Vaccine 31 (2013) 52025209

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Vaccine
j our nal homepage: www. el sevi er . com/ l ocat e/ vacci ne
An adult zebrash model for preclinical tuberculosis vaccine
development
Kaisa E. Oksanen
a,
, Nicholas J.A. Halfpenny
a
, Eleanor Sherwood
a
,
Sanna-Kaisa E. Harjula
a
, Milka M. Hammarn
a
, Maarit J. Ahava
a
, Elina T. Pajula
a
,
Marika J. Lahtinen
a
, Mataleena Parikka
a,1
, Mika Rmet
a,b,1
a
Institute of Biomedical Technology, BioMediTech, University of Tampere, FIN-33014 Tampere, Finland
b
Department of Pediatrics, Tampere University Hospital, FIN-33521 Tampere, Finland
a r t i c l e i n f o
Article history:
Received 20 May 2013
Received in revised form16 August 2013
Accepted 28 August 2013
Available online 20 September 2013
Keywords:
Zebrash
Tuberculosis
Vaccine
Mycobacteriummarinum
a b s t r a c t
Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the
current live vaccine, Bacillus CalmetteGurin. Tuberculosis vaccine research has been hampered by a
scarcity of animal models which replicate human disease and are suitable for large-scale studies. We
have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes
an infection resembling human tuberculosis in adult zebrash (Danio rerio). In the present study we
use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial
antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrash from mycobacterial infection. Using a low-
dose (2030 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the
amount of infected organs were reduced in the DNA vaccinated sh. Likewise, when infecting with a lethal
infection dose (20,00027,000 bacteria), vaccination signicantly reduced both mortality and bacterial
counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were
associated with enhanced expression of interferon gamma. Our results indicate that the zebrash is a
promising new model for preclinical tuberculosis vaccine research.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Despite an existing Bacillus CalmetteGurin (BCG) vaccine
and available antibiotic treatments, the global burden of tuber-
culosis (TB) remains enormous. TB is responsible for 1.4 million
deaths annually, and 8.7 million new cases occurred in 2011 [1].
One third of the worlds population is estimated to carry a latent
Mycobacterium tuberculosis infection, and 310% of these eventu-
ally develop active TB [2,3]. Individuals with impaired immunity
have an increased risk of disease progression.
Although BCG protects children against the disseminated forms
of TB, its efcacy in adults is inconsistent [2,4]. Moreover, BCG
does not prevent the establishment of M. tuberculosis infection or
the reactivation of the latent disease [3,5] and as a live vaccine
is unsafe among the immunocompromised [6]. Introducing vac-
cines that could replace and/or boost the BCG is a key strategy in
Abbreviations: dpi, days post infection; wpi, weeks post infection; CFU, colony
forming unit.

Corresponding author. Tel.: +358 50 3832823.

E-mail address: kaisa.oksanen@uta. (K.E. Oksanen).
1
Both authors contributed equally.
controlling the TB pandemic. Proposed newapproaches include M.
tuberculosis mutants [7,8], recombinant BCG strains [9], and DNA
[10,11] or subunit vaccines based on mycobacterial antigens [5,12].
The three main animal models used for TB vaccine develop-
ment are mice, guinea pigs and non-human primates, usually used
sequentially in this order [13]. Other common models include the
rabbit [14] and the bovine model [15,16]. All current animal mod-
els have limitations; mice, for instance, cannot completely reect
human TB as they do not form caseating granulomas or sponta-
neously develop latency [17,18]. Non-human primates reproduce
the human disease well yet their use raises ethical concerns. Gen-
erally, mammalian models are relatively expensive and laborious
to be used for large-scale studies.
The small, low-cost zebrash (Danio rerio) is gaining popularity
as a model for infectious diseases and immune function [1921].
