Download Vaccination with recombinant fusion proteins incorporating Toll

Vaccine 25 (2007) 763–775
Vaccination with recombinant fusion proteins incorporating Toll-like
receptor ligands induces rapid cellular and humoral immunity
James W. Huleatt a,∗ , Andrea R. Jacobs a , Jie Tang a , Priyanka Desai a , Elizabeth B. Kopp b ,
Yan Huang a , Langzhou Song a , Valerian Nakaar a , T.J. Powell a
b
a VaxInnate Corporation, New Haven, CT 06511, USA
Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA
Received 5 December 2005; received in revised form 27 June 2006; accepted 9 August 2006
Available online 22 August 2006
Abstract
Recognition of specific pathogen associated molecular patterns (PAMPs) is mediated primarily by members of the Toll-like receptor (TLR)
family. Stimulation through these receptors results in quantitative and qualitative changes in antigen presentation and cellular activation,
thereby linking innate and adaptive immunity. Consequently, the incorporation of TLR-ligands into vaccines could result in more potent
and efficacious vaccines. To test this hypothesis, we employed a recombinant fusion protein strategy using the TLR5 ligand flagellin fused
to specific antigens to promote protective immunity. These purified recombinant fusion proteins demonstrated potent TLR5-specific NF-␬B
dependent activity in vitro. Immunization of mice with the recombinant-flagellin-OVA fusion protein STF2.OVA resulted in potent antigenspecific T and B cell responses that were equal to or better than responses induced by OVA emulsified in Complete Freund’s adjuvant. These
included rapid and consistent antigen-specific IgG1 and IgG2a antibody responses that were detectable within 7 days of immunization, and
the development of protective CD8 T cell responses. Moreover, the enhanced immunogenicity to OVA is dependant on the direct fusion to
flagellin, as co-delivery of OVA with flagellin unlinked failed to augment antigen-specific responses in vivo. Similar results were obtained
using a recombinant fusion protein comprised of flagellin and a novel polypetide sequence containing two immuno-protective epitopes derived
from the Listeria monocytogenes antigens p60 and listeriolysin O. Animals immunized with this recombinant protein demonstrated significant
antigen-specific CD8 T cell responses and protection upon challenge with virulent L. monocytogenes. We conclude that immunization with
PAMP:antigen fusion proteins induce rapid and potent antigen-specific responses in the absence of supplemental adjuvants. Collectively our
data demonstrate that PAMP:antigen fusion proteins offer significant promise for developing recombinant protein vaccines.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Vaccination; Toll-like receptors; Antigen presentation and processing; Antibodies; B cells; T cells
1. Introduction
The development of safe and efficacious vaccines remains
a major goal in global public health [1,2]. The majority of
present day vaccines are comprised of two primary components, the antigen of therapeutic interest and components
termed adjuvants that enhance immunogenicity. The nature of
∗ Correspondence to: VaxInnate Corporation, Suite 311, 300 George
Street, New Haven, CT 09520, USA. Tel.: +1 203 785 1421;
fax: +1 203 785 1461.
E-mail address: [email protected] (J.W. Huleatt).
0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2006.08.013
adjuvants used varies greatly, but includes a variety of components including mineral oils, bacterial extracts, live and attenuated organisms and suspensions of aluminum hydroxide
metals [2–5]. Although adjuvants provide enhanced immune
responses their use can also elicit adverse side effects, as
shown for Complete Freunds adjuvant (CFA). Mycobacterial
components within this adjuvant elicit serious side effects,
including autoimmunity and arthritis [6–8]. Therefore, the
numbers of adjuvants that are approved and effective in
humans remain relatively limited.
Recent advances in the field of innate immunity have provided a better understanding of both the cellular and molecu-
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lar mechanisms governing the regulation of the host immune
response. Janeway originally postulated that the recognition
of pathogens by the host was mediated by specific receptors
on the surface of antigen presenting cells (APC) [9]. This
hypothesis has now been supported by the identification of
11 members belonging to the Toll-like receptor family (TLR)
(reviewed in [10,11]). The recognition of distinct pathogenassociated molecular patterns (PAMPs) is mediated by specific TLR receptors, including TLR2 (lipidated peptides and
lipoteichoic acids), TLR3 (double stranded RNA), TLR4
(lipopolysaccharide), TLR5 (flagellin), and TLR9 (unmethylated CpG DNA). Specific recognition of these ligands by
their cognate TLR initiates a signaling cascade through the
shared intracellular domain similar to that found in IL1 receptors. Recruitment of adaptor proteins including the
myeloid differentiation factor 88 (MyD88) promotes the signaling cascade that results in the activation of NF-␬B and
mitogen activated kinases [12]. These signals collectively
result in the activation and maturation of APC’s including
the regulated processing and presentation of antigens, upregulation of major histocompatibity complex (MHC) and
costimulatory molecules, and secretion of proinflammatory
chemokines and cytokines. These APCs then mediate the
activation of antigen-specific T and B cell responses and
thereby serve as the link between the innate and adaptive
immune response [13–15].
In this study we have examined the ability of recombinant
antigen-PAMP fusion proteins to elicit more immunogenic
responses in vivo. Flagellin is a highly conserved bacterial
protein that elicits TLR5-dependent inflammatory responses
in both animals and plants [16–18]. In situ flagellin plays
an important role in bacterial pathogenicity by facilitating
motility and adhesion to host tissues [19]. Earlier studies
have investigated the ability of flagellin to elicit antigenspecific T cell responses when delivered as live attenuated
vaccines [20–22]. Given the known potential safety risks
associated with vaccines based on attenuated microorganisms, we have examined the use of recombinant fusion proteins composed of Salmonella typhimurium flagellin fljB
(STF2) fused to specific antigenic targets to elicit enhanced
immunogenicity. In this study we demonstrate that highly
purified flagellin fusion proteins induce TLR5-specific cellular activation in the absence of endotoxin. A single subcutaneous (s.c.) immunization with the recombinant fusion
protein comprised of flagellin linked to chicken ovalbumin (STF2.OVA) results in significant cellular and humoral
OVA-specific responses. Most notable, immunization with
the fusion protein generated consistently higher and more
rapid antigen-specific IgG (IgG1 and IgG2a ) titers compared
to littermates receiving unmodified antigen in conventional
adjuvants. Moreover, similar responses were observed when
STF2 was expressed in fusion with several MHC Class I
restricted CD8 T cell epitopes derived from Listeria moncytogenes. The STF2.LIST fusion protein induced antigenspecific CD8 T cell responses and protective immunity in
vivo. The antigen-specific responses are dependent on the
direct fusion of flagellin with the antigen, as immunization
with equivalent concentrations of unlinked antigen and flagellin failed to induce detectable responses. The results of these
studies demonstrate that the use of recombinant fusion proteins encoding specific TLR ligands represents an effective
vaccination strategy that does not require the use of a conventional adjuvant.
