.3 Molecular diagnosis ............................................................................................................... 14 2.3.1.3.1 Polymerase Chain Reaction (PCR) ..................................................................................... 14 2.3.1.3.2 Loop-mediated Isothermal Amplification (LAMP) ............................................................ 15 2.3.1.4 Diagnosis to Stage Human African Trypanosomiasis ........................................................... 15 2.3.1.4.1 White blood cell count ........................................................................................................ 16 2.3.1.4.2 Protein concentration .......................................................................................................... 16 2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF.................................................... 17 2.3.1.4.4 Trypanosomes detection in the CSF ................................................................................... 17 2.3.1.5 Treatment of the disease ........................................................................................................ 18 2.4 Pyroglutamyl peptidase 1 (PGP 1) ............................................................................................... 18 2.4.1 Clan CF of Pyroglutamyl peptidase 1 ....................................................................................... 19 2.4.2 Family C15 of Pyroglutamyl peptidase 1 ................................................................................. 19 2.4.3 Trypanosoma brucei PGP 1 ...................................................................................................... 19 2.4.4 Sequence of Trypanosoma PGP 1............................................................................................. 20 2.5 Immunogenicity ........................................................................................................................... 21 2.5.1The Nature of the Protein ........................................................................................................... 21 2.5.1.1 Degree of foreignness ............................................................................................................ 21 2.5.1.2 Molecular size ........................................................................................................................ 21 2.5.1.3 Structure of the protein .......................................................................................................... 22 2.5.1.4 Ability to be processed........................................................................................................... 22 2.5.2 Route of administration............................................................................................................. 22 2.5.3 Genetic makeup of the organism .............................................................................................. 22 v

2.5.4 Adjuvant .................................................................................................................................... 23 2.5.5 Dose of protein given ................................................................................................................ 23 2.5.6 Formulation and purity of the protein ....................................................................................... 23 2.6 Overview of the techniques to be used ........................................................................................ 23 2.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ......................... 23 2.6.2 Western Blotting ....................................................................................................................... 24 2.6.3 Enzyme-Linked Immunosorbent Assay (ELISA) ..................................................................... 24 2.7 pET-28a (+) vector ....................................................................................................................... 25 CHAPTER THREE ......................................................................................................................... 26 3.0 MATERIALS AND METHODS .............................................................................................. 26 3.1.0 Study design .............................................................................................................................. 26 3.2.0 Materials: .................................................................................................................................. 26 3.2.1 Transformed BL21DE3 cells .................................................................................................... 26 3.3.0 Methods: ................................................................................................................................... 26 3.3.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells provided ............................................................................................................................................. 27 3.3.1.1 Growth of glycerol stocks containing pET28a (+) ................................................................ 27 3.3.1.2 Plasmid extraction .................................................................................................................. 27 3.3.1.3 Agarose Gel Electrophoresis.................................................................................................. 27 3.3.1.4. Restriction enzyme digestion ................................................................................................ 28 3.3.2 Expression of the recombinant Trypanosoma PGP 1 ............................................................... 28 3.3.2.1 Small scale expression ........................................................................................................... 28 3.3.2.1.1 Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................. 28 3.3.2.1.2 Western Blotting ................................................................................................................. 29 3.3.2.2 Large scale expression ........................................................................................................... 30 3.3.3 Extraction, Purification and Quantification Trypanosoma PGP 1 and bacterial protein ................................................................................................................................................ 30 3.3.3.1 Extraction ............................................................................................................................... 30 3.3.3.1.1 Extraction of Trypanosoma PGP 1 ..................................................................................... 30 vi

3.3.3.1.2 Extraction of Bacterial protein ............................................................................................ 31 3.3.3.1.2.1 Growth of competent BL21 cells from the glycerol stock ............................................... 31 3.3.3.1.2.2 Extraction of bacterial protein ......................................................................................... 31 3.3.3.2 Purification of the Trypanosoma PGP 1 and bacterial protein from inclusion bodies ................................................................................................................................................. 32 3.3.3.2.1 Purification of inclusion bodies .......................................................................................... 32 3.3.3.2.2 Purification of the recombinant Trypanosoma PGP 1 and bacterial protein ...................... 32 3.3.3.3 Quantification of the Trypanosoma PGP 1 and bacterial protein ......................................... 33 3.3.4 Immunisation of mice of the Trypanosoma PGP 1 and bacterial protein ................................. 33 3.3.5 Analysis of immune sera ........................................................................................................... 34 3.3.5.1 Western blot analysis ............................................................................................................. 34 CHAPTER FOUR ............................................................................................................................ 36 4.0 RESULTS ................................................................................................................................... 36 4.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells .............................. 36 4.2 Expression of recombinant Trypanosoma PGP 1 ........................................................................ 37 4.3 Extraction and Purification of Trypanosoma PGP 1 and bacterial protein .................................. 37 4.3.2 Purification ................................................................................................................................ 38 4.3.2.1 Purification of recombinant Trypanosoma PGP 1 ................................................................. 38 4.3.2.2 Purification of Bacterial protein............................................................................................. 38 4.4 Quantification .............................................................................................................................. 39 4.5 Analysis of sera for the different groups of mice ........................................................................ 39 4.5.1. Western blot analysis of pre-immune sera ............................................................................... 39 4.5.2 Western blot analysis of immune sera ...................................................................................... 40 4.5.3 ELISA analysis of sera.............................................................................................................. 40 4.5.2.1 Group one (Test group) .......................................................................................................... 41 4.6.2.2 Group two (Adjuvant group) ................................................................................................. 41 4.6.2.3 Group three (PBS group) ....................................................................................................... 42 4.6.2.4 Group Four (Bacterial protein group) .................................................................................... 43

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CHAPTER FIVE ............................................................................................................................. 44 5.0 DISCUSSION ............................................................................................................................. 44 CHAPTER SIX ................................................................................................................................ 48 6.0 CONLUSION AND RECOMMENDTAION .......................................................................... 48 6.1 CONCLUSION .......................................................................................................................... 48 6.2 RECOMMENDATION ............................................................................................................. 48 REFERENCES ................................................................................................................................. 49 APPENDIX I .................................................................................................................................... 56 APPENDIX II ................................................................................................................................... 59

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LIST OF FIGURES AND TABLES

Figure 1: The Life cycle of Trypanosoma brucei in the human and the tsetse fly...6

Figure 2: Vector map of pET28a (+)....23 Figure 3: A 1% agarose gel showing confirmation of insert digested from plasmid extract...34 Figure 4: A 15% SDS-PAGE gel and western blot showing successful expression of Trypanosoma PGP 1....34 Figure 5: A 15% SDS-PAGE gel showing cell lysis BL21DE3 and BL21 cells....35 Figure 6: A 15% SDS-PAGE gel showing purification of Trypanosoma PGP 1...........35 Figure 7: A 15% SDS-PAGE gel showing purification of bacterial protein..36 Figure 8: Graph for quantification .....37 Figure 9: Western blots showing analysis of pre-immune sera .....37 Figure 10: Western blots showing analysis of immune sera..37 Figure 11: ELISA for test group....38 Figure 12: ELISA for Adjuvant group..38 Figure 13: ELISA for PBS group......39 Figure 14: ELISA for bacterial protein group...39 Figure 15: Immunogenecity curve40 Table 1: Calculated concentration of the purified portions of Trypanosoma PGP 1....36 ix

ABBREVIATIONS AND ACRONYMS BBB Bst bp CAT CATT CDC CNS CSF DAB E. coli ECL ELISA EDTA HAT HRP IPTG Kda LH-RH Blood Brain Barrier Bacillus staeorothermophilus base pairs Card Agglutination Test Card Agglutination Test for Trypanosomes Center for Disease Control Central Nervous System Cerebral Spinal Fluid 3, 3-diaminobenzidine Escherichia coli Enzyme Chemiluminescence Enzyme Linked Immuno-sorbent Assay Ethylene diamine tetra acetic acid Human African Trypanosomiasis Horse Radish Peroxidase Isopropyl-beta-D-thiogalactopyranoside Kilodaltons Luteinising Releasing Hormone x

mHAET mHCT Ni-NTA O.D OPD PARP PCR PGP 1 rpm SDS SDS-PAGE spp. T. b. T. b. gambiense T. b. rhodesiense TRH WHO

Mini-anion-exchange centrifugation technique Microhematocrit Centrifugation Technique Nickel-Nitrilotriacetic acid Optical Density o-phenylenediamine dihydrochloride Procyclic acidic repetitive protein Polymerase Chain Reaction Pyroglutamyl Peptidase rotations per minute Sodium Dodecyl Sulfate SDS-Poly Acrylamide Gel Electrophoresis Species Trypanosoma brucei Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense Thyrotropin-Releasing Hormone World Health Organisation

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ABSTRACT Diagnosis of Human African Trypanosomiasis (HAT) still remains a challenge despite a number of diagnostic techniques available. There are no simple and reliable screening tests for the disease especially for T. b. rhodesiense. This therefore puts a hindrance in the control of the disease in Africa. This study was carried out to determine the immunogenic potential of Trypanosoma Pyroglutamyl peptidase type 1 which is released during intravascular destruction of Trypanosomes in blood during the infection; this would determine whether Trypanosoma Pyroglutamyl peptidase type 1can be used as a diagnostic antigen for screening HAT in endemic areas. The gene for Trypanosoma Pyroglutamyl peptidase type 1 was previously cloned in pET28a and transformed in E. coli (BL21DE3) cells. In this study, recombinant Trypanosoma Pyroglutamyl peptidase type 1 was successfully expressed in the E. coli (BL21DE3) cells. The protein was extracted by cell lysis from the E. coli cells and purified using Ni-NTA agarose column. The purified recombinant Trypanosoma Pyroglutamyl peptidase type 1 was used to immunise 7 to 8 weeks old male Swiss albino mice using 40g/ml as the initial dose (prime dose) of the protein. The first and second boosts were done using 20g/ml of the purified protein. Production of specific antibodies was determined using western blotting and ELISA. The western blots showed strong signal detection by the protein for sera after second boost at a dilution of 1:2000. However, at the same dilution of 1:2000, very weak signals were also detected on the lane of Trypanosoma Pyroglutamyl peptidase type 1 on the membrane. The ELISA results showed that the Trypanosoma Pyroglutamyl peptidase type 1 gave an antibody IgG titer of 1:486,000 for the first and second boost sera and this was depicted in the immunogenicity curve that showed no change in titers after first boosting and second boosting post imunisation. The results obtained from this study therefore show that, Trypanosoma Pyroglutamyl peptidase type 1 was capable of eliciting specific and quantifiable antibodies in mice. This means that the Trypanosoma Pyroglutamyl peptidase type 1 is immunogenic and could be a good candidate for a diagnostic antigen for screening for HAT, however, wider studies on the protein like full study of the structure, antigenecity study, evaluation to see if the protein can pick up some cases of HAT, among others, should be conducted. xii

CHAPTER ONE

1.0 INTRODUCTION
1.1 Background African trypanosomes are parasitic hemo-flagellated protozoans of the genus Trypanosoma, transmitted to the host bloodstream by the tsetse fly (Glossina spp.) and cause African Trypanosomiasis in humans and domestic animals. The different species of trypanosomes in Africa include; Trypanosoma congolense, Trypanosoma evansi, Trypanosoma vivax and Trypanosoma brucei with the sub species of T. b .brucei, T. b. gambiense, T .b .rhodesiense. T. b. brucei is one of the causative agents of Animal African trypanosomiasis, it is not human infective due to its susceptibility to lysis by human apolipoprotein L1(Vanhamme et al., 2003), T. b. gambiense causes chronic Human African Trypanosomiasis (HAT) most common in central and western Africa, while T. b. rhodesiense causes acute HAT most common in southern and eastern Africa (Barrett et al., 2003). African Trypanosomiasis is endemic in some regions of sub-Saharan Africa, covering about 37 countries and 60 million people. In 2010, it was estimated that 50,000 to 70,000 people were infected, the number showed a decline smaller compared to earlier years (WHO, 2010). The disease has devastating effects on both humans and livestock populations, contributing to poverty in some of these affected regions of Africa. Control of African Trypanosomiasis mainly depends on proper diagnosis and treatment, however HAT diagnosis is still unsatisfactory (Njiru et al., 2007); much as the current parasitological tests are cheap and simple, they are tedious, time consuming, and are of low sensitivities because of characteristic low and fluctuating parasitemia of infected individuals. The CATT detection technique is sensitive and works well in the diagnosis of HAT due T .b. gambiense, but it is not always reliable in the diagnosis of T. b. rhodesiense (Lejon et al., 2002) hence some cases may be missed out. There is therefore need to develop tests similar to CATT that can accurately diagnose the disease due to both sub species. The molecular techniques are relatively sensitive and specific but 1

involve sophisticated and expensive equipments that would need simplification, not to mention that most of the molecular techniques are still experimental and hence impracticable for field diagnosis. Identification of proteins that are released during the pathogenesis of HAT could help in identification of diagnostic antigens that can be used in diagnosis of HAT. This would for help simplify the diagnosis of HAT since the above techniques are not reliable for the diagnosis of HAT especially due to T. b. rhodesiense. Among such proteins are Cysteine peptidases like Oligopeptidase A and B as well as Pyroglutamyl peptidase 1 (PGP 1). Pyroglutamyl peptidase type 1 (PGP 1) belongs to a group of peptidases called Cysteine peptidases. Cysteine peptidases are divided into clans and further into families (Barret and Rawlings 2001); PGP 1 belongs to Clan CF and Family C15. The enzyme is intracellular and soluble. In mammals, PGP 1 has been shown to release Pyroglutamate from Thyrotropin-Releasing Hormone (TRHI), Luteinising Hormone Releasing Hormone (LH-RH), neurotensin, bombesin and leukopyrokinin (Dando et al., 2003). In Trypanosoma brucei, PGP 1 is a 25.1 Kda soluble cystosolic cysteine peptidase that is released into the host blood stream during intravascular destruction of trypanosomes in the host blood stream during the infection and is expressed in all life cycle stages of Trypanosoma brucei as well as four other African Trypanosomes (Morty et al., 2006). It is a factor involved in the pathogenesis of HAT and is known to degrade peptides including Thyrotrophin Releasing Hormone (TRH) and Gonadotropin Releasing Hormone (GnRH) (Morty et al., 2006) by removing the N-terminal Pyroglutamyl residue of these peptides that protects them from proteolysis. A study conducted on Trypanosoma PGP 1 showed that the protein can be recognised by infected human sera (Anywar, 2009), however, the study was not conclusive on the immunogenicity of Trypanosoma PGP 1. 1.2 Statement of the problem Lack of a field applicable screening test for T .b .rhodesiense is recruiting investigation of proteins predictably capable of eliciting immune response that could be exploited to develop new diagnostic tests. A previous study had already been conducted on 2