It is neurophysiologically the simplest well-established vertebrate
model organism with both innate and adaptive arms of immu-
nity [20,22,23] and can be considered an ethical choice compared
to mammalian models. Mycobacterium marinum, a natural sh
pathogen genomically closely related to M. tuberculosis, induces a
zebrash disease sharing the main histological and pathological
features of human TB [17,18,24]. A low-dose M. marinuminfection
spontaneouslydevelops intoa latent shinfection, control of which
0264-410X/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.08.093
K.E. Oksanen et al. / Vaccine 31 (2013) 52025209 5203
depends on the adaptive immune response [18,25]. Notably, pre-
infection with attenuated M. marinum has been shown to protect
zebrash against mycobacteriosis [26], conrming that zebrash
can be vaccinated against M. marinum.
In this study, we show that adult zebrash can be vaccinated
against mycobacterial infection both with BCG and a DNA vac-
cine encoding known immunoprotective mycobacterial antigens,
Ag85B, ESAT-6 and CFP-10. Zebrash thus presents itself as an
attractive model for TB vaccine research.
2. Materials and methods
2.1. Fish
Adult (58month-old) wildtypeABzebrashor adult rag1(/)
hu1999 mutant sh were used for experiments. The sh were
maintained in a ow-through system with a light/dark cycle of
14h/10h under identical husbandry and water quality conditions.
Animal studies were approved by the National Animal Experiment
BoardinFinlandandconductedinaccordancewiththeEU-directive
2010/63/EU on the protection of animals used for scientic pur-
poses.
2.2. DNA construction
The pCMV-EGFP plasmid (Addgene plasmid 11153 [27]) was
used as the basis for all plasmid constructs. Ag85B (MMAR 2777),
CFP-10 (MMAR 5449) and ESAT-6 (MMAR 0188) were amplied by
colony PCR from M. marinum grown on 7H10 Middlebrook OACD
plates (BD Biosciences, Franklin Lakes, NJ) using Phusion

High-
Fidelity PCR Kit (NEB, Ipswich, MA). PCR products were puried
and restriction cloned to be expressed with a C-terminal EGFP
tag. pCMV-plasmidwithout the EGFP-encoding gene (blank vector)
was constructed by excising the EGFP gene frompCMV-EGFP. Plas-
mids were transformed into Escherichia coli One Shot TOP10 cells
(Invitrogen) and constructs conrmed by sequencing. For in vivo
experiments, plasmidDNAwas preparedusingtheQIAGENPlasmid
Plus Maxi Kit (Qiagen, Venlo, The Netherlands).
2.3. Vaccination experiments
Before vaccination, sh were briey anaesthetized in 0.02%
3-aminobenzoic acid ethyl ester (pH 7.0) (SigmaAldrich). For
BCG vaccinations, sh were intraperitoneally (i.p.) or intramuscu-
larly (i.m.) injected with 810
3
CFU BCG Danish 1331 (Statens
Serum Institut, Copenhagen, Denmark). The i.p. injection was
performed using an Omnican 100 30G insulin needle (Braun,
Melsungen, Germany). For DNA vaccinations, 615g of plas-
mid/sh was injected in the dorsal muscle using aluminosilicate
capillary needles and PV830 Pneumatic PicoPump microinjec-
tor (World Precision Instruments, Sarasota, FL). The target tissue
was electroporated (6 pulses of 50V, 5ms each) using tweezer-
type electrodes (BTX/Harvard Apparatus, Holliston, MA) and a
GenePulser-electroporator (Bio-Rad, Hercules, CA). Injection with
sterile PBS or the pCMV-EGFP plasmid was used as negative control
for all vaccination experiments.
