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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
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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.............................................................................................................................
DEDICATION.................................................................................................................................
ACKNOWLEDGEMENT...............................................................................................................TABLE OF CONTENTS ...............................................................................................................
LIST OF FIGURES AND TABLES..............................................................................................
ABBREVIATIONS AND ACRONYMS.......................................................................................
ABSTRACT.....................................................................................................................................
CHAPTER ONE ..............................................................................................................................
1.0INTRODUCTION.....................................................................................................................
1.1Background ..................................................................................................................................
1.2 Statement of the problem ............................................................................................................
1.3 Objectives ....................................................................................................................................
1.3.1 General objective .....................................................................................................................
1.3.2 Specific objective .....................................................................................................................
1.4 Justification and significance ......................................................................................................
1.5Research Question .......................................................................................................................
CHAPTER TWO .............................................................................................................................
2.0 LITERATURE REVIEW ........................................................................................................
2.1 African Trypanosomiasis ............................................................................................................
2.2 Trypanosoma brucei ...................................................................................................................
2.2.1 Life cycle ofTrypanosoma brucei ...........................................................................................
2.3 Management of Human African Trypanosomiasis .....................................................................
2.3.1 Diagnosis of Human African Trypanosomiasis .......................................................................
2.3.1.1 Serological techniques ..........................................................................................................
2.3.1.1.1 The Card Agglutination Test for Trypanosomiasis (CATT) .............................................
2.3.1.1.2 Antibody detection .............................................................................................................
2.3.1.1.3 The LATEX agglutination test forT. b. gambiense...........................................................
2.3.1.1.4 Immunofluorescence Assays .............................................................................................
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2.3.1.2 Parasitological techniques .....................................................................................................
2.3.1.2.1 Chancre aspirate .................................................................................................................
2.3.1.2.2 Lymph node aspirate ..........................................................................................................
2.3.1.2.3 Wet and thick blood films ..................................................................................................
2.3.1.2.4 Microhematocrit centrifugation technique .........................................................................
2.3.1.2.5 Quantitative buffy coat .......................................................................................................
2.3.1.2.6 Mini-anion-exchange centrifugation technique .................................................................
2.3.1.3 Molecular diagnosis ..............................................................................................................
2.3.1.3.1 Polymerase Chain Reaction (PCR) ....................................................................................
2.3.1.3.2 Loop-mediated Isothermal Amplification (LAMP) ...........................................................
2.3.1.4 Diagnosis to Stage Human African Trypanosomiasis ..........................................................
2.3.1.4.1 White blood cell count .......................................................................................................
2.3.1.4.2 Protein concentration .........................................................................................................
2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF...................................................
2.3.1.4.4 Trypanosomes detection in the CSF ..................................................................................
2.3.1.5 Treatment of the disease .......................................................................................................
2.4 Pyroglutamyl peptidase 1 (PGP 1) ..............................................................................................
2.4.1 Clan CF of Pyroglutamyl peptidase 1 ......................................................................................
2.4.2 Family C15 of Pyroglutamyl peptidase 1 ................................................................................
2.4.3 Trypanosoma brucei PGP 1 .....................................................................................................
2.4.4 Sequence ofTrypanosoma PGP 1............................................................................................
2.5 Immunogenicity ..........................................................................................................................
2.5.1The Nature of the Protein ..........................................................................................................
2.5.1.1 Degree of foreignness ...........................................................................................................
2.5.1.2 Molecular size .......................................................................................................................
2.5.1.3 Structure of the protein .........................................................................................................
2.5.1.4 Ability to be processed...........................................................................................................
2.5.2 Route of administration............................................................................................................
2.5.3 Genetic makeup of the organism .............................................................................................
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2.5.4 Adjuvant ...................................................................................................................................
2.5.5 Dose of protein given ...............................................................................................................
2.5.6 Formulation and purity of the protein ......................................................................................
2.6 Overview of the techniques to be used .......................................................................................
2.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........................