Trypanosoma PGP 1 to determine its recognition by infected human sera using field samples (Anywar, 2009). The results obtained form that study demonstrated that Trypanosoma PGP 1 was indeed recognised by patient sera by western blotting. However, the immunogenic potential of the Trypanosoma PGP 1 was not done. This study therefore aimed to carry out an independent well controlled research in mice to show whether immunisation with Trypanosoma PGP 1 leads to generation of specific and quantifiable antibodies. 1.3 Objectives 1.3.1 General objective Determine the immunogenic potential of Trypanosoma PGP 1 1.3.2 Specific objective Determine whether recombinant Trypanosoma PGP 1 elicits specific immune response in mice. 1.4 Justification and significance Trypanosoma PGP 1 has been postulated in the pathogenesis of HAT and is released due to intravascular destruction of trypanosomes in the blood stream of the host (Morty et al., 2006). This therefore means that Trypanosoma PGP 1 could be a good candidate for a diagnostic antigen for HAT. A previous study also showed that Trypanosoma PGP 1 has a diagnostic potential (Anywar, 2009), however, its immunogenicity is not conclusively known. Conducting this study on Trypanosoma PGP 1 will demonstrate if the protein can elicit specific and quantifiable antibodies. Such a confirmation would make Trypanosoma PGP 1 a suitable candidate for further exploration as diagnostic antigen for HAT and could lead to development of a new diagnostic test for HAT which would help save time and lives that could have been lost due to poor and unreliable diagnostic techniques.

1.5 Research Question Does Trypanosoma PGP 1 elicit specific immune response in mice?

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 African Trypanosomiasis Human African Trypanosomiasis (HAT) (Sleeping sickness) and Animal African Trypanosomiasis (Nagana), is a parasitic disease that affects humans and animals respectively. The disease is caused by African trypanosomes of genus Trypanosoma and these include; Trypanosoma congolense, evansi, vivax and brucei that included the sub species of T. b. brucei, T. b. gambiense, T. b. rhodesiense. Sleeping sickness has been reported in 37 countries in sub-Saharan Africa. Many of the affected populations live in remote areas with limited access to adequate health services, which hampers the surveillance and therefore the diagnosis and treatment of cases. In addition, displacement of populations, war and poverty are important factors leading to increased transmission and this alters the distribution of the disease due to weakened or non-existent health systems (WHO Fact sheet N259, 2010). Recent estimates indicate that over 60 million people living in some 250 locations are at risk of contracting the disease. There were under 10,000 cases reported in 2009 according to WHO figures

which represents a huge decrease from the estimated 300,000 new cases in 1998 (WHO, 1998). There are two forms of the disease depending on the parasite involved; Trypanosoma brucei gambiense is found in west and central Africa. This form currently accounts for over 95% of reported cases of sleeping sickness and causes a chronic infection. A person can be infected for months or even years without major signs or symptoms of the disease. When symptoms emerge, the patient is often already in an advanced disease stage where the central nervous system is affected; Trypanosoma brucei rhodesiense is found in eastern and southern Africa. Nowadays, this form represents fewer than 5% of reported cases and causes an acute infection. First signs and symptoms are observed a few months or weeks after infection. The disease develops rapidly and invades the central nervous system. Other parasite species and sub-species of the Trypanosoma genus are pathogenic to animals and cause Animal Trypanosomiasis Nagana in cattle. Animals can host the human pathogen parasites, especially T. b. rhodesiense; thus domestic and wild animals are an important parasite reservoir. Animals can also be infected with T. b. gambiense and act as a reservoir. However the precise epidemiological role of this reservoir is not yet well known. The disease in domestic animals, particularly cattle, is a major obstacle to the economic development of affected rural areas due to its devastating effects on both humans and livestock populations hence contributing to poverty in some of these endemic areas of Africa. 2.2 Trypanosoma brucei Trypanosoma brucei species is one of the causative agents of African Trypanosomiasis (or sleeping sickness). There are 3 sub-species of T. brucei: T. b. brucei, T. b. gambiense and T. b. rhodesiense. T. brucei gambiense causes chronic Trypanosomiasis in humans most common in central and western Africa, where humans are thought to be the primary reservoirs (Barrett et al., 2003). 5

T. brucei rhodesiense causes acute Trypanosomiasis in humans most common in southern and eastern Africa, where game animals and livestock are thought to be the primary reservoir (Barrett et al., 2003). Uganda is the only country where both forms of the disease are present; most likely, a potential geographical overlap of the two endemic areas (Picozzi et al., 2005). This may therefore hinder the field identification of the correct infective sub-species of Trypanosoma brucei hence hindering treatment as well. T. brucei brucei causes Animal African Trypanosomiasis along with several other species of Trypanosoma. T. b. brucei is not human infective due to its susceptibility to lysis by human apolipoprotein L1 (Vanhamme et al., 2003).

2.2.1 Life cycle of Trypanosoma brucei

Figure 1: Life cycle of Trypanosoma brucei in the human and the tsetse fly. Image credit: Alexander J. da Silva and Melanie Moser, Centers for Disease Control Public Health Image Library.

The life cycle of a trypanosome involves various developmental stages involving a series of differentiation in both the vector (tsetse fly) and the mammalian host. During the different stages of its life cycle, the parasites changes in morphology and cell structure. The infection in the host begins when the metacyclic trypomastigotes form of the parasite is injected into the host through the skin by the infected tsetse fly (Figure 1:-1). At the site of infection, the metacyclic trypomastigotes multiply locally for a few days after they enter into the lymphatic system and pass into the blood stream. Once in the hosts blood, the metacyclic trypomastigotes go through development then transform into long slender bloodstream forms covered by the variant surface glycoprotein (VSG), (Biebinger et al., 1996) (Figure 1:-2). These bloodstream trypomastigotes are carried to other sites

throughout the body and also reach other body fluids (e.g. lymph, spinal fluid) and 7

continue replication by binary fission as well as differentiate into intermediate forms and stumpy forms which have the VSG (Figure 1:-3-4). The entire life cycle of the African trypanosomes is represented by extracellular stages. A tsetse fly becomes infected with bloodstream trypomastigotes when taking a blood meal from an infected mammalian host and it is the stumpy forms are then taken up by the vector (Figure 1:-5). Once in the midgut of the fly, they transform into procyclic trypomastigotes, the VSG coat is then shed within a few hours replaced by a coat of procyclic acidic repetitive protein (PARP) also known as procyclin (Biebinger et al., 1996) (Figure 1:-6). The procyclic

trypomastigotes then multiply by binary fission, leave the mid gut and migrate to into the ectoperitrophic space then into the foregut (mouth parts) through the proventriculus. Once in the foregut, the procyclic trypomastigotes then change into elongated and asymmetrically dividing epimastigotes (Hill, 2003) (Figure 1:-7) which then multiply actively in the proboscis and move to the salivary glands for final development. Once in the salivary glands, the epimastigotes continue to multiply by binary fission to generating short epimastigotes which attach themselves to the salivary gland epithelium (Figure 1:8). The attached epimastigotes differentiate into VSG-coated metacyclic trypomastigotes that suited for mammalian bloodstream environment (Figure 1:-1). The cycle then

continues. The cycle takes about 3 weeks in the fly, the tsetse fly then remains infective for the rest of its life (Chappuis et al., 2005). 2.3 Management of Human African Trypanosomiasis The management of the disease is basically on three steps; Screening for potential infection; which may involve use of serological techniques which are mostly available for T. b. gambiense, Diagnosing for the presence of the parasite and Staging to determine the state of the disease progression after which treatment can be effected. 2.3.1 Diagnosis of Human African Trypanosomiasis The diagnosis of HAT is based on three techniques; Serological technique, Parasitological technique and Molecular technique (WHO Fact sheet N259 2010). Diagnosis must be made as early as possible and before the neurological stage in order to 8

avoid complicated, difficult and risky treatment procedures. However, for proper, effective and accurate treatment, staging of the disease has to first be performed since the drugs used in the treatment of first stage is different from that used to treat second stage. Drugs used to treat first stage are normally effective and of low toxicity compared to drugs used to treat second stage therefore staging has to be accurate. Laboratory diagnosis range from simple procedures like microscopy to detect trypanosomes in body fluids; lymph node aspirates, chancres fluid, blood and cerebrospinal fluid (CDC 2006) to complicated procedures like most of the molecular techniques such as Polymerase chain reaction (PCR). 2.3.1.1 Serological techniques This involves using serological tests (mostly available for T. b .gambiense). Due to fluctuating parasitemia in T. b .gambiense, serological tests are important in screening for infection. 2.3.1.1.1 The Card Agglutination Test for Trypanosomiasis (CATT) Developed in the late 1970s, the CATT is a fast and simple agglutination assay for detection of trypanosome antigens however, CATT is only effective for T. b. gambiense, it is not effective and reliable for T. b. rhodesiense. It is mostly used for T .b. gambiense specific antibodies in the blood, plasma, or serum of HAT patients (Magnus et al., 1978). The antigen consists of lyophilised bloodstream forms of T. b. gambiense variable antigen type LiTat 1.3. The trypanosomes are fixed, stained with Coomassie blue, and freeze-dried. One drop the CATT reagent is mixed with one drop of blood and shaken for 5 min on the rotator, and the result is visible to the naked eye. The reported sensitivity of the CATT on undiluted whole blood (CATT-wb) varies from 87 to 98%, and the negative predictive value is excellent during mass population screening (Noireau et al., 1987; Truc et al., 2002). Nevertheless, false-negative CATT results can occur (Penchenier et al., 1991), as suspected in patients infected with strains of trypanosomes that lack or do not express the LiTat 1.3 gene (Dukes et al., 1992; Enyaru et al., 1998). Furthermore, when the CATT is performed on undiluted blood or serum with a low dilution of less than 1:4, 9

the agglutination can be inhibited, a phenomenon called prozone which can be overcome by adding EDTA to the dilution buffer (Pansaerts et al., 1988), this increases the sensitivity (Magnus et al., 2002). The test has a reported specificity of around 95% but the positive predictive value is limited because the test is used for mass screening in populations where the prevalence of HAT is usually below 5% (Robays et al., 2004). False-positive results can occur in patients with malaria and other parasitic diseases such as transient infection by nonhuman trypanosomes (Magnus et al., 1978). 2.3.1.1.2 Antibody detection Indirect evidence for trypanosome infection can be obtained by demonstrating specific antibodies in serum of infected hosts. Trypanosomes have a complex antigenic structure evoke production of a large spectrum of antibodies. T. b. gambiense specific IgG and IgM antibodies are present in high concentrations and are directed mainly against the Immunodominant surface glycoprotein antigens of the parasite. The sensitivity and specificity of the test to be used to detect these antibodies greatly depends on the antigen(s). The available current serological used to detect antibodies include Enzyme-linked immunosorbent assay (ELISA) that can antibodies after 3 to 4 weeks of infection (Vanhamme et al., 2001) with strict standardisation and quantification (Lejon et al., 1998) but sero-positivity must be interpreted with caution in previously treated patients since antibodies can persist for up to 3 years after cure (Paquet et al., 1992). ). ELISA can also detect specific antibodies in the saliva HAT (Lejon et al., 2003). However, ELISA requires time and sophiscated equipments like ELISA reader and does some of the other antibody detection techniques hence limiting their use in field diagnosis and reference laboratories for remote testing of samples collected in the field during surveys. 2.3.1.1.3 The LATEX agglutination test for T. b. gambiense The test has been developed as a field alternative to the CATT (Bscher et al., 1999). The test is based on the combination of three purified variable surface antigens, LiTat 1.3, 1.5, and 1.6, coupled with suspended latex particles. The test procedure is similar to the CATT, including the use of a similar rotator. Compared to the CATT, the LATEX shows 10

a higher specificity (96 to 99%) but a lower or similar sensitivity (71 to 100 (Penchenier et al., 2003; Magnus et al., 2002). However, the LATEX agglutination test is only available T. b. gambiense and not available for T. b. rhodesiense. 2.3.1.1.4 Immunofluorescence Assays Immunofluorescence assays have been used with success for HAT control in Equatorial Guinea, Gabon, and the Republic of Congo, where they were shown to be highly sensitive and specific (Noireau et al., 1988). The availability of standardised antigen commercially in the market at low cost has greatly improved the reliability of the test (Magnus et al., 1978). It can be used with serum but the test sensitivity has been reported to be as low as 75% when used with impregnated filter papers (Simarro et al., 1999). However this technique requires sophisticated equipment like an immunofluorescent microscope and this limits its use in remote areas. 2.3.1.2 Parasitological techniques The diagnosis of the presence of parasites has been achieved greatly through a number of parasitological tests. Parasitological diagnosis is made by microscopic examination of lymph node aspirate, blood, or CSF and this provides direct evidence for trypanosome infection thus allowing definite diagnosis. Unfortunately, it is estimated that 20% to 30% of patients are missed by the standard parasitological techniques (Robays et al., 2004). There is also always fluctuation in parasite numbers in T. b. gambiense infection 10,000 trypanosomes per ml, being easily detectable and less than 100 trypanosomes per ml, being below the detection limit of the most sensitive methods in use. This implies, failure to demonstrate parasites does not necessarily exclude infection. Parasite detection can be rather labor-intensive. Some of the available parasitological detection methods that are currently field use are mentioned below (WHO Trypanosomiasis Control Manual 1983). 2.3.1.2.1 Chancre aspirate Trypanosomes can be detected in the chancre a few days earlier than in the blood. The chancre is punctured, and the fluid obtained is microscopically examined as a fresh or 11