2. Materials and methods
2.1. Cloning of STF2.OVA and STF2.LIST fusion
constructs
S. typhimurium FljB (STF2) was cloned by PCR using the
primers: 5 -ATGGCACAAGTAATCAACACTAAC-3 and
5 -ACGTAACAGAGACAGCACGTTCTGC-3 and inserted
into the expression vector, PCRT7/CT. Chicken ovalbumin
was cloned by PCR using the primers 5 -GTTATGCTCGAGGGCTCCATCGGCGCAGCAAGC-3 and 5 -ACCTTCAAGCTTCGAAGGGGAAACACATCTGCCAAA-3 ,
digested with XhoI and HindIII and inserted in frame
downstream of STF2. STF2.LIST comprises a fusion protein
of STF2 fused at the C-terminus to a polypeptide sequences
corresponding to the L. monocytogenes virulence antigens
listeriolysin (LLO52-387 ) and p60196-319 [23,24]. To construct
STF2.LIST, a forward primer (5 -CGTCTCGAGGAATTCCCAATCGAAAAGAAACACGCG-3 ) and a reverse
primer (5 -ACGGCACTGGTCAACTTGGCCATGGTG-3 )
were used in a PCR amplification using the LIST sequence as
a template. STF2 and the LIST sequences were then ligated
and colonies were identified by PCR screening and restriction mapping. The resulting constructs were confirmed by
DNA sequencing. Both STF2.OVA and STF2.LIST encode
V5 and polyhistidine tags at the C-terminus of the fusion
proteins.
2.2. Expression and purification of recombinant fusion
proteins
BL21 (DE3)pLysS cells (Invitrogen, Carlsbad, CA, USA)
were used as the host strain for expression of the recombinant
fusion proteins. Cells transformed with DNA of pSTF2.OVA
or pSTF2.LIST were grown at 37 ◦ C in Luria broth (LB)
medium containing 0.5% glucose, carbenicillin (50 ␮g/ml)
and chloramphenicol (34 ␮g/ml). Protein expression was
induced with 1 mM IPTG. Cells were harvested at 3 h
post induction and lysed in Lysis Buffer (150 mM NaCl,
100 mM Tris–Cl, 1 mM PMSF, 1 mg/ml lysozyme, 1% glycerol, pH 8.0) followed by passing through a Microfluidizer
(Microfluidics, Newton, MA, USA). The soluble fraction was
harvested and applied to a Ni-NTA column (Sigma–Aldrich).
Eluted proteins were further purified by two gel filtration runs
using SD200 column (Amersham Biosciences, Piscataway,
NJ, USA) with and without the presence of 1% Nadeoxycholate to remove endotoxin. Identity and purity of pro-
J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
teins were evaluated by SDS-PAGE in 12% polyacrylamide
gels and western blot analysis with Tetra-HIS antibody (Qiagen, Valencia, CA, USA), followed by species-specific APconjugated secondary antibody (Pierce, Rockford, IL, USA).
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immunizations were delivered subcutaneously (s.c.) in a volume of 100 ␮l. Antigen-specific T and B cell responses were
evaluated at times stated in the text.
2.6. Measurement of antibody response by ELISA
2.3. Endotoxin measurement
Endotoxin levels were determined using the Chromogenic
Limulus Amebocyte Lysate assay (Cambrex, Walkersville,
MD, USA) as directed by the manufacturer. Endotoxin values of purified STF2.OVA and STF2.LIST proteins were
<0.03 EU/␮g.
2.4. Cells and TLR bioassays
The RAW264.7 cell line was obtained from ATCC
(Rockville, MD, USA). This cell line expresses TLR2 and
TLR4, but not TLR5 [11]. To test for TLR5-specific activity
of the fusion proteins, RAW264.7 (RAW) cells were transfected with a plasmid encoding human TLR5 (Invivogen,
San Diego, CA, USA) to generate the RAW/TLR5 cell line.
TLR5-specific activity of fusion proteins was evaluated based
on NF-␬B dependant induction of TNF␣. In brief, RAW and
RAW/TLR5 cells were cultured in 96-well microtiter plates
(Costar) at a seeding density of 3–5 × 104 cells in 100 ␮l/well
in DMEM medium supplemented with 10% FCS and antibiotics. The next day, cells were treated for 5 h with serial
dilutions of test proteins starting at 5 ␮g/ml. For positive controls RAW cells were treated with the TLR4 agonist LPS
(Sigma–Aldrich). Expression of TNF␣ was then evaluated
by ELISA (Invitrogen, Carlsbad, CA, USA).
In some experiments, TLR5-specific activity was also
evaluated using RAW and RAW/TLR5 cells transfected with
a plasmid encoding a luciferase reporter regulated by the
NF-␬B regulatory promoter. TLR mediated NF-␬B dependent luciferase activity was measured after 5 h using the
Steady-Glo Luciferase Assay System (Promega, Madison,
WI, USA) as directed by the manufacturer while. Absorbance
and luminescence were evaluated using a TECAN microplate
spectrophotometer running Magellan software (Amersham).
Results are expressed as the fold change in relative luciferase
units (RLU) or pg/ml TNF␣.