2.6.2 Western Blotting ......................................................................................................................
2.6.3 Enzyme-Linked Immunosorbent Assay (ELISA) ....................................................................
2.7 pET-28a (+) vector ......................................................................................................................
CHAPTER THREE ........................................................................................................................
3.0 MATERIALS AND METHODS .............................................................................................
3.1.0 Study design .............................................................................................................................
3.2.0 Materials: .................................................................................................................................
3.2.1 Transformed BL21DE3 cells ...................................................................................................
3.3.0 Methods: ..................................................................................................................................
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 (+) ...............................................................
3.3.1.2 Plasmid extraction .................................................................................................................
3.3.1.3 Agarose Gel Electrophoresis .................................................................................................
3.3.1.4. Restriction enzyme digestion ...............................................................................................
3.3.2 Expression of the recombinant Trypanosoma PGP 1 ..............................................................
3.3.2.1 Small scale expression ..........................................................................................................
3.3.2.1.1 Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................
3.3.2.1.2 Western Blotting ................................................................................................................
3.3.2.2 Large scale expression ..........................................................................................................
3.3.3 Extraction, Purification and Quantification Trypanosoma PGP 1 and bacterial
protein ...............................................................................................................................................
3.3.3.1 Extraction ..............................................................................................................................
3.3.3.1.1 Extraction ofTrypanosoma PGP 1 ....................................................................................
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3.3.3.1.2 Extraction of Bacterial protein ...........................................................................................
3.3.3.1.2.1 Growth of competent BL21 cells from the glycerol stock ..............................................
3.3.3.1.2.2 Extraction of bacterial protein ........................................................................................
3.3.3.2 Purification of the Trypanosoma PGP 1 and bacterial protein from inclusion
bodies ................................................................................................................................................
3.3.3.2.1 Purification of inclusion bodies .........................................................................................
3.3.3.2.2 Purification of the recombinant Trypanosoma PGP 1 and bacterial protein .....................
3.3.3.3 Quantification of the Trypanosoma PGP 1 and bacterial protein ........................................
3.3.4 Immunisation of mice of the Trypanosoma PGP 1 and bacterial protein ................................
3.3.5 Analysis of immune sera ..........................................................................................................
3.3.5.1 Western blot analysis ............................................................................................................
CHAPTER FOUR............................................................................................................................
4.0 RESULTS ...................................................................................................................................
4.1 Confirmation of the insert in pET28a (+) plasmid vector in BL21DE3 cells .............................
4.2 Expression of recombinant Trypanosoma PGP 1 .......................................................................
4.3 Extraction and Purification ofTrypanosoma PGP 1 and bacterial protein .................................
4.3.2 Purification ...............................................................................................................................
4.3.2.1 Purification of recombinant Trypanosoma PGP 1 ................................................................
4.3.2.2 Purification of Bacterial protein............................................................................................
4.4 Quantification .............................................................................................................................
4.5 Analysis of sera for the different groups of mice .......................................................................
4.5.1. Western blot analysis of pre-immune sera ..............................................................................
4.5.2 Western blot analysis of immune sera .....................................................................................
4.5.3 ELISA analysis of sera.............................................................................................................
4.5.2.1 Group one (Test group) .........................................................................................................
4.6.2.2 Group two (Adjuvant group) .................................................................................................
4.6.2.3 Group three (PBS group) ......................................................................................................
4.6.2.4 Group Four (Bacterial protein group) ...................................................................................
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CHAPTER FIVE .............................................................................................................................
5.0 DISCUSSION .............................................................................................................................
CHAPTER SIX ................................................................................................................................
6.0 CONLUSION AND RECOMMENDTAION .........................................................................
6.1 CONCLUSION ..........................................................................................................................
6.2 RECOMMENDATION ............................................................................................................
REFERENCES.................................................................................................................................
APPENDIX I ....................................................................................................................................
APPENDIX II...................................................................................................................................
