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Kollipara, Avinash, Polkinghorne, Adam, Beagley, Kenneth, & Timms, Pe-ter(2013)Vaccination of koalas with a recombinant Chlamydia pecorum major outermembrane protein induces antibodies of different specificity compared tothose following a natural live infection.PLoS One, 8(9), Article number: e74808 1-14.

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https://doi.org/10.1371/journal.pone.0074808

Vaccination of Koalas with a Recombinant Chlamydiapecorum Major Outer Membrane Protein InducesAntibodies of Different Specificity Compared to ThoseFollowing a Natural Live InfectionAvinash Kollipara, Adam Polkinghorne, Kenneth W. Beagley, Peter Timms*

Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia

Abstract

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity,infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas(particularly blindness and infertility in females) would provide an important management tool to prevent furtherpopulation decline of this species. An important step towards developing a vaccine in koalas is to understand thehost immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cellepitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that arerecognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasmaantibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located inthe variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from ananimal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not fromother genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein theyproduced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infectedkoalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserveddomains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer widecross-protection against the variety of chlamydial infections circulating in wild koala populations.

Citation: Kollipara A, Polkinghorne A, Beagley KW, Timms P (2013) Vaccination of Koalas with a Recombinant Chlamydia pecorum Major OuterMembrane Protein Induces Antibodies of Different Specificity Compared to Those Following a Natural Live Infection. PLoS ONE 8(9): e74808. doi:10.1371/journal.pone.0074808

Editor: Gilbert Greub, University of Lausanne, Switzerland

Received April 4, 2013; Accepted August 6, 2013; Published September 25, 2013

Copyright: © 2013 Kollipara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was funded by Australian Research Council Linkage Grant LP0990147 and the Queensland Government National and InternationalResearch Alliances Program (NIRAP) Scheme. The funding partners had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: p.timms@qut.edu.au

Introduction

The koala is the only surviving member of the familyPhascolarctidae and is considered an icon of Australia’s uniquebiodiversity. Despite this esteem, wild koala populations ingeographically diverse regions throughout the country continueto decline. This decline has been attributed to several variablessuch as (a) habitat loss, resulting in fragmentation of koalacolonies [1]; (b) disease [2]; (c) motor vehicle trauma [3]; and(d) dog attacks [4]. A recent study showed that addressingdisease, amongst the many variables affecting koala survivalwould have the greatest potential impact on stabilisingpopulation decline [2]. Disease caused by infections of theobligate intracellular bacterial pathogen, Chlamydia pecorum,is a major threat to the ongoing survival of this species [5]. C.

pecorum infections in koalas have been associated with aspectrum of diseases ranging from keratoconjunctivitis (oculardisease) leading to blindness, rhinitis and pneumonia, as wellas urinary and genital tract disease, resulting in inflammationand fibrosis of the bladder and the upper female genital tract[6-10]. An effective vaccine to prevent the complications ofchlamydial infections in koalas would provide a valuablemanagement tool to stop the decline in wild populations by (a)reducing the infectious load in infected animals, and (b)preventing the further development of chlamydial pathology inhealthy animals and development of pathology in alreadyinfected animals.

An ideal chlamydial vaccine should be able to induce bothcellular and humoral immune responses in the host [11]. TheMajor Outer Membrane Protein (MOMP), which constitutes

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60% of the chlamydial outer membrane, has been the mostwidely used antigen either in its native or recombinant form inseveral vaccine studies [12-15]. Initial efforts to develop aMOMP-based vaccine demonstrated a vaccine induced cell-mediated immune response lasting for more than a year aswell as a humoral immune response (Chlamydia-neutralizingantibody) lasting > 35 weeks [16]. Following this study, ourgroup further evaluated the safety and efficacy of a C. pecorumMOMP-based multi-subunit vaccine in diseased as well ashealthy koalas [17]. Strong antibody (including neutralizingantibodies) and lymphocyte proliferation responses wererecorded in all vaccinated healthy and clinically diseasedkoalas. Vaccine induced antibodies specific for MOMP G, oneof the thirteen known C. pecorum ompA genotypes (A-H;unpublished data) were observed not only in plasma but also inocular secretions.

