Documenti di Didattica
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Stephanie J Piper
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2014
17
Declaration
18
19
20I
certify that the work reported in this thesis is entirely my own effort, except where
21otherwise
acknowledged. I also certify that the work is original and has not been previously
22submitted
for assessment in any other course of study at this or any other institution.
23
24..
25Signature of
Candidate
26
27
28
29Date
30
31Endorsement
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2
Signature
Signature
Date
Date
34Acknowledgements
35
36I
would like to thank my supervisors Professor Mike Kotiw and Dr Lisa Seymour. This year
37has
been a steep learning curve and their time and patience is much appreciated. Despite
38busy
schedules and due dates, Lisa has always managed to find time to talk over lab
39procedures
40The
research team Lu, Richard, Cathy, Karren and Stephen have also been a fantastic support
41throughout
the year. I am grateful for their constant assistance and help within the practical
42side
of my project. Their advice, anecdotes and guidance made the year more enriching and
43has
better prepared me for life outside USQ. Id also like to thank the examiners Dr. John
44Dearnaley
and Dr. Noel Knight for their counsel in a variety of areas. In particular, Dr. John
45Dearnaley
has been a fantastic assistance over the year and goes above and beyond his job
46requirements.
47
48Finally,
49team,
the Manager of Residential Life Katharine Bigby and my friends and family. Life
50outside
51kept
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53
54
32
4
I am grateful for the understanding and assistance of the Steele Rudd Collegiate
my honours project has not been dull, and your constant support and shenanigans has
me motivated.
55Abstract
56Streptococcus
57patient
morbidity and mortality in the developing world and within the Australian Indigenous
58population.
59and
60scarlet
61rheumatic
heart disease are responsible for the highest morbidity. The 30-valent vaccine
62candidate
63low
64The
difficulty in creation of a suitable vaccine lies in part with the variety of GAS virulence
65factors.
66which
67the
68Furthermore,
69results
protein fragments J8 and J14 within the M protein have given encouraging
70Virus-like
this study, the use of a chimeric hepatitis B surface antigen VLP expressing M protein
73epitopes
74is
are able to induce both cell mediated and humoral immune responses.
p145, J8 and J14 for use as a dual vaccine against Hepatitis B virus (HBV) and GAS
investigated. Specifically, PCR generated DNA sequences of J8, J14 and p145 from the M
75protein
of GAS have been cloned into the highly immunogenic a determinant region of the
76HBsAg-S
VLP and transformed into human embryonic kidney (HEK293T) cells. Expressed
77recombinant
78presentation
of GAS epitopes. ELISA results showing high titres were obtained for VLP:p145
79but
53
6
80constructs,
81can
82
74
8
protein expression and antigenic screening of proteins is required before the study
83Abbreviations
GAS
Group A Streptococcus
GBS
Group B Streptococcus
RF
Rheumatic Fever
RHD
APSGN
WHO
GBM
SpeB
Streptococcal exotoxin B
VLP
Virus-Like Particles
URTIs
APCs
ELISA
TCSs
GRAB
SK
Streptokinase
HI
Humoral Immunity
CMI
MEM
PBS
HEK293T
HEPEs
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HBS
CFA
RT
Room Temperature
84
95
10
List of Figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
peoples
M protein hypervariable, variable and conserved structures
GAS Cross Section
GAS-m Protein C-region Peptides
Proposed strategy
pcDNA3.1 Vector Map
Primer design and matching agarose gel fragments
1% agarose gel of GAS-m fragments inserted into a determinant region of
5
7
21
23
30
35
36
Figure 9
Figure 10
Figure 11
HBsAg-S
1% agarose gel of pcDNA containing HBsAg-S:GAS-m fragments
ABI chromatogram of p145 sequencing
ELISA antigenic recognition of VLP:p145, VLP:J8 and VLP:J14
37
38
40
List of Tables
Table 1
Table 2
Table 3
85
86
87Contents
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
116
12
Declaration..............................................................................................................................i
Acknowledgements............................................................................................................ii
Abstract..............................................................................................................................iii
Abbreviations......................................................................................................................v
List of Figures....................................................................................................................vi
List of Tables.....................................................................................................................vi
1.0 Overview of group A streptococcus (GAS) pathogenesis............................................1
1.1 Distribution and incidence of disease...........................................................................1
1.2 Populations at risk.........................................................................................................2
1.2.1 Indigenous Australians...............................................................................................2
1.2.2 Paediatric...................................................................................................................4
2.0 The biology of group A streptococcus..........................................................................4
2.1 Virulence factors...........................................................................................................5
2.1.1 M protein....................................................................................................................5
2.1.2 Capsule.......................................................................................................................6
12
15
26
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
137
14
2.1.3 Streptokinase..............................................................................................................7
2.1.4 Streptolysin O............................................................................................................7
2.1.5 C5a Peptidase.............................................................................................................8
2.1.6 Cysteine protease SpeB.............................................................................................8
2.2 Genetics including virulence gene control...................................................................8
2.3 Nature and spectrum of GAS infections.....................................................................10
2.3.1 Common streptococcal throat infection...................................................................10
2.3.2 Rheumatic fever and rheumatic heart disease..........................................................11
2.4 Current treatment options...........................................................................................13
2.4.1 Antibiotic therapy....................................................................................................13
2.5 GAS vaccine development..........................................................................................14
2.6 Virus Like Particle (VLP) technology........................................................................19
2.6.1 Overview of the use of VLP technology for human diseases..................................19
2.6.1.1 Generating recombinant chimeras........................................................................20
2.6.1.2 Overview of use of HBsAg-S VLPs.....................................................................21
2.7 The current study proposal..........................................................................................22
2.7.1 Proposed strategy.....................................................................................................22
Hypotheses........................................................................................................................24
3.0 Bacteria and Plasmids.................................................................................................25
3.1 Molecular Analyses.....................................................................................................25
3.2 Amplification of GAS-m Epitopes.............................................................................26
3.3 Agarose Gel Electrophoresis.......................................................................................27
3.3.1 Wizard SV Gel and PCR Clean-Up......................................................................28
3.4 Digestion and Insertion of GAS-m Epitopes into HBsAg-S DNA sequence.............28
3.5 Plasmid Ligation.........................................................................................................29
3.6 Transformation of Competent Cells............................................................................30
3.7 Wizard Plus SV Minipreps DNA Purification.........................................................30
3.8 DNA Sequencing........................................................................................................31
3.9 Wizard Plus SV Midipreps DNA Purification.........................................................32
3.9.1 HEK293T Cell Culture............................................................................................32
3.9.2 HEK293T Transfection and Protein Isolation..........................................................33
3.9.3 ELISA......................................................................................................................34
4.0 GAS-m epitope Amplification....................................................................................35
4.1 GAS-m Fragment Insertion into HBsAg....................................................................35
4.2 PVLP55 Vector Insertion............................................................................................37
4.3 DNA Sequence Analysis.............................................................................................38
Significance......................................................................................................................43
Future Directions..............................................................................................................46
Conclusion........................................................................................................................47
145
146
147
148
149
150
151
Introduction
155syndromes
156the
pyogenes) is a
Australian Indigenous population and much of the developing world (Carapetis et al.
1572005).