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LIST OF FIGURES AND TABLES
Figure 1: TheLife cycle ofTrypanosoma 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 ofTrypanosoma 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 fortest 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 ofTrypanosoma PGP 1....36
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ABBREVIATIONS AND ACRONYMS
BBB Blood Brain Barrier
Bst Bacillus staeorothermophilus
bp base pairs
CAT Card Agglutination Test
CATT Card Agglutination Test for Trypanosomes
CDC Center for Disease Control
CNS Central Nervous System
CSF Cerebral Spinal Fluid
DAB 3, 3-diaminobenzidine
E. coli Escherichia coli
ECL Enzyme Chemiluminescence
ELISA Enzyme Linked Immuno-sorbent Assay
EDTA Ethylene diamine tetra acetic acid
HAT Human African Trypanosomiasis
HRP Horse Radish Peroxidase
IPTG Isopropyl-beta-D-thiogalactopyranoside
Kda Kilodaltons
LH-RH Luteinising Releasing Hormone
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mHAET Mini-anion-exchange centrifugation technique
mHCT Microhematocrit Centrifugation Technique
Ni-NTA Nickel-Nitrilotriacetic acid
O.D Optical Density
OPD o-phenylenediamine dihydrochloride
PARP Procyclic acidic repetitive protein
PCR Polymerase Chain Reaction
PGP 1 Pyroglutamyl Peptidase
rpm rotations per minute
SDS Sodium Dodecyl Sulfate
SDS-PAGE SDS-Poly Acrylamide Gel Electrophoresis
spp. Species
T. b. Trypanosoma brucei
T. b. gambiense Trypanosoma brucei gambiense
T. b. rhodesiense Trypanosoma brucei rhodesiense
TRH Thyrotropin-Releasing Hormone
W H O 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 forT. 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 peptidasetype 1 was successfully expressed in the E. coli (BL21DE3) cells. The protein was
extracted by cell lysis from theE. 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 ofTrypanosoma 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.
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CHAPTER ONE
1.0INTRODUCTION1.1BackgroundAfrican 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 ofT. 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
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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 isintracellular 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 forT .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
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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 ofTrypanosoma 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 andcould 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.
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1.5Research QuestionDoes 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 ofT. 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
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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 nervoussystem. 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 Tr ypanosoma brucei
Trypanosoma brucei species is one of the causative agents ofAfrican Trypanosomiasis
(or sleeping sickness). There are 3 sub-species ofT. 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).
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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, apotential 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
ofTrypanosoma. 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 ofTrypanosoma brucei
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Figure 1: Life cycle ofTr ypanosoma bruceiin 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 theyenter 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), (Biebingeret 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
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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
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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 cerebro-
spinal 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
detectionof trypanosome antigens however, CATT is only effective forT. b. gambiense,
it is not effective and reliable forT. b. rhodesiense. It is mostly used forT .b. gambiense
specific antibodies in the blood, plasma,or serum of HAT patients (Magnus et al., 1978).
The antigen consists of lyophilised bloodstream forms ofT. b. gambiense variable
antigen type LiTat1.3. 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 minon 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 87to 98%, and the negative
predictive value is excellent duringmass population screening (Noireau et al., 1987; Truc
et al., 2002). Nevertheless,false-negative CATT results can occur (Penchenier et al.,
1991), as suspected inpatients 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 performedon undiluted blood or serum with a low dilution of less than 1:4,
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theagglutination can be inhibited, a phenomenon called prozone which can be overcome
by adding EDTA tothe 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 predictivevalue is limited because the test is used
for mass screening in
populations where the prevalence of HATis usually below 5% (Robays et al., 2004).
