Presenter: Jennifer Wang (: STREP THROAT STREPTOCOCCAL PHARYNGITIS STREPTOCOCCAL TONSILLITIS.
Thesis: Group A Streptococcal Peptides Expressed in HBsAg-S VLPs as a Vaccine Candidate
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Transcript of Thesis: Group A Streptococcal Peptides Expressed in HBsAg-S VLPs as a Vaccine Candidate
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Group A Streptococcal Peptides
Expressed in HBsAg-S VLPs as aVaccine Candidate
Stephanie J Piper
BSc Honours candidate
Universit o! Southern "ueensland
#acult o! Health$ Engineering and Sciences
%&'(
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)eclaration
I certify that the work reported in this thesis is entirely my own effort, except where
otherwise acknowledged. I also certify that the work is original and has not been previously
submitted for assessment in any other course of study at this or any other institution.
…………………………..
Signature of Candidate
…………………………
Date
Endorsement
Supervisor !rofessor "ike #otiw Co$Supervisor Dr %isa Seymour
Signature Signature
Date Date
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Ac*no+ledge,ents
I would like to thank my supervisors !rofessor "ike #otiw and Dr %isa Seymour. 'his year
has been a steep learning curve and their time and patience is much appreciated. Despite
busy schedules and due dates, %isa has always managed to find time to talk over lab
procedures and improvements if things went wrong.
'he research team %u, (ichard, Cathy, #arren and Stephen have also been a fantastic support
throughout the year. I am grateful for their constant assistance and help within the practical
side of my pro)ect. 'heir advice, anecdotes and guidance made the year more enriching and
has better prepared me for life outside *S+. Id also like to thank the examiners Dr. -ohn
Dearnaley and Dr. oel #night for their counsel in a variety of areas. In particular, Dr. -ohn
Dearnaley has been a fantastic assistance over the year and goes above and beyond his )ob
re/uirements.
0inally, I am grateful for the understanding and assistance of the Steele (udd Collegiate
team, the "anager of (esidential %ife #atharine 1igby and my friends and family. %ife
outside my honours pro)ect has not been dull, and your constant support and shenanigans has
kept me motivated.
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AstractStreptococcus pyogenes, or 3roup 4 Streptococcus 534S6 is responsible for significant
patient morbidity and mortality in the developing world and within the 4ustralian Indigenous
population. 34S is responsible for a variety of diseases such as invasive necroti7ing fasciitis
and toxic shock syndrome, as well as non$invasive diseases, such as pharyngitis, impetigo,
scarlet fever and otitis media. 8owever, 34S se/uelae such as rheumatic fever and
rheumatic heart disease are responsible for the highest morbidity. 'he 9:$valent vaccine
candidate currently in trials is inappropriately specialised to serotypes present in areas with
low 34S incidence, such as the *nited States.
'he difficulty in creation of a suitable vaccine lies in part with the variety of 34S virulence
factors. 'he " protein is a highly abundant, multifunctional immunogenic surface protein
which confers resistance to phagocytes and complement mediated protection. 4s sections of
the " protein is highly conserved, it has been the focus of vaccination research.
0urthermore, protein fragments -; and -&< within the " protein have given encouraging
results within a mouse model.
=irus$like particle 5=%!6 technology offers a promising alternative to existing vaccination
delivery systems. =%!s are able to induce both cell mediated and humoral immune responses.
In this study, the use of a chimeric hepatitis 1 surface antigen =%! expressing " protein
epitopes p&, -; and -&< for use as a dual vaccine against 8epatitis 1 virus 581=6 and 34S
is investigated. Specifically, !C( generated D4 se/uences of -;, -&< and p& from the "
protein of 34S have been cloned into the highly immunogenic ?a determinant region of the
81s4g$S =%! and transformed into human embryonic kidney 58@#2A9'6 cells. @xpressed
recombinant 81s4g$S$34S$m protein constructs were assayed by @%IS4 to confirm
presentation of 34S epitopes. @%IS4 results showing high titres were obtained for =%!p&
but low titres were obtained for =%!-; and =%!-&
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constructs, protein expression and antigenic screening of proteins is re/uired before the study
can progress to proof$of$concept murine challenge models.
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Areviations34S 3roup 4 Streptococcus
31S 3roup 1 Streptococcus
(0 (heumatic 0ever
(8D (heumatic 8eart Disease
4!S3 4cute post$streptococcal glomerulonephritis
B8 Borld 8ealth rganisation
31" 3lomerular 1asement "embrane
Spe1 Streptococcal exotoxin 1
=%! =irus$%ike !articles
*('Is *pper (espiratory 'ract Infections
4!Cs 4ntigen !resenting Cells
@%IS4 @n7yme$linked Immunosorbent 4ssay
'CSs 'wo Component Signal 'ransduction Systems
3(41 3$(elated 2$macroglobulin$binding !rotein
S# Streptokinase
8I 8umoral Immunity
C"I Cell "ediated Immunity
"@" "inimal @ssential "edia
!1S !hosphate 1uffered Saline
8@#2A9' 8uman @mbryonic #idney Cells 2A9'
8@!@s
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List o! #igures0igure & utcomes and contributing factors to skin infections in 4ustralian Indigenous
peoples
9
0igure 2 " protein hypervariable, variable and conserved structures >
0igure 9 34S Cross Section E0igure < 34S$m !rotein C$region !eptides 2&
0igure > !roposed strategy 29
0igure F pcD49.& =ector "ap 9:
0igure E !rimer design and matching agarose gel fragments 9>
0igure ; &G agarose gel of 34S$m fragments inserted into ?a determinant region of
81s4g$S
9F
0igure A &G agarose gel of pcD4 containing 81s4g$S34S$m fragments 9E
0igure &: 41I chromatogram of p& se/uencing 9;
0igure && @%IS4 antigenic recognition of =%!p&, =%!-; and =%!-&<
'able 9 Se/uence of oligonucleotide primers used 2F
Contents
Declaration..............................................................................................................................i
4cknowledgements............................................................................................................ii
4bstract..............................................................................................................................iii4bbreviations......................................................................................................................v
%ist of 0igures....................................................................................................................vi
%ist of 'ables.....................................................................................................................vi
&.: verview of group 4 streptococcus 534S6 pathogenesis............................................&
&.& Distribution and incidence of disease...........................................................................&
&.2 !opulations at risk.........................................................................................................2
&.2.& Indigenous 4ustralians...............................................................................................2
&.2.2 !aediatric...................................................................................................................<
2.: 'he biology of group 4 streptococcus..........................................................................<
2.& =irulence factors...........................................................................................................>
2.&.& " protein....................................................................................................................>
2.&.2 Capsule.......................................................................................................................F
F
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2.&.9 Streptokinase..............................................................................................................E
2.&.< Streptolysin ............................................................................................................E
2.&.> C>a !eptidase.............................................................................................................;
2.&.F Cysteine protease Spe1.............................................................................................;
2.2 3enetics including virulence gene control...................................................................;
2.9 ature and spectrum of 34S infections.....................................................................&:2.9.& Common streptococcal throat infection...................................................................&:
2.9.2 (heumatic fever and rheumatic heart disease..........................................................&&
2.< Current treatment options...........................................................................................&9
2. 34S vaccine development..........................................................................................&<
2.F =irus %ike !article 5=%!6 technology........................................................................&A
2.F.& verview of the use of =%! technology for human diseases..................................&A
2.F.&.& 3enerating recombinant chimeras........................................................................2:
2.F.&.2 verview of use of 81s4g$S =%!s.....................................................................2&
2.E 'he current study proposal..........................................................................................22
2.E.& !roposed strategy.....................................................................................................228ypotheses........................................................................................................................2<
9.: 1acteria and !lasmids.................................................................................................2>
9.& "olecular 4nalyses.....................................................................................................2>
9.2 4mplification of 34S$m @pitopes.............................................................................2F
9.9 4garose 3el @lectrophoresis.......................................................................................2E
9.9.& Bi7ard S= 3el and !C( Clean$*p......................................................................2;
9.< Digestion and Insertion of 34S$m @pitopes into 81s4g$S D4 se/uence.............2;
9.> !lasmid %igation.........................................................................................................2A
9.F 'ransformation of Competent Cells............................................................................9:
9.E Bi7ard !lus S= "inipreps D4 !urification.........................................................9:
9.; D4 Se/uencing........................................................................................................9&
9.A Bi7ard !lus S= "idipreps D4 !urification.........................................................92
9.A.& 8@#2A9' Cell Culture............................................................................................92
9.A.2 8@#2A9' 'ransfection and !rotein Isolation..........................................................99
9.A.9 @%IS4......................................................................................................................9<
> =ector Insertion............................................................................................9E
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/ntroduction'0& 1vervie+ o! group A streptococcus 2GAS3 pathogenesis'he bacterium 3roup 4 Streptococcus 534S6 5also known as Streptococcus pyogenes6 is a
3ram positive coccus which is responsible for a variety of infections and associated
syndromes 53oering et al. 2:&26. 34S is prevalent in under$privileged communities such as
the 4ustralian Indigenous population and much of the developing world 5Carapetis et al.
