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Haemophilus influenzae Type b-Outer
Membrane Protein Complex Glycoconjugate
Vaccine Induces Cytokine Production by
Engaging Human Toll-Like Receptor 2
(TLR2) and Requires the Presence of TLR2
for Optimal Immunogenicity
Eicke Latz, Jennifer Franko, Douglas T. Golenbock and John
R. Schreiber
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 172:2431-2438; ;
doi: 10.4049/jimmunol.172.4.2431
http://www.jimmunol.org/content/172/4/2431
The Journal of Immunology
Haemophilus influenzae Type b-Outer Membrane Protein
Complex Glycoconjugate Vaccine Induces Cytokine Production
by Engaging Human Toll-Like Receptor 2 (TLR2) and
Requires the Presence of TLR2 for Optimal Immunogenicity1
Eicke Latz,* Jennifer Franko,†‡ Douglas T. Golenbock,* and John R. Schreiber2†‡
B
acterial polysaccharides (PS)3 are T cell-independent
Ags, do not induce immunological memory or yield a
booster response with repeated immunization, and are
poorly immunogenic in children ⬍24 mo of age (1– 6). Conjugation of capsular PS such as those from Haemophilus influenzae
type b (Hib) and Streptococcus pneumoniae to a carrier protein
renders them immunogenic in infants and capable of eliciting
memory, booster responses, and isotype switching of anti-PS Abs
to IgG1 (7–9). These glycoconjugate vaccines, using a variety of
carrier proteins, have had a significant role in the virtual eradication of Hib disease from the United States and in producing a
marked decline in invasive pneumococcal infections in small children (7–9).
*Department of Medicine, University of Massachusetts Medical Center, Worcester,
MA 01605; †Departments of Pediatrics and Pathology, Case Western Reserve University, and ‡Division of Infectious Diseases, Rainbow Babies and Children’s Hospital, Cleveland OH 44106
Received for publication September 23, 2003. Accepted for publication November
25, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI 32596 (to
J.R.S.), AI46667 (to J.R.S.), and AIGM54060 (to D.T.G.).
2
Address correspondence and reprint requests to Dr. John R. Schreiber, Division of
Infectious Diseases/Allergy/Immunology/Rheumatology, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address:
[email protected]
3
Abbreviations used in this paper: PS, polysaccharide; Hib, Haemophilus influenzae
type b; KO, knockout; OMPC, outer membrane protein complex; TLR, Toll-like
receptor; WT, wild type.
Copyright © 2004 by The American Association of Immunologists, Inc.
Pure PS are not processed by APC nor presented in context of
MHC II to T cells. The enhanced immunogenicity of glycoconjugate vaccines compared with native bacterial PS is thought to be
due to processing of carrier protein by the APC and presentation of
carrier protein-derived peptides in the context of MHC class II
molecules to Th cells, followed by induction of cytokine production. The cytokines produced by activated CD4⫹ T cells are presumed to stimulate PS-specific B cells to undergo clonal expansion, differentiation to memory cells, isotype switching to
predominantly IgG1, and increased production of PS-specific Abs
(2, 10). Unfortunately, glycoconjugate vaccines require multiple
doses to induce long-lasting protective levels of anti-PS Abs in
infants, greatly increasing the expense and making these vaccines
difficult to use in the developing world (11).
Despite the requirement for multiple doses of glycoconjugate
vaccines to induce protective Ab levels, the Hib vaccine in which
the PS is conjugated to a complex of outer membrane proteins
from meningococcus (OMPC) often induces protective levels (i.e.,
ⱖ1.0 ␮g/ml) of anti-Hib PS Abs after a single dose (12, 13). This
characteristic of the Hib-OMPC conjugate vaccine is clinically relevant, as breakthrough infections in infants were recently observed
when other conjugate vaccines were substituted for Hib-OMP as
the first dose in a heavily Hib-colonized Native American population (14). Different immunogenicities of glycoconjugate vaccines
that use various carrier proteins are presumed to be a result of
processing of the different carrier proteins, yielding divergence in
peptides and subsequent epitope specificity of CD4⫹ T cells that
are recruited, although the precise mechanism explaining the early
immunogenicity of Hib-OMPC compared with other Hib conjugate vaccines is unclear. Previous data have suggested that OMPC
has adjuvant-like activity due to activation of macrophages and to
0022-1767/04/$02.00
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Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to
carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States,
but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC)
conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged
human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged
mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM197 and
tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as
Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic
in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPCimmunized TLR2 KO mice also produced significantly less IL-6 and TNF-␣ than those from wild-type mice. Hib-OMPC is unique
among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose
may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the
peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant
or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines. The
Journal of Immunology, 2004, 172: 2431–2438.