False-positive results can occur in patients with malaria and other parasiticdiseases 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 obtainedby demonstrating specific
antibodies in serum of infected hosts. Trypanosomes have a complex antigenicstructure
evoke production of a large spectrum of antibodies.T. b. gambiense specific IgG and IgM
antibodies are presentin high concentrations and are directed mainly against the Immuno-
dominant 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 to4 weeks of infection
(Vanhamme et al., 2001) with strict standardisation and quantification (Lejon et al.,
1998) but sero-positivity must be interpretedwith caution in previously treated patients
since antibodiescan persist for up to 3 years after cure (Paquet et al., 1992). ). ELISA can
also detect specific antibodiesin 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
referencelaboratories 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 alternativeto the CATT (Bscheret al., 1999). The
test is based on the combination of threepurified 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
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a higherspecificity (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 forT. b. rhodesiense.
2.3.1.1.4 Immunofluorescence Assays
Immunofluorescence assays have been used with success for HATcontrol 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 greatlyimproved the reliability of the test (Magnus et al.,
1978). It can be used withserum 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 examinationof
lymph node aspirate, blood, or CSF and this provides direct evidencefor 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 infection10,000
trypanosomes per ml, being easilydetectable and less than 100 trypanosomes per ml,
being below thedetection limit of the most sensitive methods in use. This implies, failure
todemonstrate 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 earlierthan in the blood. The
chancre is punctured, and the fluid obtainedis microscopically examined as a fresh or
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fixed and Giemsa-stainedpreparation. This method is very seldom applied in the
fieldbecause 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 patientswith 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
facilitatethe reading. The wet preparation is then immediately examined under X400
magnification for the presence of motiletrypanosomes. The technique is simple and
cheap. The sensitivityvaries between 40 and 80% depending on the parasite strain,
the
stage of the disease (sensitivity is higher during the firststage), 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 isplaced on a slide, cover slipped and
examined microscopically at X400 magnification. Trypanosomes can be seen moving
betweenthe erythrocytes. Although this method has very low sensitivity of about 10,000
trypanosomes per ml, it is still usedin some centers because of its low cost and
simplicity.Examination of 20 l of stained thick blood
film slightly improves sensitivity,
with a detection thresholdof around 5000 trypanosomes per ml. It is the technique of
choicefor 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 frequentlydeformed 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 centrifugationtechnique or as the Woo test, was
developed more than 30 yearsago and is still in use in many HAT control programs
(Woo P. T., 1971, 1970).In brief, capillary tubes containing anticoagulant are filled
three-
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quarters full with finger prick blood. The dry end issealed with plasticine. By high-speed
centrifugation in a haematocritcentrifuge for 6 to 8 min, trypanosomes are concentrated
atthe 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
withthe 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 canrender the visualisation of the much smaller
trypanosomes verydifficult. 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) initiallydeveloped for the rapid assessment of
differential cell counts,has been extended to the diagnosis of hemoparasites
includingtrypanosomes (Levine et al., 1989; Bailey et al., 1992). It has the advantages of
concentratingthe 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-speedcentrifugation of the blood in special capillary tubes
containingEDTA, acridine orange, and a small floating cylinder, motile trypanosomes
can be identified by their fluorescent kinetoplastsand nuclei in the expanded buffy coat.
UV light is generatedby a cold light source connected by a glass fiber to a
specialobjective containing the appropriate filter and the procedure is done in a
darkroom. However the relative sophistication and fragilityof the material prevents its
daily use during active screeningsessions. 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 themHCT when fewer than eight capillary tubes
are used (Ancelle et al., 1997). Itis 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 atechnique developed by
Lanham and Godfrey (Lanham and Godfrey, 1970). An initial evaluationshowed that the
mAECT was more sensitive than the thick bloodfilm and the mHCT (Lumsden et al.,
1981). An updated version has been describedby Zillmann et al., 1996. The technique
consists of separatingthe trypanosomes, which are less negatively charged than
bloodcells, from venous blood by anion-exchange chromatography and
concentrating
them at the bottom of a sealed glass tube by low-speedcentrifugation. The tip of the glass
tube is then examined ina 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 arequite 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 (Bscheret al., 2004). These
methods range from Polymerase Chain Reaction (PCR) for amplifying DNA to Loop
Mediated Isothermal Amplification. However, most of the molecular methods fordiagnosis 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 beenvalidated for diagnostic
purposes. PCRs targeting repetitive sequences are in theory more sensitive than those
targetinglow-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 thatmay 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 samplethat can be applied on filter paper is small, thus limiting
the chance
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to contain enough DNA for detection. Samples shouldbe protected from sunlight to avoid
DNA degradation. PCR resultsare not always unequivocal. Unexplained false-negative
and false-positiveresults were observed in CATT-sero-positive but parasitologically
non-
confirmed 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 comparedto double centrifugation of CSF (Truc et al., 1999), but a number of
patients with positive PCR resultswith CSF were successfully treated with pentamidine,
thus showingthem to be in the first stage of the disease (Jamonneau et al., 2003). The
methods needs simplification hence PCR is not an option forfield diagnosis and for the
time being is restricted to researchpurposes.
