Oral Administration of Bacillus subtilis
Endospores Displaying Influenza Virus Matrix
Protein 1 Elicit Cellular Immune Response in Mice
Tomasz Lega1* , Paulina Weiher2,Dawid Nidzworski2
1Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Debinki ,80-211
2Department of Recombinant Vaccine, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kładki 24, 80-822
3Department of Recombinant Vaccine, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822
Tomasz Lega, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdanskand Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland, Tel: +48 58 349 14 12; Fax: +48 58 349 14 45; E-mail:
Received: November 23, 2016; Accepted:December 20, 2016; Published: January 02, 2017
Citation: Lega T, Weiher P, Nidzworski D (2016) Oral Administration of Bacillus subtilis Endospores Displaying Influenza Virus Matrix
Protein 1 Elicit Cellular Immune Response in Mice. SOJ Microbiol Infect Dis 4(4): 1-4.
Bacterium Bacillus subtilis is a gram-positive bacilli which
produce endospores. Being metabolically dormant, spores are
resistant to many environmental stressors such as UV radiation,
desiccation, heat or freezing. There are evidence that oral or
intranasal administration of spores presenting antigens induces
a specific, both cellular and humoral immune response which can
protect animals from infection. In our study, using a genetic approach
we constructed Bacillus subtilis strains producing spores presenting
influenza A virus matrix protein 1 (M1) on their surface. M1 protein
was fused to spore coat CotZ protein and was stably exposed on the
spore surface as demonstrated by the immunostaining of intact,
recombinant spores. The immunogenicity of recombinant spores was
tested by oral administration in mice. As proved by an IFN-γ ELISPOT
assay, constructed spores elicited significant cell-mediated immune
Keywords: Bacterial spores; Bacillus subtilis; Spore display;
Influenza virus; Flu
Development of an effective vaccine against infection
influenza virus is a real challenge. A key problem for the
development of effective vaccines against influenza is a high
degree of antigenic variability of the virus strains circulating
during the year. The constantly ongoing antigenic drifts and
shifts of influenza virus cause the need for annual vaccination
with a formulation against a specific strains circulating in a given
season. Most existing vaccines are effective due to the ability to
induce production of neutralizing antibodies directed against
the capsular antigen-hemagglutinin . Although the immunity
which is based on antibodies is effective it is not universal and
provides protection only against selected strains as a result of the
antigenic variability of the virus. In contrast cellular-mediatedimmune response targets conserved antigens and can provide
cross-immunity against different subtypes . Influenza virusspecific
Cytotoxic T Lymphocytes (CTLs) have been shown in
animal studies to limit influenza A virus replication and to protect
against lethal influenza A virus challenge . In last year’s a new
live antigen carrier system emerged . Recent studies show that
recombinant endospores of Bacillus subtilis can serve as antigen
carriers by fusing peptide of interest to spore coat proteins .
In our study we constructed Bacillus subtilis strain producing
spores presenting influenza M1 protein on their surface. M1 is
most abundant protein in influenza virons and it is conserved
among various strains. Moreover it has been shown that vaccine
based on M1 protein can elicit significant cellular response in
vaccinated individuals . Recombinant spores were orally
administrated to mice to evaluate the immunogenic properties of
constructs. This work indicates that spores can serve as influenza
antigen carriers and induce cell-mediated immune response.
Moreover co-administration of constructed spores with spores
presenting interleukin 2 significantly boosted observed cellmediated
Materials and Methods
Bacterial Strains and Transformation
All strains used in this study are listed in Table 1. All cloning
experiments were done using Escherichia coli DH5α . Bacteria
were transformed according to the previously described methods:
CaCl2-induced competence of E. coli cells  and transformation
of B. subtilis .
Construction of Recombinant Bacillus subtilis Strain
To generate genetic fusion, gene cotZ coding for the coat
protein was PCR-amplified together with its natural promoter
using the B. subtilis 168 chromosomal DNA as a template and oligonucleotide pair cotZ-F/ cotZ-R listed in Table 2. Amplification
product was digested with EcoRI and BamHI and cloned into the
pDL vector  yielding plasmid pDL-CotZ.