2.4. Fluorescence microscopy, Western blotting and GFP ELISA
In vivo expression of the plasmid DNA-derived protein products
(EGFP and its antigen recombinants) was veried by uores-
cence microscopy, Western blotting and ELISA. Microscopy was
done with a Lumar V.12 uorescence stereomicroscope (Carl Zeiss
MicroImaging, Jena, Germany). For Western blotting and ELISA,
sampleshwerecollectedat adesiredtimepoint andhomogenized
with a PowerLyzer
TM
24 Bench Top Bead-Based Homogenizer (MO
BIO Laboratories). Pierce

BCA Protein Assay Kit (Thermo Fisher

Scientic, Waltham, MA) was used to dene the total protein con-
centration of each lysate. For Western blotting, sh homogenates
were resolved on a 10% SDS-PAGE gel, blotted on a nitrocellulose
membrane andananti-GFP antibody NB600-303 (Novus biological,
Littleton, CO) and a HRP conjugate of goat anti-rabbit IgG (Invi-
trogen) were used for target protein detection. GFP ELISA Kit (Cell
Biolabs, San Diego, CA) was used for determining relative levels
(normalized with the total protein concentration of the samples)
of EGFP or its recombinants in sh tissue homogenates.
2.5. Bacterial infections
M. marinum(ATCC927) was culturedandpreparedfor infections
as in [18]. For experimental infections, sh were anaesthetized as
described above and i.p. injected 34 weeks after immunizations.
Toverifythebacterial dose, samples of bacterial dilutionweretaken
during infecting and plated on 7H10 plates.
2.6. RNA and DNA extraction and qPCR
RNA extraction from infected zebrash was done as previ-
ously described [18]. For cytokine gene-expression measurements,
Bio-Rad iScript One-Step RT-PCR Kit with SYBR Green was used.
The expression of GAPDH (ZDB-GENE-030115-1) was used for
normalization, and analysis of relative changes in gene expres-
sion was done using the ddCt algorithm [28]. 12wpi with a high
(20,00027,000CFU) dose of M. marinum, sh were euthanized,
the contents of their abdominal cavities were collected and DNA
extraction and M. marinum specic qPCR to quantify bacteria per
sh were carried out as reported [18].
2.7. Histology
Fish were euthanized with 0.04% 3-aminobenzoic acid ethyl
ester (SigmaAldrich) and tissue cross-sections were prepared
and stained as in [18]. In brief, the sh were xed in formalin,
decalcied with EDTAcitrate and embedded in parafn. 5mlon-
gitudinal sections were cut and every 40th section was placed
on a slide. The sh were sectioned thoroughly so that the entire
kidney tissue lining the spine was included. The slides were
stained with ZiehlNeelsen staining and analyzed with an Olym-
pus BX51 microscope. Bacterial load per sh was assayed fromthe
ZiehlNeelsen-stained tissue cross-sections by (1) counting gran-
ulomas and (2) identifying and counting the organs containing
bacteria per slide. Additionally, some sections were stained using
anti-EGFP antibody NB600-303 (Novus Biologicals) and DAKO
EnVision
TM
+ System, HRP (Dako, Glostrup, Denmark) following
standard procedures.
2.8. Survival experiments
Fish were monitored daily and humane end point criteria
approved by the national ethical board were followed. Fish were
euthanized with an anesthetic overdose if they showed any of the
following signs: abnormal swimming, gasping, observable swelling
or wasting, tissue damage, lack of response to touch.
2.9. Statistical analysis
Statistical analyses were carried out using the log rank
MantelCoxtest (for the survival experiments) or a non-parametric
MannWhitney test (one-tailed). Values of p<0.05 were consid-
ered signicant.
5204 K.E. Oksanen et al. / Vaccine 31 (2013) 52025209
3. Results
3.1. BCG protects adult zebrash against an experimental M.
marinuminfection
As BCG vaccination has been shown to restrict the progression
of mycobacteriosis in multiple animal models [13], we rst exam-
ined whether BCG is also protective in the zebrash. Groups of
adult, wild type zebrash were vaccinated with 810
3
CFU of
BCG, either i.m. or i.p. Control shwere injectedi.p. withsterile PBS.
Four weeks after vaccination, the sh were infected i.p. with a high
dose (19,500800CFU) of M. marinum. The experimental groups
were then followed for 35 weeks for survival. BCG vaccination was
unabletoprevent infectionwithmycobacteria, andmortalitydueto
mycobacterial disease occurred in all groups. However, vaccination
with BCGimproved sh survival: in the BCG-vaccinated groups the
end-point mortalities were 45.0% (i.p. route) and 57.1% (i.m. route),
whereas in the control group the mortality was signicantly higher
(84.2%) (Fig. 1). Non-vaccinated (buffer injected or non-injected,
n=70 in both groups), non-infected sh kept in the same sh unit
had mortality rates of under 10% in 70 weeks (data not shown).