2::>6. 34S can cause a number of non$invasive diseases such as pharyngitis, impetigo,
pyoderma, scarlet fever and otitis media as well as invasive diseases including necrotising
fasciitis and toxic shock syndrome. !ost$infection se/uelae, however account for the highest
global burden of 34S disease and includes rheumatic heart disease 5(8D6, rheumatic fever
5(06, related endocarditis and stroke, as well as acute post$streptococcal glomerulonephritis
54!S36 58enningham et al. 2:&96. Bhilst research activity in 34S vaccinology has been
ongoing for decades, no approved vaccine has yet become available. 4 broad 9:$valent
vaccine is under development, however the vaccine focusses on orth 4merican and
@uropean specific 34S serotypes which may not address the main serotypes present in the
developing world. ne of the main challenges in vaccine design is the incorporation of the
&>: known " protein serotypes to provide cross$serotype protection. Studies of pharyngeal
infections in South 4frican school children indicate the vaccine coverage will be between AG of isolates recorded 5Dale et al. 2:&&6. 'his indicates that the 9:$valent vaccine is an
ineffective option for a significant proportion of affected individuals in the developing world
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5Dale et al. 2:&&6. 4mong the many conserved virulence factors which have been explored
for usage in a 34S vaccine, the " protein is a promising target for vaccine research as it is a
highly conserved, abundant surface 34S protein 5J1rien et al. 2::26.
'0' )istriution and incidence o! diseaseCurrently &;.& million people worldwide suffer 34S associated infections. 34S associated
se/uelae makes up the ma)ority of cases with &>.F million individuals currently affected.
0urthermore, (8D can be fatal and is estimated to cause 299,::: deaths per year 5Carapetis
et al. 2::>6. 4!S3 affects ,::: deaths annually.
Superficial infections occur more fre/uently, where F.& million new cases of pharyngitis arise
every year and &.& million individuals currently suffer of pyoderma 5 Carapetis et al. 2::>6.
*ncertainty levels and assumptions used in the compilation of 34S data tends towards
underestimation, making the true prevalence likely to be greater than stated. Carapetis et al.
52::>6 acknowledges that issues in data collection in developing countries affects /uality of
the data. (8D specific data, however, has a higher level of /uality through rigorous data
collection. (8D has a wide geographical distribution, peaking in the following countries as a
calculated regional prevalence per thousand Sub$Saharan 4frica at >.E cases, South$Central
4sia at 2.2 cases, other 4sian areas at :.; cases, %atin 4merica at &.9 cases, "iddle @ast and
orth 4frica at &.; cases, @astern @urope at & case, !acific and Indigenous 4ustraliaHew
Kealand at 9.> cases and China at :.; cases 5Carapetis et al. 2::>6.
'0% Populations at ris* !opulations at risk for 34S disease include children and young adults within the developed
and developing countries. %ess developed countries account for EAG of (8D cases, A>G of
(0 cases, AEG of 4!S3 cases and AEG of invasive 34S cases. Indigenous 4ustralians
also experience high disease burden 5Carapetis et al. 2::>6.
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'0%0' /ndigenous Australians4s mentioned above, Indigenous 4ustralians are a demographic susceptible to 34S infection,
most commonly suffering superficial skin infections. 0urthermore, 4!S3, (0 and (8D
are heavily prevalent in the Indigenous population 5Carapetis et al. 2::>6. Indigenous
4ustralians have the highest annual mortality rate of 9:.2 per &::,::: individuals. 'his is
more than three times as high as the second highest risk population, "aori ew Kealanders at
A.F per &::,::: individuals 5Carapetis et al. 2::>6. (ural communities within the orthern
'erritory and the #imberley region in Bestern 4ustralia exhibit an annual incidence of (0 of
between 2$E cases for every &::: children between >$&< years old. 0urthermore, up to 9G of
Indigenous people in rural communities have established (8D in 4ustralia 5Carapetis L
Currie &AA;6.
34S related skin infections such as scabies and streptococcal pyoderma are endemic in
4ustralian Indigenous communities. Scabies is a parasitic infection caused by the human itch
mite or Sarcopes scabiei, and is known to spread easily in environments with poor sanitation
and overcrowding 50it73erald et al. 2:&
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'0%0% Paediatric34S pharyngitis accounts for FG of paediatric visits to a medical practitioner, with 34S
cultured from &>$9FG of children suffering a sore throat in the *S 5%inder et al. 2::>6.
4!S3 is also most commonly seen in paediatric patients, e/uating to A:G of the total
population suffering 4!S3. 'he skewed distribution towards paediatric patients is
hypothesised to be attributed to the si7e difference in the glomerular basement membrane
531"6. Children and adults have 2$9 nm and
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particular, fibrinogen is known to interact and bind with the highly conserved " protein in
the MNrepeat region near the $terminus of the protein 5Carlsson et al. 2::>6.
