Tb Vaccine

8/11/2019 Tb Vaccine

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Vaccine 31 (2013) 52025209

Contents lists available at ScienceDirect

Vaccine

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An adult zebrafish model for preclinical tuberculosis vaccinedevelopment

Kaisa E. Oksanena,, NicholasJ.A. Halfpennya, Eleanor Sherwooda,Sanna-Kaisa E. Harjulaa, Milka M. Hammarna, MaaritJ. Ahavaa, Elina T. Pajulaa,MarikaJ. Lahtinena, Mataleena Parikkaa,1, Mika Rmeta,b,1

a Institute of Biomedical Technology, BioMediTech,University of Tampere, FIN-33014 Tampere, Finlandb Department of Pediatrics, Tampere UniversityHospital, FIN-33521Tampere, Finland

a r t i c l e i n f o

Article history:

Received 20May2013

Received in revised form 16 August 2013

Accepted 28 August 2013

Available online 20 September 2013

Keywords:

Zebrafish

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 thatMycobacteriummarinum, a closerelative ofMycobacterium tuberculosis, causes

an infection resembling human tuberculosis in adult zebrafish (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 zebrafish frommycobacterial infection. Using a low-

dose (2030 bacteria) intraperitoneal M. marinum infection, both the number ofgranulomas and the

amountofinfectedorganswere reduced in theDNAvaccinatedfish. Likewise,when infectingwitha lethal

infection dose (20,00027,000bacteria), vaccination significantly reduced bothmortality and bacterial

counts in a manner dependent on the adaptive immune response. Protective effects ofvaccination were

associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish 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.

AlthoughBCGprotects childrenagainst thedisseminated forms

of TB, its efficacy in adults is inconsistent [2,4]. Moreover, BCG

does not prevent the establishment ofM. 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: [email protected] (K.E. Oksanen).1 Both authors contributedequally.

controlling theTB pandemic. Proposednewapproaches includeM.

tuberculosis mutants [7,8], recombinant BCG strains [9], and DNA

[10,11] or subunit vaccines basedon mycobacterialantigens[5,12].

The three main animal models used for TB vaccine develop-

ment aremice, guineapigs andnon-human primates, usually used

sequentially in this order [13]. Other commonmodels include the

rabbit [14] and the bovinemodel [15,16]. All current animal mod-

els have limitations; mice, for instance, cannot completely reflect

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 zebrafish (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 fish

pathogen genomically closely related toM. tuberculosis, induces a

zebrafish disease sharing the main histological and pathological

features of humanTB [17,18,24]. A low-doseM.marinum infection

spontaneouslydevelops intoa latentfishinfection,controlofwhich

0264-410X/$ seefrontmatter 2013 Elsevier Ltd. All rights reserved.

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K.E. Oksanen et al. / Vaccine31 (2013) 52025209 5203

depends on the adaptive immune response [18,25]. Notably, pre-

infection with attenuated M. marinum has been shown to protect

zebrafish against mycobacteriosis [26], confirming that zebrafish

canbe vaccinated againstM. marinum.

In this study, we show that adult zebrafish 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. Zebrafish thus presents itself as an

attractive model for TB vaccine research.

2. Materials and methods

2.1. Fish

Adult (58month-old)wild typeAB zebrafishor adultrag1(/)

hu1999 mutant fish were used for experiments. The fish were

maintained in a flow-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

Boardin FinlandandconductedinaccordancewiththeEU-directive

2010/63/EU on the protection of animals used for scientific 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 amplified by

colony PCR fromM. marinum grown on 7H10 Middlebrook OACD

plates (BD Biosciences, Franklin Lakes, NJ) using Phusion High-

Fidelity PCR Kit (NEB, Ipswich, MA). PCR products were purified

and restriction cloned to be expressed with a C-terminal EGFP

tag.pCMV-plasmidwithout theEGFP-encoding gene(blankvector)

was constructed by excising the EGFPgene from pCMV-EGFP. Plas-

mids were transformed into Escherichia coli One Shot TOP10 cells

(Invitrogen) and constructs confirmed by sequencing. For in vivo

experiments,plasmidDNAwaspreparedusingtheQIAGENPlasmid

PlusMaxi Kit (Qiagen, Venlo, TheNetherlands).