There was no signicant difference between the end-point mortal-
ities of the two vaccination routes, although sh vaccinated via the
i.m. route began to die earlier, suggesting that the i.p. BCG vacci-
nation may more efciently control the early phase of M. marinum
infection.
3.2. Mycobacterial antigens can be expressed in adult zebrash
by DNA vaccination
Next, we tested whether DNA-based vaccines could also be used
in adult zebrash. As in vivo electroporation has been shown to
enhance transgene expression and DNA vaccine immunogenicity
in animal models and humans [2931], we microinjected EGFP-
encoding plasmid (pCMV-EGFP) into the dorsal muscle of adult
zebrash and exposed the injection site to different numbers of
Fig. 1. Vaccination with the Bacillus CalmetteGurin (BCG) vaccine improves
adult zebrash survival from infection with a high mycobacterial dose. Groups of
zebrash (n=20 in each group) were immunized with BCG intramuscularly (i.m.)
or intraperitoneally (i.p.). The control group was injected i.p. with PBS. Four weeks
after immunization, the sh were infected with 19,500800CFU of M. marinum.
Fish were then followed for 35 weeks for survival. *P<0.01 (MantelCox).
50V pulses. Electroporation enhanced the plasmid-derived EGFP
expression: hardly any uorescence was detected in the non-
electroporated sh, whilst sh exposed to 6 pulses had clearly
visibleEGFPexpression(Fig. S1a). Sixpulses gavea 4-foldincrease
in EGFP levels (Fig. S1b). Plasmid-derived reporter gene expression
was detected up to 30 days post injection, with maximal reporter
proteinlevel being reachedin34 days (Fig. 2a). Insome sh, EGFP-
expressing muscle cells were observed up to 60 days (Fig. S1c).
Essentially all sh showed EGFP expression in muscle cells, but also
in other cell types including cells within the epidermis (Fig. S1d).
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.
08.093.
We then chose three extensively studied mycobacterial anti-
gens: Ag85B, CFP-10 and ESAT-6, which are immunodominant
Fig. 2. Electroporation-mediatedDNAvaccine delivery induces long-lasting reporter gene expressionand canbe used to express mycobacterial antigens inadult zebrash. (a)
EGFP expression kinetics in adult zebrash after an i.m. injection of 5g pCMV-EGFP. The injection was followed by electroporation of the injection site (6 pulses, 50V, 5ms
each). At each time point (160 days after injection), sh were collected (n=4 per each time point) and relative EGFP levels were determined fromsh tissue homogenates by
ELISA. Injection with a blank vector was used as a control (ctrl). (b and c) 6g of plasmid DNA encoding Ag85B-EGFP, ESAT6-EGFP or CFP10-EGFP was injected i.m., followed
by electroporation. The control group (ctrl) was injected with a blank vector. (b) ELISA analysis of sh tissue homogenates showing Ag85B, CFP-10 and ESAT-6 recombinant
levels 3 days after plasmid injection. n=5 in each group. (a and b) *P<0.05; **P<0.03; ***P<0.01 (One-tailed MannWhitney), error bars =mean+SD, N.S., not signicant. (c)
In vivo expression of the EGFP-tagged recombinant antigens (green) under a uorescence microscope; shown is a merge of bright eld and uorescence microscopy images.
Circle: injection site, scale bar =2000m.
K.E. Oksanen et al. / Vaccine 31 (2013) 52025209 5205
virulence factors shared between M. tuberculosis and M. marinum
and have appeared promising in vaccine trials in different exper-
imental animal models [5,11,3234], as well as in human trials
[35,36]. The corresponding genes were separately cloned into the
pCMVplasmid to be expressed with a C-terminal EGFP tag (Fig. S2).
