Content uploaded by Rachele Isticato
Author content
All content in this area was uploaded by Rachele Isticato on Jun 08, 2021
Content may be subject to copyright.
Vaccine 22 (2004) 1177–1187
Display of heterologous antigens on the Bacillus subtilis spore
coat using CotC as a fusion partner
Emilia M.F. Maurielloa,LeH.Ducb, Rachele Isticatoa, Giuseppina Cangianoa,
Huynh A. Hongb, Maurilio De Felicea, Ezio Riccaa, Simon M. Cuttingb,∗
aDipartimento di Fisiologia Generale ed Ambientale, Sezione di Microbiologia, Università Federico II, Napoli, Italy
bSchool of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK
Received 17 July 2003; received in revised form 17 September 2003; accepted 24 September 2003
Abstract
WereporttheuseofCotC,amajorcomponentoftheBacillussubtilissporecoat,asafusionpartnerfor the expressionof twoheterologous
antigens on the spore coat. Recombinant spores expressing tetanus toxin fragment C (TTFC) of Clostridium tetani or the B subunit of
the heat-labile toxin of Escherichia coli (LTB) were used for oral dosing and shown to generate specific systemic and mucosal immune
responses in a murine model. This report, expanding the previously described expression of TTFC on the spore surface by fusion to CotB [J
Bacteriol 183 (2001) 6294] and its use for oral vaccination [Infect Immun 71 (2003) 2810] shows that different antigens can be successfully
presented on the spore coat and supports the use of the spore as an efficient vehicle for mucosal immunisation.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: Vaccines; Spores; B. subtilis
1. Introduction
Strategies to control and eradicate emerging and
re-emerging pathogens are often either not available or sub-
ject to important limitations, thus prompting many studies
for the development of new, more effective and safer vacci-
nation strategies. In particular, considerable efforts have re-
cently been devoted to the development of oral vaccines, that
are able to provide better levels of local immunity against
pathogens which enter the body primarily through the mu-
cosal surface [3]. Since mucosal immunisation using soluble
antigens has long been known to generate poor immune re-
sponses due to antigen degradation in the stomach, limited
absorption and tolerance, different approaches have been
undertaken to develop carrier systems displaying heterolo-
gous antigens on the surface of microbial cells and viruses.
Delivery systems so far developed to improve the mu-
cosal immune responses fall into two general categories,
non-living and living. Non-living systems include lipo-
somes, microparticles, immune stimulating complexes
(ISCOMS), and formulations based on cholera toxin and
Escherichia coli LT toxins [4,5]. Live carrier systems in-
∗Corresponding author. Tel.: +44-1784-443760;
fax: +44-1784-434326.
E-mail address: s.cutting@rhul.ac.uk (S.M. Cutting).
clude both plants and bacteria [3,6]. The bacterial systems
for heterologous antigen presentation have attracted con-
siderable interest but because these rely largely on live
attenuated pathogens such as Salmonella and Mycobacteria
considerable safety concerns remain.
The Gram positive bacterium Bacillus subtilis has been
extensively studied as a model prokaryotic system with
which to understand gene regulation and the transcriptional
control of unicellular differentiation [7]. This organism is
regarded as a non-pathogen and is classified as a novel food
which is currently being used as a probiotic for both human
and animal consumption [8]. The distinguishing feature of
this micro-organism is that it produces an endospore as part
of its developmental life cycle when starved of nutrients.
The mature spore, when released from its mother cell can
survive in a metabolically dormant form indefinitely. The
spore offers unique resistance properties and can survive ex-
tremes of temperature, dessication and exposure to solvents
and other noxious chemicals [9]. These unique attributes
would make the spore an attractive vehicle for delivery of
heterologous antigens or, indeed, any bioactive molecule,
to extreme environments such as the gastrointestinal tract.
We have recently reported the development of a surface
display system based on the use of CotB [1], a protein
component of the spore coat, as a fusion partner to express
a highly immunogenic tetanus toxin fragment C (TTFC) on
0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2003.09.031
1178 E.M.F. Mauriello et al./Vaccine 22 (2004) 1177–1187
the spore surface. We have also shown that when admin-
istered orally spores expressing the CotB-TTFC chimera
on their surfaces can protect mice from an otherwise lethal
challenge of tetanus toxin [2]. This seminal and important
finding shows the potential of using recombinant spores as
heat stable, oral, vaccine vehicles.
Here, we have expanded our previous findings showing
that it is possible to use CotC, another protein component
of the B. subtilis spore coat [10], as fusion partner for the
expression of two heterologous antigens. In this study, we
used two model antigens, tetanus toxin fragment C from
Clostridium tetani [11] and the B subunit of the heat-labile
toxin of E. coli (LTB) [5]. Both antigens have been used
extensively to evaluate bacteria as vaccine delivery vehicles
[11–15]. Induction of local and systemic immune responses
after oral administration of recombinant spores expressing
CotC-TTFC or CotC-LTB chimeras points to the spore, and
specifically the spore coat, as a novel and potentially pow-
erful system to display heterologous antigens.
2. Materials and methods
2.1. Bacterial strains and transformation
B. subtilis wild type strain PY79 (spo+;[16]) was used.
All recombinant strains described here are isogenic deriva-
tives of PY79. Plasmid amplification for nucleotide sequenc-
ing, subcloning experiments and transformation of E. coli
competent cells were performed in the E. coli strain DH5␣
[17]. Bacterial strains were transformed according to previ-
ously described procedures: CaCl2-mediated transformation
of E. coli competent cells [17] and two-step transformation
of B. subtilis [18].