4s well as cellular adherence, 34S also has the capacity to enter epithelial cells to avoid
early host defences and antibiotics. 'his invasive virulence process is possible through
proteins on the cell surface, or invasins such as fibronectin$binding protein and the " protein
5%a!enta et al. &AA: genotypes of 34S have been found thus far
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" !rotein antigenic variation contributes to the range of 34S virulence strategies. 0or
example, the ability of " protein to bind to fibrinogen in the 1$repeat region interferes with
the complement system and contributes to phagocytic resistance 5"c4rthur L Balker 2::FO
(ingdahl et al. 2:::6. 'he " protein provides protection against complement$mediated
opsonisation and phagocytic resistance. Specifically, the " protein binds C
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minimise antibody access to bacterial surface protein 3$related 2$macroglobulin$binding
protein 53(416. 'his mechanism of evasion contributes to the difficulty in creating a
functional vaccine as it enables 34S to escape recognition by antibodies 5Dinkla et al. 2::E6.
%0'05 Strepto*inaseStreptokinase 5S#6 is a plasminogen activator and is a secreted 34S virulence factor with
four compact domains. Secretion of S# is associated with 4!S3 5Simon et al. 2:&
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plasminogen and convert into the serine protease plasmin. Control over host plasminogen is
advantageous to overcome host defences by generating unregulated soluble cell$bound
plasmin, which can degrade blood plasma proteins 5Simon et al. 2:&
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%0'07 Csteine protease SpeBCysteine protease Spe1 is a highly conserved and multi$functional pyrogenic exotoxin
hypothesised to have a role in severe invasive infection and streptococcal toxic shock
syndrome 5Collin L lsQn 2::&6. Spe1 is able to cleave human immunoglobulins, including
Ig4, Ig", IgD and Ig@ 5Collin L lsQn 2::&6. 0urthermore, it can cleave vitronectin,
fibronectin and host proteins to compromise host tissue integrity 5#agawa et al. 2::A6. It can
also spawn biologically active peptides such as interleukin$&, kinins and histamine 5#agawa
et al. 2:::6. 'hrough the degradation of host proteins, research conducted by 1arnett et al.
52:&96 suggests that a proteolytic Spe1 mechanism is utilised by 34S to evade autophagy
and enable replication in the cytosol of host cells.
%0% Genetics including virulence gene control34S has been genotyped via " protein typing in an effort to genetically categorise the
species. 'he famous %ancefield method of " protein typing of emm 34S species determines
the type of opacity factor present through an opacity factor inhibition test. emm genes are
split up into distinct subfamilies, named from 4 to @ and defined by the se/uence differences
at the 9 end 5"c3regor et al. 2::
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average of >F.2G of uni/ue genes between 34S strains is provided by prophage mediated
gene transfer.
34S has a formidable arsenal of virulence factors which enables it to persist and cause
infection in a myriad of ways. 'he control of virulence factors begins with transcription
regulators which relay information from environmental signals, usually from host$pathogen
interactions. In short, "ga and (of4$like proteins are the two global regulators which pilot
the cell as per the signals received by two$component signal transduction systems 5'CSs6
5#reikemeyer et al. 2::96. "ga is a conserved response transcriptional activator which plays
a leading role in regulating expression of surface$associated and secreted molecules. "ga
specifically regulates the " protein, streptococcal collagen$like protein, serum opacity factor,
C>a peptidase and many other virulence factors essential in host colonisation 5#reikemeyer
et al. 2::96.
If environmental conditions become hostile though lack of nutrient supply or host defence
mechanisms, 34S can switch to a stationary growth phase through downregulation of "ga
and upregulation of (of4$like protein 51eckert et al. 2::&6.
'CSs are not uni/ue to 34S and function in detection and communication of environmental
signals though the transmembrane protein sensor histidine kinase. Currently, &9 'CSs have
been identified 5#reikemeyer et al. 2::96. otably, these include ih#HIrr, which plays a role
in host$cell lysis, 34S neutrophil resistance in vitro and in mouse virulence models in vivo
5=oyich et al. 2::96. 0as1C4 is speculated to regulate expression of extracellular matrix
adhesins to promote high adherence and internalisation rates 5#lenk et al. 2::>6. Sil41
regulates I%$; expression of !rtSHScpC protease, which specialises in degradation of the
murine and human CRC chemokines I%$;, #C and "I!$2 58idalgo‐3rass et al. 2::F6.
'he 34S capsule is essential for pathogenesis. 'he production of hyaluronic acid, the main
ingredient of the capsule, is controlled by an operon made up of three genes hasA, hasB and
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hasC . (espectively, these control the production of capsular components hyaluronate
synthase, *D!$glucose dehydrogenase and *D!$glucose pyrophosphorylase, a mechanism
that is likely to be conserved 54lbert et al. &AA;6. Csr(S is a regulator of the hyaluronic acid
capsule biosynthetic operon hasABC , and is known to regulate approximately &>G of the
34S genome 5Dalton et al. 2::F6.
%05 8ature and spectru, o! GAS in!ections34S infection and se/uelae encompasses a vast array of disease, including superficial and
invasive infection and associated se/uelae 5See 'able &6.
%050' Co,,on streptococcal throat in!ectionStrep throat, or acute 34S pharyngitis, makes up approximately one third of all respiratory
tract infections in primary care 5%ittle et al. 2:&
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'he pathogenesis of (0 and (8D is poorly understood and two hypotheses currently exist
which attempt to explain the nature of these disease manifestations. 'he first hypothesis
proposes molecular mimicry and cross reactivity between sarcomeric heart myosin and
streptococcal antigen " protein. 'he second proposes collagen$mediated disease in the
valve. 'andon et al. 52:&96 proposes the following inflammatory mechanism 'he 34S$m
proteins $terminus has been shown to bind to the C19 region in collagen type I=, which in
turn initiates an antibody response against collagen, resulting in ground substance
inflammation. 8owever, Cunningham 52:&
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Cellulitis 4cute, tender, erythematous, and swollen area of skin
8ecrotising !asciitis 0ever, tender skin lesions, vomiting, diarrhoea, toxaemia,
tissue destruction
Streptococcal toxic shoc*
sndro,e
8igh fever, rapid$onset hypotension, accelerated
multisystem failure
4dapted from Carapetis et al. 52::>6
%0( Current treat,ent options4ntibiotics are the primary treatment for 34S infections. 'he advantages of penicillin
include low cost, efficacy and safety. ther drugs such as cephalosporins, macrolides,
erythromycin and clarithromycin have also proven to be effective and are in use in a clinical
environment 51isno et al. 2::26.
%0(0' Antiiotic therap4 meta$analysis of penicillin vs. cephalosporins treatment was undertaken in the context of
34S tonsillopharyngitis, and it was found that the cephalosporin cure rate was twice that of
penicillin, making it the superior choice of drug 5Casey L !ichichero 2::
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Bithin 34S strains, macrolide antibiotic resistance is beginning to spread through hori7ontal
transfer of the mef gene. 'his is a concern for individuals who are allergic to MNlactam
antibiotics as it e/uips 34S with a drug efflux pump. 'ransposon transfer has already
spread to emm types &, 2, 9, 58ad)irin et al. 2:&
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.ale %0 Current vaccination antigens$ trials$ vaccine tpes and protein
candidates
Candidates Antigen Vaccine tpe:preclinical
data
9e!erence
Cell
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mice via peptide specific
serum
C$terminal region
Bhole C$repeat
conserved region
Synthetic peptide
con)ugateHintranasal in
mice via peptide specific
serum
51essen L
0ischetti &A;;6
"inimal epitope
-;H-&
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#ironectin
Binding Protein /
with " protein
peptide -&<
delivery in mice via peptide
specific serum
Streptococcal
Protective Antigen
$Spa9F epitopes Spa
antiserumHintraperitoneal
delivery in mice
54hmed et al.