158pyoderma,
159fasciitis
160global
161(RF),
scarlet fever and otitis media as well as invasive diseases including necrotising
and toxic shock syndrome. Post-infection sequelae, however account for the highest
burden of GAS disease and includes rheumatic heart disease (RHD), rheumatic fever
162(APSGN)
(Henningham et al. 2013). Whilst research activity in GAS vaccinology has been
163ongoing
164vaccine
165European
specific GAS serotypes which may not address the main serotypes present in the
166developing
167150
world. One of the main challenges in vaccine design is the incorporation of the
168infections
16959%
in South African school children indicate the vaccine coverage will be between 40-
of isolates recorded (Dale et al. 2011). This indicates that the 30-valent vaccine is an
170ineffective
158
16
A broad 30-valent
171(Dale
172for
et al. 2011). Among the many conserved virulence factors which have been explored
usage in a GAS vaccine, the M protein is a promising target for vaccine research as it is a
173highly conserved,
makes up the majority of cases with 15.6 million individuals currently affected.
177Furthermore,
178et
RHD can be fatal and is estimated to cause 233,000 deaths per year (Carapetis
al. 2005). APSGN affects 472,000 individuals and causes a further 5,000 deaths annually.
179Superficial
180every
infections occur more frequently, where 6.1 million new cases of pharyngitis arise
year and 1.1 million individuals currently suffer of pyoderma (Carapetis et al. 2005).
181Uncertainty
levels and assumptions used in the compilation of GAS data tends towards
182underestimation,
183(2005)
184the
making the true prevalence likely to be greater than stated. Carapetis et al.
data. RHD specific data, however, has a higher level of quality through rigorous data
185collection.
186calculated
187Asia
at 2.2 cases, other Asian areas at 0.8 cases, Latin America at 1.3 cases, Middle East and
188North
Africa at 1.8 cases, Eastern Europe at 1 case, Pacific and Indigenous Australia/New
189Zealand
192and
developing countries. Less developed countries account for 79% of RHD cases, 95% of
193RF
cases, 97% of APSGN cases and 97% of invasive GAS cases. Indigenous Australians
194also
172
18
199Australians
200more
2019.6
Indigenous
have the highest annual mortality rate of 30.2 per 100,000 individuals. This is
than three times as high as the second highest risk population, Maori New Zealanders at
per 100,000 individuals (Carapetis et al. 2005). Rural communities within the Northern
202Territory
203between
2-7 cases for every 1000 children between 5-14 years old. Furthermore, up to 3% of
204Indigenous
205Currie
206GAS
1998).
related skin infections such as scabies and streptococcal pyoderma are endemic in
207Australian
208mite
or Sarcopes scabiei, and is known to spread easily in environments with poor sanitation
209and
210pyoderma
211example,
and scabies, as demonstrated by Andrews et al. (2009) (See Figure 1). For
both scabies and streptococcal pyoderma have been found in Indigenous Australian
212populations
21325%
214distinctly
lower rate of GAS throat carriage of between 0-14% in comparison to RHD and
215skin
infections (Van Buynder et al. 1992). Household crowding, access to adequate water,
216hot
weather, humidity and lack of personal hygiene are likely to contribute to a high
217prevalence
2181992).
219
220
193
20
221
222
223
224
225
226
227
228
2291.2.2 Paediatric
230GAS pharyngitis accounts
231cultured
from 15-36% of children suffering a sore throat in the US (Linder et al. 2005).
232APSGN
is also most commonly seen in paediatric patients, equating to 90% of the total
233population
suffering APSGN.
234hypothesised
235(GBM).
Children and adults have 2-3 nm and 4-4.5 nm GBM sizes, respectively, making it
236easier
for the immune complex molecule to infect the glomerulus in children rather than
237adults
(Wiwanitkit 2006).
2382.0 The
239Infection
240matrix
241process,
242electrostatic
243specificity
214
22
244components
245decorin
repulsion.
In
246particular,
fibrinogen is known to interact and bind with the highly conserved M protein in
247the
repeat region near the N-terminus of the protein (Carlsson et al. 2005).
248As
well as cellular adherence, GAS also has the capacity to enter epithelial cells to avoid
249early
host defences and antibiotics. This invasive virulence process is possible through
250proteins
on the cell surface, or invasins such as fibronectin-binding protein and the M protein
251(LaPenta
252invasion
et al. 1994).
initiates through a zipper-like mechanism where host microvilli also play a role.
2532.1
Virulence factors
2542.1.1 M protein
255M
256the
M protein is an alpha-helical coiled coil dimer anchored to the outer streptococcal cell
257wall
via the C terminus. The M protein can be divided up into the hypervariable (A-repeat
258region),
259Hydrophobic
260classification
261the
Lancefield GAS
262haemolytic
263nucleotides
as a determinant, more than 150 genotypes of GAS have been found thus far
264(Facklam
1997; McGregor et al. 2004). M proteins are divided into Class I or Class II
265dependent
upon their reaction with antibodies against the C repeat region of the M protein;
266Class
I proteins react through the presence of a surface-exposed epitope and Class II does not
267react (Bessen
235
24
et al. 1989).
268
269Figure
The M protein alpha-helical coiled coil structure with anchored domain. A to C regions of
270the M protein are multi-functional, and the protein can be divided further into conserved,
variable and hypervariable regions. Each colour-coded section can interact with the human
proteins
indicated
(McArthur
& Walker
2006).
271plasma
M Protein
antigenic
variation
contributes
to the
range of GAS virulence strategies. For
272example,
273the
the ability of M protein to bind to fibrinogen in the B-repeat region interferes with
complement system and contributes to phagocytic resistance (McArthur & Walker 2006;
274Ringdahl
275opsonisation
276which
277to
inhibits complement activation (Berggrd et al. 2001). The A-repeat region can bind
IgA, IgG, Factor H, Factor H-like protein 1, C4b binding protein and plasminogen. The C-
278repeat
region can bind Factor H and human serum albumin, further aiding GAS in phagocytic
279evasion
2802.1.2 Capsule
281The capsule structure
282strains
have been shown to have markedly decreased phagocytic resistance, and a 100-fold
283decrease in
284a
virulence when tested in mice (Wessels et al. 1991). The GAS capsule consists of
hyaluronic acid with the degree of encapsulation varying greatly across the serotypes (See
285Figure
256
26
3). GAS has been found to up-regulate hyaluronic acid production to structurally
286minimise
287protein
288functional
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
GAS virulence factors include the M Protein, C5a peptidase and capsule as outer
307 structures. Other virulence factors include streptokinase, streptolysin O, cysteine
protease SpeB and many others. Taken from Talaro & Talaro (2002).
308
309
3102.1.3 Streptokinase
311Streptokinase (SK) is a
312four
313Specifically,
314as
277
28
315plasminogen
and convert into the serine protease plasmin. Control over host plasminogen is
316advantageous
317plasmin,
318conserved
319the
within GAS and can be classified within one of nine polymorphic genotypes where
main structures such as hydrophobicity are maintained. SKs structural variability arises
320from
a region designated as V1, where it is speculated the genes for nephritis are located
321(Malke
1993).
3222.1.4 Streptolysin O
323Streptolysin O is a bacterial
324al.
2009). It is known for its ability to form large pores in cell membranes with membrane
325cholesterol
326al.
327et
SK is semi-
2009). This ability is a crucial defence mechanism as it acts to prevent phagocytosis (Feil
328membrane
329oligomerisation,
330et
al. 1986).