fixed and Giemsa-stained preparation. This method is very seldom applied in the field because most infections are detected much later, when the chancre has already disappeared. 2.3.1.2.2 Lymph node aspirate Cervical Lymph Node (CLN) palpation is done systematically in conjunction with CATT, in all patients with a positive CATT result. Enlarged CLNs are punctured and fresh aspirate is expelled onto a slide, and a cover slip is applied to spread the sample and facilitate the reading. The wet preparation is then immediately examined under X400 magnification for the presence of motile trypanosomes. The technique is simple and cheap. The sensitivity varies between 40 and 80% depending on the parasite strain, the stage of the disease (sensitivity is higher during the first stage), and the prevalence of other diseases causing lymphadenopathy (Simarro et al., 2003; Van Meirvenne, 1999). 2.3.1.2.3 Wet and thick blood films In wet blood films, 5 to 10 l of finger prick blood is placed on a slide, cover slipped and examined microscopically at X400 magnification. Trypanosomes can be seen moving between the erythrocytes. Although this method has very low sensitivity of about 10,000 trypanosomes per ml, it is still used in some centers because of its low cost and simplicity. Examination of 20 l of stained thick blood film slightly improves sensitivity, with a detection threshold of around 5000 trypanosomes per ml. It is the technique of choice for blood examination only when no centrifuge is available (Henry et al., 1981). The technique is quite time consuming and requires expertise to recognize the parasite, which is frequently deformed in this preparation. 2.3.1.2.4 Microhematocrit centrifugation technique The blood concentration microhematocrit centrifugation technique (mHCT) sometimes referred to as the capillary tube centrifugation technique or as the Woo test, was developed more than 30 years ago and is still in use in many HAT control programs (Woo P. T., 1971, 1970). In brief, capillary tubes containing anticoagulant are filled three12

quarters full with finger prick blood. The dry end is sealed with plasticine. By high-speed centrifugation in a haematocrit centrifuge for 6 to 8 min, trypanosomes are concentrated at the level of the white blood cells, between the plasma and the erythrocytes. The capillary tubes, mounted in a special holder, can be directly examined at low magnification (x100 or x200) for mobile parasites. The sensitivity of mHCT increases with the number of tubes examined, with an estimated detection threshold of 500 trypanosomes per ml. This technique is moderately time-consuming, and the concomitant presence of microfilaria in the blood can render the visualisation of the much smaller trypanosomes very difficult. Nevertheless, this relatively simple technique can be applied during mass screening by mobile teams. 2.3.1.2.5 Quantitative buffy coat The quantitative buffy coat (QBC) initially developed for the rapid assessment of differential cell counts, has been extended to the diagnosis of hemoparasites including trypanosomes (Levine et al., 1989; Bailey et al., 1992). It has the advantages of concentrating the parasites by centrifugation and, by staining the nucleus and kinetoplast of trypanosomes with acridine orange, allowing a better discrimination from white blood cells. After high-speed centrifugation of the blood in special capillary tubes containing EDTA, acridine orange, and a small floating cylinder, motile trypanosomes can be identified by their fluorescent kinetoplasts and nuclei in the expanded buffy coat. UV light is generated by a cold light source connected by a glass fiber to a special objective containing the appropriate filter and the procedure is done in a darkroom. However the relative sophistication and fragility of the material prevents its daily use during active screening sessions. QBC technique is a very sensitive technique with 95% sensitivity for trypanosome concentrations of 450 per ml. The QBC can detect more patients with low parasitemia than the mHCT when fewer than eight capillary tubes are used (Ancelle et al., 1997). It is as sensitive as the mini-anion-exchange centrifugation technique (mAECT) (Ancelle et al., 1997; Truc et al., 1998).

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2.3.1.2.6 Mini-anion-exchange centrifugation technique The mAECT was introduced by Lumsden et al, based on a technique developed by Lanham and Godfrey (Lanham and Godfrey, 1970). An initial evaluation showed that the mAECT was more sensitive than the thick blood film and the mHCT (Lumsden et al., 1981). An updated version has been described by Zillmann et al., 1996. The technique consists of separating the trypanosomes, which are less negatively charged than blood cells, from venous blood by anion-exchange chromatography and concentrating them at the bottom of a sealed glass tube by low-speed centrifugation. The tip of the glass tube is then examined in a special holder under the microscope for the presence of trypanosomes. The large blood volume (300 l) enables the detection of less than 100 trypanosomes/ml and therefore giving a high sensitivity. However, the manipulations are quite tedious and time consuming. 2.3.1.3 Molecular diagnosis Molecular diagnosis also offers another alternative in the diagnosis of HAT. The methods used in molecular diagnosis are known for being sensitive (Bscher et al., 2004). These methods range from Polymerase Chain Reaction (PCR) for amplifying DNA to Loop Mediated Isothermal Amplification. However, most of the molecular methods for diagnosis of HAT are under trial and have not yet been recommended for the diagnosis of HAT. 2.3.1.3.1 Polymerase Chain Reaction (PCR) Different assays now exist; however, none of them have been validated for diagnostic purposes. PCRs targeting repetitive sequences are in theory more sensitive than those targeting low-copy or single-copy sequences like the recently developed tests for distinguishing T. b. gambiense and T. b. rhodesiense (Jamonneau et al., 2001; Kabiri et al., 1999; Radwanska et al., 2002; Schares et al., 1996). In principle, PCR can be applied to any patient sample that may contain trypanosome DNA, such as whole blood or buffy coat, lymph node fluid, or CSF. Samples should be stabilized in special buffers. However, the amount of sample that can be applied on filter paper is small, thus limiting the chance 14

to contain enough DNA for detection. Samples should be protected from sunlight to avoid DNA degradation. PCR results are not always unequivocal. Unexplained false-negative and false-positive results were observed in CATT-sero-positive but parasitologically nonconfirmed persons and in CATT-negative controls (Garcia et al., 2000; Solano et al., 2002). Also, the significance of a positive PCR on a CSF sample is unclear. PCR is 100% sensitive compared to double centrifugation of CSF (Truc et al., 1999), but a number of patients with positive PCR results with CSF were successfully treated with pentamidine, thus showing them to be in the first stage of the disease (Jamonneau et al., 2003). The methods needs simplification hence PCR is not an option for field diagnosis and for the time being is restricted to research purposes. 2.3.1.3.2 Loop-mediated Isothermal Amplification (LAMP) LAMP is a rapid, simple and highly sensitive technique that is used for gene amplification (Notomi et al., 2000). The technique bases on autocycling strand displacement synthesis of DNA by Bacillus sterothermophillus (Bst) DNA polymerase under isothermal conditions (60-65oC). It uses 6 primers recognized by 8 selections of target DNA hence increasing specificity, rapidity and efficiency. It amplifies target DNA three fold every half cycle producing large amounts of product within 30-60 minutes (Notomi et al., 2000). Visualisation is achieved through addition of a fluorescent dye SYBR Green I to the DNA formed (Poon et al., 2006). The technique takes a short time and can be carried out in an incubator. 2.3.1.4 Diagnosis to Stage Human African Trypanosomiasis Staging of patients with HAT relies on examination of CSF obtained by lumbar puncture. This is a vital step in the diagnosis process and determination of treatment for HAT. The first stage corresponds to presence of parasites in the in the blood and lymph, the second stage involves presence of parasites in the CNS (Chappuis et al., 2005). Staging of the disease is critical and must be made as accurate as possible due to different drugs used to treat first stage and second stage. Treatment success in the second stage depends on a drug that can cross the blood-brain barrier to reach the parasite such drugs like 15

melarsoprol, an arsenical derivative associated with a 2 to 10% fatality rate (Pepin et al., 1994) are toxic ,complicated to administer and have side effects such as the brain disorder and damage like post-treatment reactive encephalopathy (PTRE). Therefore wrong staging may result not only to administering wrong treatment to the patient but also trauma and even death of the patient due to wrong drug given. A number of methods for staging the disease are available. 2.3.1.4.1 White blood cell count from the CSF The CSF white blood cell count is the most widely used technique for stage determination. After collecting the CSF sample by lumbar puncture, the cell count should be carried out as soon as possible to prevent cell lysis. Due to the small number of cells in normal CSF, a cell-counting chamber has a volume of at least 1 l, such as the FuchsRosenthal and the Neubauer devices. It is not recommended to dilute the CSF with Trck solution since this solution can lyse trypanosomes. Patients with 6 to 20 white blood cells per l in the CSF are sometimes referred to as being in the "early second stage" or "intermediate stage" of the illness. 2.3.1.4.2 Protein concentration In normal healthy individuals, proteins in the CSF consist mainly of albumin (70%) and IgG (30%), both originating from the serum. Protein concentrations in the CSF are elevated in HAT patients and range from 100 to 2,000 mg/liter (Bisser et al., 2002; Lejon et al., 2003). Protein concentrations can also be raised in first-stage illness due to the diffusion of IgG into the CSF, which can be present in very high concentrations in the serum. Recent evidence suggests that the protein concentration threshold set by WHO (370 mg/liter) is too low and should be raised to 750 mg/liter to reflect blood-brain barrier (BBB) impairment, astrocyte activation, and neuro-degeneration (Lejon et al., 2001). CSF protein concentration is simple and accurately determines the total protein concentration in CSF. However the technique is rather difficult and the CSF protein concentrations obtained by different methods and different standards are not comparable. Due to the sophistication of methods, the absence of standardisation, the instability of reagents, and 16

the limited added value compared to CSF cell count (Miezan et al., 1998), it is no longer recommended and has been virtually abandoned in field laboratories. 2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF CSF of second-stage HAT patients contains high levels of immunoglobulins, especially IgM (Bisser et al., 1997). An increased CSF IgM concentration has thus been considered by some as a strong potential marker of second-stage HAT. High IgM levels in CSF is due to intrathecal synthesis, the dominance of IgM presence is an early marker of CNS invasion whereas blood-CSF barrier dysfunction is found in late CNS involvement (Lejon et al., 2003). Despite its relevance to stage determination, IgM detection in CSF has not been carried out in the field, owing to the lack of simple and robust tests. A latex agglutination test for IgM in CSF (LATEX/IgM) has recently been developed and is designed for field use (Lejon et al., 2004). This method is however still not being used due to specific trypanosome antibodies, anti-galactocerebrosides and trypanosome DNA in the CSF, hence other methods are still undergoing evaluation and development for staging HAT (Lejon et al., 2004). 2.3.1.4.4 Trypanosomes detection in the CSF The finding of trypanosomes in CSF allows immediate classification of a patient as being in the second stage of illness. It is important to examine the CSF immediately after lumbar puncture, because trypanosomes in CSF start to lyse within 10 min. Direct detection of trypanosomes (e.g., during cell counting) is a simple and cheap technique but suffers from insufficient sensitivity. Increased sensitivity of trypanosome detection is obtained by centrifugation of the CSF sample, especially when a double centrifugation method is used (Cattand et al., 1988). The latter method is relatively timeconsuming and requires two different types of centrifuges; therefore, it is not applicable in every field setting. A modified and simplified single centrifugation of CSF using a sealed Pasteur pipette has been proposed as an alternative to double centrifugation (Miezan et al., 2000).

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2.3.1.5 Treatment of the disease Treatment is better done after staging of the disease. The drugs used in the first stage of the disease are of lower toxicity and easier to administer hence, the earlier the disease is identified, the better the prospect of a cure. Treatment success in the second stage depends on a drug that can cross the blood-brain barrier to reach the parasite. Such drugs are toxic and complicated to administer. Four drugs are registered for the treatment of sleeping sickness in endemic countries; Pentamidine; discovered in 1941, used for the treatment of the first stage of T. b. gambiense sleeping sickness. Despite non-negligible undesirable effects, it is in general well tolerated by patients. Suramin; discovered in 1921, used for the treatment of the first stage of T. b. rhodesiense though it provokes certain undesirable effects, in the urinary tract and as well as allergic reactions. Melarsoprol; discovered in 1949, it is used in second stage of both forms of infection. It is derived from arsenic and has many undesirable side effects. The most dramatic is reactive encephalopathy (encephalopathic syndrome) which can be fatal (3% to 10%). An increase in resistance to the drug has been observed in several foci particularly in central Africa. Eflornithine; is less toxic than melarsoprol and was registered in 1990. It is only effective against treatment of second stage infection due to T. b. gambiense. A combination treatment of nifurtimox and eflornithine has been recently introduced in 2009. It simplifies the use of eflornithine in monotherapy, but unfortunately it is not effective for T.b. rhodesiense. Nifurtimox is registered for the treatment of American trypanosomiasis but not for human African trypanosomiasis. Nevertheless, after safety and efficacy data provided by clinical trials, its use in combination with eflornithine has been accepted and included in the WHO List of Essential Medicine, and it is provided free of charge for this purpose by WHO (WHO Fact sheet N259, 2010). 2.4 Pyroglutamyl peptidase 1 (PGP 1) PGP 1 is a putative protein and belongs to a group of peptidase called Cysteine peptidases. Cysteine peptidases have characteristic molecular topologies in 3 dimensions and 2 dimensions (MEROPS database). They possess cysteine nucleophile and catalytic residues in the order Glutamate, Cysteine, Histidine in sequence. Cysteine peptidases are 18

