Research Article (Open access) |
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SSR Inst. Int. J. Life Sci., 6(2):
2494-2501,
March 2020
FliC-delta220-320 from Salmonella
enteritidis as an Adjuvant for Vaccine Development
Bao-Chau Thi Tran1,2, Hai-Vy Vo-Nguyen1,2, Viet-Anh Nguyen1,2,
Thuoc Linh Tran1,2, Hieu Tran-Van1,2,*
1Department of Molecular
and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam
2Vietnam
National University, Ho Chi Minh City, Vietnam
*Address for Correspondence: Dr. Hieu
Tran-Van, Associate Professor, Faculty of Biology and Biotechnology,
HCMC University of Science, Vietnam National University, HCM City, Vietnam
E-mail: tvhieu@hcmus.edu.vn; Fax Number: +842838350096
ABSTRACT- Background: High
efficacy of subunit vaccine requires immunostimulant as an adjuvant component.
Flagellin protein (FliC) in bacteria is considered as a potential candidate. However, the high antigenic property of FliC has made it difficult to apply for a variety of vaccine development. In this present study, we assessed the immunostimulant of FliCΔ220-320- a less immunogeneicity variant of FliC. In
which, 100 amino acids were deleted from the hypervariable domain of FliC in fusion with GFP (plays as a model antigen).
Methods: To generate this contract,
we isolated FliC gene from Salmonella enterica Enteritidis (S. enteritidis) serving for cloning and expressing of FliC-delta220-320 (FliC∆220-320-gfp) protein in E. coli. The gfp gene
was cloned into pET28a-FliCΔ220-320 resulting in a recombinant vector pET28a-FliCΔ220-320-gfp. The
expression of FliC∆220-320-gfp protein
was induced by 0.1 mM IPTG and confirmed by the SDS-PAGE analysis and Western blot probed with anti-6xHis
antibody. This
recombinant protein was then validated for immunostimulatory via
subcutaneous immunization in mice.
Results: FliCΔ220-320-gfp protein was expressed in soluble form and was purified by using the
immobilized-metal affinity chromatography with a purity of 56%. The present results showed that the FliCΔ220-320 variant infusing with GFP reduced its
antigenic stimulation to four times than that of FliC in the FliC-gfp and retained its ability to stimulate a humoral immune response against fused GFP.
Conclusion: This result suggested
that it was possible to apply the FliCΔ220-320 variant to subunit vaccines in a form of fusion with protein antigen(s) to increase their efficacy.
Keywords: FliCΔ220-320, Hypervariable domain, Immunoadjuvants, Salmonella enterica Enteritidis, Subunit
vaccine
INTRODUCTION- Infectious diseases remain the second leading cause of
death worldwide, but the first in infants and children [1]. Vaccination is one of the most successful public health interventions ever
undertaken and continues to have a tremendous impact in preventing the spread and death from infectious
diseases around the world by generating a pathogen-specific immune response with a long-lasting
protection [2]. Live-attenuated vaccines, inactivated
vaccines and subunit vaccines are the three different types of vaccine currently used and more popular with humans [3]. Live-attenuated vaccines are considered to be the most effective, but its side
effect reduces its vaccine of choice. Inactivated vaccines get rid of reversion to virulence, but can only stimulate a humoral
immune response with little or no cell-mediated response [4]. Subunit vaccines contain part of the pathogen
that has been purified or synthesized. Although considered safe, subunit
vaccine is not able to stimulate a strong immune response. This problem can be addressed
by the addition of immune adjuvants, which helps boost immune responses against
antigens or modulates it towards the desired immune responses [5]. In addition,
other characteristics that need to be considered when selecting an immune
adjuvant are types of activated immune response, vaccinated species, and routes
of administration.