2:&:6 5Dale et
al. &AAA6
Streptococcus
pyogenes Cell
Envelope
Proteinase:Sp&('7
Spy:
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/,,unogloulin-
Binding Protein 56
proteinHsubcutaneous
delivery in mice
2::>6
Arginine
)ea,inase:Streptoc
occal Acid
Glcoprotein
4DI epitope 4DI ad)uvanted with
C04HIntraperitoneal
delivery in mice
58enningham
et al. 2:&26
.rigger #actor '0 epitope '0 ad)uvanted with
C04HSubcutaneous delivery
in mice
58enningham
et al. 2:&26
&;4950
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%07 Virus Li*e Particle 2VLP3 technolog=accines function as a platform for the presentation of an antigen, so that the body can
formulate an immunological memory. 4ntigen presentation is a crucial part of vaccine
success and must accurately replicate the inherent immunostimulation of an infection. =%!s
are constructed from viral structural proteins such as the envelope or capsid which can self$
assemble. =%!s are produced by many viruses including hepatitis 1 and human
papillomavirus 5Khao et al. 2:&96. =%!s must sufficiently interact with innate immune cells,
professional antigen presenting cells 54!Cs6 and adaptive effectorHmemory cells without
causing host damage 5Khao et al. 2:&96 to be utilised as a vaccine. ne advantage of =%!s
are that they are able to effectively deliver an antigen to professional 4!Cs as well as
stimulate both cell$mediated 5C"I6 and humoral immune 58I6 responses. =%! success has
been demonstrated through protection against of hand$foot$and$mouth disease, influen7a,
hepatitis 1 and human papilloma virus 51right et al. 2::;O 1ryan 2::EO Chung et al. 2::;6.
%070' 1vervie+ o! the use o! VLP technolog !or hu,an diseases
'here are many advantages of =%! technology. =%!s are particulate and have been shown to
illicit immune responses without ad)uvant usage making them advantageous for vaccination
as not many ad)uvants are available for human use 50ifis et al. 2::
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technology can be engineered to induce 'h& or 'h2 through particle si7e control. %arger
particles encourage increased production of I%$< for a 'h2 response, and smaller particles
result in amplified production of I0$V for a 'h& response 5(osenthal et al. 2:&
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!ast recombinant chimeric platforms include the hepatitis 1 virus core, woodchuck hepatitis
1 virus core, hepatitis 1 virus S antigen, human papillomavirus, bovine papillomavirus,
human immunodeficiency virus 58I=6, simian immunodeficiency virus 8I= chimera, duck
hepatitis 1 virus and hepatitis @ virus 53rgacic L 4nderson 2::F6. In the commercial
setting, =%!s are synthesised through insect and yeast cell$based systems for their ease of
production, cost efficiency, post$translational modifications and ad)ustable production si7e.
1acteria, mammalian and plant cells and cell$free synthesis have been utilised in the
laboratory setting to produce =%!s 5(osenthal et al. 2:&
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pcDNA3.1:HBsAg-S pcDNA3.1:HBsAg-SpcDNA3.1:HBsAg-S
cell recombinant protein expression system. @xpressed recombinant 81s4g$S$34Sm
constructs will be isolated, purified and assayed by B1 and @%IS4. 0ollowing successful
isolation and purification recombinant molecules will be used to vaccinate 14%1HC mice in
34S challenge studies. *se of the constructs in challenge models will evaluate their ability
to generate an antibody response though =%! antigen delivery 50igure >6.
29
-&< 34S$m
protein epitope
genes
-; 34S$m
protein epitopegenes
p& 34S$m
protein epitope
genes
34S epitopes will be
amplified and cloned
into the ?a determinant
region of 81s4g$S
8@#2A9' 81s4g$S$p&, 81s4g$S$-;
and 81s4g$S$-&< =%!s
transfected into mammalian
expression cells
#igure 6 Proposed Strateg )etails
34S$m !rotein 81s4g$S pcD49.& =ector
@xpressed =%!s constructsisolated and assayed by
@%IS4
581
582
583
584
585
586
587
588
589
590
591
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593
594
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596
597
5960
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Hpotheses&. 34S antigenic peptides can be expressed in 81s4g$S =%!s to utilise =%!s as a carrier
molecule for a dual vaccine.
2. 34S =%!s which are recognised by 34S 81s4g$S sera will be good vaccine candidates
to provide protection against 34S in an animal model.
2<
598599
600
601
602
603
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605
606
6162
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Applicatio
n
.e,plate )8A Pri,er Se;uence 26>? 5>3
GAS-,
epitope
generation
p& 0ull
se/uence
334ACCGG.C''C3'C3'34C''334C3C4'C4C3
'3443C'443444C443''3444443C'''4344A
CCGG.'33
0orward!rimer
334ACCGG.C''C3'C3'34C''334C3C4'C4C3'3443C'443444C
(everse
!rimer
CC4ACCGG.''C'4443C'''''C44C''3'''C''
43C''C4C3'34'3
GAS-,
epitope
generation
-; 0ull
se/uence
33CACCGG.C433C334434'4443'3444C43'
C4C3'3443C'443444C443''3444443C'''4
444C43C'334434'4443'3C43ACCGG.33C
0orward
!rimer
33CACCGG.C433C334434'4443'3444C43'
C4C3'3443C'443444C443''3
(everse!rimer 3CCACCGG.C'3C4C'''4'C''CC43C'3''''4443C'''''C44C''3'''C''43C''C
GAS-,
epitope
generation
-&< 0ull
se/uence
33CACCGG.443C433C334434'4443'34443
C4'C4C3'3443C'443444C443''3444443C'
''4444C43C'334434'4443'3443ACCGG.3
3C
0orward
!rimer
33CACCGG.443C433C334434'4443'34443
C4'C4C3'3443C'443444C443''3
(everse
!rimer
3'3443C'443444C443''3444443C'''43'4
C43C'334434'4443'3443ACCGG.33C
A,pli!icati
on
81s4g$
S0wd
3'4344''C3CC4CC4'334344C4'C4C4'C43
A,pli!icati
on
81s4g$
S(ev
C'3C33CC3C''444'3'4'4CCC44434C
Se;uencin
g
4R & (everse
0orward
3C444'33C4''C'34C4'CC
34C'33''CC44''34C443C
50% A,pli!ication o! GAS-, EpitopesSynthesis of 34S$m D4 fragments utilised p&, -; and -&< forward and reverse primers
as shown in 'able 9. 'he se/uence ?4CC33' was included as a site for the AgeI restriction
en7yme for ease of ligation of fragments into the pcD49.&81s4g$S mammalian
expression vector. Se/uences of p&, -; and -&< were obtained from previous studies by
8ayman et al. 5&AAE6. @xpected !C( product si7es for p&, -; and -&< are E;, &:2 and &:>
bp respectively. p&, -; and -&< forward and reverse primers were used to synthesise the
2F
.ale 5 Se;uence o! oligonucleotide pri,ers used634
635
636637
638
639
640
641
642
6566
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p&, -; and -&< 34S$m D4 se/uences through !C(. 'hree primer pairs were re/uired to
create the fragments as given in 'able 9. @ach forward primer > end contained 4CC33'
with three additional terminal nucleotides to enable restriction digestion with en7yme
AgeI-H 5ew @ngland 1iolabs, 4rundel, +ueensland, 4ustralia6 , as seen in 'able 9 in bold
lettering. !C( reactions to generate each fragment contained > Wl of x&: 4ccu1uffer
51ioline6, & Wl of &: m" d'!s 5!romega, 4lexandria, SB, 4ustralia6, &.> Wl of both
appropriate forward and reverse primers at 2: pmolHWl 5'able 96, & Wl of 4ccu7ymeY D4
polymerase 51ioline6 and "illi + water to make reaction volume up to >: Wl. 'he !C( was
performed using the following parameters A>XC for 9 min, followed by 9: cycles of A>XC for
&> s, >FXC for &> s and E2XC for &> s. 'he resulting !C( products were viewed on a &G
agarose gel as described in section 9.2.&, followed by purification using the Bi7ard S= 3el
and !C( Clean$*p 5!romega6.