In the most recent study, we evaluated the immunogenicityof a vaccine consisting of either monovalent or polyvalentMOMPs [18]. Animals immunized with individual MOMPsdeveloped strong antibody and lymphocyte proliferationresponses to both homologous as well as heterologous MOMPproteins. Importantly, we also showed that vaccine-inducedantibodies effectively neutralized heterologous strains of koalaC. pecorum in an in vitro assay. Finally, we also demonstratedthat the immune responses in monovalent as well as polyvalentMOMP vaccine groups were able to recognize wholechlamydial elementary bodies, illustrating the feasibility ofdeveloping an effective MOMP-based C. pecorum vaccine thatcould protect against a range of strains. A promising aspect ofour most recent trials [17,18] was the cross-reactivity of MOMPantibody responses from vaccinated healthy and diseasedkoalas, giving hope for the generation of a MOMP-basedvaccine that will offer wide cross-protection against the varietyof genetically distinct C. pecorum strains circulating in wildkoala populations. In the present study, we further investigatedthe MOMP B cell epitopes responsible for the cross reactivity ofthe vaccine induced plasma antibodies in our previous vaccinetrials. We examined (a) the specific MOMP epitopes that wererecognized by koalas naturally infected with C. pecorum, (b)epitopes recognized by healthy animals immunized with eitherMOMP A, MOMP F, MOMP G or MOMP A plus F, and (c)epitopes recognized by the diseased animals immunized withMOMP G.

Materials and Methods

Koalas used in this studyNine koalas were analysed as part of this study, consisting of

(a) five wild animals with a current or recent C. pecoruminfection and overt signs of disease at the time of sampling,and (b) four captive healthy animals, with no evidence ofinfection or disease (Table 1). Among the diseased animals,three koalas were tested and found to be infected with C.pecorum ompA genotype F and the remaining two koalas weretested and found to be infected with C. pecorum ompAgenotype G [17]. One koala (Popeye) in the C. pecorum Ggroup and two koalas (Nixon/Felix Pitt) in the C. pecorum Fgroup were subcutaneously immunized with a vaccine

consisting of MOMP G and ISC (adjuvant), as previouslydescribed [17]. Kathy received the placebo (adjuvant only).Four healthy animals with no signs of infection or disease wereimmunized with individual MOMP types (A, F and G) ortogether (A and F), respectively [18].

The sampling and analysis of samples from wild koalasreceived for treatment at Australian Zoo Wildlife Hospital wasperformed under permission of the Queensland StateGovernment Department of Environment and HeritageProtection (Scientific Purposes Permit WISP 06056009). Allanimal work was approved by the Queensland University ofTechnology Animal Ethics Committee (Approval #0700000845& #0700000559).

Sampling of the animalsAnimals were sampled prior to immunisation (all 9 animals)

and then 20 weeks post immunisation (8 out of 9 animals).Plasma was separated from anti-coagulated bloods bycentrifugation at 233xg for 5 min at 4°C. Samples were thenstored at -80°C for further analysis.

Screening for C. pecorum infectionsOcular and urogenital tract swab samples from all animals,

prior to immunisation were screened for the presence of C.pecorum infections by 16S rRNA species-specific quantitativePCR [19] and for the presence of C. pecorum MOMP specificantibodies by immunoblotting [17].

Design of Biotinylated peptide library for screeningMOMP antibodies from koala plasma

A library of overlapping 15 amino acid peptides weredesigned by our group and constructed by Mimotopes(Melbourne, Australia). The full-length koala C. pecorumMOMP sequence for MOMP F (Figure 1) was used to design

Table 1. List of diseased as well as healthy koalas in thisstudy with their qPCR status, infecting strain and clinicaldisease observed during the study.