One of the constituents of flagella of Salmonella sp., FliC has long been recognized as a potential adjuvant due to its effective immune stimulation when
interacting with cognate receptor on the surface of innate immune cells [6]. TLR5 was identified as an
extracellular receptor of FliC [7]. When being fused to antigens, FliC favorably facilitates conditions for antigens to be presented
by antigen-presenting cells due to its affinity for TLR5s on the APC surface [8,9]. However, FliC’s antigenicity needs to be eliminated
or reduced to be applied in various vaccines [10]. Many studies
have implemented different methods to alter
the FliC protein, such as muting or eliminating some parts of the protein sequences, to discover FliC variants with less antigenicity but still retain stimulating characteristics with TLR5. In particular, Liu et al. [11] have shown that the removal of 100 amino
acids in the FliC hypervariable domain created the FliCΔ220-320
variant with the ability to stimulate the production of proinflammatory
cytokines, which is not only equivalent to the intact FliC,
but also significantly reduces its antigenicity [11]. However, in their experiments, antigen
was simply mixed with FliCΔ220-320 but not covalently linked to the variant. This could reduce immune stimulatory of the FliCΔ220-320.
In
this study, we
evaluated the immune stimulatory of recombinant FliC∆220-320
biologically linked to GFP (plays as an antigen). The model could provide more evidences for the development of highly efficient recombinant
vaccines.
MATERIALS
AND METHODS- This study performed in March, 2019 at the
Department of Molecular and Environmental Biotechnology, Faculty of Biology and
Biotechnology, University of Science, VNU-HCM, Hochiminh City, Vietnam.
Bacterial strains, plasmids,
reagents and growth conditions-
E. coli
DH5α {F- end A1 hsdR17 (rk-/mk-) supE44 thi λ-recA1 gyrA96 DlacU169 (j80 lacZ DM15)} and E. coli BL21 (DE3) (F+ ompT hsdSB (rB- mB-) gal dcm (DE3) were used as host strains for cloning and protein
expression, respectively. Both were routinely grown in Luria-Bertani (LB)
medium containing 50 μg/mL of kanamycin at 37°C. S. enterica Enteritidis and pET-gfp vector were used to obtain the gene encoding for flagellin protein and GFP protein, respectively. The pET28a plasmid was used as a vector for cloning the fusion genes FliC∆220-320-gfp and FliC-gfp, and protein expression controlled by T7
promoter via the IPTG
(Isopropyl ß-D-1-thiogalactopyranoside) (Biobasic) inducer. All strains and plasmids were provided by the Department of Molecular and
Environmental Biotechnology, University of Natural Sciences, VNU-HCM,
Vietnam. Recombinant proteins were constructed,
expressed and stored at -200C.
Construction
of recombinant pET28a-FliC∆220-320-gfp and pET28a-FliC-gfp- GFP gene obtained from pET-gfp vector and pET28a-FliCΔ220-320 vector (unpublished results) were digested to create sticky ends with HindIII
and XhoI (Thermo Scientific). The
ligation reaction containing the double-digested insert and the expression
vector, was performed in the presence of T4 DNA ligase (Thermo Scientific) and the resulting mixture was
transformed into competent E. coli DH5α. The transformants were initially screened on kanamycin-containing LB
agar plate, then re-screened by the PCR with specific primers (F: CATATGgcacaagtcattaatacaaacagcc and R: CTCGAGacgcagtaaagagaggacgttttgc) and T7pro/T7ter. Plasmids derived from positive colonies confirmed by PCR colonies were sent to the Phu Sa Biochemical Company for sequencing. The E. coli DH5α strain carrying the
target plasmid pET28a-FliC-gfp [12]
was created in a completely similar manner, but replaced pET28a-FliCΔ220-320 with
pET28a-FliC (Fig. 1).
Fig.
1: Cloning
strategies for pET28a-FliCΔ220-320-gfp
and pET28a-FliC-gfp recombinant vectors
Expression
of recombinant FliC∆220-320-gfp and FliC-gfp
in E. coli BL21 (DE3)- The
expression of recombinant FliC∆220-320-gfp and FliC-gfp
were
conducted as described with some modifications [13]. pET28a-FliCΔ220-320
and pET28a-FliC obtained
from previous steps were transformed into competent E. coli BL21 (DE3) to create vector-carrying strains. Positive
colonies were inoculated in LB media shaking tubes supplemented with kanamycin
and allowed to grow at 37°C in 16 hours. The cultures were then sub-cultured at
1:10 (v/v) and inoculated at 37°C until OD600 reached 0.8–1.0. At
this point, the cultures were induced with 0.1 mM concentration of IPTG and the
protein expressions were performed at 16oC in 16 hours. Harvested
cells proceeded for sonication on ice to obtain proteins in total, soluble and
precipitated phases. Before the induction, a sample of the bacterial culture
was taken as negative control with E.
coli BL21 (DE3)/ pET28a induced by IPTG with the same concentration.