505 Agarose Gel Electrophoresis3els were prepared as &G or 2G agarose in &R '4@ containing :.E>G 3el(edD4 Stain
51iotec, Bembley, 4ustralia6 for visualisation. 'he gel was electrophoresed using a %iberty
51iokey, California, *S46 or 1iorad minisub cell 3' electrophoresis tank, depending upon
the si7e of the gel re/uired. 'he electrophoresis tank was filled with &R '4@ 1uffer.
Samples were loaded for electrophoresis after mixing, in a ratio of >& with FR blue loading
dye. > Wl 8yper%adder I or = molecular si7e marker was loaded in a separate well to enable
estimation of the si7e and concentration of D4 in each sample. 4 !ower !ac 2:: 51io$(ad,
3ladesville SB6 was used to apply a voltage of &:: = for F: min per gel. 4 0usion 0R>
system 5=ilber %ourmat, @berhard7ell, 3ermany6 was used to visualise D4 bands under *=
light in con)unction with 0usion 0RE software 5!e/lab, @rlangen, 3ermany6. Bhen D4
bands re/uired extraction and purification from agarose gel, the Bi7ard S= 3el and !C(
Clean$*p 5!romega6 system was utilised.
2E
643
644
645
646
647
648
649
650
651
652
653
654
655656
657
658
659
660
661
662
663
664
665
666
667
6768
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5050' :: Wl of membrane wash
solution 5!romega6 and centrifuging at 9: F:: x g for > min at ('. 4fter discarding
flowthrough, the spin column was inserted into a sterile &.> ml microcentrifuge tube and >:
Wl of nuclease free water was pipetted into the spin column. 0ollowing incubation at (' for &
min, the assembly was centrifuged at 9: F:: x g for & min at ('. 'he flowthrough
containing the D4 of interest was collected in a microcentrifuge tube. 'he sample was
electrophoresed on an agarose gel for visualisation. D4 was stored at $2:XC.
50( )igestion and /nsertion o! GAS-, Epitopes into HBsAg-S )8A
se;uence4s mentioned in section 9.9.&, the primers used to create the 34S$m fragments contained an
AgeI restriction site to enable insertion into pcD49.&81s4g$S. Digestion was achieved
using > Wl of Cutsmart &x 1uffer 5ew @ngland 1iolabs6, > units of AgeI 80 restriction
en7yme 5ew @ngland 1iolabs6 and >: ng of the appropriate 34S$m fragment made up to >:
Wl. 'he mixture was then digested for 9EXC for &> min and heated to F>XC for 2: min. 'he
pcD49.& vector underwent a similar digestion, where &:: ng of pcD49.&81s4g$S, > Wl
of Cutsmart &x 1uffer 5ew @ngland 1iolabs6, > units of AgeI 80 restriction en7yme 5ew
@ngland 1iolabs6 and
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9EXC for &> min. 0ollowing digestion > units of 4ntarctic !hosphatase 5ew @ngland
1iolabs6 and > Wl of 4ntarctic !hosphatase buffer 5ew @ngland 1iolabs6 was added. 'he
mixture was further digested at 9EXC for F: min and heat inactivated at F>XC for 2: min.
@xpected !C( product si7es for 81s4g$Sp&, 81s4g$S-; and 81s4g$S-&< were EFF,
E;< and E;E bps respectively.
506 Plas,id Ligation%igation was performed at a ratio of &9 vector to insert, where pcD49.&81s4g$S was the
vector and p&, -; and -&< were the inserts respectively. 'he Invitrogen 5"ulgrave,
=ictoria6 rapid ligation protocol was utilised, where < Wl of >x ligase reaction buffer
5Invitrogen6, 9: fmol of vector D4, A: fmol of insert D4, & Wl of '< D4 ligase
5Invitrogen6 and "illi + water up to 2: Wl were added to a &.> ml microcentrifuge tube.
Contents were centrifuged briefly and incubated at (' for > min.
2A
694
695
696
697
698
699700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
7172
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507 .rans!or,ation o! Co,petent Cells'ransformations were undertaken according to ne Shot '!&: chemically competent
E.coli transformation methods 5Invitrogen, "ulgrave, =ictoria, 4ustralia6. 'he appropriate
amount of ne Shot -"&:A or '!&: E. coli was thawed on ice. >Wl of each ligation
reaction was pipetted directly into the vial of competent cells and mixed by tapping gently.
Cells were incubated on ice for 9: min followed by 9: s in a XC for & min 9: s, followed by 9: cycles
of A>XC for &> s, >>XC for &> s and E2XC for & min 9: s. 4R& primers were utilised for
se/uencing verification of plasmid preparations believed to contain 34S$m protein fragments
within the 81s4g$S se/uence 5'able 96.
50= min at ('. 'he supernatant was decanted and the pellet resuspended in 2>: Wl of cell
resuspension solution 5!romega6. 2>: Wl of cell lysis solution 5!romega6 was added to the
suspension and mixed by inversion, &: Wl of alkaline protease solution was then added and
9:
#igure 7 pc)8A50' Vector 4ap pcD4 plasmid map, detailing locations of the AgeI site, 4mpicillin resistance gene and
mammalian expression promoter. 'aken from 5Renbase 2:&
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the suspension was inverted four times. 'he suspension was incubated at (' for > min and
9>: Wl of the neutralisation solution 5!romega6 was added and the suspension was inverted
four times before centrifugation at 9:,::: x g for &: min at ('. 'he supernatant was
transferred to a spin column and collection tube assembly and centrifuged at 9: ::: x g for &
min at (', where the flowthrough was discarded. 'he membrane$bound D4 was washed
with E>: Wl of wash solution, added to the spin column and centrifuged at 9: ::: x g for &
minute at ('. 'he flowthrough was discarded and the wash step repeated using 2>: Wl of
wash solution followed by centrifugation at 9: ::: x g for 2 min at ('. 'he spin column was
re$inserted into a &.> ml microcentrifuge tube and &:: Wl of nuclease free water was pipetted
directly onto the membrane. 'he assembly was centrifuged at 9: ::: x g for & min at ('.