divided into clans and further into families (Barret and Rawlings 2001). PGP 1 belongs to Clan CF and Family C15. 2.4.1 Clan CF of Pyroglutamyl peptidase 1 The clan contains a single family, C15. The structure of PGP 1 qualifies it to clan CF because its protein fold is unlike that of any other cysteine peptidase. The tertiary structure for Pyroglutamyl peptidase 1 has been determined (Odagak et al., 1999) and shows an alpha/beta protein with an alpha/beta/alpha sandwich. PGP 1 enzyme is intracellular and soluble and in mammals, PGP 1 has been shown to release pGlu from thyrotropin-releasing hormone, luteinising hormone releasing hormone, neurotensin, bombesin and leukopyrokinin (Dando et al., 2003), but the physiological significance of this is unclear. 2.4.2 Family C15 of Pyroglutamyl peptidase 1 Peptidase family C15 contains omega peptidases that release an N-terminal pyroglutamate residue. There is a catalytic triad which occurs in the order Glu, Cys, His in the sequence. The only known activity of family C15 is removal of a pyroglutamate (pGlu) residue from the N-terminus of a peptide and typical synthetic substrates include; pyroglutamate (pGlu), 7-(4-methyl-) coumarylamide (NHMec) and pyroglutamate 4naphthylamine (pGluNHNap). The protein fold presented by PGP 1 is unlike that of any other cysteine peptidase and thus PGP 1 has a type structure for clan CF. 2.4.3 Trypanosoma brucei PGP 1 In Trypanosoma brucei, PGP 1 is present as a soluble cystosolic cysteine peptidase. It is located in chromosome Tb927_04_v4; 707328 - 708134 and is encoded by a single gene copy of 669 encoding 222 amino acids and protein of 25.1 Kda with a predicted charge of -4 and an isoelecric point of 5.4. Trypanosoma PGP 1 has no signal transmembrane domain or GPI anchor (GenDB) and is liberated into the host blood stream during intravascular destruction of trypanosomes in the host during an infection and therefore it has been postulated to take part in the pathogenesis of HAT (Morty et al., 2006). It is 19

expressed in all life cycle stages of Trypanosoma brucei and four other blood stream African trypanosomes (Morty et al., 2006). Trypanosoma PGP 1 is optically active and stable at bloodstream pH and it is insensitive to host plasma cysteine peptidase inhibitors such as cystatin C, kininogen and alpha-macroglobulin (Morty et al., 2006) and this makes it a possible factor in the pathogenesis of HAT. During infection, Trypanosoma PGP 1 is liberated into the blood stream and causes degradation of the peptides; TRH and GnRH (Morty et al., 2006) by removing the N-terminal Pyroglutamyl residue of these peptides which protects the peptides from proteolysis (Odakagi et al., 1999) and this exposes the peptides to proteolysis. 2.4.4 Sequence of Trypanosoma PGP 1 Source: http://www.genedb.org/featureSeq/Tb927.4.2670 2.4.4.1 Gene sequence
ATGAAGCCTA CAAAACCACT ACTTTACATA ACGGGATACG GACCCTTCTT GGAAGTAACG GAGAACCCCA GCGCCACCAT TGCGCAAAGT GTAGCGGAAC AGGTGAGACA AAGTGGCGAA GCGGATGTCC ATCATGAAAC ACTAGACGTG AACTTAGAGG CCGTTTCCAA ATATTTCAAC CGCCTCAATG AATCCGTCAC CGCTCATCTG GAAGCCACAC ATCCCGAGAA TCGAGTACTT CTCGTCAACG TGGGCCTTCA CAGTCGCGAA AAGGAAAAGG TACTGCGGCT GGAAGTGCGC GCCTTCAATG AACTGGAGGG AAACCCCATC GATGATGAGC TTCCCTTGAG TACATGCAAA GACAGTGCTT TCGTGAAGGG ATGCAAGCTC GAAACAACAA CAGCCCTCAT AGAGGAACTC AATGCGATTG AGAGAAATGG TAGCGATCAT CACGAAAAGC CTCGTTGGAT TATTTCTTAC GACGCGGGGC GATATTACTG CAACTATGCA CTGTACAGAG GCGTGAAGAT GCAGGAAGCT CTAAACAGCC GCGTGTTTGC CGTGTTTTTG CACATTGTAG CATCCACTGT CGTGTGCATG GAAGAGCAGG TTGCGCAGGT CCGCATGCTT GTGTCGCACC TCTTGAAACA CATGGAAGCA GTTGAATGA

2.4.4.2 Amino acid sequence

MKPTKPLLYI TGYGPFLEVT ENPSATIAQS VAEQVRQSGE ADVHHETLDV NLEAVSKY FN RLNESVTAHL EATHPENRVL LVNVGLHSRE KEKVLRLEVR AFNELEGNPI DDELPLST CK

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DSAFVKGCKL ETTTALIEEL NAIERNGSDH HEKPRWIISY DAGRYYCNYA LYRGVKM QEA LNSRVFAVFL HIVASTVVCM EEQVAQVRML VSHLLKHMEA VE

2.5 Immunogenicity Immunogenicity refers to the characteristic that endows a protein with the ability to provoke an immune response (Singh, 2011), this should not be confused with Antigenicity which is the ability of a protein to combine specifically with the final products of the immune response (i.e. secreted antibodies and/or surface receptors on Tcells) (Kuby Immunology, 2006). Several immunogenicity studies have been conducted on several proteins both therapeutic and diagnostic. A number of factors affect the immunogenicity of a protein and these include the following; 2.5.1The Nature of the Protein Under this a number of factors are examined; Degree of foreignness, Molecular size, Chemical structure and heterogeneity (structural properties) and Ability to be processed and presented by an APC 2.5.1.1 Degree of foreignness Protein capacity to induce the synthesis of specific antibodies is shown to be correlated with protein evolution rate (Ogievetskaya, 1977), the greater the phylogenetic distance between the two organisms, the higher the immunogenicity 2.5.1.2 Molecular size Proteins with high molecular weights are strong immunogens i.e. give higher levels of immunogenicity than low molecular weight proteins (Dintzis et al., 1976).Most
immunogens are large, complex molecules with a molecular weight generally greater than about 100,000 daltons. In general large molecules are better immunogens as compared to smaller molecules.

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2.5.1.3 Structure of the protein A number of structural properties affect the immunogenicity of the protein ranging from sequence variation, glycosylation, complexity in structure, and domain sites. Glycosylation is believed to interfere with antibody binding and to have an impact on auto immunity (von Delwig et al., 2006). The structure of a protein ranging from the amino acid sequence to the tertiary structure and quaternary structure to the presence domain sites and glycosylation all affect the immunogenicity of a protein. 2.5.1.4 Ability to be processed Downstream processing of a product can also influence its immunogenicity. Impurities and contaminants associated with antibody development have also been found in studies on insulin and growth hormone products (Reeves W. G., 1986). 2.5.2 Route of administration The route of administration can influence the immunogenicity of the protein (Schellekens, 2005); however there have been no published cases whereby a change in administration route completely negated immunogenicity (Schellekens H., 2003). There are different routes of administration of a protein to an organism and these include, subcutaneous, intramuscular, intravenous and topical among others. The route of administration cannot render a protein immunogenic, although it can enhance the likelihood of an immune reaction to a protein that is already immunogenic. 2.5.3 Genetic makeup of the organism The genetic background of an organism can sometimes influence immunogenicity. A well established example is with hemophilia, whereby the genetic defect determines whether an individual will or will not produce antibodies (Fakharzadeh et al., 2000). In some studies, there have been conflicting results from studies into the influence of the major histo-compatibility complex (MHC) on responses to products such as growth hormone and insulin indicating that MHC has no real effect. 22

2.5.4 Adjuvant Adjutants increase likelihood of immunogenicity by stimulating innate immune response, boosting the humoral immune responses to enhance antibody responses (O'Hagan et al., 2004). Many commonly used adjuvants are effective at elevating serum antibody titers, but do not elicit significant Th1 responses or cytotoxic T lymphocytes (CTLs) (Pashine et al., 2005). 2.5.5 Dose of protein given Proteins have been shown to induce immune responses, in particular when administered as booster doses over prolonged periods (Porter et al., 2001; Ryff et al., 2002). In some cases, increasing the dose can help increase the efficacy and immunogenicity of a given protein. 2.5.6 Formulation and purity of the protein Appropriate formulation of a protein product is highly important. Stabilisation of a protein is important since inadequacy in this may result into protein to aggregate or denature, which may affect immunogenic potential (Cleland et al., 1993). Formulation becomes even more crucial for products that may not be optimally stored or handled (EMEA data base). Purity of a protein is very important since contaminating agents like bacterial proteins can relatively increase the immunogenicity of a given protein (Gooding et al., 1985) hence giving a false positive result, therefore the protein should be as pure as possible. 2.6 Overview of the techniques to be used A few of the methods to be used during the study is mentioned below; 2.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE is a technique widely used in Molecular Biology to separate proteins according to their molecular weight. In this technique, SDS is a strong anionic detergent 23

when mixed with proteins; the proteins acquire a negative charge. In combination with other factors like -Mercaptoethanol and heat, complete denaturation of proteins with SDS can be achieved forming a SDS-polypeptide complex. When loaded onto a Polyacrylamide gel matrix which acts as the support medium for electrophoresis then an electric field applied, the polypeptide complexes migrate to the positive electrode of the electrophoretic tank and in the process, the Polyacrylamide provides the molecular sieving effect that separates the proteins basing on their molecular size/ weight. The proteins on the gels are the visualised by staining and the some of the protein can be estimated by comparing the running distance against the standard protein of known molecular weight (Sambrook et al., 1989). 2.6.2 Western Blotting Western blotting is an analytical technique used to detect specific proteins. The protein (s) are first separated by SDS-PAGE then transferred to membrane (usually nitrocellulose or PVDF) using an electric current; the gel that contains the protein is put on the negative terminal while the membrane is put on the positive terminal, the proteins are then transferred form the negative to the positive and in the process they are deposited onto the membrane. To visualise the proteins, probing is done using antibodies specific to the target protein (primary antibody) (Towbin et al., 1979; Renart et al., 1979). The primary antibody is then bound to a secondary antibody (anti-immunoglobulin) that is conjugated to an enzyme (peroxidase). When a substrate specific to the enzyme is bound on the secondary antibody, a signal inform of a band on the membrane is detected. 2.6.3 Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-linked immunosorbent assay is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. In simple terms, ELISA is technique that uses the antigen-antibody reaction. In this technique, an antigen is attached to the surface of the ELISA plate, the antibody specific to the antigen is then bound to the antigen. Visualisation is achieved by using an enzyme conjugated antibody, the antibody

24

binds to the antigen and the enzyme binds to the substrate; the end result is change in color that can be quantified by measuring Optical Density (O.D). 2.7 pET-28a (+) vector The pET-28a (+) vector carries an N-terminal His-Tag configuration plus an optional C-terminal His-Tag sequence. Unique sites are shown on the vector map below. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular vector map. The vector has a cloning/expression region of the coding strand transcribed by T7 RNA polymerase. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single stranded sequencing is performed using the T7 terminator primer (Novagen).

Figure 2: Vector map of pET28a (+)

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CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1.0 Study design This was an experimental study. The study involved use of 25 male Swiss albino mice 78 weeks old, obtained from by the Molecular Biology Laboratory, Department of Parasitology and Microbiology, College of Veterinary Medicine, Animal resources development and Biosecurity. These mice were divided into four groups; Test group, Adjuvant group, Negative control group and the bacterial protein group, each group containing 5 mice. During the study, the Trypanosoma PGP 1 as recombinant protein was obtained from previously transformed BL21DE E. coli cells through expression. The protein was then extracted from the E. coli cells, purified, quantified and immunised in mice. The results pertaining to antibody production in mice were to be got after analysis of sera collected from the mice. 3.2.0 Materials: 3.2.1 Transformed BL21DE3 cells BL21DE3 cells containing pET28a (+) carrying the gene for PGP 1 were provided as glycerol stocks by Denis Anywar. 3.3.0 Methods: 26

3.3.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells provided 3.3.1.1 Growth of glycerol stocks containing pET28a (+) Using a sterile loop, the glycerol stock was streaked on LB agar containing 30g/ ml of kanamycin under sterile conditions and incubated at 37o C overnight. A plate without kanamycin and cells and plate with kanamycin but not streaked were also incubated to determine the level of sterility of the incubated colony. A colony was picked under sterile conditions from the plate with kanamycin since it was the only one that had cells and the other plates had no cells meaning that the LB agar was sterile as well as kanamycin. The colony was cultured in 10ml of sterile terrific broth containing 30g/ ml of kanamycin, at 37oC overnight at 150 rpm shaking. 3.3.1.2 Plasmid extraction This was done to confirm if the glycerol stocks still contained the plasmid carrying the insert. From the glycerol stocks, 1.5 ml of the cells from the media were pipetted and centrifuged in microcentrifuge tubes to obtain a pellet. The plasmid was extracted from the tubes using the QIAGEN extraction kit (see Appendix II). Ten micro-liters aliquot from the plasmid extract was analyzed in a 1% Agarose Gel at 100V for 30 minutes. 3.3.1.3 Agarose Gel Electrophoresis One percent agarose gel was prepared by dissolving 0.3g of agarose powder in 30 ml of TAE buffer containing 0.005% ethidium bromide. The mixture was warmed in a microwave until the agarose dissolved. The solution was poured into a casted plate with comb and left to polymerise. The sample was prepared by mixing 10 l of sample with 1 l of the DNA sample loading dye; ten micro-liters of the prepared sample was loaded into the polymerised agarose well. The electrode terminals were connected to the electrophoretic tank and the gel ran at constant voltage of 100V for 30 minutes. The gel after running was visualised under UV-light illumination.