KoalaIdentifier

ChlamydiaPCR status

Naturallyinfected with C.pecorum(strain)

Clinical diseasestatus Immunization

Mars Bar + C. pecorum G Cystitis Not ImmunisedPopeye [19] + C. pecorum G Conjunctivitis MOMP GNixon [19] + C. pecorum F Conjunctivitis MOMP GFelix Pitt[19]

+ C. pecorum F Conjunctivitis MOMP G

Kathy [19] + C. pecorum F Cystitis Placebo onlyAmity [20] - - - MOMP ANessie [20] - - - MOMP FJaffa [20] - - - MOMP G

Guppy [20] - - -MOMP A + MOMPF

References are listed in the brackets.doi: 10.1371/journal.pone.0074808.t001

Characterisation of B cell Epitopes

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the first 63 overlapping (9 amino acid offset) 15 mer peptides.In addition to peptides representing the complete 370 aminoacids of C. pecorum MOMP F, the four variable domain regionsof C. pecorum MOMPs A, G and H, and three peptidesrepresenting two major predicted B cell epitopes in conserveddomains were also covered by the remaining 25 overlapping15mer peptides. Each peptide consisted of a biotin moleculelinked to a spacer (-SGSG-) to prevent steric hindrance,followed by the 15mer sequence with an offset of 9 aminoacids.

Biotinylated Peptide Enzyme Linked ImmunoSorbentAssay

The wells of 96 well streptavidin coated plates pre-blockedwith BSA (Thermo, Fisher Scientific, Melbourne, Australia)were coated individually with each of the 88 biotinylatedpeptides at a concentration of 2 µg/well in 1X PhosphateBuffered Saline Tween-20 and incubated for 2 hrs at roomtemperature (22°C). Post incubation, the wells were washed 3Xwith PBS-T and the individual plasma samples (diluted to1/1000) were added to the wells (100 µl/well), respectively. Theplates were incubated overnight at 4°C followed by washing 4Xwith PBS-T. The plates were then incubated with sheep anti-koala IgG (1:4000 in PBS-T) (100 µl/well) and incubated for 1hr at room temperature. Finally, after four washes, HRP-labelled rabbit anti-sheep IgG (1:1000 Southern Biotech/In vitroTechnologies, Cleveland, Australia) was added (100 µl/well) tothe wells and incubated for 1 hr at room temperature. After fivewashes with 1X PBS, 100 µl/well of ABTS

[2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid),Southern Biotech, Alabama, USA] substrate solution wasadded and incubated for 10 min at room temperature. Theoptical density was then read at 405 nm (Bio-Rad, North Ryde,Australia). Background for each sample was calculated fromthe mean plus twice the standard deviation of the negativewells (no sera added). In an attempt to minimise the inter-assay variability and truly reflect the differences in antibodyresponse profiles between samples, (a) the assay wasperformed on both pre- and post-immunised samples of eachanimal alongside on the same day, and (b) the backgroundabsorbance (green line) used for comparing the responseprofiles from pre- and post-immunisations was a result of themean plus two times the standard deviation of the negativecontrols of both samples.

Western blotWestern blots to assess the specificity of the koala plasma to

different C. pecorum MOMP proteins was performed aspreviously described [17]. In brief, 5 µg aliquots of each of theMOMP’s A, F, G and H was loaded onto 12% SDS-PAGE gelsand run at 110V for 1hr. Following blotting onto a nitrocellulosemembrane at 90V for 1hr, membranes were blocked in skimmilk solution for 1hr. Plasma samples from 4 weeks prior toimmunization and 20 weeks post immunization (1:1000) werethen analysed by incubating onto respective blots overnight at4°C. Membranes were then washed and sheep anti koala IgGwas added at 1:1000 dilution and left for an hour. Afterincubation, membranes were further washed with TBST andincubated with HRP-labelled rabbit anti-sheep IgG (1:1000) for1hr. Blots were then washed thoroughly with TBS andvisualised for bands by adding chemiluminescence substrate.