Recombinant protein expressions were analyzed by SDS-PAGE and Coomassie
Brilliant Blue stained, followed by Western blot and probed with anti-His-tag
antibody (Santa Cruz).
SDS-PAGE
and Western blotting- SDS-PAGE and Western blotting were
performed as described with some modifications [13]. Confirmation
of the FliC∆220-320-gfp and FliC-gfp
expression
was performed using 12.5% SDS-PAGE gel, followed by Western blotting. The
proteins from the gel were transferred to the nitrocellulose membrane and were probed
with mouse-anti-His-tag antibody, then detected by rabbit-anti-mouse IgG-HRP (Santa Cruz).
Purification
of recombinant FliC∆220-320-gfp and FliC-gfp-
The
purification of recombinant FliC∆220-320-gfp and FliC-gfp
were
conducted as described with some modifications [13]. The supernatant of E.
coli BL21 (DE3)/pET28a-FliC∆220-320-gfp (or pET28a-FliC-gfp)
obtained from sonicated step was used as raw material to purify FliC∆220-320-gfp
(or FliC-gfp) by affinity chromatography with HP Hitrap column (GE Healthcare).
After being equilibrated with solution A (50 mM Tris-HCl, 100 mM NaCl, 20 mM
imidazole pH 8.0), the column was loaded with soluble protein solution, then
rebalanced and washed with solution A. Finally, the target protein was eluted
with solution B (50 mM Tris-HCl, 100 mM NaCl, 200 mM imidazole, pH 8.0). Purified
FliC∆220-320-gfp (or FliC-gfp) was tested by the SDS-PAGE,
Coomassie Brilliant Blue stained and analyzed by the Image J software. Recombinant
GFP protein (pET-gfp) was also expressed, obtained and purified for the evaluation
of immune stimulatory of FliC∆220-320-gfp and FliC-gfp.
Immunizated mice with recombinant FliC∆220-320-gfp and FliC-gfp- Immunization of
mice was conducted as described with some appropriate modifications [13]. Swiss mice (Mus
musculus var. albino) male, 3–5 weeks old, healthy, average weight of
10–15 g, provided by the HCMC Drug Testing Institute, were divided into 4
experimental groups (n=5). (i) and (ii): positive control and negative control,
respectively, injected with 25 ng GFP/dose; (iii) Group 1, injected with 67 ng FliC∆220-320-gfp/dose;
(iv) Group 2, injected with 77 ng FliC-gfp/dose. FCA (Freund's Complete
Adjuvant-Santa Cruz) was used for the first dose of positive control (50
µL/dose), FIA (Freund's Incomplete Adjuvant-Santa Cruz) was used for the first
dose of negative control, test groups and booster doses (50 µL/dose). Booster dose
was injected after three weeks, and then repeated every two weeks with half the
amount of antigen, with the unchanged volume and percentage of adjuvants. The
amount of protein used for injection was calculated based on the amount of target
protein after purification and equivalent to 25 ng GFP. One week after the last
injection, 200–300 µL of blood was obtained from the orbital vein of each mouse;
serum was collected and stored at -20oC for later analysis.
Evaluation of immune stimulatory of recombinant FliC∆220-320-gfp and FliC-gfp by indirect ELISA- Specific antibody titers were determined by indirect ELISA as
described with some modifcation [13]. The 96-well microtiter plates were coated with 2
μg/mL antigen (GFP or FliC) in 100 µL carbonate buffer (pH
9.6) at room temperature in 2 hours and then blocked with 100 μL of 5% skim milk in PBST for 1 hour at room temperature. After being washed three times with PBST, 100 μL of mouse-anti-serum, diluted in 2-fold dilution manner from 1:5,000 to 1:640,000, were added and incubated at room temperature for 1 hour. Solutions were removed and 100 μL/well of rabbit-anti-mouse
IgG-HRP at 1:10,000 dilution was added and incubated for 1 hour at room temperature. The plates were developed using 100 µL of 3, 3', 5, 5'-Tetra methyl benzidine (TMB). The reaction was stopped with 100 μL of H2SO4 2N after 30 minutes, and the absorbance at 450 nm
was measured on a microplate reader (Thermo Electron Corporation). Negative control was created in a completely similar manner,
but replaced with the
serum sample collected before the first dose.