DNA sequence coding for influenza A virus matrix protein 1
( M S L L T E V E T Y V L S I I P S G P L
K A E I A Q R L E S V F A G K N T D L E A L M E W L K T R P
E Q A A E A M E V A N Q T R Q M V H A M R T I G T H P S S S A G L K
was synthesized (Life Technologies) with codon optimization
for Bacillus subtilis and restriction site BamHI was added at the
5’ and SacI at the 3’ end of the ORF. Synthesized fragment was
digested with BamHI and SacI and cloned at the 3’ end of the cotZ
gene carried by pDL-CotZ obtaining plasmid pNC02.
Plasmid pNC02 was linearized by digestion with a BsmBI
restriction enzyme and used to transform B. subtilis 168.
Transformation resulted in the integration of the plasmid into
bacterial chromosome at the amyE locus in a double homologous
recombination manner. Obtained Chloramphenicol-Resistant
(CmR) clones were PCR-tested for the incorporation of the fusion
gene at the amyE locus in B. subtilis 168 chromosome using
oligonucleotide pair AmyS/AmyA Table 2. Selected clone was
called BNC02 [Table 1].
Production of Spores
Sporulation was induced using the previously described
Table 1: Strain list
Source or reference
fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5
NEB Inc., USA
fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
Table 2: List of the PCR Oligonucleotides
nutrient depletion method in Difco Sporulation Medium (DSM)
. Briefly, 48-hour bacterial cultures were harvested and
washed in 1 M KCl, treated with lysozyme followed by washing
steps in 1 M NaCl, 0.05% (w/ v) Sodium Dodecyl Sulfate (SDS)
and water. The purified spores were titrated using Thoma
chamber and stored at -20°C.
Western Blotting Analyses
To extract spore coat proteins a buffer containing 0.1 M
NaCl, 0.1 M NaOH, 0.1 M Dithiothreitol (DTT) and 1% (w/ v)
SDS was used. 5x108 spores were resuspended in 50 μL of
decoating buffer and incubated for 30 min at 70oC with shaking
(1000 rpm). Suspension was centrifuged (10 000 RCF, 10 min,
RT) and supernatant kept for further analysis. Extracted spore
coat proteins were separated in NuPAGE® Novex® 4-12%
Bis-Tris pre-cast polyacrylamide gels (Life Technologies),
electrotransferred on a nitrocellulose using iBlot® 2 Dry
Blotting System (Life Technologies). Membranes were incubated
overnight at 4°C with GA2B anti-influenza A matrix protein 1 Mab
(Pierce). Western blots were visualized developing with BCIP/
NBT according to the manufacturer’s instructions (Thermo
Samples were fixed directly in the medium as described by
Negri A, et al. . Briefly, intact spores were incubated overnight
at 4°C with GA2B anti-influenza A matrix protein 1 mAb (Pierce),
followed by incubation with anti-mouse Cy3 (Jackson Immuno
Research) overnight at 4°C. Samples were loaded on microscope
slides and viewed using a Zeiss Axioplan fluorescence microscope.
0BALB/c mice were purchased from the breeding facilities
at the Medical University of Gdansk. The animals were kept in
polycarbonate cage, housed in well aerated rooms with a 12-h
light/12-h dark cycle at 25 ± 2°C, fed with normal pellet diet
and water ad libitum. The physical condition of the animals was
monitored daily. None of the animals exhibited clinical signs
indicative of severe illness during experiment and 100% of mice
reached humane endpoint euthanasia which was performed by
carbon dioxide inhalation followed by cervical dislocation.
Three groups of five mice (female, 8 weeks) were immunized
using intragastric gavage with water suspension of recombinant
spores expressing CotZ-M1 (BNC02), mixture of CotZ-M1 (BNC02)
CotB-IL-2 (BKH121) fusions or control, non-recombinant spores
(strain 168). Oral immunizations contained 1.0x1010 spores in a
volume of 0.2 ml in case of spores BNC02 and 168 or contained 1:1
ratio of 0.5 x 1010 spores in a volume of 0.2 ml in case of mixture
of BNC02 and BKH121 and were administered by oral gavages on
days 1, 3, 5, 22, 24, 26, 43, 45, 47. Spleens were collected from all
of the animals on day 61.
IFN-γ ELISpot Assay
Spleens were isolated and single cell suspension was
prepared as described elsewhere .
The number of IFN-γ-secreting cells was determined by using
mouse IFN-γ ELISpot kit according to manufacturer’s instructions(BD ELISpot). Splenocytes (1×105) were cultured for 48 h in
presence of recombinant M1 antigen previously described .