2432
H. influenzae TYPE B OMPC GLYCOCONJUGATE VACCINE ENGAGES TLR2
a mitogenic effect on B cells (15). In addition, it has been previously reported that porin proteins from the surface of Neisseriae
meningitidis, one of the major constituents of neisserial outer
membrane proteins, increase the expression of the B cell costimulatory molecule B7-2 and require Toll-like receptor 2 (TLR2) expression for this effect (16, 17). In the current report we show that
uniquely among the currently used glycoconjugate vaccine carrier
proteins, OMPC and OMPC conjugated to Hib PS engage human
and mouse TLR2 in a MyD88-dependent manner and induce cytokine production in vitro, and that the absence of TLR2 significantly reduces the immunogenicity of Hib-OMPC vaccine.
Materials and Methods
OMPC and other Hib vaccines
TLR-specific stimuli and controls
LPS from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich
(St. Louis, MO) and repurified to eliminate contaminating TLR2 activation
as previously described (18). Synthetic Mycoplasma-associated lipoprotein
of 2 kDa, a TLR2-engaging microbial molecule, was purchased from EMC
Microcollections (Tuebingen, Germany). Heat-killed Listeria monocytogenes
was provided by Dr. G. Teti (Universita degli Studi Messina, Messina, Italy)
and prepared as previously described (19). Porin purified from Neisseria
meningitidis was a gift from Dr. P. Massari (Boston University, Boston, MA).
Human TNF-␣ was obtained from PeproTech (Rocky Hill, NJ). The LPS
antagonist compound B1287 (also called e5564), which competitively binds to
MD-2, was provided by Dr. F. Gusovsky (20) (Eisai Research Institute,
Andover, MA).
Cell lines and constructs
Stable cell lines of human embryonic kidney (HEK) 293 cells were established as previously described (21, 22). These HEK cells naturally lack
TLR2 and TLR4, and genetic complementation with these receptors renders HEK cells responsive to the respective TLR ligands. We have previously shown that chimeric receptors consisting of TLRs C-terminally fused
to green fluorescent protein or its spectral variants (cyan or yellow fluorescent protein) are fully functional signaling molecules (21). TLR constructs C-terminally tagged with the cyan- or yellow-shifted variants of
green fluorescent protein were transfected into HEK cells by calcium phosphate transfection. Bulk populations of cells were selected by the neomycin
analog G418 (1 mg/ml). Positive selection by FACS (BD Vantage; BD
Immunocytometry Systems, San Jose, CA) was performed to enrich for
fluorescent cells. Cells were then cloned by limiting dilution in half-area,
96-well, plastic tissue culture plates (Corning, Corning, NY). TLR4 requires MD-2, a small glycosylated protein, to optimally sense LPS. We
therefore used HEK cell lines expressing TLR4-fluorescent protein and, as
a control, TLR2-fluorescent protein that coexpressed MD-2. The HEK cells
expressing TLR4 and MD-2 were generated by retroviral transduction of
HEK-TLR4 cells as described with a retrovirus encoding human MD-2
(22). The stable HEK293 cell lines were maintained in DMEM supplemented with 10% FCS and 0.5 mg of G418/ml in a 5% saturated CO2
atmosphere at 37°C. HEK cells stably transfected with the empty vector
pcDNA were used as control cells.
Cell incubation and analysis of TLR2 engagement in HEK and
mouse dendritic cells
Stable cell lines expressing fluorescent versions of TLR2, TLR4/MD-2, or
HEK-pcDNA were trypsinized and seeded into 96-well plates at a density
Mouse strains
TLR2 ⫺/⫺ and MyD88⫺/⫺ mice were generated as described previously
(23, 24) and were originally supplied by Dr. S. Akira (Osaka University,
Osaka, Japan). TLR2 knockout (KO) mice were originally a 129sv strain
and were four (TLR2⫺/⫺) or five (MyD88⫺/⫺) generations backcrossed
into C57BL/6 mice before use. C57BL/6 mice were used as the control
wild-type (WT) mice (The Jackson Laboratory, Bar Harbor, ME). Mice
were housed in microisolator cages in a pathogen-free satellite facility of
the Animal Resource Center of Case Western Reserve University. The
animal care and use committee of Case Western Reserve University approved the animal protocols that were used. Animals were fed an ad libitum
diet of autoclaved Teklad mouse chow (Harlan, Madison, WI).