2.3. Vaccination experiments

Before vaccination, fish were briefly anaesthetized in 0.02%

3-aminobenzoic acid ethyl ester (pH 7.0) (SigmaAldrich). For

BCG vaccinations, fishwere intraperitoneally (i.p.) or intramuscu-

larly (i.m.) injected with 8103 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/fish 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 PBSor thepCMV-EGFPplasmidwas used asnegativecontrol

for all vaccination experiments.

2.4. Fluorescence microscopy, Western blotting and GFP ELISA

In vivo expressionof theplasmidDNA-derived proteinproducts

(EGFP and its antigen recombinants) was verified by fluores-

cence microscopy, Western blotting and ELISA. Microscopy was

done with a Lumar V.12 fluorescence stereomicroscope (Carl Zeiss

MicroImaging, Jena, Germany). For Western blotting and ELISA,

samplefishwerecollectedat adesiredtimepointandhomogenized

with a PowerLyzerTM

24BenchTopBead-Based Homogenizer (MO

BIO Laboratories). Pierce BCA Protein Assay Kit (Thermo Fisher

Scientific, Waltham,MA)was used to define the total protein con-

centration of each lysate. ForWestern blotting, fish homogenates

were resolved on a 10% SDS-PAGE gel, blotted on a nitrocellulose

membraneand ananti-GFPantibodyNB600-303 (Novusbiological,

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 fish tissuehomogenates.

2.5. Bacterial infections

M.marinum (ATCC927)was culturedandpreparedfor infections

as in [18]. For experimental infections, fishwere anaesthetized as

described above and i.p. injected 34 weeks after immunizations.

Toverifythebacterialdose,samplesofbacterialdilutionweretaken

during infecting and plated on 7H10 plates.

2.6. RNA and DNA extraction and qPCR

RNA extraction from infected zebrafish was done as previ-

ouslydescribed[18]. For cytokinegene-expressionmeasurements,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 ofM. marinum, fish were euthanized,

the contents of their abdominal cavities were collected and DNA

extraction andM. marinum specific qPCR to quantify bacteria per

fish 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 fish were fixed in formalin,decalcifiedwithEDTAcitrate andembedded inparaffin.5m lon-

gitudinal sections were cut and every 40th section was placed

on a slide. The fish 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-

pusBX51microscope. Bacterial load perfishwas assayed from the

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

EnVisionTM+ 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

euthanizedwith an anesthetic overdose if they showed any of the

followingsigns:abnormalswimming, gasping,observable swelling

or wasting, tissuedamage, lack of response to touch.

2.9. Statistical analysis

Statistical analyses were carried out using the log rank

MantelCox test(for thesurvivalexperiments)oranon-parametric

MannWhitney test (one-tailed). Values ofp


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3. Results

3.1. BCG protects adult zebrafish against an experimental M.

marinum infection

As BCG vaccination has been shown to restrict the progression

of mycobacteriosis inmultiple animal models [13], we first exam-

ined whether BCG is also protective in the zebrafish. Groups of

adult, wild type zebrafish were vaccinated with 8103 CFU of

BCG, eitheri.m.or i.p. Control fishwereinjectedi.p.withsterilePBS.

Four weeks after vaccination, the fishwere infected i.p. with a high

dose (19,500800CFU) ofM. marinum. The experimental groups

were then followed for35weeks for survival. BCGvaccinationwas

unabletopreventinfectionwithmycobacteria,andmortalitydueto

mycobacterialdiseaseoccurredin allgroups.However,vaccination

with BCGimprovedfish survival: in theBCG-vaccinatedgroupsthe

end-pointmortalitieswere45.0% (i.p. route) and57.1% (i.m. route),

whereas in thecontrol group themortalitywassignificantlyhigher

(84.2%) (Fig. 1). Non-vaccinated (buffer injected or non-injected,

n=70 in both groups), non-infected fish kept in the same fish unit

had mortality rates of under 10% in 70 weeks (data not shown).

Therewasno significant difference between theend-pointmortal-

ities of the twovaccination routes, althoughfishvaccinatedvia the

i.m. route began to die earlier, suggesting that the i.p. BCG vacci-nationmaymore efficiently control theearly phase ofM.marinum

infection.