2.5. Mice
Female C57BL/6 and BALB/c mice at 6–8 weeks of age
were purchased from the Jackson Laboratory (Bar Harbor,
ME, USA). Animals were housed in the Yale University
Animal facility (New Haven, CT, USA). All studies were performed in accordance with the Yale University Institutional
Animal Care and Use Committee (IACUC). Animals were
immunized with purified endotoxin-free recombinant fusion
proteins in sterile phosphate buffered saline (PBS), equimolar
concentrations of OVA (Sigma–Aldrich) alone or emulsified
in aluminum hydroxide (Alum) (Pierce) or Complete Freund’s Adjuvant (CFA) (Sigma–Aldrich) or PBS alone. All
Antibody responses to OVA were analyzed by ELISA
as previously described [25]. For direct ELISAs, 96 well
plates were coated with OVA (Sigma–Aldrich) at 2 ␮g/ml
in PBS. Wells were then blocked with Assay Diluent Buffer
(BD, PharMingen) and incubated with serial dilutions of
immune sera for 1 h at room temperature. Plates were then
washed 3× with PBS + 0.05% tween and incubated with
HRP-conjugated goat anti-mouse IgG (Jackson Immunochemical) for 1 h at room temperature. Plate were washed
and developed with TMB Ultra substrate (Pierce). Where
indicated IgG isotypes were evaluated by indirect sandwich
ELISA as previously described [25]. Briefly, 96 well ELISA
plates were coated with isotype specific capture antibodies to
IgG1 and IgG2a (20 ␮g/ml) in PBS, then blocked using Assay
Diluent Buffer. Serial dilutions of serum samples were added
and incubated overnight at 4 ◦ C. OVA labeled with Digoxigenin (DIG) using the DIG labeling kit (Roche, Indianapolis,
IN, USA) was added, followed by HRP-conjugated antiDIG mAb (Roche). Plates were developed using TMB Ultra
substrate and evaluated on a TECAN microplate spectrophotometer running Magellan software. Data are expressed as
O.D.450/650 and in some instances converted to ␮g/ml based
on standard curves generated using an OVA-specific IgG1
mAb.
2.7. Antigen-specific ELISPOT assays
105 spleen or lymph node cells from animals immunized
as indicated were added to wells coated with anti-IFN␥ capture antibody diluted in PBS according to the manufacturer’s
instructions (R&D Systems, Minneapolis, MN, USA). Where
indicated, CD8 T cells were enriched (>97%) using magnetic beads on a AutoMacs cell separator (both from Miltenyi Biotec, Auburn, CA, USA). T cells were then stimulatd
overnight with naı̈ve APC (106 cells/well) in the absence or
presence of specific antigenic peptides including the OVAderived H-2Kb restricted peptide SIINFEKL(OVA257–264 )
or the L. monocytogenes-derived H2-Kd restricted peptides
listeriolysin LLO(91–99) or p60(217-225) peptides (all synthesized by GeneMed Synthesis Inc., South San Francisco, CA,
USA) at a final concentration of 0.5 ␮g/ml. Anti-CD3 (BD
Pharmingen) was used as a positive control at a final concentration of 0.25 ␮g/ml. Plates were incubated overnight
at 37 ◦ C/5% CO2 , then washed and incubated with detection antibody diluted in PBS/10% FBS according to the
manufacturer’s instructions. Plates were developed using the
ELISPOT Blue Color Development Module according to the
manufacturer’s protocol (R&D Systems). Antigen-specific
response were performed in duplicate from individual animals and quantified using an automated ELISPOT reader
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(Cellular Technology Ltd., Cleveland, OH, USA). Data is
represented as the number of antigen-specific responses/106
cells.
2.8. L. monocytogenes bacterial protection assays
Efficacy of the flagellin fusion proteins was examined
in vivo using wild type L. monocytogenes (Strain 43251,
ATCC) or recombinant OVA-expressing L. monocytogenes
strain JJL-OVA (kindly provided by Dr. Hao Shen, University
of Pennsylvania) [26]. On indicated days post-immunization
with flagellin-fusion proteins, animals (10/group) were challenged i.v. with a determined LD50 dosage of recombinant
OVA expressing L. monocytogenes strain JJl-OVA, or 43251,
respectively. Three days following bacterial challenge mice
were euthanized and bacterial burden in the spleen was determined by plating serial dilutions of spleen cell suspension
onto BHI agar plates [27]. Bacterial colonies were counted
after 30 h. Data reflect the mean and S.D. obtained from 10
animals/group. P values were determined using a paired Student’s t-test.
3. Results
A containing the S. typhimurium flagellin (fljB) gene
fused to the 5 end of the chick ovalbumin (OVA) gene
was designed and expressed in E. coli (Fig. 1A). A similar construct, STF2.LIST, comprised of flagellin fused to
polypeptide sequences corresponding to the MHC Class I
H-2d immunodominant epitopes from the L. monocytogenes
proteins listeriolysin (LLO) and p60 [23,24], was also gener-
Fig. 1. STF2.OVA and STF2.LIST flagellin fusion proteins. (A) The 3 terminus of the gene encoding the Salmonella typhimurium flagellin (fljB) was
fused to the 5 terminus of the gene for chick ovalbumin (OVA) or a recombinant sequence encoding epitopes of the Listeria monocytogenes virulence
proteins listeriolysin O (LLO52–387 ) and p60196–319 . The final constructs
were expressed in E. coli and purified by metal affinity chromatography. (B)
SDS-PAGE and (C) Western Blot with anti-HIS antibodies; M: molecular
weight markers; lane 1: STF2.OVA; lane 2: STF2.LIST.
ated. The HIS-tagged proteins were purified by metal affinity
chromatography followed by gel filtration chromatography.
The expression of STF2.OVA and STF2.LIST was confirmed
by SDS-PAGE (Fig. 1B) and Western blot analysis using antibody to the C-terminal poly-histidine tag (Fig. 1C). The purity
of the recombinant fusion proteins was >95% with endotoxin
levels of <0.03 EU/␮g, as determined by Limulus amebocyte
lysate assay (data not shown).
TLR-mediated signaling induces NF-␬B activation that
regulates the expression of multiple pro-inflammatory
cytokines, including IL-8 and TNF␣. In order to evaluate
TLR5 activity purified recocombinant proteins were examined for activity using the mouse macrophage cell line
RAW264.7. The parental line expresses TLR2 and TLR4,
and responds to stimulation with LPS that can be inhibited
with polymyxin B [11]. Notably, RAW264.7 lack expression of TLR5 and do not respond to stimulation with purified
flagellin (STF2) (Fig. 2A). To confer TLR5-specific activity, RAW264.7 cell were transfected with the gene encoding
human TLR5, to generate the RAW/hTLR5 reporter line.