The spots were counted using automated ELISpot plate reader
(CTL-ImmunoSpot S6 Micro Analyzer, USA).
ELISpot tests have been performed for each animal in
three technical repeats. Statistical significance of the data was
determined by one-way analysis of variance followed by the
Results and Discussion
Spore Coat Expression and Surface Display of M1
The presence of fusion protein in the recombinant endospore
coat was tested by western blotting with mouse monoclonal antiinfluenza
A M1 antibodies (Pierce). As demonstrated by western
blotting [Figure 1A]. CotZ-M1 fusion protein was expressed and
located in the spore coat. However, observed molecular weight
(35 kDa) of the fusion protein differ from the calculated (45 kDa).
This could be due to the reported previously instability of the
M1 protein in the bacterial expression systems [15,16]. In our
work M1 protein was fused to the C-terminus of spore coat CotZ
protein. It has been shown that M1 C-terminal domain (residues
165 to 252) is quite susceptible to degradation by endogenous
bacterial proteases. This falls with our observation as the residues
165 to 252 is 10 kDa. The surface exposition of fusion protein was
analyzed by immunofluorescence microscopy of dormant spores
of wild type and recombinant strains using mouse monoclonal
(GA2B) anti-influenza A M1 antibodies (Pierce) as primary
antibodies and anti-mouse IgG-Cy3 (Jackson Immuno Research)
as secondary antibodies. We observed a specific, fluorescent
signal only from recombinant spores [Figure 1B].
Immune Response to Recombinant Spores
To asses immunogenic properties of constructs and whether
spores presenting IL-2 will act as an adjuvant we immunized
Figure 1: (A) Western blot analysis of expression of fusion gene. Spore
coat proteins were extracted and analyzed by western blotting with
anti-influenza M1 mAb. . Numbers of lanes refer to the strain used in
experiment: 1-B. subtilis 168; 2-BNC02(CotZ-M1, ~35kDa). (B) Immunofluorescence
staining of recombinant spores. Purified spores were
incubated with anti-influenza M1 mAb, followed by anti-mouse Cy3
conjugates. Spores were visualized by phase contrast (PC) and immunofluorescence
(IF) microscopy. The same exposure time was used for
all IF images. Scale bar 10μm.
Figure 2: IFN-γ response of sensitized mouse splenocytes to M1 as assessed
by the ELISpot. The splenocytes were isolated from mice orally
immunized with 168 spores (black closed bar), BNC02 (CotZ-M1) (grey
closed bar) or 1∶1 mixture of BNC02 and BKH121(CotB-IL-2) (light-gray
closed bar). Cells were incubated with purified M1 protein for 48 h and
then the IFN-γ-secreting cells were enumerated by ELISpot procedure.
Error bars represent standard deviation. * p-value < 0.05, ** p-value <
0.01 against 168(wt).
orally mice either with spores presenting M1 antigen (BNC02),
mixture of spores presenting M1 antigen and interleukin 2
(BNC02+BKH121) or non-recombinant spores (168). To test
induction of cellular immune response we conducted IFN-γ
ELISpot using splenocytes from immunized mice. A significant
increase in the number of IFN-γ-secreting cells stimulated with
recombinant M1 was observed in case of mice immunized with
BNC02 spores [Figure 2]. These results suggest the induction
of specific cell-mediated immune response. Moreover, when
co-administered with BHK121, spores presenting M1 protein
increased the strength of observed immune response. The key
question is whether observed response would provide a robust
therapeutic or protective immunity? In our opinion spores
constructed in this research rather have the potential to serve as a
specific immunostimulant adjunctive the standard immunization
procedures than as a standalone oral vaccine.
In summary, the system presented in this work seems to be
a promising candidate as a formulation stimulating the immune
system to fight the infection with influenza virus. Although co administration
of IL-2 presenting spores as an adjuvant increases
the efficiency of immunization, further research is required to
assess protective and therapeutic potentials of such formulation.
The research was supported by the Polish National Center for
Research and Development grant no LIDER/016/489/L-4/12/
Project supported by to Foundation for Polish Science (FNP).
We express our thanks to prof. Boguslaw Szewczyk and prof.
Michal Obuchowski for their advices and encouragement during
realization of this project.
Conflict of Interests
The authors declare that they have no conflict of interests.
The animal procedures protocol was approved by the
Committee on the Ethics of Animal Experiments of the Medical
University of Gdansk (Permit Number: 3/2014).
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