2.2. Construction of gene fusions
Construction of gene fusions: In order to obtain both
cotC-based gene fusions cotC DNA was first amplified by
PCR using the B. subtilis chromosome as template and Cot-
Cp and CotCa oligonucleotides (acatgcatgcTGTAGGAT-
AAATCGTTTG and gaaagatctGTAGTGTTTTTTATGCTT,
respectively, annealing at −179−/−162 and +180/+197 of
cotC; capital and small letters indicate bases of complemen-
tarity with cotC and an unpaired tail carrying a restriction
site) as primers. An amplification product of the expected
size (395bp) was cloned into the pGem-Teasy vector
(Promega) yielding plasmid pGEM-CotC. To construct the
cotC::tetC gene fusion plasmid, pGEM-CotC was sequen-
tially digested with SphI and BglII to release cotC DNA and
the 395 bp fragment cloned in frame to the 5end of the tetC
gene carried by plasmid pGEM-TTFC [19], yielding plas-
mids pRH21. To obtain the cotC::eltB gene fusion, a 333 bp
DNA fragment coding for LTB was PCR amplified using
plasmid pSMB120 [15] as a template and oligonucleotides
LTB1 and LTB2 (tcatccagatctttcGCTCCTCAGTCTATTAC
and gcgtcgacAGTTTTCCATACTGATTGC, respectively,
annealing at +64/+90 and +355/+373 of eltB; capital and
small letters indicate bases of complementarity with eltB
and an unpaired tail carrying a restriction site) as primers.
The PCR product was cloned into the pGEM-Teasy yield-
ing plasmid pGEM-T-LTB. Plasmid pGEM-T-LTB was
sequentially digested with BglII and SalI and cloned in
frame to the 3end of the cotC gene carried by plasmid
pGEM-CotC, yielding plasmid pM10.
Plasmids pRH21 and pM10 were digested with SphI
and SalI, the fragments carrying the gene fusions were
gel-purified and ligated into plasmid pDG364 [18] previ-
ously digested with the same two restriction enzymes. E.
coli competent cells were transformed with the ligation mix-
ture and the selected ampicillin-resistant clones screened
by restriction analysis of their plasmids. Individual clones
for each transformation were selected, named pRH22 and
pIM51 (from pRH21 and pIM10, respectively), and used to
determine the nucleotide sequence of the inserted DNA.
2.3. Chromosomal integration
Plasmids pRH22 and pIM51 were linearized by diges-
tion with enzyme PstI and/or PvuII and used to trans-
form competent cells of the B. subtilis strain PY79.
Chloramphenicol-resistant (CmR) clones were the result of
a double cross-over recombination, resulting in the inter-
ruption of the non-essential amyE gene on the B. subtilis
chromosome (Fig. 1C). Several CmRclones were tested by
PCR using chromosomal DNA as a template and oligonu-
cleotides AmyS and AmyA [1] to prime DNA amplification.
Clones deriving from plasmids pRH22 showed an ampli-
fication product of 3345bp, while clones deriving from
plasmid pIM51 showed a smaller amplification product
(2265bp), thus indicating the occurrence of correct recom-
bination events. Two clones, one for each transformation,
were named RH114 (from pRH22, Fusion A in Fig. 1)
and IM201 (from pIM22, Fusion B in Fig. 1) and kept for
further studies. Both fusions were moved into a cotC null
mutant strain by chromosomal DNA-mediated transforma-
tion [18]. Chromosomal DNA extracted from strains RH114
and IM201 was used to transform the isogenic cotC null
strain RH101. RH101 was obtained by transforming strain
DL071 [10], with plasmid pJL62 carrying the cat gene in-
terrupted by the spc gene (spectinomycin resistance, SpR).
SpRclones were the result of a double recombination event
interrupting cat. Several SpRclones were selected and one
of them, RH101, used for further studies.
2.4. Preparation of spores
Sporulation of either PY79, RH114 (CotC-TTFC) or
IM201 (CotC-LTB) was made in DSM (Difco-sporulation
media) using the exhaustion method as described else-
where [20]. Sporulating cultures were harvested 24h after
the initiation of sporulation. Purified suspensions of spores
E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187 1179
(A) * * * * * *
1- MGYYKKYKEEYYTVKKTYYKKYYEYDKKDYDCDYDKKYDDYDKKYYDHDKKDYDYVVEYKKHKKHY - 66
(B) Fusion CotC-TTFC (60 kDa)
CotC TTFC
1 66
1 459
Fusion CotC-LTB (21 kDa)
CotC LTB
1 66
1 107
(C) tetC or eltB
linearised
plasmid
gene fusion
amyE back cotC cat amyE front
amyE recipient
chromosome
tetC or eltB
amyE back cotC cat amyE front recombinant
chromosome
gene fusion
Fig. 1. Cloning strategy. (A) CotC amino acid sequence [10]. (B) Schematic representation of the two fusion proteins constructed. (C) Strategy for the
chromosomal integration of the two gene fusions. Arrows indicate direction of transcription.
were made as described by Nicholson and Setlow [20] us-
ing lysozyme treatment to break any residual sporangial
cells followed by washing in 1M NaCl, 1M KCl and water
(two-times). PMSF (0.05M) was included to inhibit pro-
teolysis. After the final suspension in water spores were
treated at 65◦C for 1 h to kill any residual cells. Next, the
spore suspension was titred immediately for CFU/ml before
freezing at −20◦C. Using this method, we could reliably
produce 6×1010 spores per litre of DSM culture. Each batch
of spores prepared in this way was checked for the presence
of the 60kDa CotC-TTFC protein or the 21 kDa CotC-LTB
hybrid proteins in extracts of spore coat protein by Western
blotting using a polyclonal TTFC or LTB antiserum.
2.5. Extraction of spore coat proteins
Spore coat proteins were extracted from suspensions of
spores at high density (>1 ×1010 spores per ml) using an
SDS-DTT extraction buffer as described in detail elsewhere
[20]. Extracted proteins were assessed for integrity by
SDS-polyacrylamide gel (PAGE) and for concentration by
two independent methods: the Pierce BCA Protein Assay
(Pierce) and the BioRad DC Protein Assay kit (Bio-Rad).
2.6. Western and dot-blot analysis
Western blot filters were visualised by the ECL
(Amersham Pharmacia Biotech) method following the
manufacturer’s instruction. Serial dilutions of extracted pro-
teins and of purified TTFC or LTB were used for dot-blot
analysis. Filters were then visualised by the BCIP/NBT
Color Development Solution (Bio-Rad) or by the ECL
(Amersham Pharmacia Biotech) method and subjected to
densitometric analysis by Fluor-S Multimager (Bio-Rad).
