ORIGINAL ARTICLE

A Multi-Subunit Chlamydial Vaccine Induces Antibody and

Cell-Mediated Immunity in Immunized Koalas (Phascolarctos
cinereus): Comparison of Three Different Adjuvants
Alison J. Carey1, Peter Timms1, Galit Rawlinson2, Jacqui Brumm2, Karen Nilsson2, Jonathon M. Harris1,
Kenneth W. Beagley1
1
Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Qld, Australia;
2
Lone Pine Koala Sanctuary, Fig Tree Pocket, Brisbane, Qld, Australia

Keywords
Adjuvant, antibody, Chlamydia, koala
Correspondence
Kenneth W. Beagley, Institute of Health and
Biomedical Innovation, Queensland University
of Technology, 60 Musk Ave, Kelvin Grove,
Qld 4059, Australia.
E-mail: k2.beagley@qut.edu.au
Submitted September 1, 2009;
accepted October 9, 2009.
Citation
Carey AJ, Timms P, Rawlinson G, Brumm J,
Nilsson K, Harris JM, Beagley KW. A multi-
subunit chlamydial vaccine induces antibody
and cell-mediated immunity in immunized
koalas (Phascolarctos cinereus): comparison of
three different adjuvants. Am J Reprod
Immunol 2010; 63: 161–172
doi:10.1111/j.1600-0897.2009.00776.x
Problem
Chlamydial infections represent a major threat to the survival of the
koala. Infections caused by Chlamydia pecorum cause blindness, infertility,
pneumonia and urinary tract infections and represent a threat to the
survival of the species. Little is known about the immune response in
koalas, or the safety of commonly used adjuvants for induction of pro-
tective systemic and mucosal immunity.
Method of study
In the present study, we immunized 18 healthy female koalas subcuta-
neously with a combination of three chlamydial antigens [major outer
membrane protein (MOMP), NrdB and TC0512 (Omp85)] mixed with
one of three different adjuvants [Alhydrogel, Immunostimulating Com-
plex (ISC) and TiterMax Gold].
Results
All adjuvants induced strong neutralizing IgG responses in plasma
against the three antigens with prolonged responses lasting more than
270 days seen in Alhydrogel and ISC immunized animals. Cloacal IgG
responses lasting >270 days were also induced in ISC-immunized ani-
mals. Chlamydia-specific peripheral blood mononuclear cell proliferative
responses were elicited by both Alhydrogel and ISC, and these lasted
>270 days in the ISC group.

Conclusion
The data show that a multi-subunit chlamydial vaccine, given subcuta-
neously, can elicit Chlamydia-specific cell-mediated and antibody
responses in the koala demonstrating that the development of a protec-
tive vaccine is feasible.