331promoted
332membranes,
333(Timmer
et al. 2009).
and invasin (Cleary et al. 2004). It is capable of disrupting the complement pathway
cleavage of chemotaxin C5a at its polymorphonuclear binding site. This proteolysis halts
338recruitment
339by phagocytes
298
30
347et
(Collin & Olsn 2001). SpeB is able to cleave human immunoglobulins, including
IgM, IgD and IgE (Collin & Olsn 2001). Furthermore, it can cleave vitronectin,
345fibronectin
346also
SpeB
and host proteins to compromise host tissue integrity (Kagawa et al. 2009). It can
spawn biologically active peptides such as interleukin-1, kinins and histamine (Kagawa
al. 2000). Through the degradation of host proteins, research conducted by Barnett et al.
348(2013)
349and
350
3512.2 Genetics including virulence gene control
352GAS has been genotyped via M protein typing in an
353species.
354the
The famous Lancefield method of M protein typing of emm GAS species determines
type of opacity factor present through an opacity factor inhibition test. emm genes are
355split
up into distinct subfamilies, named from A to E and defined by the sequence differences
356at
the 3 end (McGregor et al. 2004). Classically, A to C emm pattern strains are recognised
357as
pharyngitis specific, D strains are often isolated from impetigo lesions and E strains are
358commonly found
359The
360is
size of the GAS genome ranges between 1.85-1.9 Mb, where 1.7 Mb of the GAS genome
conserved between strains (Wagner & Waldor 2002). Phage transduction is a process
361through
362integral
part of GAS survival and development, and phage genomes have the ability to alter
363the
364bacterial
adhesion, colonisation and invasion, encode bacterial toxins and alter bacterial
365susceptibility
319
32
to antibiotics (Wagner & Waldor 2002). Beres et al. (2002) found that an
366average
367gene
transfer.
368GAS
has a formidable arsenal of virulence factors which enables it to persist and cause
369infection
370regulators
371interactions.
372the
In short, Mga and RofA-like proteins are the two global regulators which pilot
cell as per the signals received by two-component signal transduction systems (TCSs)
373(Kreikemeyer
374a
375specifically
376C5a
peptidase and many other virulence factors essential in host colonisation (Kreikemeyer
377et
al. 2003).
378If
environmental conditions become hostile though lack of nutrient supply or host defence
379mechanisms,
380and
381TCSs
are not unique to GAS and function in detection and communication of environmental
382signals
383been
384in
though the transmembrane protein sensor histidine kinase. Currently, 13 TCSs have
identified (Kreikemeyer et al. 2003). Notably, these include ihK/Irr, which plays a role
host-cell lysis, GAS neutrophil resistance in vitro and in mouse virulence models in vivo
385(Voyich
386adhesins
to promote high adherence and internalisation rates (Klenk et al. 2005). SilAB
387regulates
388murine and
389The
GAS capsule is essential for pathogenesis. The production of hyaluronic acid, the main
390ingredient
3310
34
of the capsule, is controlled by an operon made up of three genes: hasA, hasB and
391hasC.
392synthase,
393that is
likely to be conserved (Albert et al. 1998). CsrRS is a regulator of the hyaluronic acid
394capsule
395GAS
402million
403al.
2005). Although major complications are rare, usage of antibiotics has been shown to
404prevent
405least 50%
joints, heart, central nervous system, skin and or subcutaneous nodules (McNamara et al.
4092008)
(Table 1). The Jones criteria (Burke & Chang 2014) states RF can be diagnosed
410through
the presence of a preceding GAS infection, plus the presence of two major
411manifestations
412the
or one major and two minor manifestations of the Jones criteria. Symptoms of
Jones criteria mainly include carditis, polyarthritis and Sydenhams chorea. Further
413research
414symptoms
415reactions
3511
36
by inflammation of
are known to cross over with diseases such as Lyme disease, serum sickness, drug
416The
pathogenesis of RF and RHD is poorly understood and two hypotheses currently exist
417which
attempt to explain the nature of these disease manifestations. The first hypothesis
418proposes
molecular mimicry and cross reactivity between sarcomeric heart myosin and
419streptococcal
420valve.
Tandon et al. (2013) proposes the following inflammatory mechanism: The GAS-m
421proteins
422turn
N-terminus has been shown to bind to the CB3 region in collagen type IV, which in
423inflammation.
424tandem.
425cardiac
The majority of autoimmune diseases involve more than one auto-antigen, thus
myosin and collagen may act as auto-antigens where one precedes the other. This is
426supported
427antibody in
428
time
poststreptococcal Oedema, hypertension, urinary sediment abnormalities,
glomerulonephritis
Invasive
Bacteremia
Puerperal sepsis
complement deficiency
High fever, nausea, vomiting
Fever, chills, abdominal pain in a pregnant or early
postpartum woman.
3712
38
Cellulitis
Necrotising fasciitis
tissue destruction
Streptococcal toxic shock High fever, rapid-onset
hypotension,
accelerated
syndrome
multisystem failure
429Adapted from Carapetis et al. (2005)
4302.4 Current treatment options
431Antibiotics are the primary treatment
432include
low cost, efficacy and safety. Other drugs such as cephalosporins, macrolides,
433erythromycin
434environment
and clarithromycin have also proven to be effective and are in use in a clinical
435
4362.4.1 Antibiotic therapy
437A meta-analysis of penicillin
438GAS
tonsillopharyngitis, and it was found that the cephalosporin cure rate was twice that of
439penicillin,
440despite
making it the superior choice of drug (Casey & Pichichero 2004). However,
the size and number of trials undertaken by Casey and Pichichero, the study has been
441criticised
442trials,
443provided
details of patients signs and symptoms at enrolment and 9 based GAS clearance on
444follow-up
throat cultures obtained in the optimal 3 to 14 day period after therapy was
445completed
(Shulman & Gerber 2004). Similar past studies (1953-1993) which retained a high
446standard
447among
448also
lies in its price and spectrum: it is 20 to 30 times cheaper than cephalosporin and has a
449smaller
target range which reduces the selection pressure and the prevalence of other
3913
40
451Within
452transfer
of the mef gene. This is a concern for individuals who are allergic to lactam
453antibiotics
454spread
455long
For example, GAS molecules with homology to human proteins could potentially
460protection
461research
462carriage,
invasive infection, asymptomatic GAS infection, RF, RHD, APSGN and toxin
463mediated
464the
following: anchored cell wall proteins, cell membrane associated and/or secreted and
465anchorless
466Vaccine
467Multi-valent
468and
30-valent GAS vaccines are in clinical or pre-clinical stages (Steer et al. 2013). Despite
469the
number of valencies covered, the 30-valent vaccine would geographically only give good
470coverage
471and
to the US, Canada and Europe, intermediate coverage to Asia and the Middle East
very poor coverage within Africa and the Pacific. However, this concern has been
472mitigated
473protein
474targeted
475vaccine
boasts protection against 98% of known serotypes responsible for pharyngitis and
476invasive infections
4114
42
Antigen
Vaccine
type/preclinical Reference
data
Cell Wall Anchored
Proteins
M Protein
N-terminal region:
6-valent,
valent
26- Multivalent
and
recombinant (Dale
30- protein/Intramuscular
valent antigen
et
al.
al.
2011)
(Kotloff et al.