Following i.m. plasmid injection, CFP-10, ESAT-6 and Ag85B-EGFP
recombinants were detectable in sh tissue by both uorescence
microscopy and GFP ELISA (Fig. 2b and c). Expression of recombi-
nant CFP10-EGFP, ESAT6-EGFP and Ag85B-EGFP was conrmed by
Western blotting (Fig. S2b).
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.vaccine.
2013.08.093.
3.3. Zebrash DNA vaccination with Ag85B, ESAT-6 and CFP-10
leads to enhanced interferon gamma expression upon
mycobacterial infection
Subsequently, we investigated whether DNA vaccination with
Ag85B, CFP-10 and ESAT-6 has an immunogenic effect in the
zebrash. Fish were immunized either with a combination of the 3
antigen-encoding plasmids (pCMV-Ag85B/CFP-10/ESAT-6), pCMV-
EGFP plasmid, or PBS. The pCMV-EGFP control was included to
verify that the observed effects are due to an antigen-specic
response rather than unspecic immune stimulation caused by
bacterial DNA [37]. Four weeks later, sh were challenged with
1500CFU M. marinum (a dose leading to high long-termmortality
but very limited mortality in the rst weeks of infection). Inter-
feron gamma (IFN1-1; ZDB-GENE-060210-1) expression levels in
the infected sh were then measured by qRT-PCR 1, 7 or 14dpi. No
differences were seen between the groups during the rst week,
although IFN1-1 expression was highly (approximately 70-fold)
upregulated in all groups 7dpi (Fig. 3). 14dpi the sh vaccinated
with pCMV-Ag85B/CFP-10/ESAT-6 showed IFN1-1 induction of
over 100-fold, whilst induction levels in the pCMV-EGFP and PBS
control groups were lower than 20-fold on average.
3.4. A DNA vaccine encoding Ag85B, ESAT-6 and CFP-10 protects
adult zebrash against low-dose M. marinuminfection
IFN- production levels or other biomarkers do not necessarily
correlate with protection against mycobacterial disease [38]. We
have previously set upmethodology to investigate mycobacteriosis
Fig. 3. Interferon gamma expression in vaccinated zebrash upon infection with
M. marinum. Adult zebrash were immunized with pCMV-Ag85B/CFP-10/ESAT-
6, pCMV-EGFP or PBS. Four weeks after immunization, the sh were infected
with 1500130CFU of M. marinum. Fish were then collected 1, 7 or 14dpi for
gene expression analysis; n=20/group at 7dpi, otherwise n=10/group. Data were
combined from two independent experiments. *P<0.03, **P<0.008 (one-tailed
MannWhitney), error bars =mean+SD, N.S., not signicant.
in adult zebrash [18], and these methods were used to study
the protective effects of DNA vaccination by determining bacterial
distribution in the sh (dened by histological staining), bacte-
rial loads in the sh (dened by qPCR) and sh survival upon
vaccination (Fig. S3). To characterize the effects of DNA vaccina-
tion against infection with a low mycobacterial dose leading to
a latent infection [18], sh were infected i. p. with 228CFU of
M. marinum 3 weeks after pCMV-Ag85B/CFP-10/ESAT-6 vaccina-
tion. At 6wpi the sh were collected, stained with ZiehlNeelsen
staining, and histologically analyzed for detection of granulomas
and affected organs. Compared to the pCMV-EGFP-injected con-
trols, the vaccinated sh had signicantly less granulomas and
fewer affected organs (Fig. 4a and b). The number of granulo-
mas in the vaccinated group was 65.9% lower and there were
37.7% less affected organs than in the control group. Organs
infected by M. marinum in the control sh included the pancreas,
gonads, liver, gut, mesentery, spleen and kidney, while in the
antigen-vaccinated sh the spleen and kidney were not affected.