urban development in particular, these combined

Introduction
Chlamydia continues to be the most important infec-
tious disease affecting the koala (Phascolarctos cinere-
us).1–4 Together with habitat destruction, because of
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
threats mean that many koala populations in Austra-
lia are under significant threat of collapse. Koalas are
infected with two species of Chlamydia, C. pecorum
and C. pneumoniae.5 Chlamydia pecorum is always the
161
 CAREY ET AL.
most prevalent and is present at higher levels in
infected animals.3 Both species can be detected at
both ocular and genital sites; however, it is generally
accepted that C. pecorum causes the most serious dis-
ease. Koalas suffer a similar spectrum of chlamydial
diseases as seen in humans. Ocular infections cause
keratoconjunctivitis leading to blindness.6,7 Rhinitis
and pneumonia are associated with respiratory infec-
tions and urinary tract infections cause cystitis and
continual urine soiling or ‘wet bottom’.2 Infections
of the genital tract can cause severe inflammation
and fibrosis resulting in cystitis and infertility.1 Many
koalas carry Chlamydia without displaying symptoms,
but increasing numbers of animals will develop overt
disease when stressed by environmental pressures
such as habitat loss. Treatment options for chlamyd-
ial infections are presently limited to antibiotics;
however, antibiotics can adversely affect the intesti-
nal microflora and health of the animals.8 Anti-
biotics can also cause persistent infections that may
evade immune responses but revert to an acute
infectious state when treatment ceases.9 Persistent
infections are also believed to enhance the inflam-
matory damage that is the hallmark of chlamydial
disease. A successful vaccine to prevent chlamydial
infection would not only overcome the limitations of
current antibiotic treatments but would also allow
for protection of animals undergoing relocation. An
effective vaccine requires both an antigen that elicits
protective immunity combined with an adjuvant
that is safe and that elicits the appropriate immune
response. To date, the only adjuvants that have been
used in koalas are Freund’s adjuvants.10–12 These
can cause severe adverse local reactions in many
species13 and are generally considered to be inappro-
priate for use outside the research laboratory. There
are currently no data on the safety of other adju-
vants in the koala.
Early trials of human vaccines for trachoma, using
whole-killed Chlamydia, resulted in exacerbated dis-
ease in many recipients following a subsequent nat-
ural infection.14,15 Since then, chlamydial vaccine
research has focused on defining the antigens that
elicit protective immunity without exacerbating
inflammatory disease. The major outer membrane
protein (MOMP) of Chlamydia is the best-studied
antigen to date and in small animal models, it is able
to elicit significant protection against infection.16,17
The major problem with MOMP-based vaccines is
the sequence diversity in the surface-exposed vari-
able domains of different C. trachomatis serovars.
162
Because MOMP is a major target of the immune
response, immunization with MOMP will only elicit
protection against homologous serovars, but not
against serovars that express a different MOMP.
Sequencing of the MOMP-encoding ompA gene of
C. pecorum from animals, including koalas,18 has
demonstrated a similar level of diversity to that seen
among human C. trachomatis serovars, suggesting
that a MOMP-based vaccine may also not be effec-
tive in koalas. Recent studies by our group have
identified a number of chlamydial antigens that elicit
protective immunity against genital infections in
mouse models and are highly conserved across
known chlamydial species. Ribonucleotide reductase
(NrdB)19 is an enzyme that catalyses the synthesis of
the four deoxynucleoside triphosphates (dNTPs)
from host cell-derived ribonucleoside triphosphates,
with its activity being the rate limiting step for chla-
mydial DNA synthesis. Comparison of NrdB
sequences across the published chlamydial genomes
demonstrates a high degree (>90%) of sequence
conservation. NrdB also contains predicted human
and mouse helper T-cell epitopes, with the region of
NrdB containing these epitopes being 100% con-
served across the published chlamydial genomes
including C. pecorum. TC0512 is a putative outer
membrane protein (omp85) from C. muridarum that
was identified using expression library immunization
as an antigen that elicits partial protection against
genital C. muridarum infection in mice.20 Comparison
of omp85 gene sequences across chlamydial species
showed >70% sequence similarity across the entire
molecule. As with NrdB, there are regions of TC0512
that contain sequences that are more highly con-
served and may contain T-cell epitopes. As vaccines
based on whole-killed Chlamydia have the potential
to exacerbate inflammatory disease15 and because
this study did not involve a challenge infection, we
combined three recombinant chlamydial antigens,
MOMP and TC0512 isolated from C. muridarum and
NrdB isolated from C. trachomatis, in our experimen-
tal vaccine. This allowed us to determine if animals
responded to each of the individual vaccine compo-
nents and also if the use of a combination of moder-
ate to highly conserved antigens would elicit
cross-protective immunity against various chlamydial
species.
The immune system of the koala has been
described as ‘lazy’,11 with humoral responses taking
significantly longer to develop than in eutherians.
This is not supported by other studies, however, and
American Journal of Reproductive Immunology 63 (2010) 161–172
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 DEVELOPING A CHLAMYDIA VACCINE FOR THE KOALA
the development of cell-mediated immunity may be
similar to that in non-metatherian mammals.21 Koa-
las have been shown to mount an antibody response
following immunization with the antigen bovine
serum albumin (BSA)10 and koala peripheral blood
leukocytes (PBL) do proliferate when cultured with
mitogens such as concanavalin A (ConA) and phyto-
haemagglutinin (PHA),22 although lymphocyte pro-
liferative responses were not detected following
skin-painting with the contact-sensitizing agent 2-4-