'he flowthrough containing plasmid D4 was harvested and a sample was electrophoresed
on a &G agarose gel for analysis. D4 was stored at $2:XC.
50 )8A Se;uencing'he 4R primer 5'able 96 was utilised to enable se/uencing of the 34S$m constructs within
the 81s4g$S$S se/uence of pcD49.&. 'he reaction mixture for each se/uencing !C(
contained 2 Wl of 4pplied 1iosystems 5"ulgrave, 4ustralia6 >R se/uencing buffer , & Wl of
1ig$Dye 'erminator se/uencing reaction 54pplied 1iosystems6, 9 Wl of the appropriate
primer at & pmolHWl, 2>: ng of plasmid D4 and "illi + water to make the total reaction
volume &2 Wl. @ach reaction was initially heated to A>XC for 2 min, followed by cycling at
A>XC for &: s, >2XC for > s and F:XC for 9 min, repeated 2> times.
0ollowing !C(, E2 Wl of E:G isopropanol was added and the reaction mixture vortexed and
incubated for &> min at ('. It was then centrifuged within a Sigma &$&> %aboratory
Centrifuge at maximum speed for 9: min at ('. 'he supernatant was removed and pellet was
briefly centrifuged to remove the remaining droplet. 'he pellet was rinsed with 9:: Wl of
E:G isopropanol and centrifuged at maximum speed for > min. 4ll li/uid was removed and
samples dried in a fume hood for approximately & h. Samples were sent to +ueensland
9&
746
747
748
749
750
751
752
753
754
755
756
757
758759
760
761
762
763
764
765
766
767
768
769
770
771
7576
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Institute of "edical (esearch 5+I"(, 1risbane, 4ustralia6 for se/uencing analysis. (esults
were analysed using 1ioedit[, a biological se/uence alignment editor 5Carlsbad, California6.
50 : Wl of Cell lysis solution 5!romega6 was
added and the suspension inverted > times, followed by incubation at (' for 9 min.
eutralisation solution 5!romega6 was added, and the suspension inverted a further &: times.
%ysate was centrifuged at 2 min. 4 #0 euberger 5(owville, =ictoria6
vacuum manifold was utilised in con)unction with blue !ure\ieldY Clearing Columns
5!romega6 and white !ure\ieldY 1inding Columns 5!romega6. %ysate was pipetted into the
column assembly and a vacuum was applied until lysate passed through the membrane, the
blue column was then discarded. > ml of @ndotoxin (emoval Bash 5!romega6 was added and
a vacuum applied. 2: ml of Column Bash 5!romega6 was added and a vacuum applied once
more. 'he membrane was dried by applying a vacuum for 9: s and the binding column was
removed from the vacuum manifold. 4n @luator vacuum elution device 54lexandria, SB6
was fitted to the vacuum manifold assembly to allow collection of D4 into a &.> ml
microcentrifuge tube. 0inally, D4 was eluted in F:: Wl of nuclease free water under a
vacuum to obtain purified plasmid D4.
500' HED%5. Cell Culture8uman @mbryonic #idney cells 2A9' 58@#2A9' 4'CC ]C(%$&>E96 were maintained in
"inimum @ssential "edia 5"@"6 culture media 5"@" containing 2> m"
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hydroxyethyl6$&$pipera7ineethanesulfonic acid 58@!@s6, 3lutamax 53ibco6 and &:G fetal
bovine serum 501S6 and incubated in a S4\ 8umidified C2 Incubator 5orth Sydney,
SB6 at 9EXC with >G C2. Cells were passaged regularly to avoid senescence and were
grown in conical flasks. 'o passage, cells were viewed under a %eica %eit7 D" I%
microscope 5orth (yde, SB6 to visually evaluate confluence. Spent media was discarded,
> ml of phosphate buffered saline 5!1S6 was added to the flask and incubated for 2 min
before being discardedO this wash was repeated. & ml of %ife 'echnologies :.:>G trypsin
5"ulgrave, =ictoria6 was added and cells were incubated for ^> min at 9EXC with >G C2.
4fter incubation, detached cells were added to > ml of "@" culture media to inhibit trypsin
activity. Cells were centrifuged at >:: x g for > min with a 9$&> %aboratory Centrifuge
5Sigma, sterode am 8ar7, 3ermany6. Supernatant was discarded and cells were re$
suspended in the "@" culture media and seeded into new culture flasks at a ratio between
&9 and &&:.
500% HED%5. .rans!ection and Protein /solation0or transfection cells were seeded at &.> x &:F cellsH&:cm dish in A ml of pre$warmed "@"
culture media. n the following day the media was replaced approximately 2 h prior to
transfection. 4 mixture of &F Wg of plasmid D4, 9F Wl of 2" CaCl2 and "illi + water at a
final volume of 9:: Wl was combined and /uickly added to 9:: Wl of 2R 8@!@S buffered
saline 581S6 5containing &: gHl 8@!@SO &F gHl aClO :.E< gHl #ClO :.2E gHl a 28!
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rotor at &:XC and &E2 E:: x g for < hours. 'he resulting supernatant was discarded and the
pellet resuspended in 2:: Wl of &R 81S over two nights, followed by sonication using an
*ltrasonic Cleaning bath 5*nisonics, 1rookvale, SB6 for > x 9: s intervals. 'he
supernatant used in subse/uent testing by @%IS4.
5005 EL/SA'wo @%IS4 tests were undertaken to detect both the presence of my 34S " proteins and
81s4g$s =%!s though usage of -; and 81s4g$s sera. !roteins were coated as a serial
dilution in triplicate wells and tested with polyvalent antibody. egative and positive control
samples were included within these tests. 'he ?mock sample referred to a protein harvest
where no D4 was transfected into the 8@#2A9' cells, and a ?no protein sample was also
coated to control discrepancies in the @%IS4 test. !ositive controls included 8eat$killed 34S
bacteria and @ngerix 1 5the current 8epatitis 1 =%! vaccine6.