Stimulation of RAW/hTLR5 cells with flagellin induced cellular activation that was not inhibited by polymyxin B, consistent with TLR5-dependent activation (Fig. 2A).
The TLR5-specific activity of STF2.OVA and STF2.LIST
was evaluated in vitro on RAW and RAW/hTLR5 cells.
Cells were incubated overnight with serial dilutions of
STF2.OVA and STF2.LIST, and secretion of TNF␣ was evaluated by ELISA. STF2.OVA induced expression of TNF␣ in
RAW/TLR5, but not RAW cells, with an apparent EC50 (concentration which yields 50% maximal activity) of 7.2 ng/ml
(Fig. 2B). Similarly, stimulation with STF2.LIST specifically induced expression of TNF␣ in RAW/hTLR5, but not
RAW cells (EC50 of 84 ng/ml) (Fig. 2C). Notably, inclusion
of polymyxin B did not inhibit the activity of STF2.OVA
or STF2.LIST further supporting the TLR5-specificity and
purity of the recombinant proteins.
The immunogenicity of purified recombinant STF2.OVA
was examined in vivo. C57BL/6 mice received a single s.c.
immunization with 25, 2.5, or 0.25 ␮g of STF2.OVA, or
PBS, and antibody responses to OVA were examined 8 days
post-immunization. OVA-specific IgG1 (Fig. 3A) and IgG2a
(Fig. 3B) responses were detected in sera of mice immunized
with as little as 0.25 or 2.5 ␮g, respectively. Notably, immunization with endotoxin-free OVA alone at concentrations
up to 90 ␮g failed to elicit detectable levels of OVA-specific
IgG on Day 8 post immunization (data not shown), although
this dose can elicit OVA-specific responses by Day 21 postimmunization [25]. Thus, presentation of OVA in the context
of a flagellin fusion protein significantly enhances its potency
as an immunogen. However, it remained unclear if the direct
fusion of flagellin to the antigen was required for the increased
immunogenicity. To evaluate this question, mice were immunized s.c. with STF2.OVA (25 ␮g), an equimolar dose of OVA
(12 ␮g) co-delivered with recombinant STF2 (12 ␮g), or PBS
alone as a negative control. Eight days following a single s.c.
immunization sera were harvested and evaluated for OVA-
J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
Fig. 2. TLR5-specific activity of recombinant flagellin fusion proteins. The
TLR5-specific activity of recombinant flagellin fusion proteins was examined on the TLR5 negative mouse macrophage cell line RAW264.7 (RAW) or
RAW cells expressing human TLR5 (RAW/hTLR5). (A) TNF␣ production
by RAW and RAW/hTLR5 cells following stimulation with LPS, or recombinant flagellin in both the absence and presence of polmyxin B. Similarly
the TLR5-specific activity of STF2.OVA (B) and STF2.LIST (C) was evaluated on RAW and RAW/hTLR5 cells in both the absence (solid lines) and
presence (dashed lines) of polmyxin B. Results reflect the levels of TNF␣
as determined by ELISA.
specific antibodies. OVA-specific IgG1 (Fig. 4A) and IgG2a
(Fig. 4B) antibody responses were detected in the sera of
mice immunized with the fusion protein STF2.OVA, but not
in mice immunized with an equivalent dose of OVA alone or
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Fig. 3. Dose dependent response to immunization with STF2.OVA. Mice
were immunized s.c. with 25, 2.5 and 0.25 ␮g of STF2.OVA. Eight days
later, OVA-specific IgG1 and IgG2a levels were examined by indirect ELISA.
Data reflect the OVA-specific IgG1 and IgG2a responses of five mice/group
at serum dilutions of 1:500 and 1:100, respectively. Similar results were
obtained in three independent experiments (* P < 0.05 vs. PBS by Student’s
t-test).
mixed with STF2. These results demonstrate that the physical fusion of the TLR5 ligand flagellin with the antigen is
required for the increased immunogenicity of the antigen.
Aluminum hydroxide (alum) is often used as an experimental adjuvant with OVA, and is in fact used in many
approved human vaccines [3]. To directly compare the
immune-potentiating capacity of flagellin fusion proteins
with this approved adjuvant, the kinetics of the OVA-specific
antibody responses were evaluated following a single s.c.
immunization with 25 ␮g of STF2.OVA or 100 ␮g of OVA
adsorbed on alum. OVA-specific total IgG responses were
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Fig. 5. Immunization with the flagellin fusion protein STF2.OVA induces
rapid antigen-specific responses in vivo. OVA-specific IgG1 responses were
examined 1, 2 and 3 weeks post immunization with 25 ␮g STF2.OVA (䊉) or
100 ␮g OVA formulated in alum (). Individual responses of three mice per
group are shown. Similar results were obtained in two independent experiments. (* P < 0.05, vs. PBS control; ‡, vs. 100 ␮g of OVA delivered in alum.)
Fig. 4. The enhanced immunogenicity of STF2.OVA is fusion-dependent.
BALB/c mice were immunized s.c. with PBS (), STF2 (), OVA (䊉),
STF2.OVA () or STF2 + OVA (). All concentrations of OVA were equivalent to 12 ␮g/mouse. Eight days following the immunization, sera were
harvested and examined for OVA-specific IgG1 (top) and IgG2a (bottom) responses by direct ELISA. Data represent the mean + S.D. of five
mice/group.
evaluated in each group at 7, 14, and 21 days following
immunization. The data in Fig. 5 show that as early as Day
7, sera from STF2.OVA-immunized mice demonstrated significant levels of OVA-specific IgG compared to control
animals (P < 0.05). These levels continued to increase on
both Days 14 and 21 post immunization. By contrast, OVAspecific IgG was not detectable in the serum of mice receiving
100 ␮g OVA in alum until 14 days following immunization. Although the levels of OVA-specific IgG continued to
increase by Day 21, OVA + alum immunized mice did not
attain the OVA-specific IgG levels observed in STF2.OVAimmunized animals at 7 days post immunization. These
results demonstrate that flagellin-fusion proteins elicit faster
immune responses in vivo in terms of both kinetics and magnitude than that observed using the conventional adjuvant
alum.