RESULTS
Molecular
cloning of recombinant pET28a-FliC∆220-320-gfp and pET28a-FliC-gfp-
PCR colonies were performed to screen for recombinant pET28a-FliC∆220-320-gfp and pET28a-FliC-gfp
from transformed E. coli DH5α.
Amplicons with sizes of approximately 1215 bp and 1515 bp were obtained by using specific primers for FliCΔ220-320 and FliC (F and R),
respectively (lane 2 and 4, Fig. 2). When using T7pro/ T7ter primers, the resulted bands were approximately 2230 bp and 2530 bp (lane 3 and 5, Fig. 2), including the FliCΔ220-320 gene in case of pET28a-FliC∆220-320-gfp, the FliC
gene
in case of pET28a-FliC-gfp,
respectively, and the gfp gene incorporated with
the length from T7 pro/ter to the target genes. The recombinant vectors of
desire were confirmed.
Fig. 2: PCR colonies of E. coli (DH5α)/pET28a-FliCΔ220-320-gfp and E. coli (DH5α)/ pET28a-FliC-gfp. M: DNA maker 1 kb;
1: Negative control; 2: FliCΔ220-320 gene; 3:
FliCΔ220-320-gfp gene; 4: FliC gene; 5: FliC-gfp gene
Expression of recombinant FliC∆220-320-gfp and FliC-gfp- The pET28a-FliC∆220-320-gfp
and pET28a-FliC-gfp vectors were transformed into E. coli BL21 (DE3) cells. Positive clones were induced by IPTG to
produce recombinant proteins. The cultures of both strains after induction showed
a green color of GFP, which meant the GFP retained its fluorescent properties
when combined with FliC∆220-320 and FliC, facilitating further
experiments. To verify the recombinant FliC∆220-320-gfp and FliC-gfp proteins, the E. coli cells
were lysed, subjected to 12.5% SDS-PAGE and stained with coomassie blue. The separated bands on gel showed over expression of two proteins of
about 73 kDa (lane 2, Fig. 3A) and 82 kDa (lane 2, Fig. 3B), which were exactly
the predicted sizes of FliC∆220-320-gfp and FliC-gfp,
respectively. There was no over expression band in the negative control (lane
1, Fig. 3). In addition, the FliC∆220-320-gfp and FliC-gfp
were designed to fuse with the His-tag on the C-terminal, so the presence of
FliC∆220-320-gfp and FliC-gfp were confirmed by using
anti-His-tag antibody in Western blot. The results indicated that the proteins
excessively expressed in the SDS-PAGE gels were FliC∆220-320-gfp
and FliC-gfp (lane 3, Fig. 3) and these proteins expressed mainly in the
soluble phase. Thus, recombinant FliC∆220-320-gfp and FliC-gfp fused to the His-tag were successfully expressed
in E. coli BL21 (DE3).
Fig.
3: Expression of FliC∆220-320-gfp (A) and FliC-gfp (B) analyzed by SDS-PAGE and
Western blot with anti-His-tag antibody. M: Protein maker; 1: E.
coli BL21 (DE3)/pET28a
(+IPTG); 2-4, E. coli BL21 (DE3)/pET28a-FliCΔ220-320-gfp (+IPTG) (A)
and E. coli BL21 (DE3)/pET28a-FliC-gfp
(+IPTG) (B); 2: Total phase; 3: Soluble phase; 4: Insoluble
phase
Purification
of recombinant FliC∆220-320-gfp and FliC-gfp-
With
the fused His-tag, FliC∆220-320-gfp and FliC-gfp were purified
using affinity chromatography with Ni2+ column. The proteins those
carry the 6xHis tag were retained through the interaction between the 6xHis and
the Ni2+ ions presented in the column. The target proteins were then
eluted from the column by the imidazole. Recombinant GFP protein was also
expressed, obtained and purified for the evaluation
of immune stimulatory of FliC∆220-320-gfp and FliC-gfp.