Results

Response profile of plasma from Chlamydia negativekoalas

Plasma from four healthy, pre-immunised, Chlamydianegative koalas (Amity, Nessie, Jaffa and Guppy) wereevaluated against the 88 peptides in the MOMP peptide library.Figure 2 shows each individual animal’s response (indicated bythe blue lines; A, B, C and D). There were weak absorbancelevels to all 88 peptides (except for peptide 22 in the conserveddomain) in all four healthy, Chlamydia-free animals. Theresponse profiles for each pre-immunisation sample was usedto establish a background response profile for analysis of post-immunisation samples.

Vaccination elicits cross-reactive MOMP antibodies inChlamydia free animals immunized with recombinantMOMP proteins A, F and G

Figure 2A shows the specificity of the antibodies producedby a Chlamydia negative koala (Amity) following immunizationwith recombinant MOMP A. Interestingly, when compared toanimals immunised with other MOMPs (Figure 2), Amity’s postimmunised sample had a better (but not extremely high)response profile against the majority of the same peptidesrecognized by her own pre-immunised sample. Immunizationalso induced cross-reactive antibody against three otherMOMPs, as shown in the immunoblots of Figure 2A. AnotherChlamydia negative koala (Nessie) was immunized withrecombinant MOMP F protein and by 20 weeks post

Figure 1. Layout of C. pecorum MOMP peptide library. Peptides 1 to 63 represent five conserved domains (straight lines)interspersed by four variable domains (boxes) of MOMP F. Peptides 64 to 88 represent four variable domains of MOMP A, MOMPG and MOMP H, respectively.doi: 10.1371/journal.pone.0074808.g001

Characterisation of B cell Epitopes

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vaccination produced plasma antibodies (Figure 2B) thatreacted strongly against epitopes in (a) the conserved domains(peptides 16 and 27), (b) variable domains of MOMP F

(peptides 13, 14 and 38) and; (c) variable domain two ofMOMP H (peptide 84) (Table 2). Immunisation of a Chlamydianegative animal (Jaffa) with MOMP G protein resulted in

Figure 2. B cell epitope mapping across four MOMPs in Chlamydia free koalas after vaccination. Response profiles andimmunoblots of four Chlamydia free koalas vaccinated with MOMP A (A), MOMP F (B), MOMP G (C) and MOMP A as well asMOMP F (D), respectively. The blue line indicates the absorbance of pre immunized samples collected from individual Chlamydia-negative animals. The red line indicates the 20 weeks post immunized sample. The green line indicates the background (Mean andtwo times standard deviation of negative controls).doi: 10.1371/journal.pone.0074808.g002

Characterisation of B cell Epitopes

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antibodies that primarily recognise various linear epitopes inthe conserved domains of MOMP (peptides 35, 44, 55 and 87)(Figure 2C). Finally, when a Chlamydia negative animal wasimmunised with a combination of two MOMP proteins (A plusF) it produced antibodies that recognised the epitopes inconserved domains (peptides 2 and 19) as well as the variabledomain one of MOMP F (peptides 13 and 14) (Figure 2D)(Table 2). Both the animals immunised with MOMP F (one incombination with MOMP A) gave the strongest reaction topeptide 13 (DSVSLQERENPAYGK) and its overlappingpeptide 14 (ERENPAYGKHMHDAE). Additionally, tocorroborate the Pepscan data, we performed immuno-blotassays on both pre- and post-immunised plasma at the sametime points, using similar concentrations of plasma (1:1000)used in Pepscan assay for each animal. Although the pre-immunised samples were negative against four MOMPvariants, the samples collected 20 weeks after immunisationdeveloped cross-reacting antibodies to various MOMPs (Figure2)

Response profiles of plasma antibodies in koalas withnatural Chlamydia infections