3.2. Mycobacterial antigens can be expressed in adult zebrafish

by DNAvaccination

Next,wetestedwhetherDNA-basedvaccines couldalsobeused

in adult zebrafish. 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

zebrafish and exposed the injection site to different numbers of

Fig. 1. Vaccination with the Bacillus CalmetteGurin (BCG) vaccine improves

adult zebrafish survival from infection with a high mycobacterial dose. Groups of

zebrafish (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 fish were infected with 19,500800CFU ofM. marinum.

Fish were then followed for35 weeks forsurvival. *P


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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 tobeexpressedwitha C-terminal EGFP tag(Fig. S2).

Following i.m. plasmid injection, CFP-10, ESAT-6 andAg85B-EGFP

recombinants were detectable in fish tissue by both fluorescence

microscopy and GFP ELISA (Fig. 2b and c). Expression of recombi-

nant CFP10-EGFP, ESAT6-EGFP and Ag85B-EGFPwas confirmed 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. Zebrafish 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

zebrafish. Fish were immunizedeitherwith a combination of the3

antigen-encodingplasmids(pCMV-Ag85B/CFP-10/ESAT-6),pCMV-

EGFP plasmid, or PBS. The pCMV-EGFP control was included toverify that the observed effects are due to an antigen-specific

response rather than unspecific immune stimulation caused by

bacterial DNA [37]. Four weeks later, fish were challenged with

1500CFUM. marinum (a dose leading to high long-termmortality

but very limited mortality in the first weeks of infection). Inter-

ferongamma (IFN1-1;ZDB-GENE-060210-1) expression levels in

the infectedfishwere thenmeasuredbyqRT-PCR 1, 7 or14dpi. No

differences were seen between the groups during the first week,

although IFN1-1 expression was highly (approximately 70-fold)

upregulated in all groups 7dpi (Fig. 3). 14dpi the fish 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 groupswere lower than 20-fold on average.

3.4. A DNA vaccine encoding Ag85B, ESAT-6 and CFP-10 protects

adult zebrafish against low-doseM. marinum infection

IFN- production levels or other biomarkers do not necessarily

correlate with protection against mycobacterial disease [38]. We

havepreviously setupmethodology to investigatemycobacteriosis

Fig. 3. Interferon gamma expression in vaccinated zebrafish upon infection with

M. marinum. Adult zebrafish were immunized with pCMV-Ag85B/CFP-10/ESAT-

6, pCMV-EGFP or PBS. Four weeks after immunization, the fish were infected

with 1500130CFU ofM. 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


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5206 K.E. Oksanen et al. / Vaccine31 (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 zebrafish survival and reduces

mycobacterial burden during an active, rapidly progressing

infection

To assay the effect of fish DNA vaccination on an active, rapidly

progressing mycobacterial infection, DNA-vaccinated fish were

infected with a high dose (20,5004300CFU) ofM. marinum 3

weeks after immunization. Survival was then monitored for 12

weeks. Vaccination with the combination ofAg85B, ESAT-6 and

CFP-10 increased fish survival rates upon infection by 29.8% com-pared to pCMV-EGFP-vaccinated controls (Fig. 5a). 12wpi, M.

marinum-specific qPCR was used to determine the mycobacterial

loads both in vaccinated and control groups. The mean bacterial

loadin themock-vaccinatedfishwas2.8-fold(64.5%)higherthan in

the vaccinated group (Fig. 5b). No bacteria could be detected from

non-infected fish (n=10; data not shown). Taken together, these

results support the observation that zebrafish can be protected

against anM.marinum infection by DNA-vaccination.

3.6. Adaptive immunity is required for the protection gained by

zebrafish vaccination

Aszebrafishare knowntohavestronginnateimmuneresponses

[39], we next examined whether the vaccine-induced protection
http://dx.doi.org/10.1016/j.vaccine.2013.08.093http://dx.doi.org/10.1016/j.vaccine.2013.08.093http://dx.doi.org/10.1016/j.vaccine.2013.08.093http://dx.doi.org/10.1016/j.vaccine.2013.08.093


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Fig. 5. Effect of adult zebrafish 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/fish). The control group (GFP) was

injected with 10g of pCMV-EGFP. Three weeks later, the fish were infected with

20,5004300CFUofM. marinum. (a)Survival analysis of DNA-vaccinated (Ag) and

control (GFP) fish after infection. Data was averaged from two independent experi-

ments; n(Ag)=40, n(GFP)= 38.*P


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