While a single immunization with a flagellin fusion protein appears to induce potent, rapid-onset antibody responses,
the utility of this approach might be limited if the host
also generates antibody responses to flagellin that neutralize its TLR5 activity. Such antibodies might be expected
to prevent a boost response to the same fusion protein or
a primary response to a different flagellin fusion protein.
This type of ‘vector effect’ has hampered the development
of some viral vectors for recombinant vaccines, including
some adenoviruses [28,29]. To address this possibility, antisera from mice immunized with STF2.OVA were examined for
the ability to specifically inhibit TLR5-mediated activation
by flagellin in vitro. Prior to incubation with RAW/hTLR5
cells, purified flagellin was first pre-incubated with naı̈ve
or immune sera from mice immunized with STF2.OVA or
OVA in CFA. Following incubation, samples were added
to RAW/hTLR5 cells containing a luciferase reporter under
the regulation of the NF-␬B promoter. After 5 h TLR5specific NF-␬B-mediated luciferase activity was examined.
The results show that sera from STF2.OVA-immune animals
did not inhibit TLR5-specific recognition and activation by
flagellin in vitro (Fig. 6A). Similarly the potential ability of
preexisting anti-flagellin immune responses to inhibit subsequent immune responses to STF2 fusion protein in vivo
was examined. C57BL/6 mice (n = 5/group) were immunized
with PBS, STF2 (12 ␮g) or STF2.OVA (25 ␮g). Three weeks
later, all animals were challenged with 25 ␮g of STF2.OVA,
and OVA-specific IgG antibody responses were compared
by ELISA 7 days later. As expected, animals primed with
STF2.OVA exhibited the highest levels of OVA-specific IgG.
Interestingly, animals primed with STF2 alone demonstrated
higher OVA-specific IgG than animals receiving only PBS in
J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
Fig. 6. Antibody responses to flagellin do not inhibit responses to recombinant flagellin fusion proteins in vitro or in vivo. (A) Antisera from animals
immunized with PBS (NI), STF2.OVA, or CFA + OVA were diluted 1:100
and pre-incubated for 1 h with (solid bars) or without (open bars) recombinant STF2. Samples were then examined for TLR5 mediated activity on
RAW/hTLR5 cells transfected with a NF-␬B-dependant luciferase reporter.
Results reflect the fold increase in relative luciferase units (RLU) in duplicate
samples. (B) The effect of anti-flagellin antibodies on a subsequent vaccination with flagellin recombinant fusion proteins in vivo. C57BL/6 mice
animals were primed with PBS (), STF2 () or STF2.OVA (×). All mice
then received a single immunization with 25 ␮g of STF2.OVA 21 days later,
and OVA-specific IgG responses were evaluated 7 days later by ELISA.
Response of animals receiving PBS only are shown as a control (). Data
reflects the mean + S.D. obtained from five mice/group.
the primary immunization (Fig. 6B). These results demonstrate that immune responses elicited to flagellin do not
inhibit subsequent TLR5-mediated activation or immunogenicity to recombinant flagellin fusion proteins in vitro or
in vivo.
Having demonstrated that fusion to flagellin converted
OVA into a potent immunogen for antibody responses,
we investigated the ability of STF2.OVA to induce potent
OVA-specific T-cell responses. We focused on the wellcharacterized H-2Kb -restricted CD8 epitope, SIINFEKL
(OVA257–264 ). C57BL/6 mice received a single s.c. immunization with PBS, STF2.OVA (25 ␮g) or an equimolar dose
of OVA (12 ␮g) emulsified in the adjuvant CFA. Antigenspecific T and B cell responses to OVA were examined 8
days following immunization. Similar to the results presented
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above, mice immunized with STF2.OVA demonstrated consistently higher OVA-specific total IgG responses than mice
receiving OVA delivered in the conventional adjuvant CFA
(Fig. 7A). Similar antibody responses were obtained following immunization of several different strains including
BALB/c, C3H/HeN, and TLR4 unresponsive C3H/HeJ (data
not shown). To examine T-cell responses, spleen and draining lymph node cells from the immunized C57BL/6 mice
were restimulated overnight in the presence of the SIINFEKL
peptide, and IFN␥ ELISPOT responses were measured. The
results demonstrate that immunization with STF2.OVA in
PBS induces antigen specific T cell responses in the spleen
(Fig. 7B) and draining lymph node (Fig. 7C) similar in magnitude to those observed following immunization with an
equivalent concentration of OVA delivered in CFA. Collectively, these data demonstrate that flagellin fusion proteins
are highly immunogenic and induce antigen-specific T and
B cell responses in vivo.
The ultimate test of a vaccine design is its ability to
induce protective immunity in a host organism. To determine if responses to flagellin fusion proteins could impart
protective immunity, challenge studies using the Gram positive bacteria L. monocytogenes were performed. Protective
immunity to this intracellular pathogen is primarily attributed
to the CD8 T cell response. Immunity to L. monocytogenes is evident by reduced bacterial burden in the spleens
and liver following an i.v. challenge with virulent L. monocytogenes. Using this model, the ability of STF2.OVA to
induce protective immunity was examined in C57BL/6 mice.
All mice were immunized with the equivalent of 12 ␮g
OVA as OVA alone or STF2.OVA. Three weeks following immunization, mice were challenged i.v. with 2 × 104
CFU of the OVA-expressing recombinant L. monocytogenes
strain JJL-OVA [26]. Antigen-specific CD8 T cell responses
were examined in IFN␥ ELISPOT assays both before and
after an i.v. challenge with the recombinant OVA-expressing
L. monocytogenes strain JJL-OVA. Mice immunized with
STF2.OVA demonstrated higher antigen-specific IFN␥ CD8
T cell responses than animals immunized with STF2 + OVA
following both prime alone (88.3/106 versus 0.42/106 ) and
post-challenge (310.8/106 versus 65.2/106 ) (Fig. 8A). Thus,
immunization with STF2.OVA induced a potent CD8 T cell
response to the surrogate antigen OVA, confirming the results
presented above. To evaluate efficacy in vivo STF2.OVA
immunized animals were challenged i.v. on Day 21 with
1 × 104 CFU of OVA-expressing L. monocytogenes JJL-OVA
and bacterial burden in the spleen was examined 3 days
post challenge. Animals immunized with OVA alone developed bacterial bacterial burden in the spleen (1.5 × 105 CFU)
that was not significantly lower than that observed in PBSimmunized (naı̈ve) mice (Fig. 8B). In contrast, STF2.OVAimmunized mice exhibited significantly lower bacterial burden (8 × 103 CFU, P = 0.0014) in the spleen compared to
naı̈ve mice. These results demonstrate that immunization
with STF2.OVA primes robust antigen-specific and protective CD8 T cell responses in vivo.