8igh$binding AF well 3reiner 1io one plates were coated with > Wl of protein in carbonate
coating buffer 5:.& " a8C9 at p8 A.F6, in &::Wl of coating buffer per well. 'he plate was
sealed and incubated over night at G !hosphate 1uffered Saline with 'ween2:6 which contains
9.2 m" a28! m" #82!G 'ween 2:. &::Wl of blocking
buffer 52G skim milk in !1S6 was added to each well and incubated for &.> h. 'he plate was
washed twice with !1S' and &::Wl of serially diluted primary rabbit sera 5anti$81s4g$S or
-; sera6. &G skim in !1S was added per well and incubated at for & h. 'he plate was washed
four times with !1S', and &::Wl of anti$rabbit con)ugated antibody 5Sigma6 diluted at
&9,::: in &G skim in !1S was added per well and incubated at (' for & h. 'he plate was
washed six times with !1S' and &::Wl of '"1 was added to each well. 4fter development
in a dimly$lit environment, the reaction was stopped with the addition of >:Wl of 2" 82S
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9esults(0& GAS-, epitope A,pli!ication
'he p&, -; and -&< D4 se/uences were generated by !C( amplification. !rimers were
designed with an overlap of approximately 2: base pairs between forward and reverse
primers. AgeI restriction en7yme sites were also included on each end for insertion into the
81s4g se/uence, an overhang of 9 nucleotides was included to enable better efficiency of
restriction digestion downstream 50igure E46. 3enerated p&, -; and -&< 34S$m
fragments had expected si7es of E; bp, &:2 bp and &:> bp respectively and these si7es were
confirmed through agarose gel visualisation 50igure E16.
(0' GAS-, #rag,ent /nsertion into HBsAg81s4g$s and pcD49.& were digested and ligated first in readiness for 34S$m epitope
insertion into the ?a determinant region of 81s4g$s. 0irstly, pcD49.&81s4g$s post
9>
A
B
& 2 9
&:: $
E> $
#igure = Pri,er design and ,atching agarose gel !rag,ents4. !rimer design for generation of p&, -; and -& bp. Smear above bands is present as D4 had not been purified as detailed in
section 9.2.
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869870
871872
873
8384
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ligation vector was transformed into -"&:A& '!&: Escherichia coli 5!romega6 for selection
of positive uptake through !C( screen as described in section 9.9. !lasmid was visualised on
agarose gel 50igure E6 and se/uence integrity was confirmed through se/uencing. 34S$m
constructs p&, -; and -&< fragments were inserted into the ?a determinant of the 81s4g$s
within pcD49.& through AgeI digestion followed by ligation. @xpected product output si7e
was EFF bp, E;< bp and E;E bp respectively for p&, -; and -&< fragments selectively
amplified from within pcD49.&81s4g 50igure E6.
9F
& 2 9 < >
&, ::: $
;:: $
F:: $
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10,000 -
6,000 -5,000 -
4,000 -
3,000 -
2,000 -
1 2 3 4 5 6
(0% PVLP66 Vector /nsertion
#igure Agarose gel o! pc)8A50' containing HBsAgGAS-, !rag,ents!lasmids were purified from @. coli utilising the midiprep techni/ue as described in section
9.2. Bells & and 2 contain pcD49.&81s4g$sp&, 9 and < contain pcD49.&81s4g$
s-; and > and F contain pcD49.&81s4g$s-&6 and & Wl 5lanes 2, < and F6.
9E
900901
902
903
904
905
906
907
908
909910911912913914
916
8788
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(05 )8A Se;uence AnalsisSe/uencing analysis was carried out to verify correct insertion of the 34S$m epitopes into
the 81s4g se/uence. Se/uencing results from the pcD49.&81s4gp& plasmid
confirmed that the inserted p& se/uence is F: base pairs in length, in the correct orientation
and se/uence integrity was maintained 50igure A6. Se/uencing results for -; and -&< inserts
were not completed.
9;
p'(6
>''C'4443C'''''C44 C ' 'riginal reverse complement se/uence
9 bpSe/uencing results
bp
#igure '& Chro,atogra, o! pc)8A50'HBsAg-sp'(6 se;uencingSe/uencing results for the region covering the p& insert are displayed alongside the
matching original reverse compliment se/uences for comparison.
917918
919
920
921
922
923924925
926927
928929930931932
933
934
935
936
937
938
939
940
941
942
943
944
8990
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(0( )etection o! HBsAgGAS, VLPs EL/SA
@%IS4 was undertaken to confirm expression of 81s4g =%! and 81s4g34Sm =%!
constructs. 'hese proteins have been referred to as =%!, =%!p&, =%!-; and =%!-&<
throughout this study. 'he @%IS4 plates were pre coated with protein in carbonate coating
buffer, and serial dilutions of 81s4g and -; sera polyvalent rabbit sera were performed in
triplicate. 0ollowing incubation with the secondary 8(! antibody and the addition of '"1
substrate absorbance was measured at : nm 50igure &:6. 'itres of =%!p& from both
81s4g and -; sera tests were similar to the heat$killed 34S positive control and higher than
the =%! expression control and @ngerix 1. 'his indicates that _2: WgHm% of =%!p&
resulted from protein expression. @ngerix 1 is the current 8epatitis 1 vaccine and is a
formulation containing 81s4g =%!s at 2: WgHm%. 8owever, analysis of the =%!-; and
=%!-&< protein samples resulted in a reading lower than the mock 5no transfection6 negative
control.
9A
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
9192
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)iscussion34S is responsible for a substantial global disease burden with an estimated &;.& million
individuals currently suffering disease due to 34S infection and se/uelae 5Carapetis et al.
2::>6. Despite 34S susceptibility to penicillin, the disease burden has not been shown to
decrease and effective treatment of se/uelae such as (8D can re/uire monthly penicillin
in)ections over many years 53erber et al. 2::A6. !reventative measures such as improved
living conditions and vaccination are the superior solutions in terms of reducing mortality,
morbidity and economic costs, including within the developed world.
In this study, 34S " protein epitopes p&, -; and -&< were amplified by !C( through
custom primers and inserted into the ?a determinant region of 81s4g within a mammalian
expression vector.
Se/uencing results displayed successful insertion of the p& gene 50igure A6. Se/uencing
results for 81s4g-; and 81s4g-&< fragments are incomplete, however no errors have been
observed in preliminary se/uence data to date. Correct se/uencing data is of paramount
importance as errors such as double or backwards inserts can occur in recombinant D4
manipulation. 'he ?a determinant region of 81s4g$s is located within a double$looped
structure where one 22 nm particle contains about &:: 81s4g$s molecules. Incorrect
insertion in this area could result in unfavourable assembly of proteins for antibody
recognition 5 etter et al. 2::&6. 4s well as se/uencing results, D4 purification techni/ues
can also be employed to reach a /uality D4 output and remove short primers,
unincorporated d'!s, en7ymes, short failed !C( products and salts from !C( reactions.
'echni/ues described in sections 9.2.2, 9.2.< and 9.2.> of D4 purification and clean up
assisted in this process. etter et al. 52::&6 undertook a similar study utilising 81s4g$s
=%!s where proteins were purified through a 2:G sucrose cushion followed by a CsCl
density gradient, which was further measured by the !rism 81s4g assay. @xamination by
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electron microscopy was also performed and compared with wild type 81s4g. 0urther
development on this study could be conducted by mirroring etters purification methods.
0urther techni/ues such as hydroxyapatite chromatography for the purification of plasmid
D4 and affinity tagging for protein purification could be considered 5 8ilbrig L 0reitag
2:&2O \oung et al. 2:&26.