27

3.3.1.4. Restriction enzyme digestion The plasmid extracted was digested using BamHI and HindIII enzymes in a reaction mixture containing; 0.5 l of BSA, 4.5 l of PCR water, 3.0 l of 10X buffer, 10.0 l of extracted plasmid DNA template, 1.0 l of Bam HI and 1.0 l of Hind III. The mix was incubated for 2 hours and 30 minutes at 37oC in a water bath. Ten micro-liters aliquot from the digest was analyzed in a 1% Agarose Gel at constant voltage of 100V for 30 minutes. 3.3.2 Expression of the recombinant Trypanosoma PGP 1 3.3.2.1 Small scale expression A small scale expression in 100 ml terrific broth was done to confirm whether the protein can be expressed and also to standardize the expression protocol. Glycerol stocks were grown as in 3.4.1. The cells were transferred under sterile conditions to 100 ml of terrific broth containing 30 g/ml of kanamycin and grown to an OD > 0.6 at 37oC at 200 rpm shaking. An aliquot from the pre-induced expression was taken centrifuged mixed with protein sample loading buffer and stored for analysis. The expression was then induced with 1mM IPTG for 2 hours and 30 minutes; an aliquot from the expression was taken, centrifuged and mixed with protein sample loading buffer. From the pre-induced and induced prepared samples, 10 l was picked and analyzed together in a 15% SDS-PAGE gel at 200V for 1 hour. Western Blot analysis using anti-His antibody was also done to confirm the expressed protein. 3.3.2.1.1 Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) A 15% SDS-PAGE gel was prepared; the casting plates were prepared before preparing the gel. During the preparation of the gel, 15% Separating gel was prepared by adding the following volumes in a 15 ml tube; 2.5 ml of (30%) monomer, 1.1 ml of distilled water, 1.3 ml of separating buffer, 60 l of (10%) SDS, 30 l of APS and 10 l of TEMED total volume 5 ml (the last two reagents were added last). The mix was immediately cast into the plates, leveled with distilled water and left to polymerise for 30 minutes. The distilled 28

water used to level was drained off after polymerisation. The 4% stacking gel was then prepared by adding the following volumes in a 15 ml tube; 680 l of (30%) monomer, 3.0 ml of distilled water, 1.2 ml of stacking buffer, 100 l of (10%) SDS, 60 l of APS and 5 l of TEMED in a total volume of 5 ml (the last two reagents were added last). The mix was immediately cast into the plated and the combs inserted immediately and left to polymerise for 30 minutes. The protein samples were prepared by mixing with sample loading buffer (see Appendix III) in a ratio of sample loading buffer to sample of 1:4. The combs were removed, the plates loaded into the electrophoretic tank and running buffer (see Appendix III) poured into the tank. Ten micro-liters of the prepared samples were loaded into the wells in two parts. The electrodes were connected to the tank the gel ran at constant volume of 200V for 1 hour. One part of the gel was stained with Coomasie brilliant blue, the other part of the gel was transferred to the nitrocellulose membrane by western blotting. 3.3.2.1.2 Western Blotting After SDS-PAGE, the gel and the membrane were equilibrated in transfer buffer (see Appendix III). The gel and the membrane were fixed on the western blot cassette with the gel on the negative (black) and the membrane on the positive (white). The cassette containing the gel and membrane and a dummy cassette were then fixed into the transfer tank; the transfer buffer was then poured into the tank. An ice pack was inserted into the tank; the electrodes were then connected to the tank. The transfer was done at constant voltage of 100V for 1 hour. After transfer, the membrane was blocked in 5% skimmed milk overnight. The membrane was washed three times in PBS-T buffer with shaking for 15 minutes per wash. The wash was poured off and primary antibody added in a dilution of 1:2000 in PBS-T and incubated for 1 hour with shaking at room temperature. The membrane was washed three times using PBS-T buffer with shaking for 10 minutes per wash. The wash was poured off and secondary antibody added in a dilution of 1:10,000 in PBS-T and incubated for 1 hour with shaking at room temperature. The membrane was then washed three times using PBS-T buffer with shaking for 10 minutes per wash and the wash poured off. A solution of Diaminobenzidine (DAB) in PBS was then added and 100 l of hydrogen peroxide added to visualize the bands. 29

3.3.2.2 Large scale expression The small scale expression confirmed that the protein could be expressed; however, the purification from of the protein form the small scale expression gave very low amount and hence the need for a large scale expression in 1000 ml. Glycerol stocks were grown as in 3.4.1; the cells were then transferred under sterile conditions to 100 ml terrific broth containing 30 g/ml of kanamycin and grown at 37oC overnight at 150 rpm shaking. The cells from the 100 ml were transferred under sterile conditions to 1000 ml of terrific broth containing same concentration of kanamycin and grown to an OD>0.6 at 37oC at 200 rpm shaking. An aliquot from the pre-induced sample was taken and treated as in small scale expression. The expression was then induced using 1 mM of IPTG for 2 hours and 30 minutes. An aliquot from the induced sample was treated as in small scale expression. From the pre-induced and induced prepared samples, 10 l was picked and analyzed together in a 15% SDS-PAGE gel at constant voltage of 200V for 1 hour. 3.3.3 Extraction, Purification and Quantification Trypanosoma PGP 1 and bacterial protein 3.3.3.1 Extraction 3.3.3.1.1 Extraction of Trypanosoma PGP 1 The protein was extracted using cell lysis protocol described in Sambrook et al, 1989. The cells after the large scale expression were centrifuged at 10,000 rpm for 10 minutes in a pre-weighed empty 50 ml tube. A pellet of 3.2943 g was obtained in the 50 ml tube. Ten milliliters of the lysis buffer (Appendix III) was added followed by 264 l of 1 mg/ml of lysozyme and protease cocktail inhibitor (according to manufacturers instructions). To the mixture, 13 mg of deoxycholic acid was also added to aid breakage of membrane of the cells. When the lysate was viscous, it was sonicated for 10 seconds four times at interval of 30 seconds. The lysate was placed at room temperature until it was no longer viscous. The lysate in the 50 ml tube was centrifuged at 10,000 rpm for 30 minutes; the supernatant was separated from the pellet. Aliquots from both the pellet and the supernatant were analysed on SDS-PAGE. The bulks were stored at -80oC. 30

3.3.3.1.2 Extraction of Bacterial protein 3.3.3.1.2.1 Growth of competent BL21 cells from the glycerol stock Competent BL21 cells (without insert) were grown in a volume 1000 ml of terrific broth for extraction of bacterial protein; using a sterile loop, the glycerol stock was streaked on LB agar containing no antibiotic under sterile conditions and incubated at 37o C overnight. A plate with kanamycin and competent BL21 cells and plate without cells or kanamycin were also incubated to determine the level of sterility of the incubated colony. A colony was picked under sterile conditions from the plate with no antibiotic since it was the only one that had cells and the other plates had no cells meaning that the LB agar was sterile as well as there were no contaminations in the BL21 cells glycerol stock. The colony was cultured in 10ml of sterile terrific broth containing no antibiotic, at 37oC overnight at 150 rpm shaking. The cells were transferred under sterile conditions to 100 ml terrific broth without kanamycin and grown at 37oC overnight at 150 rpm shaking. The cells from the 100 ml were transferred under sterile conditions to 1000 ml of terrific broth without kanamycin and grown at 37oC at 200 rpm shaking overnight. An aliquot was taken and treated with protein sample loading dye, 10 l of the aliquot was ran and analyzed in a 15% SDS-PAGE gel at 200V for 1 hour. 3.3.3.1.2.2 Extraction of bacterial protein The protein was extracted as the Trypanosoma PGP 1 using cell lysis protocol described in Sambrook et al, 1989. The cells from the 1000 ml terrific broth were centrifuged at 10,000 rpm for 10 minutes in a pre-weighed empty 50 ml tube. A pellet of 2.9734 g was obtained in the 50 ml tube. Nine milliliters of the lysis buffer was added followed by 237 l of 1 mg/ml of lysozyme and protease cocktail inhibitor (according to manufacturers instructions). To the mixture, 12 mg of deoxycholic acid was also added to aid breakage of membrane of the cells. When the lysate was viscous, it was sonicated for 10 seconds four times at interval of 30 seconds. The lysate was placed at room temperature until it was no longer viscous. The lysate in the 50 ml tube was centrifuged at 10,000 rpm for 30

31

minutes; the supernatant was separated from the pellet. Aliquots from both the pellet and the supernatant were analysed on SDS-PAGE. The bulks were stored at -80oC. 3.3.3.2 Purification of Trypanosoma PGP 1 and bacterial protein from inclusion bodies 3.3.3.2.1 Purification of inclusion bodies The results from the extraction showed that Trypanosoma PGP 1 was expressed as an inclusion body; the protein was therefore purified from the pelleted inclusion bodies. In this case, the pellets were re-weighed to re-determine their weight after extraction; the weight had reduced, for the BL21DE3 cells (Trypanosoma PGP 1) was 2.9345 g and the competent BL21 (bacterial protein) cells was 2.7365 g. The two pellets were treated differently. To the pellet; 9 ml of buffer A (see Appendix III) was added and pellet was fully resuspended. The resuspended pellet was transferred to a clean 50 ml tube. Three hundred microliters of 10X stock of cocktail protease inhibitor was added followed by 50 l of 50 mg/ml of lysozyme. The mixture was placed in water bath set at 37oC until the solution was viscous. The mixture was sonicated to reduce the viscosity and chop up DNA. The solution was span at 10,000 rpm for 30 minutes and the supernatant transferred to a clean 15 ml tube. Nine milliliters of buffer B (see Appendix III) was added to the pellet, the pellet was fully resuspended. One hundred microliters of 10 X stock of cocktail protease inhibitor (SigmaFast) was added followed by, 90 l of Triton X-100. The mixture was then span at 10,000 rpm for 30 minutes and transferred the supernatant to a new 15 ml tube. To the pellet remaining in the tube, 10 ml of 8.0M urea (added DTT to 8.0M urea) was added to dissolve the pellet. An aliquot from the dissolved pellet was picked and ran on SDS-PAGE. 3.3.3.2.2 Purification of the recombinant Trypanosoma PGP 1 and bacterial protein The two samples once again were treated separately: To the dissolved pellet, 1.5 ml of Ni-NTA agarose was added and the mixture incubated at room temperature for 1 hour with shaking. The mixture was passed through a Ni-NTA fast start column to hold the agarose and allow the solution to flow through. The column was washed using 10 ml of 32

wash buffer (see Appendix III) while collecting 1ml aliquot from the flow through. The protein was eluted using increasing concentrations of in the elution buffers; one, two and three. The protein was eluted with 5 ml of elution buffer one, followed by buffer two and lastly buffer three (see Appendix III) while collecting 1 ml aliquot flow through. Portions were got form all the aliquots and analysed in SDS-PAGE. The aliquots were stored at 20oC. 3.3.3.3 Quantification of the Trypanosoma PGP 1 and bacterial protein The most pure aliquots from Trypanosoma PGP 1 purification with less background were pooled and for the bacterial protein, all the aliquots from the elution buffers were pooled. The two were quantified using Bradford dye quantification protocol: Ten different standards with varying concentration were prepared using 1 mg/ml of BSA (0.00, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5) mg/ml. The Bradford dye was prepared in distilled water in ratio of 1: 4 of Bradford dye to distilled water. Ten microliters of each standard was loaded in duplicates into a 96 well plate. Ten microliters of the pooled purified Trypanosoma PGP 1 and bacterial protein were as well loaded into the plate in duplicates. Two hundred microliters of the prepared Bradford dye in distilled water was added to each well with a sample; the plate was incubated at room temperature for 2 minutes. The optical density (O.D) was read at 595 nm using the plate reader. To estimate the concentration of the proteins, the results of the standards from the plate reader were analysed using Microsoft Excel 2007 by plotting a graph of O.D against concentration. From the equation of the line of best fit from the standard, the concentrations of the two proteins were estimated. 3.3.4 Immunisation of mice Swiss albino male mice 6-8 weeks old were used. The mice were divided in four groups, each group containing 5 mice. Group 1 (Test group) received the PGP 1 protein, Group 2 (Adjuvant group ) received only Quil A adjuvant, Group 3( Negative control group) did not receive the PGP 1 protein nor the adjuvant but only phosphate buffered saline (PBS), Group 4 (Bacterial protein group ) received the purified bacterial protein. Before the 33

immunisation, all the mice in the respective groups were first tail bled for pre-immune sera four days after they were acquired. The pre-immune sera were collected and stored ar 20oC. At immunising, the groups were first given a prime dose then two boosts were done after the prime dose. The test group was immunised with 200l of 40g prime dose of the Trypanosoma PGP 1 protein per mouse subcutaneously and 200l of 20g boost doses of protein for the first boost and second boost. The adjuvant group received 200l of same dosage subcutaneously for prime and boosts but without the Trypanosoma PGP 1 protein i.e. the calculated volume for the protein was replaced with PBS. The Negative control group received 200l of same dosage subcutaneously for the prime and the boosts but the adjuvant and Trypanosoma PGP 1 protein volumes were replaced with PBS. The bacterial protein group received 200l of 40g prime dose of bacterial protein per mouse subcutaneously and 200l of 20g boost doses of bacterial protein for the first boost and second boost.. Ten days after, the mice were given the prime dose, after ten days they were tail bled for prime sera, twenty days after the prime dose, the mice were given the first boost dose and ten days later tail bled for first boost sera, twenty days after the first boost, the mice were given the second boost dose and ten days later, tail bled for second boost sera. 3.3.5 Analysis of sera for the different groups of mice Immune sera from all the mice in the groups after a given phase were got from the mice by tail bleeding to collect about 0.3 ml of blood per mouse into a 1.5ml eppendorf tube. The blood in the tubes were then left to clot for one hour on bench after collection and then at 4oC overnight. To collect the sera, the tubes containing clotted blood were then centrifuged at 3000 rpm using micro-centrifuge, the sera (supernatant) was then carefully pipetted out and put into a clean 1ml eppendorf tube and stored at -20oC. The sera were then analyzed using both western blotting and ELISA. 3.3.5.1 Western blot analysis Pre-immune, prime, first boost and second boost sera obtained from all the groups from each mice were all also analyzed using western blotting; A 15% SDS-PAGE gel were 34

first ran containing; Protein marker, PGP 1, Trypanosoma whole cell lysate, Competent bacterial whole cell lysate and plasma from infected mice with. T. b. brucei in duplicates; the gel cut into half, one part of the gel was stained with Coomasie brilliant blue; the other part was transferred to a nitrocellulose membrane and probed with antibodies from sera collected as primary antibody and anti-mouse as the secondary antibody. After transfer, the nitrocellulose membrane was blocked with 5% BSA in PBS overnight at 4oC. The membrane was then washed with PBS-T three times for 5 minutes each time, the membrane was probed with primary antibody at a dilution of 1:2000 in PBS-T for 1 hour with shaking. After this, the membrane was washed with PBS-T three times 5 minutes per wash with shaking. The membrane was then probed with peroxidase conjugated anti-mouse secondary antibody at a dilution 1:10,000 in PBS-T for 1 hour at room temperature with shaking. After this, the membrane was washed as described. The membrane was incubated in PBS solution containing DAB (3, 3-Diaminobenzidene) for about 1 minute then added 100 l of hydrogen peroxide.