dinitrofluorobenzene.11 It must be said, however,
that knowledge of the koala immune response is
scarce and this is mirrored by the lack of reagents
available to measure immunity in the koala. As a
first step toward developing a safe and effective chla-
mydial vaccine for the koala, we immunized groups
of healthy female koalas with three different adju-
vants [Immuno stimulating complex (ISC), Alhydro-
gel and TiterMax Gold] currently used in veterinary
and animal vaccines, admixed with a combination of
three chlamydial antigens (MOMP, NrdB, TC0512).
Signs of adverse reactions at the injection sites were
monitored after each immunization. Antigen-specific
lymphocyte proliferation was measured in peripheral
blood leukocytes over a 10-month period, together
with plasma and reproductive tract antibody levels.
Plasma antibody was also assayed for neutralizing
activity against three different species of Chlamydia.
Two of the three adjuvants were well-tolerated by
koalas. High levels of antigen-specific peripheral
blood mononuclear cell (PBMC) proliferation were
observed in animals immunized with ISC and plasma
antibody against all three antigens and were present
in all animals in the ISC and Alhydrogel groups
throughout the study. Plasma antibody was able to
inhibit in vitro chlamydial infections of two species of
Chlamydia, to varying degrees. Our data show that a
multi-subunit vaccine approach may be able to pro-
vide protection against chlamydial infection in the
koala.
Materials and methods
Animals
Eighteen female koalas aged 2–5 years were assigned
to one of three groups, each of six animals. Animals
were housed at the Lone Pine Koala Sanctuary
(Brisbane, Australia). All procedures were approved
by the Queensland University Animal Ethics Com-
mittee.
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
Antigens
Three recombinant chlamydial proteins were used.
A recombinant maltose binding protein-major outer
membrane protein (MBP-MOMP) from C. murida-
rum, produced in Escherichia coli, was generously pro-
vided by Dr. Harlan Caldwell (Rocky Mountain
Laboratories)23 and was purified as described.16
Ribonucleotide reductase small chain protein (NrdB)
from C. trachomatis serovar D was cloned in pRSET-A
(InVitrogen, Sydney, Australia), expressed in E. coli
and purified as described.19 TC0512 (Omp85), a
putative outer membrane protein from C. muridrum,
was produced as a recombinant His-tagged protein
in E. coli using the gene fragment identified by
McNeilly et al.20 The three antigens were chosen
because each was able to elicit partial protection
against genital challenge in the mouse model with
either C. muridarum16,20 or C. trachomatis.19
Adjuvants
Three adjuvants were evaluated in this study. Immu-
nostimulating complex (ISC, lot #08141a6) was a
generous gift from Dr. John Walker, Pfizer Animal
Health, VMRD, Parkville, Australia. Alhydrogel (Alu-
minum hydroxide Gel, Batch #3957; Accurate
Chemical and Scientific Corporation, Westbury, NY,
USA) was obtained from Auspep Pty Ltd, Parkville
Australia. TiterMax Gold adjuvant was obtained
from TiterMax USA, Norcross, GA, USA.
Immunizations
All animals were immunized subcutaneously on days
0, 30 and 60 with 50 lg each of MBP-MOMP, TC0512
and NrdB combined with 50 lg of ISC in 1 mL of PBS
(group 1), Alhydrogel (final concentration 20%) in
1 mL PBS (group 2) or suspended in PBS and emulsi-
fied in an equal volume of TiterMax Gold (group 3).
Blood sample was collected into EDTA vacutainer
tubes from all animals at 4-week intervals, beginning
2 weeks after the second immunization, up to
270 days. Cloacal swabs were collected at monthly
intervals following the third immunization.
Lymphocyte Stimulation Assay
Blood samples were centrifuged at 41 · g (Beckman
Coulter GS-6R, centrifuge with a GH3.8 rotor) for
10 min. Plasma was collected and stored frozen at
163
 CAREY ET AL.
)20C until further use. An equal volume of Hanks
Balanced Salt Solution (HBSS) was added to the pel-
leted blood and mixed by inverting the tube several
times. The diluted blood was layered over Ficoll-
paque premium (GE Healthcare, Rydalmere, Austra-
lia) and the tubes were centrifuged at 650 · g for
25 min with the brake off. PBMC at the interface
were removed and transferred to a new tube,
washed twice with HBSS and counted. Cells were
then suspended at 2 · 106 cells ⁄ mL in PBS supple-
mented with 1% fetal calf serum. Carboxyfluoresce-
in succinimidyl ester (CFSE; Sigma-Aldrich, Castle
Hill, Australia) was added to give a final concentra-
tion of 5 lm and cells were incubated in the dark at
room temperature for 5 min. Cells were then
washed 3· in HBSS and suspended at 2 · 106
cells ⁄ mL in culture medium (DMEM containing 5%
foetal calf serum, 2 mm l-glutamine, 100 lg ⁄ mL
streptomycin sulfate, 2 lg ⁄ mL gentamycin and
50 lm 2 b-mercaptoethanol). An aliquot of non-
CFSE treated cells was retained as control. A total of
100 lL ⁄ well (2 · 105 cells) was aliquoted into wells
of 96-well tissue culture plates (In Vitro Technolo-
gies, Noble Park North, Australia). Antigens (MBP-
MOMP, NrdB and TC0512) were added to give a
final concentration of each antigen of 50 lg ⁄ mL.
PHA (10 lg ⁄ mL) was added as a positive control and
culture medium as a negative control. Plates were
incubated for 4 days at 37C in 5% CO2. After
4 days, cells were washed once in PBS, then resus-
pended in PBS containing 4% paraformaldehyde
and stored in the dark at 4C until analysis. Prolifer-
ation of PBMC was determined using a Beckman
Coulter flow cytometer (FC500, Gladesville, NSW,
Australia). Proliferation was expressed as the % of
PBMC that had undergone >3 cell divisions.
ELISAs
Wells of 96-well ELISA plates (Greiner bio-one med-
ium binding; InterPath, West Heidelberg, Australia)
were coated overnight at 4C with MBP-MOMP
(0.5 lg ⁄ mL), NrdB (40 lg ⁄ mL) or TC0512 (80 lg ⁄ mL)
in borate-buffered saline (BBS). Wells were then
washed 3· with PBS containing 0.05% Tween-20
(PBS-T) then blocked for 1 hr at 37C with 5% skim
milk in PBS-T. After blocking, samples were serially
diluted twofold in PBS-T and added to wells, then
incubated for 1 hr at 37C. For TC0512 antibody
detection, plasma was initially diluted 1:100. For
detection of MOMP and NrdB-specific antibodies,
164
plasma was initially diluted 1:500. For detection of