N-terminal
2004)
(Brandt et al.
Peptide-protein
/Subcutaneous 2000)
tetanus toxoid
delivery
N-terminal
opsonic antibodies
Lipopeptides: IgG
peptides
via
in
mice
via
and (Olive
linked opsonisation/Subcutaneous
lipid
specific serum
Peptide
4315
44
2003)
(Brandt et al.
al.
peptide
Heteropolymer
and
et
C- delivery
in
mice
2000)
via
terminal peptides)
N-terminal
antibodies
Amino
Plasminogen-
residues/subcutaneous
binding group
delivery in
acid (SandersonSmith
quackenbush 2008)
et
al.
C-repeat Synthetic
conserved region
peptide (Bessen
conjugate/intranasal
&
in Fischetti 1988)
serum
epitope Self-adjuvanting
J8/J14/p145
(Zaman et al.
C-repeat
serum
epitope C-terminal
amino
(StreptInCor
residues/subcutaneous
vaccine)
delivery
in
mice
StreptInCor suspension
Conserved region C-repeat
amino
acid (Mannam et al.
proteins expressed residues/nasal
in
Lactococcus
lactis
and 2004)
subcutaneous delivery in
mice via peptide specific
serum
FbaA Recombinant
Fibronectin-
Seven
Binding Protein A
epitopes
co- protein/Intraperitoneal
Protein
fragments
Recombinant H12 Recombinant
F1/Streptococcal
co-administered
4516
46
multivalent (Georgousakis
protein/subcutaneous
et al. 2009)
Fibronectin
with
Binding Protein I
Streptococcal
peptide J14
N-Spa36 epitopes
Protective Antigen
specific serum
Spa
(Ahmed et al.
antiserum/intraperitoneal
2010) (Dale et
Streptococcus
Spy0416/SpyCEP
delivery in mice
Recombinant
pyogenes Cell
epitopes
protein/Intramuscular
Envelope
Proteinase/Spy0416
Cell Membrane
Associated and/or
Secreted GAS
Candidates
C5a Peptidase
al. 1999)
CEP (Turner et al.
2009)
delivery in mice
C5a
epitopes
(functional
C5a
2004)
in peptidase/Subcutaneous
(GBS))
Shr (CFA/IFA
Hemoprotein
Receptor
expressing
Lactococcus
in mice.
SpeB catalytic site Chimeric
co-administered
SpeB
SpeA/Intramuscular
Sib35 epitope
rSib35
(Okamoto et al.
Immunoglobulin-
protein/subcutaneous
Binding Protein 35
Arginine
delivery in mice
ADI
adjuvanted
ADI epitope
Deaminase/Streptoc
CFA/Intraperitoneal
occal Acid
delivery in mice
Glycoprotein
Trigger Factor
TF epitope
TF
adjuvanted
2005)
with (Henningham
et al. 2012)
with (Henningham
4918
50
constructed from viral structural proteins such as the envelope or capsid which can self-
482assemble.
483papillomavirus
484professional
485causing
486are
(Zhao et al. 2013). VLPs must sufficiently interact with innate immune cells,
host damage (Zhao et al. 2013) to be utilised as a vaccine. One advantage of VLPs
that they are able to effectively deliver an antigen to professional APCs as well as
487stimulate
488been
both cell-mediated (CMI) and humoral immune (HI) responses. VLP success has
489hepatitis
B and human papilloma virus (Bright et al. 2008; Bryan 2007; Chung et al. 2008).
shown to
immune responses without adjuvant usage making them advantageous for vaccination
not many adjuvants are available for human use (Fifis et al. 2004). VLP Adjuvant
494properties
stem from the small size of the VLP, which allows easy uptake by dendritic cells
495for
major histocompatibility complex class II (MHC II) cells and subsequent stimulation of
496the
497Traditional
498a
aluminium based adjuvants are not well paired with all vaccines as they generate
Th2 bias. Th2 humoral responses typically result from infections caused by bacteria and
499multicellular
500infections
501distinct
(Rosenthal et al. 2014). Th1 responses are directed at inducing CMI and have a
502antibody
5119
52
Versatile VLP
503technology
504particles
505result
encourage increased production of IL-4 for a Th2 response, and smaller particles
506Licenced
507Gardasil
508used
can be engineered to induce Th1 or Th2 through particle size control. Larger
and Cervarix against HPV (Henningham et al. 2013). VLPs are most commonly
to prevent viral pathogens, but there has been evidence of successful delivery of
choice for vaccine development. The highly conserved C-repeat region within the
514environment
with high GAS exposure (see Figure 4). However, challenges for the use of this
515peptide
516epitope
with the human heart protein myosin (Hayman et al. 1997). A study by Hayman et
517al.
(1997) utilising mouse anti-p145 sera determined that the M protein peptides containing
518minimal
519showed
520mice
cross-reactivity were J8 and J14. A follow up study cited by Good and Olive (2003)
protection following challenge with GAS and production of opsonic antibodies when
521vaccine
522Australia (Dale
523
524
525
526
527
528
5320
54
et al. 2013)
529
530
531
532
533
534
535
536Research
recombinant chimeric platforms include the hepatitis B virus core, woodchuck hepatitis
542human
543hepatitis
544setting,
B virus and hepatitis E virus (Grgacic & Anderson 2006). In the commercial
VLPs are synthesised through insect and yeast cell-based systems for their ease of
545production,
546Bacteria,
mammalian and plant cells and cell-free synthesis have been utilised in the
547laboratory
setting to produce VLPs (Rosenthal et al. 2014). VLP technology has proven to
548be
safe, effective, long lasting and cost efficient. In particular, the HBsAg-S surface protein
549is
the most common molecule used in VLP technology due to its non-infectious nature and
550worldwide
dual-vaccination regimens.
554determinant
5521
56
region, a highly immunogenic tertiary structure (Netter et al. 2003). The most
555recent
556Mosquirix,
557vaccine,
558worldwide.
559B,
currently in phase 3 trials (Wilby et al. 2012). Similarly, the original hepatitis B
For example, 18% of Vietnamese children in 1998 were infected with hepatitis
compared to a drastic reduction to 2.7% in 2013 (Nguyen et al. 2014). New HBsAg-S
560VLP
research has also explored the possibility of incorporating bacterial vaccine epitopes.
561Notably,
562with
Kotiw et al. (2012) has utilised Helicobacter pylori KatA epitopes in conjunction
563increased
564research.
5652.7 The current
566J8, J14 and p145
567Furthermore,
study proposal
from the M protein of Streptococcus pyogenes are highly conserved.
568reactivity
to human proteins (Dale et al. 2013). In this study J8, J14 and p145 DNA
569sequences
will be generated by PCR and cloned into the a determinant region of HBsAg-S.
570Recombinant
571system and
per 100,000 annually (Carapetis et al. 2005). The purpose of the study is to target
Australian Indigenous population and developing countries through the development and
576evaluation
of a HBsAg-S-M protein dual vaccine. The HBsAg-S surface protein VLP has
577proven
578protein
epitopes J8, J14 and p145 have demonstrated antigenic and safety properties.