3.3.5.2 ELISA Pre-immune, prime, first boost and second boost sera obtained from all the groups from each mouse were all analyzed by indirect ELISA. The plate wells were coated with 100 ng per well of purified protein in PBS overnight at 4oC. The coating material was discarded and the plate was washed three times with 200 l of PBS-T buffer, with shaking for 5 minutes per wash. The wells were blocked with 200 l of 0.5% I-block in PBS-T for 2 hours. Blocking solution was poured off. In the first 12 wells in the A-row, 150 l of the primary antibody in sera from the individual mice in a specific group diluted 1:2000 in I-block, was added in duplicates. The remaining 12 wells in B to H rows were added 100l of PBS. A serial dilution of factor three was then performed by picking 50l from the 12 wells in the A row and adding and pippetting downwards up to 35

G row and discarding the last 50 l leaving H row as blank and the final volume in all the wells as 100 l. The primary antibody was incubated for 1 hour with shaking. The plate was washed six times with 200 l of PBS-T buffer with shaking for 5 minutes per wash. The wash material was poured off, 100 l of secondary antibody (anti-mouse) diluted 1:10,000 in PBS was added and incubated for 1 hour with shaking. The plate was washed three times with 200 l of PBS-T buffer with shaking for 5 minutes per wash. One hundred microlitres of OPD (o-phenylenediamine dihydrochloride) tablets (SigmaFast) in 20 ml of distilled water was then added to all the wells and the plate covered with aluminium foil and incubated at room temperature for 10 minutes. The reaction was stopped using 100 l of 1X sulphuric acid. The optical density (O.D) was measured at 490 nm using ELISA plate reader. The ELISA results were then further analyzed using Microsoft Excel.

CHAPTER FOUR

4.0 RESULTS
4.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells The Agarose gel showed confirmation of the insert digested from the extracted plasmid (Figure 2: panel A, lane 1). A unique band between 700 bp and 600 bp approximately 669 bp (Figure 2: panel B, lane 2) was observed. This was indeed the insert (cloned gene) for Trypanosoma PGP 1. 36

1 Plasmid extract 700 bp 600 bp

669 bp

Figure 3: Confirmation of presence of insert in pET28a (+) plasmid vector. A is a 1% Agarose gel showing successful plasmid extraction. B is a 1 % Agarose gel showing result of R.E digestion. In both, lane 1 is undigested plasmid and lane 2 is digested plasmid. In B, lane M is Bio-rad 1Kb DNA ladder.

4.2 Expression of recombinant Trypanosoma PGP 1 SDS-PAGE and western blot showed successful expression of the recombinant Trypanosoma PGP 1. A unique band slightly above 25 Kda (Figure 4: panel A, lane 2) was the recombinant Trypanosoma PGP 1 and this was further confirmed by a western blot (Figure 4: panel B, lane 2) using anti-His primary antibody at a dilution of 1:2000.
M 1 2 1 2

25 Kda A A

PGP 1 B

PGP 1

Figure 4: Successful expression of recombinant Trypanosoma PGP 1 in BL21DE3 cells. A is a 15% SDS-PAGE gel showing recombinant Trypanosoma PGP 1 successfully expressed. B is western blot showing detection of Trypanosoma PGP 1 by anti-His antibody. In both, lane 1 is pre-induced expression, lane 2 is induced expression. In A, lane M is the Bio-rad protein marker (lane M).

4.3 Extraction and Purification of Trypanosoma PGP 1 and bacterial protein 4.3.1 Extraction The recombinant Trypanosoma PGP 1 expressed was an insoluble protein since it was observed in the BL21DE 3 cell pellet (Figure 5: lane 2) and not in the supernatant (Figure 5: lane 3). The bacterial protein was therefore also purified from the BL21 cell pellet (Figure 5: lane 4). 37

25 Kda

Figure 5: Trypanosoma PGP 1 and bacterial protein localized in the cell pellet after cell lysis. A 15% SDS-PAGE showing the recombinant Trypanosoma PGP 1 and bacterial protein successfully extracted from BL21DE3 after expression and BL21 competent cells respectively. Lane 1 is BL21DE3 whole cell lysate, lane 2 is pellet from the BL21DE3 whole cell lysate, lane 3 is supernatant from BL21DE3 whole cell lysate, lane 4 is pellet from the BL21 whole cell lysate and lane M is the Bio-rad protein marker.

4.3.2 Purification 4.3.2.1 Purification of recombinant Trypanosoma PGP 1 The different portions of the recombinant Trypanosoma PGP 1 eluted with increasing concentrations of imidazole in elution buffer gave different purity levels, 100 nM of imidazole (Figure 6: lane 4 to lane 9 ), 200 nM of imidazole (Figure 6: lane 10 to lane 13) and 300 nM (Figure 6: lane 14 to lane 16) in that order of elution.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

25 Kda

Figure 6: Recombinant Trypanosoma PGP 1 successfully eluted from the Ni-NTA column using increasing concentration of imidazole in elution buffer. A 15% SDS-PAGE showing the recombinant Trypanosoma PGP 1 successfully purified. Lane 1is dissolved pellet, lane 2 is first flow through, lane 3 is the wash, lane 4 to lane 16 are the eluted protein portions and lane M is the Bio-rad protein marker.

4.3.2.2 Purification of Bacterial protein The bacterial protein was eluted using only one concentration of 300 nM of imidazole in elution buffer (Figure 7: lane 3). The recombinant Trypanosoma PGP 1 (Figure 7: lane 4) was ran alongside for comparison.
1 2 3 4

38

25 Kda PGP I

Figure 7: Bacterial protein eluted form the Ni-NTA column using 300nM imidazole in elution buffer. A 15% SDS-PAGE gel showing bacterial protein purified using Ni-NTA column. Lane 1 is the dissolved pellet, lane 2 is the wash, lane 3 is the eluted protein and lane 4 is the recombinant Trypanosoma PGP 1, gel was stained with coomasie brilliant blue.

4.4 Quantification Data obtained from the micro-plate reader was analysed using Microsoft Excel 2007 to obtain the concentration of the proteins.
A graph showing concentration plotted against OD 0.6 0.5 0.4 Concetration 0.3 0.2 0.1 0 0 0.5 1 13 Pooled (14,15,16) y = 0.6061x - 0.0437 R = 0.9712 Bacterial protein 0.726 0.529 0.443 0.4 0.28 0.225 5 6 0.525 0.691 0.28 0.38 Purified portion of Trypanosoma PGP 1 3 OD 0.823 Calculated conc. (mg/ml) 0.46

OpticalDensity (OD)

Figure 8: Graph showing concentration plotted against OD using data obtained from for the standards

Table 1: Calculated concentration of the purified portions of Trypanosoma PGP 1 and bacterial protein from the graph. raph showing concentration plotted against OD

4.5 Analysis of sera collected from the mice The sera collected from the animals were analyzed using western blotting and ELISA; 4.5.1. Western blot analysis of pre-immune sera The recombinant Trypanosoma PGP 1 was not detected by the pre-immune sera at a dilution of 1:2000 as this was shown by absence of band detection on western blot 39

(Figure 8: panels B-E at lane 1) for all the groups, test group (panel B), Adjuvant group (panel C ), PBS group (panel D) and Bacterial protein group (panel E).
M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

25 Kda

Figure 9: Western blots showing no antibodies in pre-immune sera. A is a 15% SDS-PAGE gel showing the recombinant Trypanosoma PGP 1; B, C, D, E are western blots from the test group, adjuvant group, PBS group, bacterial protein group respectively. In all, lane 1 is the recombinant Trypanosoma PGP 1, lane 2 is the trypanosome whole cell lysate, lane 3 is the bacteria cell lysate. In A, lane M is the Biorad protein marker.

4.5.2 Western blot analysis of immune sera The recombinant Trypanosoma PGP 1 was detected by the immune sera from the test group at a dilution of 1:2000 as shown by a signal detection on western blot (Figure 9: panel B, lane 1). The rest of the groups did not detect any signals.
M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

25 Kda

Figure 10: Western blots showing specific antibodies produced only against the recombinant Trypanosoma PGP 1. A is a 15% SDS-PAGE gel showing the recombinant Trypanosoma PGP 1; B, C, D, E are western blots from the test group, adjuvant group, PBS group, bacterial protein group respectively. In all, lane 1 is the recombinant Trypanosoma PGP 1, lane 2 is the trypanosome whole cell lysate, lane 3 is the bacteria cell lysate. In A, lane M is the Bio-rad protein marker.4.5.2

ELISA analysis of sera.

4.5.3 ELISA analysis of sera The immune sera from prime, first boost and second boost from all the groups were all analysed against the pre-immune sera. The first boost and second boost sera from the test

40

group gave a cut off beyond 1:486,000 meaning that the titers were 1:486,000 but the prime sera gave no titers. All the other groups did no give any titers at all the phases. 4.5.2.1 Group one (Test group)
Prime sera
Average OD vs Dilution
A v e r a g e O D

First boost sera

Average OD vs Dilution
A v e r a g e

Second boost sera

Average OD vs Dilution
A v e r a g e

1 0.5 0 162000d 486000d 1458000d 1458000d

0.5

0.5

2000d

6000d

18000d

162000d

486000d

1458000d

2000d

18000d

54000d

6000d

162000d

486000d

1458000d

2000d

6000d

18000d

54000d

O D

O D

Dilution

Dilution Dilution

Figure 11: Antibody titer of 1:486,000 shown by the first and second boost but no titer for the prime sera. ELISA data for the test group analysed by Micosoft excel, A is the prime sera, B is the first boost sera and C is the second boost sera.

4.6.2.2 Group two (Adjuvant group)

Prime sera
Average O.D Vs Dilution 1
A v e r a g e O . D A v e r a g e

First boost sera

Average O.D Vs Dilution 1 0.5 0 1458000d

Second boost sera

Average O.D Vs Dilution
A v e r a g e O . D

1 0.5
486000d
162000d 18000d 54000d 2000d 6000d

0.5 0 1458000d 162000d 486000d 18000d 54000d 2000d 6000d

54000d

162000d

Dilution

O . D

Dilution

486000d

18000d

2000d

6000d

Dilution

41

54000d

Figure 12: No antibody titers shown by the ajuvant group for all phases. ELISA data for the adjuvant group analysed by Micosoft excel, A is the prime sera, B is the first boost sera and C is the second boost sera.

4.6.2.3 Group three (PBS group)

Prime sera
Average O.D Vs Dilution
A v e r a g e O . D

First boost sera

Average O.D Vs Dilution
A v e r a g e

Second boost sera

Average O.D Vs Dilution
A v e r a g e

1 0.5 0

1 0.5 0 1458000d 162000d 486000d 18000d 54000d 2000d 6000d

1 0.5 0 1458000d 162000d 486000d 18000d 54000d 2000d 6000d

1458000d

162000d

486000d

18000d

54000d

2000d

6000d

O . D

O . D

Dilution

Dilution

Dilution

Figure 14: No antibody titers shown by the PBS group for all phases. ELISA data for thePBSt group analysed by Micosoft excel, A is the prime sera, B is the first boost sera and C is the second boost sera.

42

4.6.2.4 Group Four (Bacterial protein group)

Prime sera
Average O.D Vs Dilution
A v e r a g e O . D

First boost sera

Average O.D Vs Dilution
A v e r a g e

Second boost sera

Average O.D Vs Dilution
A v e r a g e

1 0.5 0 1458000d 162000d 486000d 18000d 54000d 2000d 6000d

1 0.5 0 1458000d 162000d 486000d

1 0.5 0 1458000d

54000d

162000d

O . D

O . D

Dilution

Dilution

Dilution

Figure 14: No antibody titers shown by the bacterial protein group for all phases. ELISA data for the bacterial protein group analysed by Micosoft excel, A is the prime sera, B is the first boost sera and C is the second boost sera.

4.6.2.5 Immunogenicity curve

43

486000d

18000d

54000d

18000d

2000d

2000d

6000d

6000d

A graph showing titer plotted against days post immunisation

600000 500000 T 400000 i t 300000 e 200000 r 100000 0 2nd boost (day 40) 1st boost (day 20)

Prime (day 0)

Days post immunisation

Figure 15: No change in the antibody titers after the second boost. ELISA data for the titers from test group analysed by Micosoft excel.