CotC-, TTFC-and LTB-specific antibodies were raised
against 15 amino acid synthetic peptides designed on the
base of the C-terminal region of the respective proteins
(IGtech, Salerno, Italy).
2.7. Indirect ELISA for detection of antigen-specific serum
and mucosal antibodies
Plates were coated with 50l per well of the specific
antigen (2g/ml in carbonate/bicarbonate buffer) and left
at room temperature overnight. Antigen was either TTFC
or LTB purified protein. After blocking with 0.5% BSA in
PBS for 1h at 37 ◦C serum samples were applied using
a two-fold dilution series starting with a 1/40 dilution in
ELISA diluent buffer (0.1M Tris–HCl, pH 7.4; 3% (w/v)
NaCl; 0.5% (w/v) BSA; 10% (v/v) sheep serum (Sigma);
0.1% (v/v) Triton-X-100; 0.05% (v/v) Tween-20). Every
plate carried replicate wells of a negative control (a 1/40
diluted pre-immune serum), a positive control (serum from
mice immunised parentally with either TTFC or LTB puri-
fied protein). Plates were incubated for 2h at 37 ◦C before
addition of anti-mouse HRP conjugates (Sigma). Plates were
1180 E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187
incubated for a further 1h at 37 ◦C then developed using
the substrate TMB (3,3,5,5-tetramethyl-benzidine; Sigma).
Reactions were stopped using 2M H2SO4. Dilution curves
were drawn for each sample and end-point titres calculated
as the dilution producing the same optical density as the
1/40 dilution of a pooled pre-immune serum. Statistical com-
parisons between groups were made by the Mann–Whitney
U-test. A P-value of >0.05 was considered non-significant.
For ELISA analysis of faecal IgA, we followed the pro-
cedure of Robinson et al. [21] using approximately 0.1g
faecal pellets that had been suspended in PBS with BSA
(1%) and PMSF (1mM), incubated at 4 ◦C overnight and
then stored at −20◦C prior to ELISA. For each sample,
the end-point titre was calculated as the dilution producing
the same optical density as the undiluted pre-immune faecal
extract.
2.8. Immunisations
Groups of eight mice (female, C57 BL/6, 8 weeks)
were immunised by either the intra-peritoneal or oral route
with suspensions of either spores expressing CotC-TTFC
(RH114), CotC-LTB (IM201) or control, non-expressing,
spores (strain PY79). For oral dosings mice were lightly
anaesthetised with halothane using a regime based on pre-
vious work optimising mucosal immunisations [21,22].
Ana
¨
ıve, non-immunised control group was included.
Intra-peritoneal injections contained 1.5×109spores in a
volume of 0.1ml administered on days 0, 14 and 28. Serum
samples were taken on days −1, 13, 27, 43 and 82. Oral
immunisations contained 1.0×1010 spores in a volume
of 0.15ml and were administered by intra-gastric lavage
on days 0–2, 16–18, 33–35. Serum samples were collected
on days −1, 15, 32 and 68 and faeces on days −1, 15, 32
and 53.
2.9. Toxin binding inhibition (ToBI) test for measurement
of neutralising antibody levels
ELISA plates (Immuno Maxisorp, Nunc) were coated
with 80 l per well of horse tetanus antitoxin (1 IU/ml in car-
bonate/bicarbonate buffer) and incubated at 37◦C overnight.
Neutralising reaction was carried out using another set of
plates that had been blocked with 1% BSA in PBS at 37◦C
for 1h. In the reaction, serum samples were applied using a
two-fold dilution series, mixed with equal volumes of tetanus
toxoid (0.005 IU/ml in PBS, R.I.V.M., The Netherlands) and
then incubated at 37◦C overnight. Dilutions starting with
1IU/ml of a tetanus antitoxin reference (WHO) were also
included. After the reaction, corresponding wells were trans-
ferred from neutralising plates to ELISA plates which were
then incubated for 2 h at 37◦C. Remaining tetanus toxoid af-
ter neutralisation was detected by further incubation of plates
with tetanus antitoxin peroxidase conjugate (R.I.V.M., The
Netherlands) for 1h at 37 ◦C, and development using TMB
substrate (Sigma). The levels of tetanus antitoxin in serum
samples were calculated from the standard curve drawn with
tetanus antitoxin reference data.
3. Results
3.1. CotC as a fusion partner
At least 25 polypeptides are organised to form the B.
subtilis spore coat [23]. Some of these polypeptides have
been associated with the outer layer of the coat but only
one of them, CotB, has so far been localised on the spore
surface and, based on this surface display, employed as a
fusion partner to express the tetanus toxin fragment C on
the spore surface [1,2]. In order to identify additional routes
for expressing heterologous antigens on the spore surface,
we searched for other components of the spore coat to be
used as fusion partner. Based on its association with the
outer layer of the spore coat and on its relative abundance
within the coat structure we focused our attention on CotC
(Fig. 1A), a 8.8kDa component of the outer coat, rich in
tyrosine (30.3%), lysine (28.8%) and aspartic acid (18.2%)
residues [10].
As model antigens, we used: (i) the 459 amino acid
C-terminal fragment of the tetanus toxin, a well charac-
terised and highly immunogenic [24] 51.8kDa peptide, en-
coded by the tetC gene of C. tetani; and (ii) the 103 amino
acid B subunit of the heat-labile toxin of E. coli,a12kDa
peptide, encoded by the eltB gene [5] (Fig. 1B). The strategy
to obtain recombinant B. subtilis spores expressing TTFC or
LTB on their surface was based on (i) use of the cotC gene
and of its promoter for the construction of translational fu-
sions and on (ii) chromosomal integration of the cotC-tetC
or cotC-etlB gene fusions into the coding sequence of
the non-essential gene amyE [18]. Both CotC-TTFC and
CotC-LTB gene fusions were obtained by cloning tetC
or eltB in frame with the 3codon of cotC (Fig. 1B)in
the integrative vector pDG364 [18]. Both fusions were
integrated into the B. subtilis chromosome at the amyE
locus by double cross-over recombination events (Fig. 1C;
Section 2). Individual clones for each transformation were
tested by PCR (not shown), named RH114 (CotC-TTFC)
and IM201 (CotC-LTB) and used for further analysis. The
two recombinant strains and their isogenic parental strain
PY79 showed comparable sporulation and germination
efficiencies and their spores were equally resistant to chlo-
roform and lysozyme treatment (not shown), indicating that
the presence of CotC-TTFC or CotC-LTB fusion did not
significantly affect spore structure and/or function.