Figure 3 shows the specificity of plasma antibodies producedin two animals [Popeye (Figure 3A) and Mars Bar (Figure 3B)]naturally infected with C. pecorum genotype G when comparedto the response of four Chlamydia negative animals. Bothanimals had a similar response profile to the Chlamydia freeanimals for 84 out of 88 peptides, however, plasma from thesetwo diseased animals reacted strongly with epitopes fromvariable domain four of MOMP G (298’-TLLGEATQVDNSNKF;peptide 79) and the equivalent epitopein MOMP H (298’-TLLGQATQVDNSNKF;peptide 85),respectively. Interestingly, when this epitope of 15 amino acidlength was aligned with all four MOMPs (Figure 3C), there arenine amino acid differences between MOMP G and MOMP A,eight amino acid differences between MOMP G and MOMP Fbut only one amino acid difference between MOMP G andMOMP H, which presumably explains the cross reactivity ofplasma antibodies to MOMP G and MOMP H. Apart from theepitopes in the variable domains, plasma antibodies from MarsBar also recognised two unique B cell epitopes in theconserved regions of MOMP (peptides 16 and 27), consistentwith differences in immune responses in out-bred animals.

Figure 4 shows the specificity of the plasma antibodies in twoanimals [Nixon (Figure 4A), and Felix Pitt (Figure 4B)] naturallyinfected with C. pecorum genotype F, again compared to theresponse of the four Chlamydia negative animals. Althoughboth these wild koalas were infected with C. pecorum F, theyhad different responses to the MOMP peptides. Nixon (Figure4A) produced antibodies to the three epitopes in the variabledomains, SMGQAPSTNSPADSV (variable domain one;peptide 11), GLIGISGSDLNSKVP (variable domain two;peptide 23) and LNMTTWNPTLLGEAT (variable domain four;peptide 49) whereas Felix Pitt (Figure 4B) produced antibodiesto epitopes GLIGISGSDLNSKVP (variable domain two; peptide23) and EATSVNAGNKYADVL (variable domain four; peptide51). For the epitope recognized by both animals in variabledomain two (Figure 4C), we found four amino acid differences

between MOMP F and the other three MOMPs. Interestingly,within the MOMP variable domain four (Figure 4D), bothanimals recognised two different epitopes VD4 (1) and VD4 (3),respectively, although the epitopes shared three amino acidsbetween them. At VD4 (1) there are 3-5 amino acid differencesbetween the various MOMPs and 5-8 amino acid differencesbetween various MOMPs at VD4 (3). Another animal (Kathy),naturally infected with C. pecorum F also produced antibodiesto epitopes DSVSLQERENPAYGK (variable domain one;peptide 13) and NQTLPLPITAGTATD (variable domain three;peptide 37) of MOMP F (Figure 5A).

Evaluation of MOMP antibody responses in aChlamydia infected animal administered with placebo(adjuvant only)

A C. pecorum F infected koala when vaccinated with aplacebo (ISC) did not produce any antibodies to conserveddomains, but retained a similar reactivity pattern to the pre-immunization sample (Figure 5A) by producing antibodiespredominantly to the variable domains one and three of MOMPF.

Enhancement of a broader repertoire of MOMPantibodies in naturally diseased animals followingimmunization with recombinant MOMP protein

Figure 5B illustrates the vaccine induced production ofantibodies to various epitopes in a koala that was previouslynaturally infected with C. pecorum G. Interestingly, afterimmunization with recombinant MOMP G protein there was asignificant production of antibodies against previouslyrecognized epitopes (induced by natural infection) in variabledomain four of MOMP G and H and additionally to theconserved domains (Table 2). Figure 5C shows theenhancement of antibodies in a C. pecorum F infected koalaafter immunization with MOMP G protein. The major epitopesrecognized fall in the conserved domains as well as thevariable domain four of MOMP F and variable domain one ofMOMP H (Table 2).