CHAPTER FIVE

5.0 DISCUSSION
Diagnosis of Human African Trypanosomiasis still remains a challenge despite a number of diagnostic techniques available. There are no simple and reliable screening tests for T. 44

b. rhodesiesne. The parasitological technique which is reliable is cumbersome and time consuming (Njiru et al., 2007). The Card agglutination test, the serological test used for screening, is not reliable for T. b. rhodesiesne (Lejon et al., 2002) since some cases are sometimes missed out. There is therefore need to identify diagnostic antigens that could be used to screen for HAT. A previous study conducted on Trypanosoma PGP 1 to determine its recognition by infected human sera showed that it a promising candidate for a diagnostic antigen for HAT. However, there was no data to on its immunogenicity. In this study the immunogenic potential of Trypanosoma PGP 1 was investigated. To do this, previously, the gene for Trypanosoma PGP 1 was successfully cloned in pET28a vector and transformed into BL21DE3 cells (Anywar, thesis) and this was confirmed by the results from the plasmid extraction and restriction enzyme digestion. Trypanosoma PGP 1 was successfully expressed in BL21DE3 cells as this was confirmed by SDSPAGE and western blot. In SDS-PAGE, the expression gave a protein slightly above the 25 Kda mark compared to the 25.1Kda the expected weight of the Trypanosoma PGP 1. This could be explained by the fact that the sample loading buffer containing the SDS was added to the protein in a ratio of 1:4 other than the recommended ratio of 1:1 and since inappropriate amount of SDS added to a protein can slow down its mobility, this could have affected the mobility of the protein on the gel. The recombinant Trypanosoma PGP 1 was also cloned between BamHI and HindIII and there was an addition of approximately 1.3 Kda on the weight of the protein. All these factors combined could have attributed to the observed weight of the Trypanosoma PGP 1 on the gel. Western blotting using anti-His antibody confirmed that the protein expressed was indeed recombinant Trypanosoma PGP 1 since it was cloned with six Histidine (His) sequences. The recombinant Trypanosoma PGP 1 was purified using Ni-NTA agarose column since Nickel present in the column has high affinity for Histidine. However, from the purification, there was varying purity levels in the different aliquots collected during elution. This was because increasing concentration of imidazole in elution buffers was used. The first elution buffers with low concentration of imidazole gave low pure proteins (50%-60%) while the last elution buffer high concentration of imidazole gave relatively high pure protein (70%-85%). Imidazole is used to break the bond between the His and 45

Nickel hence releasing the protein. Therefore, its concentration affects the process of purification. Further more, failure to standardize the purification protocol and luck of amicon tubes also hindered the chances to produce highly purified protein (95%-99% purity). The western blot results from showed that; the antibodies from the pre-immune sera of all the groups did not give any signal on the membrane at a dilution of 1:2000 as expected since there were no antibodies against the recombinant Trypanosoma PGP 1. The immune sera from the test group contained specific antibodies to the recombinant Trypanosoma PGP 1 since a band was detected at a dilution of 1:2000 by recombinant Trypanosoma PGP 1 but none for the bacterial cell lysate. The intensity of the band was greater in the second boost as shown in the results than the first boost and the prime (data not shown). Very weak signals at lower regions in the Trypanosoma PGP 1 lane were also detected; this was because the protein was not absolutely pure. Surprisingly, the native Trypanosoma PGP 1 in the trypanosome whole cell lysate was not detected by the immune sera from the test group. Previous study shows that Trypanosoma PGP 1 is intracellular and is released in the blood stream of infected mice due to intravascular destruction of trypanosomes and is expressed at all life stages (Morty et al, 2006). This therefore means that the protein should be detected in the trypanosome whole cell lysate but this was not the case. This could have been due to very low concentrations of the native Trypanosoma PGP 1 in the trypanosome whole cell lysate that could have caused its luck of detection in the trypanosome whole cell lysate. The immune sera from all the other groups did produce specific antibodies against the recombinant Trypanosoma PGP 1 as this was shown by absence of signal detection by Trypanosoma PGP 1. The western results therefore showed that the recombinant Trypanosoma PGP 1 was capable of eliciting specific antibody production in mice. The ELISA results from the sera analysis showed that the sera from the test group gave high IgG titers of 1:486,000 for the first and second boosts against the recombinant Trypanosoma PGP 1 and no titers for after priming. Absence of antibody titers at priming could have been due to the fact that, at the priming, it is mainly IgM antibodies produced and very few IgG antibodies. Failure to observe antibodies at priming was due to the fact 46

that, anti-mouse IgG antibodies were used as the secondary antibody instead of the antimouse IgM hence there was no binding between primary antibody and the secondary antibody. The ELISA results also showed that the priming, first boosting and second boosting for adjuvant group and bacterial protein group did not produce specific antibodies against Trypanosoma PGP 1. There were no titers observed for these groups, though the bacterial protein group showed a very low titer at a very low optical density at second boost but was insignificant compared to the test group. This therefore meant that the adjuvant and the bacterial protein had no effect in the production of specific antibodies against the recombinant Trypanosoma PGP 1. The immunogenicity curve showed that, there was no change in the titers at the first boosting and second boosting. This could have been because the dose for the second boost was maintained as the first boost. Results from the sera analysis from both the western blot and ELISA showed that Trypanosoma PGP 1 was capable of producing specific and quantifiable antibodies in mice.

47

CHAPTER SIX

6.0 CONLUSION AND RECOMMENDTAION

6.1 CONCLUSION The recombinant Trypanosoma PGP 1 was successfully expressed in BL21DE3 cells. There was high anti- Trypanosoma PGP 1 antibodies detected in the immune sera for the test group after first boosting and second boosting. The recombinant Trypanosoma PGP 1 also produced specific antibodies at a very high titer. The demonstrated ability of the Trypanosoma PGP 1 to produce specific and quantifiable antibodies in mice showed that Trypanosoma PGP 1 is immunogenic. 6.2 RECOMMENDATION The study shows promising results for usage of Trypanosoma PGP 1 as a candidate for a diagnostic antigen for screening for HAT. However, broader studies should be conducted on Trypanosoma PGP 1. Incase Trypanosoma PGP 1 is to be used for any study, the purification protocol should be standardised in order to obtain a highly purified protein. The same study could be conducted using native Trypanosoma PGP 1 to compare the antibody titers with the recombinant Trypanosoma PGP 1. Lack of detection of the native Trypanosoma PGP 1 can be overcome by using more sensitive methods like the Enzyme Chemiluminiscence.

48

REFERENCES Ancelle T., A. Paugam, F. Bourlioux, A. Merad, and J. P. Vigier (1997): Detection of trypanosomes in blood by the Quantitative Buffy Coat (QBC) technique: experimental evaluation. Med. Trop. 57:245-248 Anywar Dennis Arony (2009): Makerere University Kampala. Thesis; Determination of antigenic recognition of Trypanosoma; Oligopeptidase A and B and Pyroglutamyl peptidase 1 by infected human sera Bailey J. W and D. H. Smith (1992): The use of the acridine orange QBC technique in the diagnosis of African Trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 86:630 Barrett M. P., R. J. Burchmore, A. Stich, J. O. Lazzari, A. C. Frasch, J. J. Cazzulo, S. Bisser, Z. Ayed, B. Bouteille, A. Stanghellini, J. C. Breton, M. Dumas and M. O. Jauberteau (2000): Central nervous system involvement in African Trypanosomiasis: presence of anti-galactocerebroside antibodies in patients' cerebrospinal fluid. Trans. R. Soc. Trop. Med. Hyg. 94:225-226 Barrett M. P., R. J. Burchmore, A. Stich, J. O. Lazzari, A. C. Frasch, J. J. Cazzulo, S. Krishna (2003): The Trypanosomiases. Lancet 1; 362 (9394):1469-80 Bisser S., B. Bouteille, J. Sarda, A. Stanghellini, D. Ricard, M. O. Jauberteau, F. Marchan, M. Dumas and J. C. Breton (1997): Contribution of biochemical tests in the diagnosis of the nervous phase of human African Trypanosomiasis. Bull. Soc. Pathol. Exot. 90:321-326 Bisser S., V. Lejon, P. M. Preux, B. Bouteille, A. Stanghellini, M. O. Jauberteau, P. Bscher and M. Dumas (2002): Blood-cerebrospinal fluid barrier and intrathecalimmunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness. J. Neurol. Sci. 193:127-135 Bscher P., V. Lejon, E. Magnus and N. Van Meirvenne (1999): Improved latex agglutination test for detection of antibodies in serum and cerebrospinal fluid of Trypanosomabruceigambiense infected patients. Acta Trop. 73:11-20 Cattand P., B.T. Miezan and P.deRaadt (1988): Human African Trypanosomiasis: use of double centrifugation of cerebrospinal fluid to detect trypanosomes. Bull. W. H. O. 66:83-86 49

Chappuis F., A. Pittet, P. A. Bovier, K. Adams, V. Godineau, S. Y. Hwang, E. Magnus and P. Bscher (2002): Field evaluation of the CATT/Trypanosomabruceigambiense on blood-impregnated filter papers for diagnosis of human African Trypanosomiasis in southern Sudan. Trop. Med. Int. Health 7:942-948 Chappuis F., E. Stivanello, K. Adams, S. Kidane, A. Pittet and P. A. Bovier (2004): Card agglutination test for Trypanosomiasis (CATT) end-dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosomabruceigambiense) among serologically suspected individuals in southern Sudan. Am. J. Trop. Med. Hyg. 71:313-317 Cleland J. L., M. F. Powell, S. J. Shire (1993): The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst 1993; 10: 307377 Dando P. M, M. Fortunato, G. B. Strand (2003). "Pyroglutamyl-peptidase I: cloning, sequencing, and characterisation of the recombinant human enzyme". Protein Expr. Purif. 28 (1): 1119 Dintzis H. M., R. Z. Dintzis, and B. Vogelstein (1976): The molecular determinants of immunogenicity: the immunon model of immune response. Proc. Natl. Acad. Sci. USA. 73:3671 Enyaru J. C., E. Matovu, M. Akol, C. Sebikali, J. Kyambadde, C. Schmidt, R.Brun, R. Kaminsky, L. M. Ogwal and F. Kansiime (1998): Parasitological detection of Trypanosomabruceigambiense in serologically negative sleeping-sickness suspects from north-western Uganda. Ann. Trop. Med. Parasitol. 92:845-850 Fakharzadeh S. S, H. H. Kazazian Jr. (2000): Correlation between factor VIII genotype and inhibitor development in hemophilia A.Semin Thromb Hemostasis 2000; 26: 167171 Garcia A., V. Jamonneau, E. Magnus, C. Laveissiere, V. Lejon, P. N'Guessan, L. N'Dri, N. Van Meirvenne and P. Bscher (2000): Follow-up of Card Agglutination Trypanosomiasis Test (CATT) positive but apparently aparasitaemic individuals in Cote d'Ivoire: evidence for a complex and heterogeneous population. Trop. Med. Int. Health 5:786-793

50

Henry M. C., P. Kageruka, J. F. Ruppol, H. Bruneel and Y. Claes (1981): Evaluation of field diagnosis of Trypanosomiasis caused by Trypanosomabruceigambiense. Ann. Soc. Belg. Med. Trop. 61:79-92 Hill L.K (2003): The eukaryotic cell: pg. 200-208 Volume 2, Number 2 Holm and Sander, (1999): Protein folds and families: sequence and structure alignments. Nucl Acid Res, 27, 244-247 Jamonneau V., P. Truc, A. Garcia, E. Magnus, and P. Bscher (2000): Preliminary evaluation of LATEX/T. b. gambiense and alternative versions of CATT/T. b. gambiense for the serodiagnosisofhuman African Trypanosomiasis of a population at risk in Cote d'Ivoire: considerations for mass-screening. Acta Trop. 76:175-183 Jamonneau V., P. Solano and G. Cuny (2001): Use of molecular biology in the diagnosis of human African Trypanosomiasis. Med. Trop. 61:347-354 Jamonneau V., P. Solano, A. Garcia, V. Lejon, N. Dje, T. W. Miezan, P. N'Guessan, G. Cuny and P. Bscher (2003): Stage determination and therapeutic decision in human African Trypanosomiasis: value of polymerase chain reaction and immunoglobulin M quantification on the cerebrospinal fluid of sleeping sickness patients in Cote d'Ivoire.Trop. Med. Int. Health 8:589-594 Kabiri M., J. R. Franco, P. P. Simarro, J. A. Ruis, M. Sarsa and D. Steverding (1999): Detection of Trypanosomabruceigambiense in sleeping sickness suspects by PCR amplification of expression-site-associated genes 6 and 7. Trop. Med. Int. Health 4:658-661 Kuby Immunology (2006), 6th edition, Macmillan, pg. 77 Lanham S. M. and D. G. Godfrey (1970): Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 28:521-534 LejonV., M. Boelaert, J. Jannin, A. Moore and P. Bscher (2003): The challenge of Trypanosomabruceigambiense sleeping sickness diagnosis outside Africa. Lancet Infect. Dis. 3:804-808 Lejon V., P. Bscher, E. Magnus, A. Moons, I. Wouters and N. Van Meirvenne (1998): A semi-quantitative ELISA for detection 51

of Trypanosomabruceigambiense specific antibodies in serum and cerebrospinal fluid of sleeping sickness patients. Acta Trop. 69:151-164 Lejon V., P. Bscher, N. H. Sema, E. Magnus and N. Van Meirvenne (1998): Human African Trypanosomiasis: a latex agglutination field test for quantifying IgM in cerebrospinal fluid. Bull. W. H. O. 76:553-558 Lejon V., D. Legros, L. Rosengren, M. Gastellu Etchegorry and P. Bscher (2001): Biological data and clinical symptoms as predictors of astrogliosis and neurodegeneration in patients with second-stage Trypanosomabruceigambiense sleeping sickness. Am. J. Trop. Med. Hyg. 65:931-935 Lejon V., D. Legros, M. Richer, J. A. Ruis, V. Jamonneau, P. Truc, F. Doua, N. Dje, F. X. N'Siesi, S. Bisser, E. Magnus, I. Wouters, J. Konings, T. Vervoort, F. Sultan and P. Bscher (2002): IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test. Trop. Med. Int. Health 7:685-692 Lejon V., J. Kwete and P. Bscher (2003): Towards saliva-based screening for sleeping sickness. Trop. Med. Int. Health8:585-588 Levine R. A., S. C. Wardlaw and C. L. Patton (1989): Detection of haematoparasites using quantitative buffy coat analysis tubes. Parasitol. Today 5:132-134 Magnus E., N. Van Meirvenne, T. Vervoort, D. Le Ray and M. Wery (1978): Use of freeze-dried trypanosomes in the indirect fluorescent antibody test for the serodiagnosis of sleeping sickness. Ann. Soc. Belg. Med. Trop. 58:103-109 Magnus E., T. Vervoort and N. Van Meirvenne (1978): A card-agglutination test with stained trypanosomes (CATT) for the serological diagnosis of T. b. gambiense Trypanosomiasis. Ann. Soc. Belg. Med. Trop. 58:169-176 Magnus E., V. Lejon, D. Bayon, D. Buyse, P. Simarro, D. Verloo, T. Vervoort, R. Pansaerts, P. Bscher and N. Van Meirvenne (2002): Evaluation of an EDTA version of CATT/Trypanosomabruceigambiense for serological screening of human blood samples. Acta Trop. 81:7-12