3.2. Expression of TTFC or LTB in the spore coat
Western blot analysis of the coat protein fraction purified
from spores of wild type and recombinant strains revealed
the presence of an approximately 60kDa polypeptide which
reacted with both TTFC- and CotC-specific antibodies and
E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187 1181
Fig. 2. Western blot of proteins extracted from purified spores of strains IM201 (CotC-LTB) and RH114 (CotC-TTFC) and reacted against anti-CotC,
anti-LTB and anti-TTFC antibodies. Fifteen micrograms of total proteins from strains PY79 (lanes 1 in all panels), IM201 (lanes 2, Panel CotC-LTB)
and RH114 (lanes 2 Panel CotC-TTFC) were fractionated on 10% polyacrylamide gel and upon electro-transfer on nitrocellulose membranes, reacted
with primary anti-rabbit antibodies, then with secondary antibodies and visualised as described in Section 2. Arrows point to fusion proteins. Molecular
weight markers (kDa) are indicated.
a 21kDa polypeptide which reacted with both LTB- and
CotC-specific antibodies (Fig. 2). Additional polypeptides
of 12 and 21kDa, specifically reacting with CotC-specific
antibodies, were present in extracts from wild type and re-
combinant spores (Fig. 2). These additional polypeptides are
due to the ability of CotC to assemble in multiple forms in
the spore coat [25].
CotC and the two chimeric proteins were not found by
western blot analysis with anti-CotC antibodies of the coat
protein fraction extracted from spores of strains PY79,
RH114 and IM201 in which the cotE gene had been deleted
(data not shown). Since cotE is known to encode a morpho-
genetic protein required for outer coat assembly [26], this
CotE requirement suggests that CotC-containing chimeras
are assembled within the outer spore coat. In order to anal-
yse whether an intact copy of CotC was needed for surface
expression of CotC-based fusions we moved, by chro-
mosomal DNA-mediated transformation, the gene fusions
Fig. 3. Western blot of proteins extracted from purified spores of strains carrying the CotC-LTB or CotC-TTFC fusion but deleted of the cotC gene.
Fifteen micrograms of total proteins from strains PY79 (lanes 1 in all panels), IM201 (lanes 2, Panel CotC-LTB) and RH114 (lanes 2 Panel CotC-TTFC)
were fractionated on 10% polyacrylamide gel and upon electro-transfer to nitrocellulose membranes, reacted with CotC-specific rabbit antibodies, then
with secondary antibodies and visualised as described in Section 2. Molecular weight markers (kDa) are indicated.
into a cotC mutant strain (RH101). Western blot experi-
ments, performed with coat proteins from strains carrying
the gene fusions in the absence of an intact copy of cotC
showed the presence of CotC-TTFC and CotC-LTB specific
polypeptides (Fig. 3). However, in the presence of an in-
tact copy of cotC both fusion proteins were expressed with
higher efficiency (Fig. 3), thereby indicating that the cotC
gene product is needed to obtain optimal expression of the
CotC-based fusion proteins within the spore coat structure.
Based on this result, strains RH114 and IM201 carrying
the CotC-based chimera in the presence of an intact copy
of cotC were selected for further analysis. We also verified
that TTFC and LTB were surface exposed by immunofluo-
rescence. Using polyclonal sera against TTFC and LTB we
could detect both antigens in sporulating cultures harvested
at 6h following the initiation of spore formation but this
labelling did not occur in the isogenic non-recombinant
strain PY79 (data not shown).
1182 E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187
Fig. 4. Quantification of expressed antigen. Dot blot experiments performed with the indicated concentrations of coat proteins (in g) extracted from
spores carrying the CotC-TTFC or CotC-LTB fusion in otherwise wild type background (lanes 3) and from wild type spores (lanes 2). Purified TTFC
or LTB (in ng, lanes 1) were also utilised. Anti-TTFC and anti-LTB primary antibodies and secondary anti-rabbit peroxidase-conjugated antibodies were
used. Reactions were visualized by NCP/BCIP or ECL as described in Section 2.
3.3. Efficiency of TTFC and LTB presentation
A quantitative determination of the amount of TTFC or
LTB exposed on B. subtilis spores was obtained by dot
blot experiments using serial dilutions of purified TTFC
or LTB and of coat proteins extracted from spores of the
wild type and the recombinant strains. Proteins were reacted
with anti-TTFC and anti-LTB antibody, then with alkaline
phosphatase-conjugated secondary antibodies and colour de-
veloped by the BCIP/NBT or ECL system (Bio-Rad). Fig. 4
shows the results obtained with strains RH114 and IM201,
carrying fusion CotC-TTFC or CotC-LTB, respectively. A
densitometric analysis indicated that both fusion proteins
amounted to 0.3% of total coat proteins extracted. Since in
our experimental conditions, an average of 0.032 (±2%) pg
of total coat proteins was reproducibly extracted from each
spore of the recombinant strains by SDS-DTT treatment at
65◦C (see Section 2), we calculated that 9.6×10−5pg of
CotC-TTFC or CotC-LTB fusion protein was extracted from
each spore. Based on that and on the deduced molecular
weight of 60 and 21kDa for the two recombinant proteins,
we estimated that ca. 9.7×102and 2.7×103molecules
of CotC-TTFC and CotC-LTB, respectively, were extracted
from each spore.