antibody in reproductive tract secretions, cloacal
swabs were collected in 1 mL PBS-T and frozen at
)20C. Prior to assay, swabs were thawed and
vortexed. Swab eluate was serially diluted twofold
starting with undiluted sample. Plates were then
washed 4· in PBS-T and sheep anti-koala IgG
(1:8000 in PBS-T) added and plates incubated for a
further hour at 37C. After further three washes
(PBS-T), HRP-conjugated rabbit anti-sheep IgG
(1:1000, Southern Biotech ⁄ Millipore, North Ryde,
Australia) was added to wells and plates incubated
for a further hour. After five washes with PBS,
50 lL ⁄ well of TMB substrate (Sigma-Aldrich) in cit-
rate buffer was added and after 10 min, the reactions
were stopped by adding 50 lL ⁄ well 1 m H2SO4. Opti-
cal density was then read at 450 nm (Bio-Rad, North
Ryde, NSW, Australia). Endpoint titers (EPT) were
calculated as the inverse of the dilution, where
absorbance values were equivalent to that of the
mean of the no sample (PBS-T) control wells + 2
standard deviations.
Preparation of Sheep Anti-Koala IgG
Serum from three healthy koalas was pooled and
the IgG fraction isolated using a Protein A ⁄ Protein G
column (Zymed Laboratories, South San Francisco,
CA, USA) according to the manufacturer’s instruc-
tions. The purified IgG fraction was sent to the Insti-
tute of Medical and Veterinary Science (IMVS,
Adelaide, Australia) where it was used to immunize
a sheep. The sheep antiserum was used at a dilution
of 1:8000 in ELISA assays.
In Vitro Neutralization Assay
McCoy cells were seeded in 48-well plates and
grown overnight until 70–80% confluent. Plasma
samples were diluted 1:10, 1:50, 1:250 and 1: 1250.
Chlamydia muridarum (1000 ifu) or a koala isolate of
C. pneumoniae24 was added to 0.1 mL of diluted
plasma or cloacal eluate and incubated for 1 hr at
37C. Medium was removed from the McCoy cell
monolayers and replaced by the plasma or cloacal
eluate samples and the plates incubated for 4 hr.
Samples were removed and replaced with fresh med-
ium containing 1 lg ⁄ mL cycloheximide. Cells were
incubated for further 30 hr and then fixed with
100% methanol. Inclusions were identified by stain-
ing with FITC-labeled anti-chlamydial LPS (CelLabs,
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
 DEVELOPING A CHLAMYDIA VACCINE FOR THE KOALA
Brookvale, Australia) according to the manufac-
turer’s instructions.
Statistics
All statistical analyses were performed using Graph-
Pad Prism version 5 (GraphPad Software, LaJolla,
CA, USA). All data presented are the mean of six
koalas ± SE of the mean. Two-way anova with Bon-
ferroni’s post test with the P value set at <0.05 was
used to analyze the PBMC proliferation and the
plasma and cloacal swab antibody samples. Neutral-
ization studies were analyzed using unpaired t-test
with the P value set at 0.05.
Results
Female koalas were immunized with a combination
of three chlamydial antigens combined with one of
three different adjuvants. No adverse effects were
noted in animals immunized with either the ISC or
Alhydrogel containing vaccine formulations and all
six animals in each group completed the three
immunization schedule. In the group receiving the
TiterMax Gold-formulated vaccine, however, three
of the six animals developed abscesses at the vacci-
nation site following the second immunization.
These were obviously painful and impaired mobility
for several days. These animals were treated with
non-steroidal anti-inflammatories for 2–3 days after
the second immunization. For reasons of the adverse
reactions, animals in this group only received two
immunizations on days 0 and 30 and no samples
were collected beyond day 158 of the trial.
PBMC Proliferation
The PBMC were stimulated in vitro with the antigen
combination and the proliferation measured by
CFSE dye-dilution assay (Fig. 1). At days 74 and
102, equivalent levels of proliferation were observed
in the animals immunized with the ISC or Alhydro-
gel-formulated vaccines. At day 270, only PBMC
from animals that had received the ISC-formulated
vaccine showed in vitro proliferation with approxi-
mately 15% of cells proliferating. Proliferation in
this group was significantly higher (P < 0.05) than
in either of the other groups. Low-levels of prolifera-
tion in both the Alhydrogel and ISC groups were
observed at day 158. The reason for this remains
unknown. No significant proliferation above media
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
* *
30
Iscomatirx
Cells proliferating (%)
Alhydrogel
20 Titre Max Gold
10
0
0
74
10
15
27
ay
ay
ay
ay
D
D
Fig. 1 Koala peripheral blood mononuclear cells were isolated by
centrifugation on Ficoll-paque gradients and then labeled with CFSE as
described in methods. Cells were cultured in vitro for 4 days with
50 lg ⁄ mL of MOMP, CT0512 and NrdB and proliferation determined
by analysis of CFSE fluorescence using a Beckman Coulter FC500. The
Y-axis shows the percent of PBMC that has undergone ‡3 cell divi-
sions. n = 6 animals per group. Two-way ANOVA was performed with
Bonferroni post tests. *P < 0.05.
background was seen in PBMC from animals immu-
nized with TiterMax Gold at any time-point. The
data show that the strongest cell-mediated response
is induced by immunization with ISC and that this is
sustained for >200 days after the third immuniza-
tion. Stimulation with an irrelevant antigen (BSA)
resulted in <1% of PBMC proliferating and stimula-
tion of PBMC from non-immunized koalas with the
chlamydial antigen combination did not exceed pro-
liferation levels seen in medium controls (data not
shown).
Antibody Responses in Plasma
IgG antibodies against the individual antigens,
TC0512, MOMP and NrdB were measured by ELISA.
TC0512 Antibody Response
At day 74, animals in all three groups had equiva-
lent levels of TC0512-specific IgG with EPT of 7000–
10,000 (Fig. 2a). At day 102 plasma from animals
immunized with Alhydrogel and TiterMax Gold for-
mulated vaccines had significantly higher (P < 0.05,
P < 0.01 respectively) plasma levels of IgG than the
animals immunized with ISC. At day 158, there was
no significant difference in TC0512 IgG levels among
the three groups, while at day 270, antibody titres
>5000 were still present in animals immunized with
the ISC or Alhydrogel formulations.
165
 CAREY ET AL.