Discussion

Studies in various animal models (mice, guinea pigs andnon-human primates) of Chlamydia infection, have suggested asignificant requirement for antibody-mediated immuneresponses (neutralising antibodies) along with cellular immuneresponses (Th1) for an effective vaccine. Morrison et al. [20]demonstrated a significant role for B cells and CD4+ T cells butnot CD8+ T cells, in resistance to C. trachomatis genital tractreinfection. In a follow up study, Morrison et al. [21]subsequently showed that antibody-mediated immuneresponses were critical in the resolution of secondary genitaltract infection in mice but this resolution was independent ofboth CD4+ and CD8+ T cells. Hence, optimising the antibodyresponse is essential for effective chlamydial vaccinedevelopment.

Our previous koala Chlamydia vaccine trials [17,18] havedemonstrated strong vaccine induced humoral immuneresponses in healthy koalas as well as vaccine enhanced

Characterisation of B cell Epitopes

PLOS ONE | www.plosone.org 5 September 2013 | Volume 8 | Issue 9 | e74808

Tabl

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Lis

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by

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Characterisation of B cell Epitopes

PLOS ONE | www.plosone.org 6 September 2013 | Volume 8 | Issue 9 | e74808

Tabl

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Characterisation of B cell Epitopes

PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e74808

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Characterisation of B cell Epitopes

PLOS ONE | www.plosone.org 8 September 2013 | Volume 8 | Issue 9 | e74808

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Characterisation of B cell Epitopes

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immune responses in naturally infected koalas, at bothsystemic and mucosal sites. One particularly encouragingaspect of these studies was the observation of cross-reactiveantibodies in vaccinated healthy and diseased animals [17] thatrecognized MOMP from different C. pecorum strains. Thefunctionality of these antibodies was demonstrated by theirability to cross-neutralize heterologous C. pecorum infections,in vitro [18].

In our current study, we used Pepscan methodology toinvestigate linear epitopes along the entire MOMP protein todetermine the specificity of the antibody responses producedfollowing, (a) natural live infection, (b) recombinant MOMPvaccination, (c) recombinant MOMP vaccination on top ofnatural live infection. The 15mer peptides (overlapping by 9amino acids) covered the full length of koala C. pecorumMOMP F protein, as well as the variable domain regions for

koala C. pecorum MOMPs A, G and H. When we evaluatedimmune sera from four naturally infected koalas, interestingly,only six linear epitopes were recognised. Five of these epitopes(peptides 23, 49, 51, 79 and 85) were located in the variabledomains, while the other epitope (peptide 53) was in aconserved domain. These naturally recognised epitopespresumably represent the surface-exposed regions on intactchlamydial EBs/RBs that are recognised during a natural liveinfection. Of these six naturally recognised MOMP epitopes,none were recognised by all four koalas. One possibleexplanation for this could be due to major histocompatibilitycomplex restriction in these out bred koalas [22]. We found thatdifferent epitopes were recognised in koalas infected withdifferent C. pecorum genotypes (peptides 79 and 85 wererecognised by C. pecorum G infected koalas only; peptides 23,

Figure 3. B cell epitope mapping across four MOMPs in naturally, C. pecorum genotype G infected koalas. Responseprofiles of two wild koalas naturally infected with C. pecorum G, Popeye (A) and Mars Bar (B) across MOMP peptide library andamino acid alignment of four MOMPs at VD4 epitope (C) (dots represent the consensus). Representative blot of plasma from thekoalas infected with C. pecorum G against four recombinant full length MOMP proteins (D). The blue line indicates the mean ofabsorbance of four Chlamydia free animals. The red line indicates the Chlamydia infected animal. The green line indicates thebackground (Mean and two times standard deviation of negative controls).doi: 10.1371/journal.pone.0074808.g003

Characterisation of B cell Epitopes

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49 and 51 were recognised by C. pecorum F infected koalasonly).