Examples of granulomas in different target organs are shown
in Fig. 4c. No granulomas or individual bacteria were detected
in healthy, non-infected sh (n=8) that were included in the
experiment to rule out the possibility of background infection
with any mycobacterial species (data not shown). These data sug-
gest that the progression of a mycobacterial infection in adult
zebrash can be reduced with DNA vaccination. The presence of
fewer granulomas and the restricted distribution of mycobacteria
Fig. 4. Effect of DNA vaccination with Ag85B, CFP-10 and ESAT-6 on mycobacterial disease progression in adult zebrash after infection with a lowbacterial dose. Fish were
vaccinated with a cocktail of plasmids encoding 3 selected mycobacterial antigens: Ag85B, ESAT-6 and CFP-10 (pCMV-Ag85B/CFP-10/ESAT-6), 610g of plasmid altogether.
The control group (pCMV-EGFP) was injected with 610g of pCMV-EGFP. Three weeks later, the sh were i.p. infected with 228CFU of M. marinum, and 6wpi collected
for histological analysis. Each sh was sectioned into 5mslides, out of which every 40th was stained and analyzed. The total number of granulomas per sh equals the sum
of granulomas counted fromall stained slides of the corresponding sh. DNA vaccination reduced (a) the number of granulomas and (b) the number of affected organs. The
following organs or types of tissue were examined: pancreas, gonads, muscle, liver, gut, mesentery, spleen, kidney, swimbladder, heart. (a and b) *P<0.005 **P <0.003 (One-
tailed MannWhitney). Error bars =mean+SD, n(Ag) =21, n(GFP) =13; data were combined fromtwo independent experiments. (c) ZiehlNeelsen staining of mycobacteria
(bright red) in tissue sections of different target organs of infected control sh. Granulomas are indicated by arrowheads.
5206 K.E. Oksanen et al. / Vaccine 31 (2013) 52025209
Fig. 4. (Continued).
have been linked to a reduced chance of developing an active dis-
ease [3].
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.vaccine.
2013.08.093.
3.5. DNA vaccination improves zebrash survival and reduces
mycobacterial burden during an active, rapidly progressing
infection
To assay the effect of sh DNA vaccination on an active, rapidly
progressing mycobacterial infection, DNA-vaccinated sh were
infected with a high dose (20,5004300CFU) of M. marinum 3
weeks after immunization. Survival was then monitored for 12
weeks. Vaccination with the combination of Ag85B, ESAT-6 and
CFP-10 increased sh survival rates upon infection by 29.8% com-
pared to pCMV-EGFP-vaccinated controls (Fig. 5a). 12wpi, M.
marinum-specic qPCR was used to determine the mycobacterial
loads both in vaccinated and control groups. The mean bacterial
loadinthe mock-vaccinatedshwas 2.8-fold(64.5%) higher thanin
the vaccinated group (Fig. 5b). No bacteria could be detected from
non-infected sh (n=10; data not shown). Taken together, these
results support the observation that zebrash can be protected
against an M. marinuminfection by DNA-vaccination.
3.6. Adaptive immunity is required for the protection gained by
zebrash vaccination
As zebrashare knowntohave strong innate immune responses
[39], we next examined whether the vaccine-induced protection
K.E. Oksanen et al. / Vaccine 31 (2013) 52025209 5207
Fig. 5. Effect of adult zebrash DNA vaccination on the course of infection with a
high mycobacterial dose. Fish were vaccinated with a cocktail of plasmids encod-
ing Ag85B, ESAT-6 and CFP-10 (Ag) (10g DNA/sh). The control group (GFP) was
injected with 10g of pCMV-EGFP. Three weeks later, the sh were infected with
20,5004300CFU of M. marinum. (a) Survival analysis of DNA-vaccinated (Ag) and
control (GFP) sh after infection. Data was averaged fromtwo independent experi-
ments; n(Ag) =40, n(GFP) =38. *P<0.03 (MantelCox). (b) 12 weeks after infection,
sample sh from both groups (Ag and GFP) were collected and the mycobacterial
load in each sh was assessed by qPCR (n=10 in both groups, combined data from
two independent experiments). The control sh had bacterial loads 2.8-fold higher
than the vaccinated sh. *P<0.03 (one-tailed MannWhitney).
is dependent on the adaptive immune response. Recombination
activation protein 1 (Rag1) decient zebrash lacking functional
T and B cells [40] were vaccinated with either pCMV-Ag85B/CFP-
10/ESAT-6or withpCMV-EGFP. Four weeks after immunization, the
vaccinated sh along with a group of non-injected rag1(/) group
were infected with 27,3002400CFU of M. marinumand followed
for their survival. Within 9wpi, all sh from both the pCMV-
Ag85B/CFP-10/ESAT-6 and the control groups had died (Fig. 6).