(a) † TiterMax group (EPT 80,000) than in the other

15 000 Iscomatirx groups. At day 158, antibody levels in the TiterMax
Alhydrogel group had dropped to levels equivalent to those of
*
Titre Max Gold
the ISC and Alhydrogel groups and at day 270,
10 000
EPT value

animals in the ISC and Alhydrogel groups still had

titres of 20,000. Similar to the MOMP-specific
5000 response, the highest NrdB titres were seen in ani-
mals immunized with TiterMax but longer-lasting
responses were observed in the ISC and Alhydrogel
0 groups (Fig. 2b).
74

0
10

15

27
ay

ay

ay

ay
D

# MOMP Antibody Response

(b)
100 000 # At day 74, animals immunized with TiterMax-
formulated antigens had significantly higher MOMP-
80 000
specific IgG titres (EPT of 80,000) than animals
EPT value

60 000 immunized with either the Alhydrogel or ISC formu-

lations (EPT 40,000–55,000, Fig. 2c; P < 0.001). At
40 000
day 102, MOMP IgG titres were still significantly
20 000 higher in the TiterMax animals, although EPT had
dropped to approximately 60,000, while antibody
0
titres in the Alhydrogel and ISC groups remained at
74

0
10

15

27

an EPT of approximately 40,000 (P < 0.05 and

ay

ay

ay

ay
D

P < 0.001 respectively). A similar pattern was seen

(c) # # # at day 158 while at day 270 MOMP-specific IgG was
* † still detected in plasma of animals immunized with
100 000
ISC or Alhydrogel with titres in the ISC group
80 000 slightly higher, although this was not significant
(Fig. 2c). Overall, the highest MOMP-specific IgG
EPT value

60 000
titres were observed in the animals immunized with
40 000 TiterMax; however, antibody responses were
sustained for longer periods in the ISC and Alhydro-
20 000
gel groups, possibly because these received three
0 immunizations.
74

0
10

15

27
ay

ay

ay

ay
D

Genital Tract Antibody Responses

Fig. 2 Plasma was collected on the indicated days from animals
immunized with MOMP ⁄ CT0512 ⁄ NrdB, combined with ISC, Alhydrogel
or TiterMax Gold. Plasma IgG specific for CT0512 (a), NrdB (b) and
MOMP (c) were determined by ELISA as described in methods. n = 6
animals per group. Two-way ANOVA was performed with Bonferroni
post tests. *P < 0.05, !P < 0.01, #P < 0.001.
NrdB Antibody Response
At day 74, titres of NrdB-specific IgG of 50,000
were seen in the TiterMax group and this was sig-
nificantly higher (P < 0.001) than IgG levels in
either the ISC or Alhydrogel groups (Fig. 2b). At
day 102, titres were still significantly higher in the
166
Cloacal swabs were eluted in 1 mL of PBS-T and
assayed by ELISA for IgG antibodies against TC0512,
MOMP and NrdB.
TC0512 Antibody Response
The highest TC0512-specific IgG responses in swab
eluate at all time-points were observed in the
ISC-immunized animals (Fig. 3a). These levels were
significantly higher than in the Alhydrogel and Titer-
Max-immunized groups at day 74 (P < 0.05), and
the Alhydrogel group at day 158 (P < 0.05) and
were continuing to increase at day 270 (Fig. 3a),
reaching EPT of 300–400.
American Journal of Reproductive Immunology 63 (2010) 161–172
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 DEVELOPING A CHLAMYDIA VACCINE FOR THE KOALA

(a) ISC
MOMP Antibody Response
400 * * #
Alhydrogel
At days 74, 102 and 158, there was no difference in
Titre Max Gold
300 MOMP-specific IgG antibodies in swab eluates
EPT value

among the three experimental groups (Fig. 3c). At

200 day 270, antibody levels in samples from ISC-immu-
nized animals were significantly greater than those
100 in the Alhydrogel-immunized group (P < 0.01;
Fig. 3c). Antibody levels in all groups peaked at day
0 102, but in the ISC-immunized animals, the anti-
2

0
74

body levels were maintained, while in the other two

10

15

27
y

y
Da

Da

Da

Da

groups, levels declined beyond 6 months.