579PCR
generated DNA sequences of J8, J14 and p145 from the M protein of GAS will be
580cloned
5722
58
into the a determinant region of the HBsAg-SS and transformed into a mammalian
581cell
582constructs
583isolation
584GAS
585to
challenge studies. Use of the constructs in challenge models will evaluate their ability
586
587
588
p145 GAS-m
589 protein epitope
genes
J8
pcDNA3.1:HBsAg-S
GAS-m
pcDNA3.1:HBsAg-S
J14 GAS-m pcDNA3.1:HBsAg-S
protein epitope
protein epitope
genes
genes
590
591
592
593
594
595
596
HEK293T
597
HBsAg-S-p145, HBsAg-S-J8
and HBsAg-S-J14 VLPs
transfected into mammalian
expression cells
Expressed VLPs constructs
isolated and assayed by
ELISA
GAS-m Protein
5923Figure
60
HBsAg-S
pcDNA3.1 Vector
598Hypotheses
5991. GAS antigenic
600molecule for
6012.
602to
603
604
605
606
6124
62
a dual vaccine.
GAS VLPs which are recognised by GAS HBsAg-S sera will be good vaccine candidates
provide protection against GAS in an animal model.
607
608
609
610Escherichia
611Bertani
612liquid
coli JM109 (Promega) and TOP10 (Life Technologies) was grown in Luria
(LB) broth or on LB agar containing 100 g/ml ampicillin. Incubation of plated and
613Alexandria,
614mammalian
615by Hans
The
were purchased from Invitrogen, and all PCR reactions were carried out in a PTC-
Applicatio
n
GAS-m
epitope
generation
GAS-m
epitope
generation
GAS-m
epitope
generation
Amplificati
on
Amplificati
on
Sequencin
g
634Table
Template DNA
Primer Sequence (5 3)
p145
Forward
Primer
GGAACCGGTCTTCGTCGTGACTTGGACGCATCACG
TGAAGCTAAGAAACAAGTTGAAAAAGCTTTAGAAA
CCGGTTGG
GGAACCGGTCTTCGTCGTGACTTGGACGCATCACG
TGAAGCTAAGAAAC
CCAACCGGTTTCTAAAGCTTTTTCAACTTGTTTCTT
AGCTTCACGTGATG
GGCACCGGTCAGGCGGAAGATAAAGTGAAACAGT
CACGTGAAGCTAAGAAACAAGTTGAAAAAGCTTTA
AAACAGCTGGAAGATAAAGTGCAGACCGGTGGC
GGCACCGGTCAGGCGGAAGATAAAGTGAAACAGT
CACGTGAAGCTAAGAAACAAGTTG
Reverse
Primer
GCCACCGGTCTGCACTTTATCTTCCAGCTGTTTTAA
AGCTTTTTCAACTTGTTTCTTAGCTTC
Full
sequence
Forward
Primer
GGCACCGGTAAGCAGGCGGAAGATAAAGTGAAAG
CATCACGTGAAGCTAAGAAACAAGTTGAAAAAGCT
TTAAAACAGCTGGAAGATAAAGTGAAGACCGGTG
GC
GGCACCGGTAAGCAGGCGGAAGATAAAGTGAAAG
CATCACGTGAAGCTAAGAAACAAGTTG
Reverse
Primer
GTGAAGCTAAGAAACAAGTTGAAAAAGCTTTAGTA
CAGCTGGAAGATAAAGTGAAGACCGGTGGC
J8
J14
HBsAgSFwd
HBsAgSRev
AOX 1
Full
sequence
Forward
Primer
Reverse
Primer
Full
sequence
GTAGAATTCGCCACCATGGAGAACATCACATCAG
CTGCGGCCGCTTAAATGTATACCCAAAGAC
Reverse
Forward
GCAAATGGCATTCTGACATCC
GACTGGTTCCAATTGACAAGC
635
6363.2 Amplification of GAS-m Epitopes
637Synthesis of GAS-m DNA fragments utilised
638as
shown in Table 3. The sequence ACCGGT was included as a site for the AgeI restriction
639enzyme
640expression
641Hayman
642bp
6526
66
vector. Sequences of p145, J8 and J14 were obtained from previous studies by
et al. (1997). Expected PCR product sizes for p145, J8 and J14 are 78, 102 and 105
respectively. p145, J8 and J14 forward and reverse primers were used to synthesise the
643p145,
J8 and J14 GAS-m DNA sequences through PCR. Three primer pairs were required to
644create
the fragments as given in Table 3. Each forward primer 5 end contained ACCGGT
645with
646AgeI-HF
647lettering.
648(Bioline),
649appropriate
650polymerase
(Bioline) and Milli Q water to make reaction volume up to 50 l. The PCR was
651performed
65215
using the following parameters: 95C for 3 min, followed by 30 cycles of 95C for
s, 56C for 15 s and 72C for 15 s. The resulting PCR products were viewed on a 1%
653agarose
654and
gel as described in section 3.2.1, followed by purification using the Wizard SV Gel
657(Biotec,
Wembley, Australia) for visualisation. The gel was electrophoresed using a Liberty
658(Biokey,
659the
size of the gel required. The electrophoresis tank was filled with 1X TAE Buffer.
660Samples
661dye.
were loaded for electrophoresis after mixing, in a ratio of 5:1 with 6X blue loading
662estimation
of the size and concentration of DNA in each sample. A Power Pac 200 (Bio-Rad,
663Gladesville
NSW) was used to apply a voltage of 100 V for 60 min per gel. A Fusion FX5
664system (Vilber
665light
in conjunction with Fusion FX7 software (Peqlab, Erlangen, Germany). When DNA
666bands
required extraction and purification from agarose gel, the Wizard SV Gel and PCR
667Clean-Up
6727
68
PCR products.
671agarose
The PCR product or the desired DNA band was removed from the
gel and dissolved within 1 l of membrane binding solution per mg of agarose gel.
672The
solution was vortexed and incubated at 60C until fully dissolved and transferred to a
673spin
column and collection tube assembly for incubation at RT for 1 min. The assembly was
674centrifuged
675flowthrough
was discarded. DNA bound to the column membrane was washed with 700 l
676of
membrane wash solution and centrifuged further at 30 600 x g for 1 min at RT. The
677flowthrough
678solution
679flowthrough,
680l
was discarded and the step was repeated using 500 l of membrane wash
the spin column was inserted into a sterile 1.5 ml microcentrifuge tube and 50
of nuclease free water was pipetted into the spin column. Following incubation at RT for 1
681min,
the assembly was centrifuged at 30 600 x g for 1 min at RT. The flowthrough
682containing
the DNA of interest was collected in a microcentrifuge tube. The sample was
683electrophoresed
6843.4
mentioned in section 3.3.1, the primers used to create the GAS-m fragments contained an
687AgeI
688using
689enzyme
690l.
The mixture was then digested for 37C for 15 min and heated to 65C for 20 min. The
691pcDNA3.1
692of
Cutsmart 1x Buffer (New England Biolabs), 5 units of AgeI HF restriction enzyme (New
693England
6928
70
Biolabs) and 40 l of Milli Q Water was combined. The mixture was digested for
69437C
695Biolabs)
and 5 l of Antarctic Phosphatase buffer (New England Biolabs) was added. The
696mixture
was further digested at 37C for 60 min and heat inactivated at 65C for 20 min.