The SDS-PAGE results and the evaluation
of purity by Image J software showed the fractions in lane 2, 4 and 6 (Fig. 4A)
for a protein band at each lane with the appropriate sizes of FliC-gfp, FliC∆220-320-gfp
and GFP with a purity of 79%, 56%, and 98%, respectively (Figure 4B-D). Thus,
it was initially purified and successfully acquired the target recombinant
proteins to serve for immunological evaluation. However, the recovery
efficiency of FliCΔ220-320-gfp and FliC-gfp proteins was not
high, compared to GFP. Therefore, further investigation in purification is
necessary for the production of a large number of these proteins.
Fig.
4: Evaluation of
purity of recombinant FliC-gfp, FliC∆220-320-gfp, and GFP by SDS-PAGE and Coomassie Brilliant Blue stained (A): M,
protein maker; 1, Total sample of FliC-gfp; 2, Purified
FliC-gfp; 3, Total sample of FliC∆220-320-gfp; 4, Purified
FliC∆220-320-gfp; 5, Total
sample of GFP; 6, Purified
GFP and analysis by Image J software of FliC-gfp (B), FliC∆220-320-gfp (C), and GFP (D)
Evaluation of immune stimulatory
of FliC∆220-320 and FliC in humoral immunity to GFP- To
evaluate the ability of FliC∆220-320 and
FliC in humoral immunity stimulation to GFP antigen,
GFP specific antibody titers and antigenic characteristic of FliCΔ220-320 variances to FliC were determined by using serum samples obtained from experimental mice groups via
indirect ELISA.
In case of GFP specific
antibody titers, OD450nm of group 1 (FliC∆220-320-gfp
+ FIA) and group 2 (FliC-gfp + FIA) at 1/20000 dilution were 0.602 and 0.799,
respectively, which were 1.3 times and 1.8 times higher compared to the negative
control (GFP + FIA) (Fig. 5A). These results suggested that, the combination of
GFP protein with FliCΔ220-320 and FliC have increased the
production of GFP-specific antibodies. The stimulatory effects of FliCΔ220-320
and FliC could have come from their abilities to stimulate the innate immune
cells’ functions, which led to the secretion of pro-inflammatory cytokines
activating the adaptive immune system. In particular, when in the fusion form
with GFP, FliCΔ220-320 and FliC could enhance GFP presentation
through the interaction of these two proteins with TLR5 on the surface of
antigen-presenting cells. Thus, in the form of fusion with GFP, the FliCΔ220-320
variant still can retain the immune stimulatory of the intact FliC.
Fig. 5: Evaluation
of GFP specific antibody titers (A) and antigenic characteristic of FliCΔ220-320 variant to FliC (B)
To assess the antigenic characteristic of FliC∆220-320
variant to FliC, the presence of FliC specific antibodies in the antisera sample
obtained from group 1 was
determined
through indirect ELISA. The results showed that the amount of
FliC specific antibody of the FliCΔ220-320-gfp immunized mice were much lower than that of the FliC-gfp, specifically, at the 1/20000 dilution, OD450nm of FliC-gfp was 3.013, which was 4 times higher compared to FliCΔ220-320-gfp
(0.761) (Fig. 5B). These results indicated that the removal of 100 amino
acids (from 220 to 320) in the hypervariable domain of FliC had significantly reduced the antigenic
properties of this protein.