!rotein expression was achieved using 8@#2A9 cells. 4 similar study, undertaken by #otiw
et al. 52:&26, utilised epitopes from the H. pylori #at4 gene inserted into 81s4g$s. In this
study, =%!s were expressed using the 8u8E hepatocellular carcinoma cell line for use in
animal vaccination models. Sufficient yield was obtained for animal model testing and
81s4g$s conformation was confirmed through electron micrographs. 0urthermore, this
method of =%! expression has also shown success in studies by etter et al. 52::96 with
8epatitis C =%!s and Schumacher et al. 52::E6 in tumor therapy =%!s. 'his study, however,
utilised 8@#2A9 cells, a predominant cell line used for transient expression of recombinant
proteins, where a foreign gene is expressed for a period of time but not integrated into the
genome. 'he 8@#2A9 cell line has the clear advantage of rapid production for usage within
a time$restricted study 53eisse L 0ux 2::A6.
@%IS4 testing was undertaken to confirm expression and measure antigenic recognition of
=%! constructs through serial dilution of 81s4g and -; sera. (esults indicated a high
=%!p& yield for both -; and 81s4g sera tests. Similar results of the positive control
heat$killed 34S and standard =%! controls in comparison to =%!p& were obtained with
an estimated yield of 2: WgHm% 50igure &:4 and &:16, indicating immunogenicity. Studies
by Burm et al. 52::
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negative controls. %ow titres of =%!-; and =%!-&< indicate that either the original D4
se/uence was incorrect or the correct =%! was unfavourable for antibody detection. 'he
presence of an incorrect D4 se/uence is possible as it is still unverified by se/uencing
results. 8owever, if the latter conclusion is correct then this would suggest that placement of
these 34Sm genes for use within =%! may be unsuitable due to incompatibility. Similar
findings by #otiw et al. 52:&26 and etter et al. 52::96 support this conclusion, where data
has suggested that interference may occur when the 81s4g$s ?a determinant is disrupted by
foreign se/uences. 'his could be due to minor epitopes remaining within the ?a determinant
or elsewhere in the 81s4g molecule, misfolding or unstable expression of 81s4g$s proteins.
@%IS4 testing within this study is a potential limitation as actual expression level may vary
as peptides have been inserted into the ?a determinant region which is highly antigenic.
0urther protein verification work and higher yields are re/uired before the pro)ect could be
continued. 'his study is further limited by the lack of protein purification and SDS$!43@
results, which could further indicate protein /uality.
'o increase protein expression yields for animal studies a yeast expression system could be
considered. \east systems utilising Saccharomyces cerevisiae in the development and
production of the 8epatitis 1 vaccine were successful obtaining high yields and successfully
demonstrated protection in grivet monkeys 5"c4leer et al. &A;
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other ad)uvants are not re/uired within the vaccine preparation. =%!s are non$infectious,
non$replicating and have higher stability than soluble antigens in extreme environmental
conditions, this makes =%!s favourable for use in developing countries 5Khao et al. 2:&96.
=%! and foreign antigen compatibility make recombinant proteins potentially useful for
usage within dual vaccine regimens. Bithin this study, peptides of 34S " protein were
inserted into the ?a determinant region of 81s4g$s se/uence which is highly immunogenic
5 etter et al. 2::9O =ietheer et al. 2::E6.
4pproximately 2: 34S vaccine prototypes have been created across the spectrum of 34S
cell wall and secreted proteins, yet few have progressed to clinical trials. @vidence has
shown that there is a biological feasibility for such a vaccine to exist. 0or example, 34S
pharyngeal studies undertaken in the &AE:s successfully demonstrated protection against
challenge with a homologous strain of 34S after immunisation with purified " proteins
5!olly et al. &AE>6. !reclinical murine studies have demonstrated protection against
challenge infections when vaccinated with purified " proteins 5Dale et al. 2:&&O 3uerino et
al. 2:&&6. 0urthermore, patterns of 34S infection in school aged children who are
repetitively exposed to 34S indicate that a threshold level of protective immunity can be
achieved 5"artin et al. 2::
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Conclusion4 successful 34S vaccine has the potential to save over >::,::: premature deaths annually,
greatly improve /uality of life and reduce the economic burden of common childhood
diseases caused by 34S 5Carapetis et al. 2::>6.
@%IS4 assays showed that 34S antigenic peptides can be expressed in 81s4g =%!s for use
as a dual vaccine. Specifically, from preliminary results =%!p& obtained high protein
titres. 0urther testing of these vaccine candidates still needs to occur and use of proof$of$
concept murine challenge models will be essential in assessing their efficacy. 34S vaccines
are possible, but not in the foreseeable future despite a long history of developments.
Incorporation of 34Sm peptides into a dual or combination vaccine may offer a more
appealing solution for widespread 34S protection across developed and developing countries
alike.
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9e!erencesAhmed, EA, e!"#$!d, %A, B&e'e&, S(, %e!!)!*, A, (h+)!g, E D)e, /B 2010,S*&ep*#c#cc) p*ec*+e )!*+ge!s Sp): ) !e' ")m+ #" *pe-spec+c p*e+!s #" g$p A s*&ep*#c#cc+, European Journal of Clinical Microbiology & InfectiousDiseases, #. 29, !#. 1, pp. 51-7.
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B+s!#, A=, >e&e&, A, >')*!e, /, F)p)!, E= Sch')&*?, H 2002, &)c*+ceg$+de+!es "#& *he d+)g!#s+s )!d m)!)geme!* #" g$p A s*&ep*#c#cc)ph)&!g+*+s, Clinical Infectious Diseases, #. 35, !#. 2, pp. 113-25.
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(#+!, KsM!, A 2001, ECec* #" SpeB )!d E!d#S "m S*&ep*#c#cc$sp#ge!es #! h$m)! +mm$!#g#$+!s, Infection and immunity , #. 69, !#. 11,pp. 7187-9.
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128612871288128912901291129212931294129512961297
12981299130013011302130313041305130613071308
130913101311131213131314131513161317131813191320
1321
115116
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13221323132413251326132713281329133013311332133313341335133613371338
133913401341134213431344134513461347134813491350
13511352135313541355135613571358135913601361
136213631364136513661367136813691370137113721373
13741375
117118
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H+d)g#‐>&)ss, (, +sh)+)!, , D)!‐>##&, , Be#*se&
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c>&eg#&, F, Sp&)**, B>, F)+), A, Be!!e**, A, B+eh#sh, 2008, (#+ed-(#+ &&eg$)&+*+es )!d !s*)++*+es +! >$p AS*&ep*#c#cc$s 1 A&e eG$+&ed "#& +&$e!ce, Science, #. 319, !#. 5868, pp.1405-8.
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%)!d#!, , Sh)&m), , (h)!d&)sheh#ch)!+, , &e, %>, F)&+!, ,S)ese!, >S N+?e*, 2009, S*&ep*#s+! K m#*es >$p A S*&ep*#c#cc$smm$!e E)s+#! Accee&)*ed )cph)ge Ap#p*#s+s, Journal of 0iologicalChemistry , #. 284, !#. 2, pp. 862-71.
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