52

Miezan T. W., H. A. Meda, F. Doua, F. B. Yapo and T. Baltz (1998): Assessment of central nervous system involvement in gambienseTrypanosomiasis: value of the cerebrospinal white cell count. Trop. Med. Int. Health 3:571-575 Morty R. E, P. Bulau, R. Pelle, S. Wilk and K. Abe (2006): Pyroglutamyl peptidase type I from Trypanosomabrucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts. Biochem Journal (2006) Mar 15; 394(Pt 3): 635-45 Noireau F., J. P. Gouteux and J. P. Duteurtre (1987): Diagnostic value of a card agglutination test (Testryp CATT) in the mass screening of human Trypanosomiasis in the Congo. Bull. Soc. Pathol. Exot.Fil. 80:797-803 Noireau F., P. Force-Barge and P. Cattand (1991): Evaluation of Testryp CATT applied to samples of dried blood for the diagnosis of sleeping sickness. Bull. W. H. Or. 69:603-608 O'Hagan D. T., R. Rappuoli (2004): Novel approaches to vaccine delivery. Pharm Res; 21(9):1519-30 Odagaki Y, A. Hayashi, K. Okada, K. Hirotsu, T. Kabashima,K.Ito,T.Yoshimoto, D. Tsuru, M. Sato and J.Clardy (1999): The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease. Structure, Volume 7, Number 4, 15 April 1999, pp. 399411(13) Ogievetskaya, M. M. (1977): Method for the determination of protein evolution rates by amino acid composition. Evolution rate of actins in: Origins of life and evolution of the biospheres, ISSN 1573-0875, Vol. 8 (2. 1977), p. 145-154 Pashine A., N. M. Valiante, J. B. Ulmer (2005): Targeting the innate immune response with improved vaccine adjuvants. Nature Medicine; 11(4 Suppl):S63-8

Papadopoulos M. C., P. M. Abel, D. Agranoff, A. Stich, E. Tarelli, B. A. Bell, T. Planche, A. Loosemore, S. Saadoun, P. Wilkins and S. Krishna (2004): A novel and accurate diagnostic test for human African Trypanosomiasis. Lancet 363:1358-1363

53

Picozzi K., E. M Fvre, M. Odiit, M. Carrington, M. C Eisler, I. Maudlin, S. C. Welburn (2005): Sleeping sickness in Uganda: a thin line between two fatal diseases. BMJ 331 : 1238 doi: 10.1136/bmj.331.7527.1238 Porter S. (2001): Human immune response to recombinant human proteins. J Pharm Sci 2001; 90: 111 Radwanska M., M. Chamekh, L. Vanhamme, F. Claes, S. Magez, E. Magnus, P. de Baetselier, P. Bscher and E. Pays (2002): The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosomabruceirhodesiense. Am. J. Trop. Med. Hyg. 67:684-690, of Trypanosomabruceigambiense. Am. J. Trop. Med. Hyg. 67:289-295 Renart J, J. ReiserandG. R. Stark (1979): Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. ProcNatlAcadSciU S A.76 (7): 31163120 Reeves W. G., K. G. Alberti, L. P. Krall (1986): The immune response to insulin: characterisation and clinical consequences. Diabetes Annual 2. Elsevier Science Publishers, Amsterdam Ryff J. C., Schellekens H (2002): Immunogenicity of rDNA-derived pharmaceuticals. Trends Pharmacol Sci; 23: 254256 Sambrook J., E. F. Fristsch and T. Maniatis (1989): Molecular cloning. A laboratory manual. Cold spring harbor laboratory press. Second edition: 18.47, 18.60 Schellekens H (2003): Immunogenicity of therapeutic proteins. Nephrol Dial Transplant; 18: 12571259 Schellekens H (2005): Factors influencing the immunogenicity of therapeutic proteins Nephrol Dial Transplant; 20 [Suppl 6]: vi3vi9 doi:10.1093/ndt/gfh1092 Simarro P., J. A. Ruis, J. R. Franco and T. Josenando (1999): Attitude towards CATT-positive individuals without parasitological confirmation in the African Trypanosomiasis (T.b. gambiense) focus of Quicama (Angola). Trop. Med. Int. Health4:858-861

Singh S. K (2011): Impact of product-related factors on immunogenicity of biotherapeutics, J. of Pharm. Sci. Vol. 100, Iss. 2, pg. 354387

54

Solano P., V. Jamonneau, P. N'Guessan, L. N'Dri, N. N. Dje, T. W.Miezan, V. Lejon, P. Bscher and A. Garcia (2002): Comparison of different DNA preparation protocols for PCR diagnosis of human African trypanosomosis in Cote d'Ivoire. Acta Trop.82:349-356 Towbin H, T. Staehelin and J. Gordon (1979): Electrophoretic transfer of proteins from Polyacrylamide gels to nitrocellulose sheets: procedure and some applications. ProcNatlAcadSci U S A.76 (9): 43504354 Truc P., D. Aerts, J. J. McNamara, Y.Claes, R. Allingham, D. Le Ray and D. G. Godfrey (1992): Direct isolation in vitro ofTrypanosomabrucei from man and other animals, and its potential value for the diagnosis of gambianTrypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 86:627-629 Truc P., J. W. Bailey, F. Doua, C. Laveissiere and D. G. Godfrey (1994): A comparison of parasitological methods for the diagnosis of Gambian Trypanosomiasis in an area of low endemicity in Cote d'Ivoire. Trans. R. Soc.Trop. Med. Hyg. 88:419-421 Truc P., V. Jamonneau, P. N'Guessan, P. B. Diallo and A. Garcia (1998): Parasitological diagnosis of human African Trypanosomiasis: a comparison of the OBC and miniature anion-exchange centrifugation techniques. Trans. R. Soc. Trop. Med. Hyg. 92:288-289 Truc P., V. Jamonneau, G. Cuny and J. L. Frezil (1999): Use of polymerase chain reaction in human African Trypanosomiasis stage determination and follow-up. Bull. W. H. Or. 77:745-748 Truc P., V. Lejon, E. Magnus, V. Jamonneau, A. Nangouma, D. Verloo, L. Penchenier and P. Bscher (2002): Evaluation of the micro-CATT, CATT/Trypanosomabruceigambiense, and LATEX/T. b. gambiense methods for serodiagnosis and surveillance of human African Trypanosomiasis in West and Central Africa. Bull. W. H. O. 80:882-886 Vanhamme L., E. Pays, R. McCulloch and J. D. Barry (2001): An update on antigenic variation in African trypanosomes. Trends Parasitol. 17:338-343 von Delwig A., D. M. Altmann , J. D. Isaacs , C. V. Harding , R. Holmdahl , N. McKie , J. H. Robinson (2006 ): The impact of glycosylation on HLA-DR1-restricted T cell recognition of type II collagen in a mouse model, Arthritis Rheum. 54(2):482-91

55

Woo P. T. (1970): The haematocrit centrifuge technique for the diagnosis of African Trypanosomiasis. Acta Trop. 27:384-386 World Health Organisation (1998): Control and surveillance of African Trypanosomiasis: report of a WHO expert committee, World Health Organ Tech Rep Ser. 1998;881:I-VI, 1-114 World Health Organisation (2010): Fact sheet N259, African trypanosomiasis (sleeping sickness), October 2010

APPENDIX I Protocols used Plasmid extraction using QIAprep Spin Miniprep Kit procedure Resuspended the pelleted bacterial cells in 250l of buffer P1 and transferred to a microcentrifuge tube Added 250l of buffer P2 and mixed thoroughly by inverting the tube 6 times Added 350l of buffer N3 and mixed immediately and thoroughly by inverting the tube 6 times Centrifuged for 10minutes at 13,000rpm in a table top microcentrifuge Applied the supernatant to the QIAprep spin column by pipetting Centrifuged for 60 seconds, discarded the flow through Washed the spin column by adding 0.5ml of buffer PB and centrifuged for 60 seconds. Discarded the flow through Washed the QIAprep spin by adding 0.75ml off buffer PE and centrifuged for 6- seconds Discarded the flow through and centrifuged fro an additional 1 min to remove residual wash buffer. 56

Eluted DNA by placing the QIAprep column in a clean 1.5ml microcentrifuge tube and added buffer EB to the center of each QIAprep spin column, left to stand fro 1min then centrifuged fro 1 minute. Plasmid DNA extraction using Alkali lysis method Grew 10 ml of cells as described in 3.1 Transferred the cells to a sterile clean 15ml tube, centrifuged bacterial cells at 6000 rpm for 10 minutes Resuspended the cells in 250l of Solution I Then added 250l of Solution II, mixed gently by inverting tube and left to stand for 5 minutes at room temperature To the contents added 350l of Solution III, mixed by shaking the bottle several times Centrifuged at 10.000 rpm for 10 minutes Pipetted 800l of supernatant a clean 15ml tube and added 2 volumes of absolute ethanol and span at 7,000 rpm for 10 minutes Poured off the supernatant and inverted the tube on paper towel for 10 minutes Rinsed the pellet with 2ml of 70% ethanol, vortexed gently to resuspended the pellet and span at 7,000 rpm for 10 minutes Poured off the supernatant and inverted the tube on paper towel for 10 minutes Eluted the plasmid DNA with 100l of distilled water Analyzed the 10l of the sample with 2l of loading dye in a 2% agarose gel

Restriction enzyme digestion In PCR tube or 0.5ml eppendorf tubes the following are added in order; work done in the lamina flow hood BSA 0.5l

Distilled water (PCR water) 4.5 l 10X buffer DNA template Bam HI 3.0 l 10.0 l 1.0 l 57

Hind III

1.0 l

Tapped the tube several times to mix Incubated at 37oC for 21/2 hours

Quantification PGP 1 Prepared standards

BSA (vol. in l) Cocn (mg/ml) 1X (vol. l) PBS in

0 0.00

50 0.05

100 0.1 900

150 0.15 850

200 0.2 800

250 0.25 750

300 0.3 700

350 0.35 650

400 0.4 600

450 0.45 550

500 0.5 500

1000 950

Preparation of samples: very concentrated samples were diluted in ration 1:2 using 1X PBS Preparation of Bradford dye: the dye was mixed with distilled water in the ratio of 1:4 The microplate reader was switched n 15 minutes before reading The standards and samples were then loaded in the microplate wells and read at 595 nM

58

APPENDIX II Preparation of reagents Preparation of LB and Terrific broth Terrific broth 12 g of Pancreatic digest of casein 24 g of Yeast extract 9.4 g of Di-potassium phosphate 2.2 g of Mono-potassium phosphate In a total of 1000 mls Preparation of reagents used in PGP 1 expression

LB broth 10 g of Tryptone salt 5 g of Yeast extract 10 g of NaCl

1.0M IPTG stock

59

Dissolved 1.9115g of IPTG powder in 5ml of distilled water; from the stock a working concentration of 1mM was used Transfer buffer 3.03 g Tris (25 mM Tris) 14.4 g Glycine (192 mM Glycine) 200 ml of methanol (20 % w/v) or without Final pH 8.3 to 1 liter Stacking buffer (0.5 M Tris-HCl (pH 6.8)) 6.0 g Tris base 60 ml of distilled water Adjusted pH to 6.8 with 6N HCl; made the mark to 100 ml with distilled water; stored at 4oC

Separating buffer (1.5 M Tris-HCl (pH 8.8) 27.23 g Tris base (18.15 g per 100 ml) 80 ml distilled water Adjusted pH to 8.8 with 6N HCl; made 150 ml with distilled water; stored at 4oC 10% SDS Desolved 10 g SDS powder in 90 ml of distilled water with gentle stirring and made to 100 ml with distilled water. Sample loading buffer Distilled water 0.5 M Tris-HCl (pH 6.8) Glycine 5.8 ml 1.0 ml 0.8 ml 60

10% (w/v) SDS 2-mercaptoethanol 1% (w/v) bromophenol blue

1.6 ml 0.4 ml 0.4 ml

Dilute 1:4 with sample buffer and heat at 95oC for 4 minutes 5X electrode buffer Tris base Glycine SDS 9 g (15 g/l) 43.2 g (72 g/l) 3 g (5 g/l)

Topped, 600 ml of water; stored at Rtp; for 1X, added 60 ml of 5X to 240 ml of distilled water Staining buffer (Coomasie) 0.1 % (w/v) coomasie blue 40% (v/v) methanol 10% (v/v) Glacial acetate Distilled water Distaining solution I 5% (v/v) methanol 7% (v/v) Acetic acid 0.3 g 120 ml 30 ml 150 ml Distaining solution II 40 % (v/v) methanol 10% (v/v) Acetic acid

Preparation of reagents used in Extraction and purification of PGP 1

Lysis buffer 50 mM Tris.Cl (pH 8.0) 1 mM EDTA 100 mM NaCl 61

8.0M Urea Dissolved 48.05g of Urea crystals in 100ml distilled water Urea buffer NaH2PO4.H2O Tris-base Urea 1.38g 0.12g 48.05g

Dissolved in 100ml distilled water; Adjust pH to 8.0 using NaOH

Wash buffer (used during protein elution from Ni-NTA column) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml 10l 500l 4.44l 50l 0.7l

Elution buffer one (100mM) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml 10l 500l 3.989l 500l 0.7l

Elution buffer two (200mM) 0.2% Tween 20 10% glycerol Urea buffer Imidazole 10l 500l 3.4893l 1000l 62

 mercaptoethanol Total volume is 5ml

0.7l

Elution buffer three (300mM) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml Buffer A 50 mM Tris.Cl pH 8.0 5 mM EDTA 10 mM NaCl Buffer B 20 mM Na2HPO4 (pH 7.2) 20 mM NaCl 5mM EDTA 25% w/v sucrose 10l 500l 2.989l 1500l 0.7l

63