The in silico B cell prediction algorithm has predicted majorepitopes in all four variable domains across C. pecorumMOMPs [18], however koalas naturally infected with C.pecorum G genotype produced antibodies only to the linearepitopes of variable domain four of MOMP G and MOMP H.This cross reactivity to MOMP G and MOMP H was similar tothe immunoblot data we observed (Figure 3D). Interestingly, analignment of the amino acids at this epitope, revealed only oneamino acid difference between them i.e. Glutamic acid (E) inMOMP G and Glutamine (Q) in MOMP H (Figure 3C). Aminoacids at variable domain four between MOMP G and H weremore similar (93.3% consensus) when compared to thevariable domain three (73.3% consensus), variable domain two(69.2% consensus) and variable domain one (62.5%consensus) regions, which explains the cross-reactivity ofantibodies to MOMP G and H. Among the koalas infected

naturally with C. pecorum F, one animal (Nixon) producedplasma antibodies to three epitopes at the variable domains ofMOMP F (VD1, VD2 and VD4) whereas the other animal (FelixPitt) produced antibodies to epitopes in two variable domains ofMOMP F (VD2 and VD4). Both the animals (Nixon and FelixPitt) shared a common epitope located in variable domain two.Even though there was 73.3% similarity between MOMP F andMOMP A at this linear epitope, and 66.6% between MOMP Fand MOMP G/H, both koalas specifically produced antibodiesonly to MOMP F (Figure 4C). Surprisingly, although bothtargeting variable domain four of MOMP F, antibodies fromeach animal recognised two different overlapping epitopeswithin this domain (Figure 4D).

Pre-immunised plasma samples from four Chlamydianegative koalas, with the exception of Amity, did not recogniseany of the epitopes except for peptide 22 (Figure 2). When weevaluated sera from four Chlamydia negative koalas that wereimmunised either with individual recombinant MOMP proteins

Figure 4. B cell epitope mapping across four MOMPs in naturally, C. pecorum genotype F infected koalas. Responseprofiles of two wild koalas naturally infected with C. pecorum F, Nixon (A) and Felix Pitt (B) across MOMP peptide library. Alignmentof four MOMPs at an epitope at variable domain two (C), and two overlapping epitopes at variable domain four (D) (dots representthe consensus). The blue line indicates the mean of absorbance of four Chlamydia free animals. The red line indicates theChlamydia infected animal. The green line indicates the background (Mean and two times standard deviation of negative controls).doi: 10.1371/journal.pone.0074808.g004

Characterisation of B cell Epitopes

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(A, F and G) or a combination of two recombinant MOMPproteins (A and F), we observed an epitope response that wasdifferent to the response seen with naturally infected koalas(Figure 2) (Table 2). In these four immunised koalas, a total of15 epitopes were recognised and eight of these epitopes were

different to the epitopes recognised during a natural liveinfection. Only five of these vaccine induced epitope antibodieswere in the variable domains of MOMP F and MOMP H, while10 were in conserved domains. Furthermore, a koalaimmunised with both recombinant MOMPs (A and F) did not

Figure 5. B cell epitope mapping across four MOMPs in Chlamydia infected koalas after administration with placebo orvaccine. Response profile of a C. pecorum F infected koala administered with a placebo (adjuvant only) across our MOMP peptidelibrary (A). Response profiles of a naturally infected C. pecorum G koala immunized with recombinant MOMP G (B), and a naturallyinfected C. pecorum F koala immunized with recombinant MOMP G (C) across our MOMP peptide library. The blue line representsthe absorbance of the pre-immunized sample. The red line represents the 20 weeks post-immunized sample. The green lineindicates the background (Mean and two times standard deviation of negative controls).doi: 10.1371/journal.pone.0074808.g005

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show any extra boosting effect to the conserved domains inparticular, when compared to animals immunized with a singleMOMP. This data strongly correlates with the observation of nosignificant difference in the cross-reactivity of antibodies post-vaccination between the individual and dual MOMP groups, asshown in Figure 2 and previously observed [18]. None of the insilico predicted B cell epitopes in the conserved domain [18]were recognized by the koalas; however antibodies to variousother epitopes in conserved domains were elicited.