No signicant differences in the course of infection were detected
between the vaccinated sh and the control groups. These results
indicate that vaccine-induced protection depends on the adaptive
immune response mediated by T and/or B lymphocytes.
4. Discussion
An improved vaccine is needed to relieve the global TB burden.
Vaccine researchanddevelopment, however, is still highly depend-
ent on the use of animal models. Although various experimental
organisms have been used to model mycobacterial infection,
M. tuberculosis naturally only infects primates, complicating TB
research. Moreover, mammalian models are not optimal for large
screens due to logistical, budgetary and ethical reasons and novel
models for preclinical testing of TBvaccines are warranted. As there
is currentlynovaccine preventingthe formationandreactivationof
latent mycobacterial disease, there is a particular search for models
mimicking latent TB and its reactivation to an active disease.
Fig. 6. DNA vaccination does not affect the course of infection in Rag(/) mutant
sh infected with a high mycobacterial dose. Recombination activation protein 1
(Rag1) decient zebrash lacking functional T and B cells were vaccinated with
either pCMV-Ag85B/CFP-10/ESAT-6 or with pCMV-EGFP, n=15 in both groups. Four
weeks after immunization, the vaccinated sh along with a group of non-injected
rag1(/) group were infected with 27,3002400CFUof M. marinumand followed
for their survival. No signicant differences were detected between the groups.
We have recently shown that M. marinum, a close relative of
M. tuberculosis that infects a broad range of hosts including sh,
is a valid model for studying mycobacterial infection, its latency
and reactivation [18]. In the present study, we established adult
zebrash as a platform for DNA-based vaccine antigen screening.
Vaccination both with the BCG and with plasmid DNA encoding
mycobacterial antigens was protective against mycobacteriosis in
adult zebrash. Based on the close genetic relationship between M.
marinumand M. tuberculosis genomes and the extensive amount of
orthologous genes shared by the two species [24], we postulate
that the M. marinum model can be used to nd protective vac-
cine antigens that are relevant also for the resistance against M.
tuberculosis. As a latent mycobacterial disease can be assessed and
experimentally reactivated in adult zebrash, our ndings open up
newhorizons for vaccine development against TB.
The assessment of vaccine efcacy requires reliable correlates
of vaccine-induced protection. In humans and many experimen-
tal TB models, the effects of vaccination are dened indirectly
by characterizing putative mediators of protection, for example
IFN--secreting CD4
+
T cells [38,41]. However, there are still no
unanimously proven biomarkers for protection against TB [2,38],
and even strong, apparently appropriate immune responses some-
times fail to provide protection [42]. One clear-cut advantage of
a small, non-mammalian model organismlike the zebrash is the
possibility of directly following the protective effects of vaccina-
tion. Besides measuring IFN- or other biomarkers, the course of a
mycobacterial infection can easily be monitored by following sh
survival, determining bacterial loads in the whole sh or individ-
ual organs, or by visualizing mycobacteria and granulomas in the
sh by ZiehlNeelsen staining. Due its small (5cm) size, a whole
sh can be examined on a single longitudinal section, facilitating
histological analysis of disease progression.