DISCUSSION-
Vaccine invention is one of the
most crucial and cost-effective medical and public health achievements of all
time,
which helps repel dangerous diseases threating human health. An effective
vaccine included a strong immunogen (antigen) and a potent adjuvant (immuno stimulant)
that can activate early innate defense mechanisms to aid in the generation of
robust and long-lasting immune responses [14]. Nevertheless,
most known adjuvants such as Freund’s complete adjuvant (FCA) and Freund’s
incomplete adjuvant (FIA) are inappropriate for human and animal utilization
due to their toxicity and side effects [15]. Previous researches
indicated that antigenicity of FliC made it potential candidate vaccine
adjuvants [6-8]. However, its
strong antigenicity needs to be eliminated
or reduced. In the present study,
molecular cloning and recombination expression of FliC∆220-320-gfp
and FliC-gfp were performed, and immune stimulatory of FliC∆220-320
and FliC in
humoral immunity to GFP were determined to
evaluate their adjuvant efficacy. Previous publications of Clement Nempont [16] and Liu et al. [11] on the adjuvant
activity of different FliC variants come
into the conclusion
that immunodominant epitopes, which are responsible for antigenicity of the FliC are in the hypervariable domain. Sequencing and analyzing results from various Salmonella sp. and other bacterial
flagellin genes proved that the main antigenic epitopes of flagellin are mainly
detected at residues from 200 to 350 [16-18] explaining why FliC∆220-320 lost its antigenicity but
still remained mucosal adjuvancy [11]. However, the FliCΔ220-320 variant that Liu et al. [11] created had anti-FliC antibody titers 80 times lower than FliC,
compared to FliCΔ220-320 variant in fusion with GFP in this
study. This dissimilarity might come from the differences between fusion forms
with GFP in this study and single forms in Liu’s [11] study of the two protein FliCΔ220-320 and
FliC; or might also be caused by certain test conditions. Additional researches
are needed to clarify this. The
results suggested that even though
the high antigenicity of the adjuvants may be a barrier to its utility for different vaccine
applications, but in an immune response
activated by FliC or FliCΔ220-320 along
with a certain antigen protein, the antigenic properties of the adjuvant neither has a significant correlation with the immune complementary support of these proteins, nor a significant effect to the humoral response towards the target antigen.
Further
experiments need to be conducted to confirm the ability of stimulating the
protective response against antigens of FliCΔ220-320's immune
complementation support, and to determine the ability of retaining immunity
after being repeatedly exposed. In
addition, there have been studies showing that the low antigenicity of 6x-His tag causes no affect
on the immune response in mice
[19,20], so 6x-His tag could not interfere with the present data.
CONCLUSIONS-
The recombinant vectors carrying the genes FliCΔ220-320-gfp and FliC-gfp
(pET28a-FliCΔ220-320-gfp and pET28a-FliC-gfp) encoding for FliCΔ220-320-gfp and FliC-gfp, respectively, had been successfully constructed, in which FliCΔ220-320 and FliC were derived from S. enterica Enteritidis. Transformed E.
coli BL21 (DE3) with
recombinant vectors
pET28a-FliCΔ220-320-gfp and pET28a-FliC-gfp were successfully
created. Expression and purification of recombinant FliCΔ220-320-gfp and FliC-gfp were performed with a purity of 56% and 79%, respectively.
FliCΔ220-320 variant in the fusion form
with GFP had four times less antigenicity than FliC (in the FliC-gfp fusion) and still retained the FliC's immune stimulation.
Further experiments should be conducted to compare the
stimulating effect to
the humoral immune response of the mixture of GFP antigen and FliCΔ220-320 protein
individually with FliCΔ220-320-gfp fusion protein.
ACKNOWLEDGEMENTS- This research did not receive any specific grant
from funding sagencies in the public, commercial, or not-for-profit sectors.
CONTRIBUTION OF AUTHORS
Research concept- Hieu
Tran-Van
Research design- Bao-Chau Thi Tran, Viet-Anh Nguyen, Hieu Tran-Van
Supervision-
Hieu Tran-Van
Materials-
Hieu Tran-Van, Thuoc
Linh Tran
Data collection- Bao-Chau Thi Tran, Viet-Anh Nguyen, Hieu Tran-Van
Data analysis and Interpretation- Bao-Chau Thi Tran, Viet-Anh
Nguyen, Hai-Vy Vo-Nguyen, Hieu Tran-Van
Literature search- Bao-Chau Thi Tran, Hai-Vy
Vo-Nguyen,
Writing article- Bao-Chau Thi Tran, Hai-Vy Vo-Nguyen, Hieu Tran-Van
Critical review-
Hieu Tran-Van
Article editing-
Hai-Vy Vo-Nguyen, Hieu Tran-Van
Final approval-
Hieu Tran-Van
REFERRENCES
1.
Fauci AS. Infectious diseases:
considerations for the 21st century. Clin. Infect. Dis., 2001;
32(5): 675-85.