3.4. Serum anti-TTFC and anti-LTB responses following
intra-peritoneal injection of recombinant spores expressing
TTFC or LTB
Immunogencity of recombinant spores was determined
by intra-peritoneal injection of groups of eight C57 mice
(Fig. 5). Our immunisation schedule used three injections
(containing 1.5×109spores per dose) of either recom-
binant RH114 spores (expressing hybrid CotC-TTFC),
recombinant IM201 spores (expressing hybrid CotC-LTB)
or non-recombinant PY79 spores. Since each recombinant
spore was shown to carry approximately 9.6×10−5pg
of CotC-LTB or CotC-TTFC, our immunising dose would
contain 0.14 g of CotC-LTB or CotC-TTFC fusion protein,
which would correspond to 0.12g of TTFC or 0.08g
of LTB per injection. Immunisation with RH114 spores
resulted in peak anti-TTFC IgG titres (∼5×102) at day 43
(Fig. 5A). Similarly, immunisation with IM201 spores re-
sulted in peak anti-LTB titres (∼2×102) at day 43 (Fig. 5B)
although these levels were somewhat lower than that ob-
served with CotC-TTFC but significantly different from
control groups (P<0.05). This demonstrated that both
TTFC and LTB were stably expressed and appropriately
immunogenic when displayed on the spore surface.
3.5. Serum anti-TTFC and anti-LTB responses following
oral immunisation
Groups of eight mice were dosed orally with 1 ×1010
spores per dose (0.96g of CotC-TTFC or CotC-LTB,
i.e. 0.82g TTFC or 0.55 g LTB per oral dose). As
shown in Fig. 6A oral immunisation of mice with RH114
(CotC-TTFC) spores gave anti-TTFC IgG titres greater than
2×102on day 68. These were found to be significantly
above (P<0.05) those of mice dosed with non-recombinant
spores (PY79) or the control na¨
ıve group. Oral immu-
nisation of mice with IM201 (CotC-LTB) spores though
only produced an anti-LTB serum IgG response, not sig-
nificantly above (P>0.05) those of mice dosed with
non-recombinant spores (PY79) (Fig. 6B).
3.6. Mucosal anti-TTFC and anti-LTB IgA responses
Fresh faecal pellets from mice immunised orally with
RH114 or IM201 spores were tested for the presence of
TTFC-specific or LTB-specific secretory IgA (sIgA) by
ELISA. Immunisation with spores expressing CotC-TTFC
or CotC-LTB elicited, respectively, clear TTFC- (Fig. 6C)
or LTB-specific (Fig. 6D) sIgA responses. In the case of
E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187 1183
(A)
10
100
1000
0 102030405060708090
Days post primary vaccination
End-point titers
(B)
10
100
1000
0 102030405060708090
Days post primary vaccination
End-point titers
Fig. 5. Serum IgG titers following intra-peritoneal immunisation with recombinant B. subtilis spores. Individual samples from groups of eight mice
immunised intra-peritoneally (↑) with 1.5×109wild type (䊉), CotC-TTFC (䉱), or CotC-LTB expressing B. subtilis spores (䊏) were tested by ELISA
for TTFC- (Panel A) or LTB-specific IgG (Panel B). Sera from a na¨
ıve control group (䊊) were also assayed. The end-point titre was calculated as the
dilution of serum producing the same optical density as a 1/40 dilution of a pooled pre-immune serum. Data were presented as arithmetic means ±
standard deviations.
anti-LTB responses, these were 10-fold higher than those
directed against TTFC. The end-point titres of faecal TTFC-
or LTB-specific sIgA were shown to be significantly higher
than the control groups (P<0.05) while there was no sig-
nificant difference between the control groups (P>0.05).
3.7. Analysis of IgG subclasses
We analysed the anti-spore specific IgG subclasses, IgG1,
IgG2a and IgG2b, present in the serum following parenteral
as well as oral immunisations. The low anti-TTFC and
anti-LTB IgG titres did not allow any significant indication
of the levels of individual IgG subclasses (data not shown).
Following intra-peritoneal immunisations however, analysis
of anti-TTFC responses revealed substantial levels of all
three subclasses with the IgG2a subclass predominating and
appearing first (Fig. 7A). In contrast, anti-LTB responses
showed only an IgG2a response with no significant levels
of IgG1 or IgG2b (Fig. 7B).
3.8. Prediction of tetanus neutralising antibodies
The presence of neutralising antibodies against tetanus
can be measured by a number of in vitro assays as an al-
ternative to in vivo testing [27]. We used the tetanus toxin
binding inhibition test (ToBI; [28]) to determine the levels
of neutralising IgG TTFC antibodies in groups of mice
immunised orally or parenterally with CotC-TTFC spores
on the final day post-immunisation (Table 1). As controls
we also measured sera from naive mice and mice dosed
with non-recombinant (PY79) spores. Finally, we examined
Table 1
Average neutralising antibody unitsa
Route GroupaDose (g TTFC
per dose) International units
(IU) per ml
IpbCotC-TTFC (RH114) 0.12 0.017 ±0.006
CotB-TTFC (RH103) 0.15 0.014 ±0.008
Non-recombinant – <0.009
Na¨
ıve – <0.009
OralcCotC-TTFC (RH114) 0.82 0.016 ±0.006
CotB-TTFC (RH103) 1.65 0.022 ±0.011
Non-recombinant <0.009
Na¨
ıve <0.009
aAntitoxin levels obtained at the final day of immunisation with
recombinant (expressing TTFC) or non-recombinant spores. Expressed in
international units/ml.
bMice dosed on days 0, 14 and 28 by the intra-peritoneal route and
serum tested at day 82.
cMice dosed on days 0–2, 16–18, 33–35 by intra-gastric gavage and
tested at day 68.
1184 E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187
Fig. 6. Serum IgG and faecal sIgA titers following oral immunisation with recombinant B. subtilis spores. Groups of eight mice were immunised (↑)
orally with spores expressing CotC-TTFC (䉱), CotC-LTB (䊏) or non-recombinant spores (䊉). A dose of 1 ×1010 spores was used for each oral dose
and individual serum samples from groups were tested by ELISA for TTFC- (Panel A) or LTB-specific IgG (Panel B). Sera from a na¨
ıve control group
(䊊) were also assayed. Fresh faecal pellets were collected from immunised mice as well as a na¨
ıve group and tested for the presence of TTFC- (Panel
C) or LTB-specific IgA (Panel D) as described in Section 2. For IgG (Panels A and B), the end-point titre was calculated as the dilution of serum
producing the same optical density as a 1/40 dilution of a pooled pre-immune serum. For sIgA (Panels C and D), the end-point titre was calculated
as the dilution of the faecal extract producing the same optical density as the undiluted pre-immune faecal extract. Data were presented as arithmetic
means ±standard deviations.