697Expected
698784
PCR product sizes for HBsAg-S:p145, HBsAg-S:J8 and HBsAg-S:J14 were 766,
and p145, J8 and J14 were the inserts respectively. The Invitrogen (Mulgrave,
702Victoria)
703(Invitrogen),
704(Invitrogen)
705Contents
706
707
708
709
710
711
712
713
714
715
716
717
718
719
7129
72
720
721
pcDNA
3.6 Transformation
plasmid map, detailing
of Competent
locations of the
Cells
AgeI site, Ampicillin resistance gene and
mammalian
expression
Taken
from (Xenbase
722
Transformations
werepromoter.
undertaken
according
to One 2014).
Shot TOP10 chemically competent
723E.coli
724amount
of One Shot JM109 or TOP10 E. coli was thawed on ice. 5l of each ligation
725reaction
was pipetted directly into the vial of competent cells and mixed by tapping gently.
726Cells
727was
were incubated on ice for 30 min followed by 30 s in a 42C water bath. The reaction
placed back into ice and 250 l of pre-warmed S.O.C medium (Super Optimal Broth,
728details?)
729each
was added using sterile technique. Cells were shaken at 37C for 1 h and 150 l of
transformation was pipetted directly onto labelled LB agar plates containing 1 g/mL of
730Ampicillin
731vector
732repatched
733PCR
734of
and incubated at 37C overnight. The ampicillin gene contained in the plasmid
Colonies were then
onto fresh plates for colony identification. Possible clones were selected through
performed using the following parameters: 95C for 1 min 30 s, followed by 30 cycles
95C for 15 s, 55C for 15 s and 72C for 1 min 30 s. AOX1 primers were utilised for
735sequencing
736within
737
7383.7 Wizard Plus SV Minipreps DNA Purification
739The Promega (Alexandria, NSW) Wizard Plus SV Miniprep
740plasmid
741inoculated
742shaking
743for
with single colonies of bacteria. The culture was grown overnight at 37C with
5 min at RT. The supernatant was decanted and the pellet resuspended in 250 l of cell
744resuspension
745suspension
7330
74
solution (Promega). 250 l of cell lysis solution (Promega) was added to the
and mixed by inversion, 10 l of alkaline protease solution was then added and
746the
suspension was inverted four times. The suspension was incubated at RT for 5 min and
747350
l of the neutralisation solution (Promega) was added and the suspension was inverted
748four
times before centrifugation at 30,000 x g for 10 min at RT. The supernatant was
749transferred
to a spin column and collection tube assembly and centrifuged at 30 000 x g for 1
750min
at RT, where the flowthrough was discarded. The membrane-bound DNA was washed
751with
750 l of wash solution, added to the spin column and centrifuged at 30 000 x g for 1
752minute
753wash
at RT. The flowthrough was discarded and the wash step repeated using 250 l of
solution followed by centrifugation at 30 000 x g for 2 min at RT. The spin column was
754re-inserted
755directly
756The
757on
into a 1.5 ml microcentrifuge tube and 100 l of nuclease free water was pipetted
onto the membrane. The assembly was centrifuged at 30 000 x g for 1 min at RT.
flowthrough containing plasmid DNA was harvested and a sample was electrophoresed
HBsAg-S-S sequence of pcDNA3.1. The reaction mixture for each sequencing PCR
761contained
762Big-Dye
763primer
at 1 pmol/l, 250 ng of plasmid DNA and Milli Q water to make the total reaction
764volume
12 l. Each reaction was initially heated to 95C for 2 min, followed by cycling at
76595C
766Following
PCR, 72 l of 70% isopropanol was added and the reaction mixture vortexed and
767incubated
for 15 min at RT. It was then centrifuged within a Sigma 1-15 Laboratory
centrifuged to remove the remaining droplet. The pellet was rinsed with 300 l of
isopropanol and centrifuged at maximum speed for 5 min. All liquid was removed and
771samples
7531
76
for 30 min at RT. The supernatant was removed and pellet was
772Institute
773were analysed
to the quality and amount required for transfection of mammalian cells. All solutions
materials for Wizard Plus Midiprep kit were obtained from Promega. 100 ml of LB
778containing
100 g/ml ampicillin was inoculated with a colony of freshly grown E. coli
779containing
the plasmid of interest. The culture was grown overnight at 37C with shaking.
780Cells
781ml
of cell resuspension solution (Promega). 250 l of Cell lysis solution (Promega) was
782added
783Neutralisation
784Lysate
785vacuum
786(Promega)
787column
788blue
789a
solution (Promega) was added, and the suspension inverted a further 10 times.
and white PureYield Binding Columns (Promega). Lysate was pipetted into the
assembly and a vacuum was applied until lysate passed through the membrane, the
column was then discarded. 5 ml of Endotoxin Removal Wash (Promega) was added and
vacuum applied. 20 ml of Column Wash (Promega) was added and a vacuum applied once
790more.
The membrane was dried by applying a vacuum for 30 s and the binding column was
791removed
792was
from the vacuum manifold. An Eluator vacuum elution device (Alexandria, NSW)
fitted to the vacuum manifold assembly to allow collection of DNA into a 1.5 ml
793microcentrifuge
794vacuum to
tube. Finally, DNA was eluted in 600 l of nuclease free water under a
798hydroxyethyl)-1-piperazineethanesulfonic
799bovine
800NSW)
at 37C with 5% CO2. Cells were passaged regularly to avoid senescence and were
801grown
in conical flasks.
802microscope
8035
(North Ryde, NSW) to visually evaluate confluence. Spent media was discarded,
ml of phosphate buffered saline (PBS) was added to the flask and incubated for 2 min
804before
being discarded; this wash was repeated. 1 ml of Life Technologies 0.05% trypsin
805(Mulgrave,
806After
incubation, detached cells were added to 5 ml of MEM culture media to inhibit trypsin
807activity.
808(Sigma,
Cells were centrifuged at 500 x g for 5 min with a 3-15 Laboratory Centrifuge
Osterode am Harz, Germany).
809suspended
8101:3
Victoria) was added and cells were incubated for <5 min at 37C with 5% CO 2.
in the MEM culture media and seeded into new culture flasks at a ratio between
and 1:10.
media. On the following day the media was replaced approximately 2 h prior to
814transfection.
815final
(HBS) (containing 10 g/l HEPES; 16 g/l NaCl; 0.74 g/l KCl; 0.27 g/l Na 2HPO4.2H2O;
g/l dextrose). The mixture was vortexed and incubated at 37C for 3 min before being
818added
81937C
820and
volume of 300 l was combined and quickly added to 300 l of 2X HEPES buffered
816saline
8172.0
drop wise to the cells with gentle swirling to facilitate mixing. Cells were incubated at
with 5% CO2. At 6 days post-transfection, media was taken from the transfection plates
821Harz,
822sucrose
823ultracentrifuge
7933
80
in 9 ml of pre-warmed MEM
824rotor
at 10C and 172 700 x g for 4 hours. The resulting supernatant was discarded and the
825pellet
826Ultrasonic
827supernatant used
8283.9.3 ELISA
829Two ELISA tests
830HBsAg-s
The
VLPs though usage of J8 and HBsAg-s sera. Proteins were coated as a serial
831dilution
in triplicate wells and tested with polyvalent antibody. Negative and positive control
832samples
were included within these tests. The mock sample referred to a protein harvest
833where
no DNA was transfected into the HEK293T cells, and a no protein sample was also
834coated
to control discrepancies in the ELISA test. Positive controls included Heat-killed GAS
835bacteria
836High-binding
837coating
96 well Greiner Bio one plates were coated with 5 l of protein in carbonate
buffer (0.1 M NaHCO3 at pH 9.6), in 100l of coating buffer per well. The plate was
838sealed
and incubated over night at 4C. The following day, plates were washed twice with
839PBST
(0.05% Phosphate Buffered Saline with Tween20) which contains 3.2 mM Na2HPO4,
8400.5
mM KH2PO4, 1.3 mM KCl, 135 mM NaCl and 0.05% Tween 20. 100l of blocking
841buffer
(2% skim milk in PBS) was added to each well and incubated for 1.5 h. The plate was
842washed
843J8
twice with PBST and 100l of serially diluted primary rabbit sera (anti-HBsAg-S or
sera). 1% skim in PBS was added per well and incubated at for 1 h. The plate was washed
844four
times with PBST, and 100l of anti-rabbit conjugated antibody (Sigma) diluted at
8451:3,000
in 1% skim in PBS was added per well and incubated at RT for 1 h. The plate was
846washed
six times with PBST and 100l of TMB was added to each well. After development
847in
a dimly-lit environment, the reaction was stopped with the addition of 50l of 2M H 2SO4.