2.
Leroux-Roels G. Unmet needs in modern
vaccinology: Adjuvants to improve the immune response. Vaccine, 2010; 28:
C25-C36.
3.
Peggy R, Kai S, Thomas E, Blair P, Carlos
AG. Vaccine adjuvants: key tools for innovative vaccine design. Curr. Top. Med.
Chem., 2013; 13(20): 2562-80.
4.
Lee S, Nguyen MT. Recent advances of
vaccine adjuvants for infectious diseases. Immune network, 2015; 15(2): 51-57.
5.
Guideline on adjuvants in vaccines for
human use, Committee for Medicinal Products for Human Use (CHMP), The European
Medicines Agency Evaluation of Medicines for Human Use, 2005; pp. 1-18.
6.
Andersen NE, Smith KD, Strobe KL,
Barrett SLR, Cookson BT, et al. Evasion of Toll-like receptor 5 by flagellated
bacteria. Proce. Natl. Acad. Sci., 2005;
102(26): 9247-52.
7.
Hayashi F, Smith KD, Ozinsky A, Hawn TR,
Eugene CY, et al. The innate immune response to bacterial flagellin is mediated
by Toll-like receptor 5. Nature, 2001; 410(6832): 1099.
8.
Boyd S. Design and Production of a
Recombinant FliC-Antigen Co-Expression Platform for Increased Vaccine Efficacy
(Thesis), 2014; pp. 1-59.
9.
Karam MRA, Oloomi M, Mahdavi M, Habibi
M, Bouzari S. Vaccination with recombinant FimH fused with flagellin enhances
cellular and humoral immunity against urinary tract infection in mice. Vaccine,
2013; 31(8): 1210-16.
10. Wang
G, Shi B, Li T, Zuo T, Wang B, et al. Linear antigenic mapping of flagellin
(FliC) from Salmonella enterica
serovar Enteritidis with yeast surface expression system. Vet. Microbial., 2016;
184: 20-46.
11. Liu
F, Yang J, Zhang Y, Zhou D, Chen Y, et al. Recombinant flagellins with partial
deletions of the hypervariable domain lose antigenicity but not mucosal
adjuvancy. Biochem. Biophys. Res. Commu., 2010; 392(4): 582-87.
12. Bao-Chau
TT, Viet-Anh N, Hieu TV. Cloning, expression and purification of the
recombinant FliC from Salmonella
enteritidis. Sci. Technol. Dev., 2016; 19: 62-69.
13. Ausubel
F, Brent R, Kingston R, Moore D, Seidman J, et al. Current protocols in
Molecular Biology. John. Wiley. Sons, 2003.
14. Kaur
G, Chitradevi S, Nimker C, Bansal A. rIL-22 as an adjuvant enhances the
immunogenicity of rGroEL in mice and its protective efficacy against S. Typhi and S. typhimurium. Cellular Mol. Immunol., 2015; 12(1): 96-106.
15. Billiau
A, Matthys P. Modes of action of Freund’s adjuvants in experimental models of
autoimmune diseases. J. Leukoc. Biol., 2001; 70(6): 849-60.
16. Nempont
C, Cayet D, Rumbo M, Bompard C, Villeret V, et al. Deletion of flagellin’s
hypervariable region abrogates antibody-mediated neutralization and systemic
activation of TLR5-dependent immunity. J. Immunol., 2008; 181(3): 2036-43.
17. He
X-S, Rivkina M, Stocker B, and Robinson WS. Hypervariable region IV of
Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing
factor for functional flagella. Journal of bacteriology, 1994; 176(8): 2406-14.
18. Kuwajima
G. Construction of a minimum-size functional flagellin of Escherichia coli.
Journal of bacteriology, 1988; 170(7): 3305-09.
19. Goldsby
RA, Kindt TJ, Osborne BA. Kuby immunology, 4th USA. W, 2000.
20. Watson
DS, Platt VM, Cao L, Venditto VJ, Szoka FC. Antibody response to
polyhistidine-tagged peptide and protein antigens attached to liposomes via
lipid-linked nitrilotriacetic acid in mice. Clin. Vaccine Immunol., 2011;
18(2): 289-97.