E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187 1185
(A) (B)
10
100
1000
0102030405060708090
Days post primary vaccination
End-point titers
10
100
1000
0102030405060708090
Days post primary vaccination
End-point titers
Fig. 7. Serum anti-TTFC and anti-LTB IgG subclasses following parenteral delivery. Sera from na¨
ıve and immunised groups (outlined in Fig. 5) were
taken at different days post-immunisation (↑) and analysed for IgG1, IgG2a and IgG2b isotypes. Panel A shows anti-TTFC subclasses and Panel B
anti-LTB subsclasses. IgG subclasses from mice immunised with spores, IgG1 (䊉), IgG2a (䊏) and IgG2b (䉱). Na¨
ıve groups, IgG1 (䊊), IgG2a (䊐)
and IgG2b (). Data were presented as arithmetic means ±standard deviations.
the sera of mice immunised orally and parentally with
CotB-TTFC recombinant spores. This was reported recently
in a study using C57 BL/6 mice and, most importantly,
orally immunised mice were protected against challenge
witha20LD
50 dose of tetanus toxin [2]. We established
the sensitivity of the test to be 0.009IU/ml and groups im-
munised by the oral or intra-peritoneal route gave antitoxin
levels above this baseline.
4. Discussion
It has been recently reported that recombinant spores ex-
pressing the C-terminal fragment of the tetanus toxin fused
to the spore coat protein CotB elicit specific anti-TTFC im-
mune responses following mucosal immunisation of a mouse
model as well as protection to a lethal challenge with tetanus
toxin [1,2]. This paper expands on this work by showing
that it is possible to use at least one other spore coat compo-
nent, CotC a small 8.8 kDa. polypeptide, to display heterolo-
gous antigens, TTFC (51.8 kDa) and LTB (12 kDa). TTFC is
non-toxic and immunogenic [21,29–32] and expression in E.
coli [12], yeast [33],Salmonella [34] and Lactococcus lactis
[21] has been shown to provide protection against tetanus
toxin challenge. LTB is the B subunit of the heat-labile toxin
produced in enterotoxigenic strains of E. coli. LTB has been
used extensively for studies of mucosal immunity and oral
administration of LTB has been shown to be a potent inducer
of serum and mucosal (sIgA) anti-LTB antibodies [5,35–38].
We have found that the presence of either TTFC or LTB on
the surface of spores does not significantly affect spore struc-
ture and/or function. It would have been possible that when
fused to CotC, a large molecule, such as TTFC, may enable
surface expression by disruption and protrusion through the
coat layers while a smaller antigen, like LTB, may be hid-
den. Disruption of the apparently, rigid and compact, spore
coat layer might damage spore function, affecting either its
resistance properties or its ability to germinate correctly. Our
data is therefore encouraging and emphasises the flexibility
of the spore coat structure and therefore of the spore-based
presentation system. The malleability and functional redun-
dancy of both CotC and, in a separate study, CotB [1], makes
the spore coat an attractive route for heterologous antigen
presentation. One exciting possibility is that, in principle,
two different antigens could be displayed simultaneously on
the spore surface, using CotC and CotB for presentation.
The second part of this study was to address the im-
mogenicity of the TTFC and LTB monomeric antigens
expressed on the spore surface. Our results show that both
monomers are immunogenic when delivered parenterally
to mice with serum IgG titres that were significantly dif-
ferent from mice receiving non-recombinant spores. This
1186 E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187
demonstrates that the TTFC and LTB monomers were stable
when displayed on the spore coat. When delivered mucos-
ally CotC-TTFC spores were able to generate both systemic
and local anti-TTFC responses. In contrast, systemic IgG
responses to LTB were low and barely significant although
clear sIgA anti-LTB responses were seen. LTB has been
shown to induce strong sIgA responses when given orally
and has also been shown to exhibit significant adjuvant ac-
tivity when administered orally with other antigens [35,39].
Our work implies that when LTB is expressed on the spore
it is more efficient at inducing local immunity than sys-
temic, at least with the spore dose and immunisation regime
we have chosen here. LTB forms part of the labile toxin of
E. coli and is made in a pentameric form [40]. In this pen-
tameric form, LTB has adjuvant properties. We reasoned
that attached to the CotC protein and displayed on the spore
coat it was unlikely that LTB could oligomerise although
this was not tested using the in vitro binding assay of LTB
to epithelial M1 ganglioside receptors (GM1) [41].
Our subclass analysis also revealed that using the par-
enteral route different responses to TTFC and LTB were
obtained. Th1 cells are involved in cellular immunity while
Th2 cells co-ordinate B-cell responses [42,43]. It is gen-
erally accepted that a predominance of the IgG2a subclass
is indicative of a Th1 response (T-cell responses leading
to CTL recruitment as well as IgG synthesis) while the
predominance of IgG1 is more indicative of a Th2 response
[15,21,29–32]. We found that parenteral immunisation with
CotC-LTB spores induced only IgG2a and at relatively high
levels. The absence of any IgG1 suggests a biased, Th1
response and involvement of cellular immunity. Confirma-
tion must await analysis of specific cytokine and cellular
responses but a straightforward explanation would be that
recombinant spores are phagocytosed and that LTB anti-
gens were expressed on the cell surface and indeed IgG2a
is the dominant antibody response to a large number of vi-
ral infections. Interestingly, when LTB is expressed on the
surface of Streptococcus gordonii the dominant response
following parenteral immunisation was IgG1 inicative of a
Th2 response [15]. Responses to TTFC consisted of roughly
equivalent amounts of IgG1, IgG2a and IgG2b which could
imply a mixed Th1/Th2 response. This mixed response
could be unique to spore presentation of the TTFC antigen.
