Thesis: Group A Streptococcal Peptides Expressed in HBsAg-S VLPs as a Vaccine Candidate

8/9/2019 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

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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

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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

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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<

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604

605

606

6162


33/66


34/66

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


35/66

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


36/66

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


37/66

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


38/66

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:&


39/66

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


40/66

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"


41/66

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!


42/66

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


43/66

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


44/66

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:: $


45/66

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


46/66

(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


47/66

(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


48/66


49/66

)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


50/66

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::


51/66

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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Documents

Thesis: Group A Streptococcal Peptides Expressed in HBsAg-S VLPs as a Vaccine Candidate