(b)
250

200
In Vitro Neutralization
Two of the three chlamydial antigens in this study
EPT value

150
(MOMP and TC0512) were isolated from the mouse
100 strain C. muridarum while the third antigen (NrdB)
50
was isolated from the human strain C. trachomatis
serovar D. We therefore tested the ability of plasma
0 from immunized animals to neutralize C. muridarum
in vitro and also to neutralize in vitro infectivity of
2

0
74

27
10

15
y
Da

the unrelated C. pneumoniae species LPCoLN isolated

Da

Da

Da

(c) from koalas.24 Plasma from Alhydrogel- and ISC-

400 * * #
immunized animals, at a 1 ⁄ 10 dilution, neutralized
300
in vitro infection of McCoy cells by 80–90% at day
74 and this was sustained up to day 270 (Fig. 4).
EPT value

200 Neutralization by plasma from Alhydrogel-immu-

nized animals was slightly higher at days 74, 102
100 and 158, although this difference was not significant.
Plasma from TiterMax-immunized animals inhibited
0 in vitro C. muridarum infection by >95% at day 74;
2

0
74

10

15

27
y

y
Da

Da

Da

Da

100
Iscomatirx
Fig. 3 Cloacal swabs were collected on the indicated days from
Neutralisation (%)

90 Alhydrogel
animals immunized with MOMP ⁄ CT0512 ⁄ NrdB, combined with either Titre Max Gold
ISC, Alhydrogel or TiterMax Gold. Swabs were eluted in 1 mL of 80
PBS-T and IgG specific for CT0512 (a), NrdB (b) and MOMP (c) were
determined by ELISA as described in methods. n = 6 animals per 70
group. Two-way ANOVA was performed with Bonferroni post tests.
60
*P < 0.05, #P < 0.01.
50
74

0
10

15

27
ay

ay

ay

ay
D

NrdB Antibody Response

At days 74, 102 and 158, there was no difference in
NrdB-specific IgG in swab eluates among the three
experimental groups (Fig. 3b). On day 270, antibody
was still evident in animals immunized with ISC and
Alhydrogel, but was undetectable in animals immu-
nized with TiterMax.
Fig. 4 Plasma from animals immunized with MOMP ⁄ CT0512 ⁄ NrdB,
combined with either ISC, Alhydrogel or TiterMax Gold was diluted
1:10 and 0.1 mL incubated with 1000 ifu of Chlamydia muridarum for
1 hr as described in methods. Samples were then plated on McCoy
cell monolayers and incubated for a further 30 hr. Cells containing
inclusions were identified by staining with FITC-labeled anti-chlamydial
LPS. Unpaired t-tests were performed to determine the significance of
differences between groups at the indicated time-points.