Alternatively, release of TTFC, by proteolytic cleavage,
from the spore would release soluble antigen which might
account for the IgG1 levels. In recent work, we have exam-
ined the fate of spores when administered orally to mice [2].
This showed that a small number of intact spores are able
to cross the mucosa and are found in the Peyer’s Patches.
The ability of the whole spore to interact with the GALT is
interesting and might be important for cellular immunity.
We did not determine whether the levels of anti-tetanus
antibodies obtained in this experiment are protective in vivo
but using an established ToBI test, we can state they are
certainly within the desired range. In studies using guinea
pigs vaccinated by injection, serum tetanus antitoxin levels
of between 0.05 and 0.1IU/ml provided protection against
challenge with 50 LD50 units of toxin [44]. In a recent study,
we have shown that oral immunisation of mice (C57 BL/6)
with spores expressing TTFC fused to the spore coat protein
CotB (CotB-TTFC) protected seven out of eight mice against
a challenge dose of 20 LD50 [2]. In the CotB-TTFC study,
the actual dose of TTFC was about twice that of TTFC
when fused to CotC reported here but the antitoxin levels
determined from the ToBI test appear to correlate well with
the dose given. We would predict from our data that tetanus
antitoxin levels in mice immunised with CotC-TTFC could
be protective but to a challenge dose less than 20 LD50.
In conclusion, this study has opened the way for the devel-
opment of bacterial spores for heterologous antigen presen-
tation and vaccine development. Clearly, the spore has po-
tential for heterologous antigen presentation and ultimately
for use as a vaccine system.
Acknowledgements
We thank Jan Hendriks (R.I.V.M., The Netherlands) for
advice and reagents for the TOBI tests. This work was sup-
ported by grants from the European Union (QLK5-CT-2001-
01729) to SMC and ER, from The Wellcome Trust to
SMC and from the Italian Ministry of the University
(MIUR-Progetto FIRB ) to ER.
References
[1] Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D, Oggioni
MR, et al. Surface display of recombinant proteins on Bacillus
subtilis spores. J Bacteriol 2001;183:6294–301.
[2] Duc LH, Hong HA, Fairweather N, Ricca E, Cutting SM. Bacterial
spores as vaccine vehicles. Infect Immun 2003;71:2810–8.
[3] Fischetti VA, Medaglini D, Pozzi G. Gram-positive commensal
bacteria for mucosal vaccine delivery. Curr Opin Biotechnol
1996;7:659–66.
[4] Michalek SM, O’Hagan DT, Gould-Fogerite S, Rimmelzwaan
GF, Osterhaus ADME. Antigen delivery systems: nonliving
microparticles, liposomes, cochleates, and ISCOMS. In: Ogra PL,
Wells JM, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors.
Mucosal immunology. 2nd ed. San Diego, USA: Academic Press;
1999. p. 759–78.
[5] Douce G, Turcotte C, Cropley I, Roberts M, Pizza M, Domenghini
M, et al. Mutants of Escherichia coli heat-labile toxin lacking
ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.
Proc Natl Acad Sci USA 1995;92:1644–8.
[6] Mason HS, Lam DM-K, Arntzen CJ. Expression of hepatitis B
surface antigen in transgenic plants. Proc Natl Acad Sci USA
1992;89:11745–9.
[7] Errington J. Bacillus subtilis sporulation: regulation of gene
expression and control of morphogenesis. Microbiol Rev 1993;
57(1):1–33.
[8] Mazza P. The use of Bacillus subtilis as an antidiarrhoeal micro-
organism. Bull Chim Farm 1994;133:3–18.
[9] Nicholson WJ, Munakata N, Horneck G, Melosh HJ, Setlow
P. Resistance of Bacillus endospores to extreme terrestial and
extraterrestrial environments. Microbiol Mol Biol Rev 2000;64:
548–72.
E.M.F. Mauriello et al. / Vaccine 22 (2004) 1177–1187 1187
[10] Donovan W, Zheng L, Sandman K, Losick R. Genes encoding spore
coat polypeptides from Bacillus subtilis. J Mol Biol 1987;196:1–10.
[11] Makoff AJ, Ballantine SP, Smallwood AE, Fairweather NF.
Expression of tetanus toxin fragment C in E. coli: its purification
and potential as a vaccine. Biotechnology 1989;7:1043–6.
[12] Fairweather NF, Lyness VA, Maskell DJ. Immunisation of mice
against tetanus with fragments of tetanus toxin synthesised in
Escherichia coli. Infect Immun 1987;55:2541–5.
[13] Fairweather N, Chatfield S, Makoff A, Strugnell R, Bester J, Maskell
D, et al. Oral vaccination of mice against tetanus by use of a live
attenuated Salmonella carrier. Infect Immun 1990;58:1323–6.
[14] Figueiredo D, Turcotte C, Frankel G, Li Y, Dolly O, Wikin G, et
al. Characterization of recombinant tetanus toxin derivatives suitable
for vaccine development. Infect Immun 1995;63:3218–21.
[15] Ricci S, Medaglini D, Rush CM, Marcello A, Peppoloni S,
Manganelli R, et al. Immunogenicity of the B monomer of
Escherichia coli heat-labile toxin expressed on the surface of
Streptococcus gordonii. Infect Immun 2000;68:760–6.
[16] Youngman P, Perkins J, Losick R. Construction of a cloning site
near one end of Tn917 into which foreign DNA may be inserted
without affecting transposition in Bacillus subtilis or expression of
the transposon-borne erm gene. Plasmid 1984;12:1–9.
[17] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory
manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory; 1989.
[18] Cutting SM, Vander-Horn PB. Genetic analysis. In: Harwood CR,
Cutting SM, editors. Molecular biological methods for bacillus.
Chichester, UK: Wiley; 1990. p. 27–74.
[19] Medaglini D, Ciabattini A, Spinosa MR, et al. Immunisation
with recombinant Streptococcus gordonii expressing tetanus toxin
fragment C confers protection from lethal challenge in mice. Vaccine
2001;19:1931–9.