848Absorbance
was read at 450 nm with a 200rt Biochrom Zenyth Anthos ELISA machine
849(Cambridge,
UK).
8134
82
850
Results
8514.0
852The
p145, J8 and J14 DNA sequences were generated by PCR amplification. Primers were
853designed
854primers.
AgeI restriction enzyme sites were also included on each end for insertion into the
855HBsAg
856restriction
857fragments
had expected sizes of 78 bp, 102 bp and 105 bp respectively and these sizes were
858confirmed
859
860
861
862
B
863
864
865
100 75 -
866
867
868
869
870
874ligation
875of
vector was transformed into JM1091 TOP10 Escherichia coli (Promega) for selection
positive uptake through PCR screen as described in section 3.3. Plasmid was visualised on
876agarose
gel (Figure 7) and sequence integrity was confirmed through sequencing. GAS-m
877constructs
878within
879was
p145, J8 and J14 fragments were inserted into the a determinant of the HBsAg-s
pcDNA3.1 through AgeI digestion followed by ligation. Expected product output size
766 bp, 784 bp and 787 bp respectively for p145, J8 and J14 fragments selectively
880amplified
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
8536
86
9004.2
901
902
903
10,000 -
904
6,000 5,000 -
905
4,000 -
906
907
3,000 -
2,000 -
908
909
910Figure 9 Agarose gel of pcDNA3.1 containing HBsAg:GAS-m fragments
911Plasmids were purified from E. coli utilising the midiprep technique as described in section
9123.2. Wells 1 and 2 contain pcDNA3.1:HBsAg-s:p145, 3 and 4 contain pcDNA3.1:HBsAg913s:J8 and 5 and 6 contain pcDNA3.1:HBsAg-s:J14, where each sample was run on the gel in
9140.4 l (lanes 1, 3 and 5) and 1 l (lanes 2, 4 and 6).
916
8737
88
HBsAg sequence.
920confirmed
921and
that the inserted p145 sequence is 60 base pairs in length, in the correct orientation
sequence integrity was maintained (Figure 9). Sequencing results for J8 and J14 inserts
922were not
completed.
p145
GTTTCTTAGCTTCACGTGATGCGTCCAAGTCACGACGAA
926
927
928
929 Figure 10 Chromatogram of pcDNA3.1:HBsAg-s:p145 sequencing
930 Sequencing results for the region covering the p145 insert are displayed alongside the
931 matching original reverse compliment sequences for comparison.
932
933
934
935
936
937
938
939
940
941
942
943
944
8938
90
3 bp
bp
945
946ELISA
947constructs.
These proteins have been referred to as VLP, VLP:p145, VLP:J8 and VLP:J14
948throughout
this study. The ELISA plates were pre coated with protein in carbonate coating
949buffer,
and serial dilutions of HBsAg and J8 sera polyvalent rabbit sera were performed in
950triplicate.
Following incubation with the secondary HRP antibody and the addition of TMB
951substrate
absorbance was measured at 450 nm (Figure 10). Titres of VLP:p145 from both
952HBsAg
953the
and J8 sera tests were similar to the heat-killed GAS positive control and higher than
VLP expression control and Engerix B. This indicates that >20 g/mL of VLP:p145
954resulted
955formulation
956VLP:J14
957control.
958
959
960
961
962
963
964
965
966
967
968
969
9139
92
protein samples resulted in a reading lower than the mock (no transfection) negative
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
Serial dilutions of primary J8 serum (A) or HBsAg-s serum (B) were utilised where the graph
994 displays mean standard error for triplicate wells at 450 nm absorbance. Primary sera dilution
refers to the ratio at which the protein was diluted. High titres of VLP:p145 were obtained for both
995 J8 and HBsAg-s sera in comparison to the positive controls. Low titres of VLP:J8 and VLP:J14
were obtained in comparison to the negative controls.
996
9340
94
997
Discussion
998GAS
is responsible for a substantial global disease burden with an estimated 18.1 million
999individuals
10002005).
Despite GAS susceptibility to penicillin, the disease burden has not been shown to
1001decrease
and effective treatment of sequelae such as RHD can require monthly penicillin
1002injections
1003living
currently suffering disease due to GAS infection and sequelae (Carapetis et al.
over many years (Gerber et al. 2009). Preventative measures such as improved
conditions and vaccination are the superior solutions in terms of reducing mortality,
1004morbidity and
1005In
this study, GAS M protein epitopes p145, J8 and J14 were amplified by PCR through
1006custom
primers and inserted into the a determinant region of HBsAg within a mammalian
1007expression
vector.
1008Sequencing
1009results
results displayed successful insertion of the p145 gene (Figure 9). Sequencing
for HBsAg:J8 and HBsAg:J14 fragments are incomplete, however no errors have been
1010observed
1011importance
1012manipulation.
1013structure
1014insertion
1015recognition
1016can
also be employed to reach a quality DNA output and remove short primers,
1017unincorporated
1018Techniques
1019assisted
1020VLPs
dNTPs, enzymes, short failed PCR products and salts from PCR reactions.
described in sections 3.2.2, 3.2.4 and 3.2.5 of DNA purification and clean up
in this process. Netter et al. (2001) undertook a similar study utilising HBsAg-s
where proteins were purified through a 20% sucrose cushion followed by a CsCl
1021density
9541
96
Incorrect
gradient, which was further measured by the Prism HBsAg assay. Examination by
1022electron
microscopy was also performed and compared with wild type HBsAg. Further
1023development
1024Further
1025DNA
and affinity tagging for protein purification could be considered (Hilbrig & Freitag
10262012;
1027Protein
1028et
expression was achieved using HEK293 cells. A similar study, undertaken by Kotiw
al. (2012), utilised epitopes from the H. pylori KatA gene inserted into HBsAg-s. In this
1029study,
VLPs were expressed using the HuH7 hepatocellular carcinoma cell line for use in
1030animal
vaccination models. Sufficient yield was obtained for animal model testing and
1031HBsAg-s
1032method
Furthermore, this
of VLP expression has also shown success in studies by Netter et al. (2003) with
1033Hepatitis
1034utilised
C VLPs and Schumacher et al. (2007) in tumor therapy VLPs. This study, however,
HEK293 cells, a predominant cell line used for transient expression of recombinant
1035proteins,
where a foreign gene is expressed for a period of time but not integrated into the
1036genome.