American Journal of Reproductive Immunology 63 (2010) 161–172

ª 2010 John Wiley & Sons A/S 167
 CAREY ET AL.
however, the percent neutralization steadily declined
and was approximately 70% at day 158 (Fig. 4).
Thus, at early stages, plasma from TiterMax-immu-
nized animals showed the greatest neutralizing activ-
ity, but this was not sustained throughout the
10 months of the trial compared with that seen in
the other two groups. At a 1 ⁄ 250 dilution, plasma
from the Alhydrogel-immunized animals showed the
greatest levels of in vitro neutralization (data not
shown) at day 74. The in vitro neutralization level
dropped to around 50% at day 102 and was main-
tained at 50–60% throughout the remainder of the
trial. At this dilution, plasma from ISC-immunized
animals neutralized infectivity by approximately
65% at day 74 and neutralization activity was sus-
tained at 35–55% for the rest of the study. Plasma
from TiterMax-immunized animals at a dilution of
1 ⁄ 250 caused a 40–50% reduction in infectivity at
days 74, 102 and 158.
Plasma was also tested for neutralization of C.
pneumoniae infection, using the LPCoLN strain,
which was isolated from koalas. Plasma (1 ⁄ 10 dilu-
tion) from Alhydrogel-immunized koalas neutral-
ized C. pneumoniae infection by approximately 30%
across the entire time course of the study (Fig. 5).
Similar levels of C. pneumoniae neutralization were
seen using plasma from ISC-immunized animals at
days 74, 102 and 158, but had dropped to 10–12%
neutralization at day 270 (Fig. 5). Plasma from
TiterMax-immunized animals also neutralized C.
pneumoniae infection by 25–30% at days 74 and
102. At a 1 ⁄ 50 dilution, only plasma from the
Alhydrogel-immunized animals contained C. pneu-
moniae-neutralizing activity throughout the entire
study. At early time-points, this plasma neutralized
30–40% of C. pneumoniae infection and this had
dropped to approximately 15% by day 270 (data
not shown). Plasma from ISC-immunized animals
neutralized infectivity by 15% at day 74, but this
had dropped to baseline at day 102 while plasma
from TiterMax-immunized animals did not signifi-
cantly inhibit in vitro infectivity throughout the
entire study period. Plasma from immunized
animals, of any group, was not able to neutralize
in vitro infectivity of a koala isolate of C. pecorum
(data not shown).
Discussion
Immunization of koalas with ISC and Alhydrogel
containing vaccines were well tolerated, whereas
168
80
Iscomatirx
Alhydrogel
Neutralisation (%)
60
Titre Max Gold
40
20
0
74
0
10
15
27
ay
ay
ay
ay
D
Fig. 5 Plasma from animals immunized with MOMP ⁄ CT0512 ⁄ NrdB,
combined with either ISC, Alhydrogel or TiterMax Gold was diluted
1:10 and 0.1 mL incubated with 1000 ifu of a koala isolate of Chla-
mydia pneumoniae for 1 hr as described in methods. Samples were
then plated on McCoy cell monolayers and incubated for a further
30 hr. Cells containing inclusions were identified by staining with FITC-
labeled anti-chlamydial LPS. Unpaired t-tests were performed to deter-
mine the significance of differences between groups at the indicated
time-points.
50% of koalas immunized with TiterMax developed
abscesses at the injection site after the second vacci-
nation. Animals in this group therefore only
received two of the three planned immunizations
and samples were not collected from these animals
beyond day 158. Strong PBL proliferation was still
present at 270 days in the ISC-immunized koalas
with almost 20% of PBL proliferating (>3 cell divi-
sions) as determined by CFSE dye-dilution assay. At
the early time-points (days 74 and 102), plasma
antibody specific for all three antigens was the high-
est in the animals immunized with TiterMax; how-
ever, no samples were collected beyond day 158
from this group because of the adverse reactions to
immunization. Antibody against all 3 antigens was
sustained throughout the study in animals immu-
nized with ISC or Alhydrogel. Overall, NrdB and
MOMP antibody titres in plasma were 4–8 fold
higher than the TC0512 antibody titres. Plasma-neu-
tralizing activity for C. muridarum was the highest in
plasma from animals immunized with Alhydrogel
and ISC with slightly higher neutralizing activity
seen in the Alhydrogel group. Neutralizing activity
for C. pneumoniae was the greatest in plasma from
animals immunized with Alhydrogel. Significantly
higher levels of inhibition of C. muridarum infection,
compared with C. pneumoniae infection were
observed, probably because both MOMP and TC0512
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
 DEVELOPING A CHLAMYDIA VACCINE FOR THE KOALA
were derived from C. muridarum. It is tempting to
speculate that MOMP-specific antibody and, to a les-
ser extent, TC512-specific antibody were responsible
for neutralizing C. muridarum infection, while anti-
body against the highly conserved antigen NrdB
neutralized C. pneumoniae infection. This is perhaps
not surprising and confirms the immunodominance
of MOMP as the major antigen recognized during
the humoral response to chlamydial infection in
most species.25 No neutralization of in vitro infection
of a koala isolate of C. pecorum was observed with
any of the plasma samples (data not shown). This
lack of neutralization of C. pecorum does, however,
suggest that the koala response to NrdB, even
though this antigen is highly conserved, was not
neutralizing. The three antigens we chose to evalu-
ate in the trial had different levels of amino acid
conservation across the chlamydial species. NrdB
had the highest level of conservation, estimated
between 90 and 92% across the seven chlamydial
species for which full genome sequences are avail-
able. We have previously predicted both human and
mouse T-cell epitope regions in this protein and
these were 100% conserved across the chlamydial
species. TC0512 (omp85) had the next highest level
of amino acid conservation, 70–75%, with several
regions of higher level variation observed. As previ-
ously well known, ompA (MOMP) had the lowest
level of conservation, between 60 and 65%, again
with regions of conservation interspersed with
regions of high variability.
Of the three adjuvants used, TiterMax Gold
caused severe local reactions at the injection site
in 50% of the animals following the second
immunization. At the day 74 time-point, plasma
IgG levels against two of the three antigens were
the highest in the animals immunized with Titer-
Max Gold, whereas PBMC proliferation was the
lowest in this group at all time-points. This is
consistent with the manufacturer’s claims that
TiterMax adjuvants elicit antibody responses supe-