[20] Nicholson WL, Setlow P. Sporulation, germination and outgrowth.
In: Harwood CR, Cutting SM, editors. Molecular biological methods
for bacillus. Chichester, UK: Wiley; 1990. p. 391–450.
[21] Robinson K, Chamberlain LM, Schofield KM, Wells JM, Le Page
RWF. Oral vaccination of mice against tetanus with recombinant
Lactococcus lactis. Nat Biotechnol 1997;15:653–7.
[22] Challacombe SJ. Salivary antibodies and systemic tolerance in mice
after oral immunisation with bacterial antigens. Ann N Y Acad Sci
1983;409:177–92.
[23] Driks A. Bacillus subtilis spore coat. Microbiol Mol Biol Rev
1999;63:1–20.
[24] Helting TB, Zwisler O. Structure of tetanus toxin. I. Breakdown of the
toxin molecule and discrimination between polypeptide fragments. J
Biol Chem 1977;252:194–8.
[25] Isticato R, Esposito G, Zilhao R, Nolasco S., Cangiano G., De Felice
M., et al. Assembly of multiple CotC forms into the Bacillus subtilis
spore coat. J Bacteriol, in press.
[26] Zheng L, Donovan WP, Fitz-James PC, Losick R. Gene encoding a
morphogenic protein required in the assembly of the outer coat of
the Bacillus subtilis endospore. Genes Dev 1988;2:1047–54.
[27] Galazka, AM. The immunological basis for immunisation series.
Module 3: tetanus. Geneva: World Health Organisation; 1993.
[28] van der Gun J, Akkermans A, Hendriksen C, van de Donk
H. Validation of the toxin-binding inhibition (ToBI) test for the
estimation of the potency of the tetanus toxoid component in vaccines.
Dev Biol Stand 1996;86:199–206.
[29] Roberts M, Li J, Bacon A, Chatfield S. Oral vaccination against
tetanus: comparison of the immunogenicities of Salmonella strains
expressing fragment C from the nirA and htrA promoters. Infect
Immun 1998;66:3080–7.
[30] Isaka M, Yasuda Y, Mizokami M, Kozuka S, Taniguchi T, Matano K,
et al. Mucosal immunisation against hepatitis B virus by intranasal
co-administration of recombinant hepatitis B surface antigen and
recombinant cholera toxin B subunit as an adjuvant. Vaccine
2001;19:1460–6.
[31] VanCott JL, Staats HF, Pascual DW, Roberts M, Chatfield SN,
Yamamoto A, et al. Regulation of mucosal and systemic antibody
responses by T helper cell subsets, macrophages, and derived
cytokines following or al immunisation with live recombinant
Salmonella. J Immunol 1996;156:1504–14.
[32] Balloul J-M, Grzych J-M, Pierce RJ, Capron A. A purified 28,000
dalton protein from Schistosoma mansoni adult worms protects
rats and mice against experimental schistosomiasis. J Immunol
1987;138:3448–53.
[33] Romanos MA, Makoff AJ, Fairweather NF, Neesley KM, Slater DE,
Rayment FB, et al. Expression of tetanus toxin fragment C in yeast:
gene synthesis is required to eliminate fortuitous polyadenylation
sites in AT-rich DNA. Nucleic Acids Res 1991;19:1461–7.
[34] Chatfield SN, Charles IG, Makoff AJ, Oxer MD, Dougan G, Pickard
D, et al. Use of the nirB promoter to direct the stable expression of
heterologous antigens in Salmonella oral vaccine strains: development
of a single-dose oral tetanus vaccine. Biotechnology 1992;10:888–92.
[35] Takahashi I, Marinaro M, Kiyono H, Jackson RJ, Nakagawa I,
Fujihashi K, et al. Mechanisms for mucosal immunogenicity and
adjuvancy of Escherichia coli labile enterotoxin. J Infect Dis
1996;173:627–35.
[36] Clements JD, Hartzog NM, Lyon FL. Adjuvant activity of
Escherichia coli heat-labile enterotoxin and effect of oral tolerance
in mice to unrelated protein antigens. Vaccine 1988;6:269–77.
[37] Walker RI, Clements JD. Use of heat-labile toxin of enetrotoxigenic
Escherichia coli to facilitate mucosal immunisation. Vaccine Res
1993;2:1–10.
[38] Rollwagen FM, Pacheco ND, Clements JD, Pavlovskis O, Rollins
DM, Walker RI. Killed Campylobacter elicits immune response
and protection when administered with an oral adjuvant. Vaccine
1993;11:1316–20.
[39] Nakagawa I, Takahashi I, Kiyono H, McGhee JR, Hamada S. Oral
immunisation with the B subunit of the heat-labile enterotoxin of
Escherichia coli induces early Th1 and Th2 cytokine expression in
Peyer’s Patches. J Infect Dis 1996;173:1428–36.
[40] Sixma TK, Kalk KH, van Zanten BAM, Dauter Z, Kingma J, Witholt
B, et al. Refined structure of Escherichia coli heat-labile enterotoxin,
a close relative of cholera toxin. J Mol Biol 1993;230:890–918.
[41] Biet F, Kremer L, Wolowczuk I, Delacre M, Locht C. Immune
response induced by recombinant Mycobacterium bovis BCG
producing the cholera toxin B subunit. Infect Immun 2003;71:
2933–7.
[42] Coffman RL, Seymour BWP, Lebman DA, Hiraki DD, Christiansen
JA, Shrader B, et al. The role of helper T cell products in mouse cell
differentiation and isotype regulation. Immunol Rev 1988;102:5–28.
[43] Mossmann TR, Coffman RL. TH1 and TH2 cells: different patterns
of lymphokine secretion lead to different functional properties. Annu
Rev Immunol 1989;7:145–73.
[44] Winsnes R, Hendriksen C, Sesardic D, Akkermans A, Daas A.
Serological assays as alternatives to the Ph Eur challenge test for
batch release of tetanus vaccines for human use. In: Brown F,
Hendriksen C, Sesardic D, editors. Developmental of biological
standardization, vol. 101. 1999, p. 277–88.