The HEK293 cell line has the clear advantage of rapid production for usage within
1037a
1038ELISA
1039VLP
constructs through serial dilution of HBsAg and J8 sera. Results indicated a high
1040VLP:p145
yield for both J8 and HBsAg sera tests. Similar results of the positive control
1041heat-killed
GAS and standard VLP controls in comparison to VLP:p145 were obtained with
1042an
estimated yield of 20 g/mL (Figure 10A and 10B), indicating immunogenicity. Studies
1043by
Wurm et al. (2004) indicate that baseline values of 20-40 g/mL yields can be obtained
1044at
1045yields
within this study are on par with research standards. Within both primary sera tests,
1046VLP:J8
9742
98
and VLP:J14 proteins were detected at similar or lower titres in comparison to the
1047negative
controls. Low titres of VLP:J8 and VLP:J14 indicate that either the original DNA
1048sequence
was incorrect or the correct VLP was unfavourable for antibody detection. The
1049presence
1050results.
1051these
However, if the latter conclusion is correct then this would suggest that placement of
GAS:m genes for use within VLP may be unsuitable due to incompatibility. Similar
1052findings
1053has
suggested that interference may occur when the HBsAg-s a determinant is disrupted by
1054foreign
1055or
sequences. This could be due to minor epitopes remaining within the a determinant
1056ELISA
1057as
by Kotiw et al. (2012) and Netter et al. (2003) support this conclusion, where data
testing within this study is a potential limitation as actual expression level may vary
peptides have been inserted into the a determinant region which is highly antigenic.
1058Further
protein verification work and higher yields are required before the project could be
1059continued.
1060results,
1061To
This study is further limited by the lack of protein purification and SDS-PAGE
increase protein expression yields for animal studies a yeast expression system could be
1062considered.
1063production
1064demonstrated
1065are
favourable as they can obtain high protein yields of 1000 g/mL (Young & Robinson
10662014).
Additionally, yeast systems hold the advantage of being single cells with fast growth
1067capabilities
1068correct protein
1069Significance
1070VLPs have a number
1071and
able to carry foreign epitopes. Particulate vaccines are advantageous as they can be
1072efficiently
9943
100
taken up by APCs, this enables the VLP to act as an adjuvant and may mean that
1073other
adjuvants are not required within the vaccine preparation. VLPs are non-infectious,
1074non-replicating
1075conditions,
this makes VLPs favourable for use in developing countries (Zhao et al. 2013).
1076VLP
and foreign antigen compatibility make recombinant proteins potentially useful for
1077usage
within dual vaccine regimens. Within this study, peptides of GAS M protein were
1078inserted
1079(Netter
1080Approximately
1081cell
wall and secreted proteins, yet few have progressed to clinical trials. Evidence has
1082shown
that there is a biological feasibility for such a vaccine to exist. For example, GAS
1083pharyngeal
1084challenge
1085(Polly
et al. 1975).
1086challenge
1087al.
20 GAS vaccine prototypes have been created across the spectrum of GAS
infections when vaccinated with purified M proteins (Dale et al. 2011; Guerino et
2011). Furthermore, patterns of GAS infection in school aged children who are
1088repetitively
1089achieved
1090Despite
1091vaccine
is not yet in the foreseeable future. The World Health Organisations roadmap for
1092GAS
1093step
vaccine development outlines past and present research for future developments is a
1094challenges
1095about
are still present (Dale et al. 2013). Remaining challenges include safety concerns
the theoretical risk of autoimmune reactions and a necessity for further understanding
1096of
the basis for immunological protection in humans. For example, a greater understanding
1097of
10144
102
1098immune
1099(WHO
protection against GAS skin infection and the role of T-cell immunity are necessary
2014).
1100Suitable
1101serotypes
1102successful
peptides to overcome these challenges have arisen from the M protein. Early
1103studies
1104refined
attempts such as StreptInCor, J8 and J14 minimal epitope vaccines and the 30-valent
1105vaccine
have shown promising results in animal and clinical trials (Dale et al. 2011; Guerino
1106et
al. 2011; Massell et al. 1968). However, serotype-specific prototypes such as these have
1107been
criticised due to low coverage of strains prevalent in developing countries (Steer et al.
11082009).
Thus, the previous 26-valent vaccine which had progressed to Phase II clinical trials
1109in
adult human volunteers was re-worked into a 30-valent vaccine to allow greater coverage
1110in
the Asia-Pacific region. Criticism regarding coverage has been met with a counter-
1111argument
1112inferred
1113high
valency vaccines such as these may incur a higher production costs making the vaccine
1114prohibitive
for widespread use. Both the 30-valent and the minimal epitope J8 vaccine are
1115anticipated
to begin phase I trials within adult volunteers as of 2014 (WHO 2014). The main
1116advantage
of the 30-valent vaccine lies in the wide coverage through utilisation of fused
1117recombinant
1118protein
vaccines such as StreptInCor and the minimal epitope J8 and J14 vaccines have the
1119greater
1120autoimmune reaction
1121Use
1122to
10345
104
of virus-like particles in conjunction with minimal epitope J8 and J14 vaccines are likely
broaden the immune response, making combination vaccines are a more viable approach in
1123the
long term. Despite the multitude of functional GAS vaccine candidates, no commercial
1124vaccine
is yet available.
1125companies
1126vaccine
1127adverse
1128a
more effective and reliable investment for usage within developed and developing countries
1129alike.
1130prevention
1131as
and control framework for global action (WHO 2012). A successful vaccine such
this could allow smarter investing, improve quality of life and reduce mortality across
1132developing
1133Future
1134A
Directions
combination vaccine for both GAS and Hepatitis B could be an important piece of the
1135puzzle within
1136To
global health, and this study had success in the synthesis of VLP:p145.
progress this study it is essential to verify the sequencing of VLP:J8 and VLP:J14, to
1137confirm
1138region.
Further protein analysis such as SDS-PAGE and western blot and other testing to
1139verify protein
1140Proof-of-concept
1141Isolated
1142would
and purified vaccine candidates would be evaluated in GAS challenge studies. Mice
1143mice
1144mice
serum would be assayed for a specific antibody. GAS load in pharyngeal tissue would
1145be
determined by culture and histology 4 days post bacterial challenge. Generation of this
1146data using
would evaluate the ability of GAS:m proteins to generate an antibody response and
1147protection
from GAS infection when delivered as a dual vaccine within the HBsAg VLP.
10546
106
1148
10747
108
1149
Conclusion
1150A successful
GAS vaccine has the potential to save over 500,000 premature deaths annually,
1151greatly
improve quality of life and reduce the economic burden of common childhood
1152diseases
1153ELISA assays
1154as
a dual vaccine. Specifically, from preliminary results VLP:p145 obtained high protein
1155titres.
Further testing of these vaccine candidates still needs to occur and use of proof-of-
1156concept
1157are
murine challenge models will be essential in assessing their efficacy. GAS vaccines
possible, but not in the foreseeable future despite a long history of developments.
1158Incorporation
1159appealing
1160alike.
1161
10948
110
showed that GAS antigenic peptides can be expressed in HBsAg VLPs for use
solution for widespread GAS protection across developed and developing countries
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