Early studies by Girjes et al. [23] reported in vitroneutralisation with sera from naturally infected koalas.However, in their study, less than 50% of the sera showed invitro neutralisation activity and this varied between koalas. Bycomparison, the four koalas that were given a recombinantMOMP vaccine in our current study were all previously shownby Kollipara et al. [17] to produce in vitro neutralisingantibodies. Although animals were immunized with singlerecombinant C. pecorum MOMPs, they still effectively crossneutralized the heterologous infections. Interestingly, there wasno significant difference between individual and combinedMOMP groups (MOMP A and MOMP F) in cross neutralizationof C. pecorum infections. This is very encouraging from avaccine development perspective, for the fact that seven of therecognised epitopes were in conserved domains and thereforeconserved across all 13 C. pecorum MOMP genotypes (datanot shown) that we currently have sequence information for,bodes well for wider cross neutralisation. It has also becomeevident from studies in naturally infected koalas that strong,long lasting protection from the neutralising antibodies does notresult in natural infections [18]. Hence, an effective vaccine willneed to improve on the existing natural immune response.

In the final aspect of our study, we evaluated the specificityof the response of koalas naturally infected with C. pecorumand then administered the recombinant MOMP vaccine. This iscritical because, if the vaccine has to be delivered to wildpopulations, which already have 20-50% C. pecorum infectionslevels (Kollipara et al., 2013; unpublished data), it has to beeffective in these groups. In the naturally infected vaccinatedanimals, a total of eight vaccine induced epitopes wererecognised (Table 2). Out of eight epitopes, six were

recognised in conserved domains and the remaining two in thevariable domains (peptides 50 and 83).

In conclusion, although this study has dealt with thespecificity of the polyclonal antibodies against MOMP in wild(outbred) koalas, known to have highly variable MHC Class IIgenes [22]. We have primarily focussed on expanding thefindings (highly specific as well as cross-recognising humoralimmune responses) from our previous studies [17,18]. In thisstudy, we have shown, (a) specific MOMP B cell linearepitopes recognised in C. pecorum G and F genotype infectedwild koalas and (b) MOMP linear epitopes responsible for theproduction of cross recognition antibodies in post-vaccinatedhealthy and diseased koalas. The findings from this studyprovides further insights into (a) screening various potential Bcell epitopes using the Pepscan methodology in otherchlamydial antigens, such as chlamydial heat shock protein(hsp60) [24] and thereby (b) developing diagnostic assayswhich aid in screening various healthy as well as diseasedkoalas. Using such findings as a proof of concept, a recentstudy [25] has identified new peptide antigens from C.trachomatis infected women and demonstrated the potential todevelop an epitope-based serological diagnostic assay. Sinceboth humoral and cell mediated responses are critical for aprotective immune response against persistent chlamydialinfections, our next step is to apply this screening assay toidentify several potential T cell epitopes in healthy anddiseased koalas.

Acknowledgements

We thank Charles Wan for providing assistance with designingthe Peptide library.

Author Contributions

Conceived and designed the experiments: AK AP KB PT.Performed the experiments: AK. Analyzed the data: AK PT.Contributed reagents/materials/analysis tools: AK PT. Wrotethe manuscript: AK AP KB PT.

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24. Yi H, Zhong G, Brunham RC (1993) Continuous B-cell epitopes inChlamydia trachomatis heat shock protein 60. Infect Immun 61:1117-11120. PubMed: 7679373.

25. Stansfield SH, Patel P, Debattista J, Armitage CW, Cunningham K etal. (2013) Proof of concept: A bioinformatic and serological screeningmethod for identifying new peptide antigens for Chlamydiatrachomatis related sequelae in women. Results in Immunology 3:33-39.

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