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 obtainedby 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
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melarsoprol, an arsenicalderivative 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 techniquefor stage
determination. After collecting the CSF sample by lumbar puncture, thecell count should
be carried out as soon as possible to preventcell 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 Fuchs-
Rosenthal and the Neubauer devices. It isnot recommended to dilute the CSF with Trck
solution sincethis solution can lyse trypanosomes. Patients
with 6 to 20 white blood cells
per l in the CSF are sometimes referredto 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 mainlyof albumin (70%) and
IgG (30%), both originating from the serum.Protein concentrations in the CSF are
elevated in HAT patientsand range from 100 to 2,000 mg/liter (Bisseret al., 2002; Lejon
et al., 2003). Protein concentrationscan also be raised in first-stage illness due to the
diffusionof IgG into the CSF, which can be present in very high concentrations
in the
serum. Recent evidence suggests that the protein concentrationthreshold set by WHO
(370 mg/liter) is too low and should beraised 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 totalprotein concentration
in CSF. However the technique is rather difficult and the CSF proteinconcentrations
obtained by different methods and different standardsare not comparable. Due to the
sophistication of methods, the absence of standardisation, the instabilityof reagents, and
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the limited added value compared toCSF cell count (Miezan et al., 1998), it is no longer
recommended and has been virtually abandoned infield laboratories.
2.3.1.4.3 Antibody (IgM) detection and concentration in the CSF
CSF of second-stageHAT patients contains high levels of immunoglobulins,
especiallyIgM (Bisseret al., 1997). An increased CSF IgM concentration has
thus been
considered by some as a strong potential marker ofsecond-stage HAT. High IgM levels
in CSF is due to intrathecalsynthesis, the dominance of IgM presence is an early
markerof CNS invasion whereas blood-CSF barrier dysfunction is found in
late CNS
involvement (Lejon et al., 2003). Despite its relevance to stage determination, IgM
detectionin CSF has not been carried out in the field, owing to the lack
of simple and
robust tests. A latex agglutination test for IgMin CSF (LATEX/IgM) has recently been
developed and is designedfor 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 classificationof a patient as being
in the second stage of illness. It isimportant to examine the CSF immediately after
lumbar puncture,because trypanosomes in CSF start to lyse within 10 min.
Directdetection 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 bycentrifugation of the CSF sample, especially when a double
centrifugation method is used (Cattand et al., 1988). The latter method is relatively time-
consuming
and requires two different types of centrifuges; therefore,
it is not applicable in
every field setting. A modified andsimplified single centrifugation of CSF using a sealed
Pasteurpipette 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 ofT. 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
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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 4-
naphthylamine (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 bruceiPGP 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
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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 ofTrypanosomaPGP 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 T-
cells) (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 ofimmunogenicity 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.
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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 (Porteret 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
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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 (Sambrooket 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
orPVDF) 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
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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 7-
8 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 theE. 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:
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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.
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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 TrypanosomaPGP 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
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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.
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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 TrypanosomaPGP 1 and bacterial
protein
3.3.3.1 Extraction
3.3.3.1.1 Extraction ofTrypanosomaPGP 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 minute
Recommended