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J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
Fig. 8. STF2.OVA mediated protection from bacterial challenge in vivo.
C57BL/6 mice were immunized s.c. with OVA (12 ␮g, hatched bars) or
STF2.OVA (25 ␮g, solid bars) or PBS. (A) On day 9 CD8 T cells were isolated and restimulated in vitro with the OVA-derived MHC Class I restricted
peptide SIINFEKL in IFN␥ ELISPOT assays. (B) On Day 21 post immunization, a second cohort of mice was challenged i.v. with 2 × 104 CFU
of recombinant OVA-expressing L. monocytogenes. Bacterial burden in the
spleen was measured 3 days post-challenge. Data depict the mean ± S.D. of
10 individual mice per group performed in duplicate. The data are representative of results from three experiments with similar results (* P < 0.05 by
paired Student’s t-test).
Fig. 7. STF2.OVA fusion protein induces potent OVA-specific antibody and
T-cell responses. Antigen-specific responses to OVA were examined following a single s.c. immunization with 25 ␮g of STF2.OVA (), 12 ␮g
(equimolar) of OVA emulsified in CFA () or PBS alone (䊉). Eight days following immunization antigen-specific antibody and cellular responses were
examined in ELISA (A) and IFN␥ ELISPOT assays (B, C), respectively.
ELISA results reflect the OVA-specific total IgG serum titer. ELISPOT values denote the number of antigen-specific IFN␥ responses per 106 total
lymph node and spleen cells following stimulation with the OVA-derived
peptide SIINFEKL. Data depict the mean ± S.D. of five mice/group, performed in duplicate (* P < 0.05 vs. control animals). Similar results were
obtained in five independent experiments.
Given that STF2.OVA is limited as a model antigen system, the ability of recombinant flagellin fusion vaccines to
impart protection against a natural pathogen was also examined following immunization with STF2.LIST. STF2.LIST
is comprised of flagellin fused to a de novo antigen (LIST)
sequence that includes two dominant MHC Class I H-2Kd
restricted peptides derived from L. monocytogenes, LLO91–99
and p60217–225 . Antigen-specific T cell responses to these
peptides confer protection to L. monocytogenes in vivo
[23,24].
BALB/c (H-2Kd ) mice were immunized s.c. with 25 ␮g
of STF2.LIST or PBS alone. An additional group of mice
was immunized with 1 × 103 CFU of L. monocytogenes as
a positive control. Antigen-specific CD8 T cell responses
J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
were examined 9 days following immunization. Immunization with STF2.LIST elicited antigen-specific CD8 T cell
responses to both LLO91–99 and p60217–225 that were equal
to or greater than responses elicited by immunization with the
intact pathogen, L. monocytogenes (Fig. 9A and B). To evaluate the priming of antigen-specific CD8 T cell responses,
a second cohort of animals was challenged on Day 21 with
2 × 104 CFU of L. monocytogenes and CD8 T cells responses
771
were evaluated 5 days later. As seen following the prime, mice
immunized with STF2.LIST demonstrated markedly higher
antigen-specific CD8 T cell responses following a challenge
with L. monocytogenes (Fig. 9A and B). These responses
were similar in magnitude to those elicited by exposure to
the intact pathogen.
To evaluate the ability of STF2.LIST to confer protective immunity, BALB/c mice were immunized s.c. with
PBS, STF2 or STF2.LIST. Three weeks following immunization animals received a challenge with a 50% lethal
effective dose (LD50 ) of 2 × 104 CFU of virulent L. monocytogenes. Three days post challenge protection was evaluated
based on bacterial burden in the spleen, as described above.
While STF2 alone demonstrated no significant protection
compared to PBS control animals, STF2.LIST-immunized
animals exhibited a marked reduction (P = 0.06) in bacterial burden in the spleen 3 days following immunization
(Fig. 9C). These results demonstrate that STF2.LIST effectively induces antigen-specific CD8 T cell responses that
correlate with protective immunity in vivo.
4. Discussion
Traditionally, the most successful vaccines were composed of inactivated or attenuated whole organisms, such as
polio or smallpox virus, or partially purified preparations of
pathogen components, such as the split influenza virion seasonal vaccines. With the advent of recombinant technology,
the focus of vaccine development shifted to highly-purified
recombinant proteins. Unfortunately, many recombinant proteins lose their ability to induce a potent and protective
immune response when presented in highly-purified form,
unless formulated with components broadly termed adjuvants
[3–5], which increase the immunogenicity of antigens by
mechanisms that were previously unknown. Recent advances
in our understanding of the innate immune system have
uncovered the mechanisms of actions of many adjuvants. It
is now understood that the majority of adjuvants with the
exception of alum activate the host immune system via TLRmediated signaling through the recognition of PAMPs [9,30].
Fig. 9. Immunization with STF2.LIST induces antigen-specific CD8 T cell
responses and protective immunity. (A) BALB/c mice were immunized
s.c. with PBS, STF2.LIST (25 ␮g), or 1 × 103 CFU of L. monocytogenes
(L.m.) as positive controls. Antigen-specific CD8 T cells responses to the
H-2Kd restricted immunodominant epitopes LLO91-99 (A) and p60217-225
(B) were evaluated in IFN␥ ELISPOT assays on Day 9 post immunization () and 3 days following a challenge with 1 × 104 CFU of live L.
monocytogenes on Day 21 (). (C) Antigen-specific protection in vivo was
examined in BALB/c mice following immunization with PBS, STF2 (12 ␮g)
or STF2.LIST (25 ␮g). On Day 21 post immunization animals were challenged i.v. with 2 × 104 CFU of L. monocytogenes. Bacterial burden in the
spleen was measured 3 days post-challenge. Data depict the mean ± S.D. of
10 individual animals (performed in duplicate) per group. The data are representative of results from three experiments with similar results (* P < 0.05
by paired Student’s t-test).