So far, there is limited information on zebrash vaccina-
tion. However, other teleost sh are known to have an adaptive
immunity that can be primed with foreign molecules, leading to
immunity upon re-exposure to the same molecules [43], and sh
vaccinations are routinely carried out on sh farms around the
world. Previous reports also suggest that zebrash can be specif-
ically immunized against infectious disease [26,44,45], implying
that they develop immunological memory. Furthermore, zebrash
5208 K.E. Oksanen et al. / Vaccine 31 (2013) 52025209
models for different infections and diseases have been success-
fully created (reviewed in [1922]) and these models can be used
to assess the possible protective effects of vaccination. No proof
of principle of antigen-specic protective effects of DNA-based
vaccination of zebrash has been published so far. Plasmid DNA
vaccines, however, have proven efcient in specically protecting
other sh species against infectious diseases, including mycobac-
teriosis [4649]. In hybrid striped bass, for example, an i.m. DNA
vaccination was shown to be immunogenic and it could delay the
destructive effects of an acute high-dose infection [48,49].
Inadditionto newpreclinical vaccinationmodels, efcient ways
to replace live attenuated vaccines with other vaccine modali-
ties have to be developed for safer TB prevention. Potential new
methods include subunit vaccines or DNA-basedvaccines delivered
through a viral vector or as plasmid DNA. DNA vaccines are a pre-
sumably safe method to evoke both T and B cell responses [50,51],
and they augment the need for peptide production. As DNA vac-
cines are stable as well as easy to produce and modify, they are
a promising tool for preclinical vaccine antigen identication. In
this study, we established adult zebrash as a platform for DNA-
based vaccine antigen screening. It remains to be seen whether
the use of DNA vaccines will spread fromthe experimental animal
models also to use in humans. However, they are a useful approach
for identifying molecules withgood immunoprotective or adjuvant
properties that might be utilized in conventional vaccines.
In conclusion, our results indicate that DNA-based vaccines
provide protection against an M. marinum infection in zebrash.
The zebrash thus is an attractive vertebrate model for screening
and identication of protective mycobacterial vaccine antigens. As
DNA vaccination can induce immune responses against various
pathogens or other immunomodulated diseases, it is plausible that
zebrash DNA vaccination can also be used in combination with
other disease models, including many sh diseases for which no
vaccines exist. Furthermore, the attributes of the model provide
an excellent opportunity for other applications including vaccine
adjuvant screening. Therefore, the adult zebrashDNA-vaccination
model is a promising platformfor future vaccine research.
Acknowledgements
We thank Ilmari Larivuo, Matilda Martikainen, Ilmari Tammi-
nen, Pirjo Kerola and Aref Jalili-Monir for technical assistance. In
addition, we thank Antti Iivanainen for scientic discussion and
constructive comments.
Contributors: Conception and design of the study: K.E. Oksanen,
M. Parikka, M. Rmet. Acquisition of data: K.E. Oksanen, N.J.A. Half-
penny, E. Sherwood, S.-K.E. Harjula, M.M. Hammarn, M.J. Ahava,
E.T. Pajula, M.J. Lahtinen. Analysis or interpretation of data: K.E.
Oksanen, E. Sherwood, M. Rmet. Drafting the article or revising
it critically for important intellectual content: All authors. Final
approval of theversiontobesubmitted: All authors. Conict of inter-
est statement: None. Funding: This work was supported by grants
fromthe Academy of Finland(projects 121003, M. Parikka; 139225,
M. Rmet), the Jane and Aatos Erkko Foundation (M. Rmet), the
Sigrid Juselius Foundation (M. Rmet), the Competitive Research
Funding of the Tampere University Hospital (M. Parikka, M. Rmet),
the Tampere Tuberculosis Foundation (K. Oksanen, M. Parikka, M.
Rmet), Foundationof theFinnishAnti-Tuberculosis Association(K.
Oksanen, M. Parikka, M. Hammaren, S.-K. Harjula), Finnish Cultural
Foundation, Pirkanmaa Regional Fund (K. Oksanen, M. Ahava), the
Jalmari and Rauha Ahokas Foundation (K. Oksanen) and the Vin
and Laina Kivi Foundation (K. Oksanen). The Tampere Zebrash
facility is supported by Biocenter Finland. The funding sources had
no involvement in study design; in the collection, analysis and
interpretationof data; inthe writing of the report; or inthe decision
to submit the article for publication.
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