rior to Freund’s complete adjuvant (CFA) in most
species. TiterMax Gold is a water-in-oil emulsion
comprising the block co-polymer CRL-8300, the
metabolizable oil squalene and a microparticle
stabilizer. Whilst TiterMax adjuvants are claimed to
cause less severe side effects than CFA in many
species, the development of abscesses at the injec-
tion site that were obviously painful and impaired
mobility of animals for several days make this
adjuvant unacceptable for use in koalas.
American Journal of Reproductive Immunology 63 (2010) 161–172
ª 2010 John Wiley & Sons A/S
Aluminum-based adjuvants have been used in
both human and veterinary vaccines for more than
half a century. Veterinary vaccines containing alumi-
num adjuvants have been developed to target viral,
bacterial and parasite infections (reviewed in26). Alu-
minum adjuvants induce primarily Th2 immunity,
driving strong antibody responses, but usually do
not elicit strong cell-mediated immunity. Mecha-
nisms of action include formation of an antigen
depot, production of particulate antigen to facilitate
targeting to APCs, activation of complement and
stimulation of monocyte ⁄ macrophages.26,27 Recently,
it has been shown that Alum, following intraperito-
neal injection, induces the differentiation of mono-
cytes into CD11c+ inflammatory dendritic cells that
migrate to draining lymph nodes. Stimulation of
these dendritic cells involves activation of the NALP3
inflammasome, the production of IL-1b and induc-
tion of the endogenous danger signal uric acid28,29
all of which are required for adjuvant action. Plasma
IgG levels in koalas immunized with Alhydrogel
were less than those in the TiterMax-immunized
group at the early time-points and equivalent to
those in the ISC-immunized animals at the later
time-points. Chlamydia-neutralizing activity in
plasma from Alhydrogel-immunized animals was
maintained at high levels for the duration of the
study. Animals immunized with Alhydrogel also
maintained a local genital IgG response up to at least
day 158, while PBMC proliferation in this group was
not as great or long lasting as that seen in ISC-
immunized animals, consistent with the expected
Th2 predominance of these adjuvants.
Immune stimulating complex (ISCOMATRIX) is
composed of purified saponin (ISCOPREP) com-
plexed with cholesterol and phospholipid (reviewed
in.30,31 ISCOMATRIX induces both strong antibody
and cell-mediated responses, including cytotoxic
T-lymphocyte (CTL) responses30,32,33 in most species
tested. ISCOMATRIX has also been shown to reduce
the antigen dose required to elicit an antibody
response34 and can be delivered by the intranasal
route, where it is able to elicit significant mucosal
responses (reviewed in35). The mechanism of action
seems to depend on the induction of multiple cyto-
kines including IL-1, IL-2, IL-4, IL-5, IL-6, IL-10,
IL-8, IL-12 and IFN-c, which occurs in the draining
lymph nodes.31 Up-regulation of CD86 and MHC
class II on APC may also be important for adjuvant
function.36 As ISCOMATRIX is cleared rapidly from
the injection site, formation of an antigen depot is
169
 CAREY ET AL.
probably not important for adjuvant function.30 In
our studies, animals immunized with ISC mounted
robust PBMC proliferative responses that were both
superior to those induced by Alhydrogel and Titer-
Max and were maintained for the entire length of
the study (>270 days). The longevity of this response
is encouraging as healthy koalas have a life span of
10–12 years in the wild. Plasma IgG levels in ISC
immunized animals were equivalent in magnitude
and duration to those in the Alhydrogel-immunized
group, while genital IgG responses were the highest
in ISC-immunized koalas. This combination of a
robust, long-lived cell-mediated response, together
with a sustained systemic and mucosal antibody
response, suggests that immunization with ISC
should elicit protective immunity against chlamydial
infection.37 Studies are currently underway to
develop assays to determine if the PBMC prolifera-
tive response is also characterized by secretion of
IFN-c, a cytokine essential for protection against
chlamydial infection. Future studies will also investi-
gate intranasal immunization of koalas using ISC
to determine if protective immunity is elicited by
this route.
Despite the fact that all animals were immunized
subcutaneously, antibody against all 3 antigens was
detected in cloacal swab eluates. These antibodies
were sustained for 270 days in animals immunized
with Alhydrogel and ISC. Swab antibodies were of
the IgG class, consistent with findings in humans
and rodents that serum-derived IgG plays a major
protective role in the female reproductive tract.38
We are currently developing an IgA-specific antise-
rum to determine if IgA is also present in the koala
reproductive tract.
In summary, our studies have shown that subcu-
taneous immunization of koalas with a multi-sub-
unit chlamydial vaccine, adjuvanted with either ISC
or Alhydrogel, is well tolerated and elicits both
humoral and cell-mediated immune responses that
last >270 days. Plasma antibodies from both groups
were able to neutralize chlamydial infection in vitro.
PBMC proliferative responses and genital IgG levels
were the highest in animals immunized with ISC
suggesting that this adjuvant is best suited for induc-
tion of protective immunity against chlamydial infec-
tions in the koala. The studies also highlight the
need to use antigens from Chlamydia species that
infect koalas. We now have a collection of C. pecorum
strains isolated from koalas in South-East Queens-
land. Antigens have been prepared from these and
170
are currently being used in ongoing immunization
and challenge studies. However, the induction of
long-lived systemic and mucosal antibody and cell-
mediated immunity by immunization with a safe
ISC-adjuvanted vaccine, that can be boosted in cap-
tive animals by repeat vaccination and in wild ani-
mals could be boosted by natural challenge, is
extremely encouraging and provides evidence that
protective immunity against chlamydial infection in
the koala can be induced by vaccination.
Acknowledgments
We thank Dr. John Walker VMRD, Pfizer, Australia
for provision of the Immunostimulating Complex
(ISC) adjuvant, Ms Candice Mitchell for provision of
the C. pneumoniae LPCoLN strain, Dr. Adam Polking-
horne for assistance with chlamydial gene alignment
studies and Dr. Kelly Cunningham for review of the
manuscript. We also gratefully acknowledge the gen-
erous financial support provided by Lone Pine Koala
Sanctuary.
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