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J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
Due to the critical role played by TLRs in initiating and regulating the adaptive immune response, and the contribution of
PAMPs to adjuvant activity, we hypothesized that physically
linking PAMP and antigen would significantly increase the
immunogenicity of the antigen. We tested this hypothesis by
generating two recombinant fusion proteins, each of which
fuses nominal antigen to the C-terminus of bacterial flagellin, the natural ligand for TLR5. The purified recombinant
flagellin fusion proteins STF2.OVA and STF2.LIST demonstrated TLR5 specific activity, with no TLR2 or TLR4 activity
in vitro (Fig. 2 and data not shown). A single immunization with as little as 0.25 ␮g of STF2.OVA elicited antigenspecific antibody responses that were detectable as early as
8 days post-immunization (Fig. 3); the antibody response
clearly involved isotype switching as both IgG1 and IgG2a
isotypes were detected. It is clear that the fusion of flagellin
to OVA significantly increases the immunogenicity of OVA,
since doses of OVA up to 90 ␮g failed to induce detectable
antibody responses (data not shown). Fusion of the flagellin and antigen components was essential for the induction
of antigen-specific antibody responses, as immunizing mice
with a simple mixture of STF2 and OVA did not induce
detectable OVA-specific antibodies (Fig. 4). This point is an
important one since it confirms the importance of physical
association of PAMP and antigen in subcellular trafficking
and presentation of the antigen, as reported by Blander and
Medzhitov [31,32]. Those authors argue that co-delivery of
the two components separately does not result in optimal
antigen processing and presentation due to the absence of an
active TLR signal in the subcellular vesicle containing the
antigen, a deficiency which can be overcome by delivering
physically linked PAMP and antigen such that the TLR signal
occurs in the same vesicle that contains the antigen payload.
We have demonstrated on the whole animal level that the
fusion protein strategy is superior to the co-mixture strategy,
in the current study and in other antigen systems using either
flagellin (TLR5 ligand) or Pam3Cys (TLR2 ligand) (unpublished observations).
Comparison of the flagellin fusion strategy with standard approved and experimental adjuvants showed that
STF2.OVA was at least as potent as equivalent doses of
OVA in alum (Fig. 5) or CFA (Fig. 7). In fact, immunization with STF2.OVA generated not only higher titer
antibody responses than OVA/alum, but also a faster-onset
response (Fig. 5). A single immunization with STF2.OVA
elicited near-maximal antibody responses within 7 days,
while immunization with OVA/alum did not elicit measurable OVA-specific antibody responses until day 14 postimmunization. While these responses increased by Day 21,
they remained lower than those observed in STF2.OVAimmunized animals as early as Day 7. Therefore, the use
of recombinant fusion proteins encoding a specific TLR
ligand(s) represents a potentially valuable strategy in the
development of new vaccines, particularly in scenarios where
protective immunity is required in terms of days rather than
weeks.
In addition to inducing OVA-specific antibody responses,
we found that immunization with STF2.OVA induced potent
T-cell responses, particularly a CD8 response specific for the
H-2Kb -restricted epitope OVA257–264 , or SIINFEKL (Fig. 7).
A single immunization with STF2.OVA induced potent CD8
IFN␥ ELISPOT responses in as little as 8 days. These
results encouraged us to hypothesize that recombinant flagellin fusion proteins could provide CD8-mediated protection
to a pathogenic challenge in vivo. This hypothesis was tested
by immunizing mice with STF2.OVA and challenging them
with L. monocytogenes engineered to express OVA as a surrogate antigen (JJL-OVA). In addition to antibody responses,
immunization with STF2.OVA elicited antigen-specific CD8
T cell responses that were comparable to those observed in
animals following a challenge with the live pathogen, and
this T cell response correlated with protection from a challenge with live JJL-OVA (Fig. 8). Since natural immunity
to L. monocytogenes is attributed predominately to CD8 T
cells, we similarly evaluated protection following vaccination with flagellin fusion protein STF2.LIST that contains the
immuno-dominant MHC Class I H-2Kd epitopes (LLO91–99
and p60217–225 ) identified for L. monocytogenes. Mice immunized with STF2.LIST demonstrated antigen-specific CD8 T
cell response that were quantitatively equivalent to animals
immunized with live L. monocytogenes following a single
immunization (Fig. 9A and B), and the mice were protected
against a challenge with virulent L. monocytogenes (Fig. 9C).
Based on these results, we conclude that recombinant fusion
proteins are efficient in eliciting CD8 T cells responses that
are capable of imparting protective immunity. Thus, recombinant flagellin fusion proteins also offer a new approach in
the development of vaccines that induce enhanced CD8 T cell
responses.
In addition to this study, the potential utility of flagellin in
of vaccines has been investigated by others [33–35]. McSorley et al. [33] demonstrated that co-delivery of a mixture
of the OVA derived peptide (323–339) and flagellin resulted
in enhanced Th1 responses in vivo. Interestingly, Didierlaurent et al. [35] reported co-immunization with antigen and
flagellin promotes the development of Th2 responses in vivo
in a MyD88 dependent manner. While the absence of IL12 p70 expression following TLR5 stimulation with flagellin
has been implicated in Th2-polarization, we have not examined the expression of IL-12 p70 in our studies. In addition
to the co-delivery of antigen with flagellin, the incorporation
of antigens into flagellin has also been evaluated. Insertion
of specific CTL epitopes into the flagellin of Salmonella has
been investigated when delivered as a live vaccine [20–22].
In light of the potential risks of live vaccines, recombinant
fusion protein strategies offer a significant advantage in vaccine development and safety. Here we demonstrate that two
distinct fusion proteins induce significantly enhanced antigen specific cellular and humoral responses in vivo. Similar
to our findings, Cuadros et al. [34] recently reported that a
fusion protein comprised of flagellin and the enhanced green
fluorescent protein (EGFP) was highly effective in eliciting
J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
antigen specific T cell responses in vivo. In their study, immunization with the flagellin fusion protein resulted in enhanced
EGFP uptake and processing by APCs in vivo, including CTL
responses more potent than seen when EGFP was delivered
alone.
Studies examining the immunogenicity of flagellin offer
conflicting findings [33–36]. Cuadros et al. [34] reported
immunization with flagellin fusion proteins resulted in little to no detectable flagellin specific responses. Similar to
the finding of Didierlaurent et al. [35], we observe detectable
anti-flagellin responses beginning around 1 week post immunization. This raises the possibility that preexisting antiflagellin immune responses could interfere or neutralize the
TLR5 stimulatory activity and thereby inhibit or diminish subsequent responses to recombinant flagellin fusion
vaccines. Given this possibility we examined whether preexisting anti-flagellin responses inhibited responses to subsequent encounters with flagellin fusion proteins both in vitro
and in vivo. Our results demonstrated that anti-flagellin antibodies do not inhibit the development of OVA-specific IgG
responses following immunization with STF2.OVA (Fig. 6).
These results are similar to those of Ben-Yedidia and Arnon
[37], who reported anti-flagellin antibodies failed to interfere
with subsequent antigen-specific responses. It is interesting
that established responses to flagellin do not significantly
interfere with the TLR5 activity of flagellin. Flagellin is characterized by highly conserved N and C-terminal structures
with an intervening hypervariable region [38–40]. Structure
function studies suggest that specific sequences in both the
N and C-termini are necessary for TLR5-mediated monocyte pro-inflammatory responses in vitro [38]. Although it
is unknown if inhibitory or neutralizing antibodies to TLR
ligands are specifically regulated, such a mechanism would
have obvious evolutionary benefits to the host. Tolerance or
immunological privilege to these moieties would ensure efficient responses in the event of repeated re-exposures to these
ligands.
While the specific mechanism by which the flagellin–
antigen fusion proteins induce immune responses is unclear,
several possibilities exist. First, TLR-ligand interactions may
affect the trafficking and processing of antigens following
endocytosis [31,41]. In the case of flagellin fusion proteins,
TLR5 binding could result in enhanced antigen processing and presentation. The flagellin–antigen fusion proteins
likely enhance the activation and maturation of naı̈ve APCs
resulting in more efficient stimulation of adaptive immune
responses in vivo. Thus, TLR5-mediated activation by flagellin fusion proteins would result in the enhanced activation
of antigen-bearing APC, as the antigen and maturation signal are delivered simultaneously. A third and somewhat less
likely possibility is that the flagellin–antigen fusion proteins
are capable of driving adaptive immune response by directly
interacting with antigen-specific TLR5+ lymphocytes and
thereby provide the necessary signals for activation. However, failure of flagellin to activate purified B cells ([35]
and data not shown) and the requirement of antigen pro-
773
cessing and presentation for T cells, would make this an
unlikely possibility in vivo. Consequently, we favor the possibility that flagellin–antigen fusion proteins promote faster
and stronger immunogenicity by linking antigen delivery
directly to the activation and maturation signal of the APC
in the periphery. This conclusion is supported by the demonstration that efficient processing and presentation of antigen
is promoted by the physical association of the antigen with
a PAMP–TLR complex during internalization in dendritic
cells [31,32]. This would contrast vaccination with unlinked
antigen and PAMP, as occurs with adjuvants, in which the
presentation of antigen and immunostimulatory PAMP are
unlinked physically or mechanistically. This regimen results
in a dichotomy of less efficient populations of APC: those
that have encountered PAMP, but not antigen, and vice versa.
Subsequently the use of recombinant fusion protein vaccines
ensures a significantly lower PAMP to antigen ratio than seen
using conventional adjuvants. Thus, the enhanced immunogenicity to the fusion proteins may not simply be due to
activating more APC, but by ensuring the more selective
activation of APC that have encountered the vaccine antigen
in situ.
While our results demonstrate the TLR5-dependent activity of the fusion proteins in vitro, we cannot rule out the
possibility that the flagellin fusion protein is capable of interacting with additional receptors in vivo. Indeed flagellin also
is reported to interact with the mammalian host protein glycolipid asialoganglioside monosialic acid 1 [42]. While our
flagellin-fusion proteins elicit TLR5 dependant responses at
concentrations <1 ng/ml on TLR5 + cell lines in vitro, we
do not observe significant cellular activation of bone marrow derived DC or spleen APC ex vivo at concentrations of
≥25 ␮g/ml (data not shown). This contrasts with the rapid upregulation of MHC Class II and CD80 observed in the same
populations in response to <1 ng/ml concentrations of LPS
and peptidoglycan (data not shown). Several recent studies
have examined the expression of TLR5 in mice and humans
[36,43,44]. Our results are similar to those of Means et al.
[36] who reported murine DCs appear unresponsive to flagellin and could not detect TLR5 expression on spleen DCs via
RT-PCR. Similarly, Didierluarent et al. [35] did not observe
activation of murine DC or B cells when stimulated with
purified flagellin ex vivo. However, immunization with flagellin resulted in increased numbers of a F4/80low /CD11c+
gated APC population in spleen 6 h following immunization.
The authors postulated that the discrepancy between the in
vivo and ex vivo results relates to the method of APC isolation. However, it is also possible that immunization with
flagellin induced the selective activation and migration of
a peripheral TLR5+ APC population(s). In this regard, it
should be noted that TLR5 expression is detected on a variety
of cells including macrophages and osteoblasts, in addition
to specific tissues including the lungs and liver [43,45,46].
Thus, the population of activated APC observed in the spleen
6 h following immunization with flagellin could reflect the
migration of a peripheral TLR5+ APC population.
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J.W. Huleatt et al. / Vaccine 25 (2007) 763–775
Collectively the results of our study demonstrate
recombinant flagellin fusion proteins provide enhanced
immunogenicity and efficacy as vaccines. The responses
elicited are characterized by faster and stronger cellular and
humoral immune responses than that observed following
immunization with antigen alone or when antigen and flagellin are co-delivered unlinked. Moreover, immunization with
the flagellin fusion proteins efficiently elicits antigen-specific
protective immunity in vivo. While the results of this study
are limited to the TLR5 ligand flagellin, it will be interesting
to similarly evaluate the potential of additional TLR-ligands
in hopes of developing safer and more efficacious vaccines.
Acknowledgments
The authors would like to thank Dr. Hao Shen at the University of Pennsylvania for generously providing the recombinant OVA-expressing L. monocytogenes strain JJL-OVA,
and Dr. Ruslan Medzhitov and Dr. Richard Flavell for their
thoughtful and critical review of this manuscript.
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