Immunization of mice with glycoconjugate vaccines and OMPC
WT mice and TLR2 KO mice, aged 8 –13 wk, were injected i.p. with
Hib-OMPC (5.0 ␮g Hib capsular PS, 83.5 ␮g OMPC in 100 ␮l), OMPC
(83.5 ␮g in 100 ␮l), or PBS (100 ␮l) on day 0. These doses were chosen
based on dose-response experiments yielding the amount of conjugated
Hib PS that generated optimal Hib PS Ab levels (data not shown). The WT
group contained seven mice, and the KO group contained 13 mice. Mice
were reimmunized on wk 2, 4, and 6 with the same doses of Hib-OMPC,
OMPC, or PBS previously administered to mimic the multiple dosing that
is used in human infant immunization with Hib-OMPC. Tail bleeds were
performed each week to obtain serum samples to measure Hib PS-specific
and OMPC-specific Ab levels by ELISA. Two days after the fourth injection, a subset of seven WT and six KO mice immunized with OMPC were
euthanized by inhalation of CO2, and their spleens were removed. Splenocytes from both WT and TLR2 KO mice were isolated, seeded at a density
of 1 ⫻ 106 cells/well in 12-well polystyrene tissue culture plates (BD
Biosciences, Franklin Lakes, NJ), and stimulated for 48 h with Hib-OMPC
(5.0 ␮g/ml Hib PS, 83.5 ␮g/ml OMPC), 83.5 ␮g/ml OMPC, 5.0 ␮g/ml
purified Hib PS, 5.0 ␮g/ml Hib PS conjugated to CRM197 (HibTITER;
Wyeth-Lederle, Pearl River, NY), or 5.0 ␮g/ml Hib PS conjugated to inactivated tetanus toxoid (ActHIB; Aventis Pasteur, Swiftwater, PA). Cytokines produced by mouse splenocytes after Ag stimulation were measured by ELISA on cell supernatants as described below.
ELISA measurement of Hib PS and OMP Ab titers
Hib PS-specific capsular polysaccharide Abs were measured by ELISA as
previously described (25, 26). Hib oligosaccharide-human serum albumin
conjugate-coating Ag (provided by Dr. M. Nahm, University of Alabama,
Birmingham, AL) was diluted in PBS to 4 ␮g/ml, and 100 ␮l/well was
adsorbed onto the wells of polystyrene microtiter plates (Polysorp; Nunc,
Naperville, IL). Dilutions of the test serum were made in PBS containing
0.05% Tween and 1% BSA, and 100 ␮l of a 1/50 diluted serum from each
mouse at each time point postimmunization was added to coated wells in
duplicate after washing, then incubated overnight. After washing with
PBS-Tween (0.05%), goat anti-mouse, class-specific Abs conjugated to
alkaline phosphatase (Southern Biotechnology Associates, Birmingham,
AL) diluted in PBS-Tween (0.05%) with 1% BSA were added to each well.
Plates were washed again after a 2-h incubation, and p-nitro-phenyl phosphate (disodium; Sigma-Aldrich) was then added. Absorbance was read at
415 nm on a Microplate ELISA reader (model 550; Bio-Rad, Hercules,
CA). Plates were normalized by reading each plate at the time when control
sera (obtained from Hib-PS hyperimmunized mice) were at identical OD.
OMPC-specific Abs in mouse sera before and after immunization were
detected using a similar ELISA in which microtiter plates were coated with
1.0 ␮g/ml purified OMPC (Merck, West Point, PA) on sera from a subset
of five WT and nine immunized KO mice. After washing, 1/50 diluted
mouse sera from each mouse at each time point were added in duplicate,
followed by goat anti-mouse, class-specific Abs conjugated to alkaline
phosphatase (Southern Biotechnology Associates). The assay plates were
developed and read as described for the Hib PS ELISA above.
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Purified OMPC from Neisseriae meningitidis was supplied by Dr. A. Shaw
(5.0 mg/ml 0.9% sodium chloride; Merck, West Point, PA) and was prepared as previously described (12). Hib-OMPC vaccine (Merck) was purchased commercially and contained 7.5 ␮g of Hib capsular PS, 125 ␮g of
OMPC randomly cross-linked to Hib PS, and 225 ␮g of aluminum hydroxyphosphate sulfate in 0.5 ml of 0.9% sodium chloride. Hib-CRM197
vaccine (HibTITER; Wyeth-Lederle, West Henrietta, NY) containing 10
␮g of Hib PS covalently linked to 25 ␮g of CRM197 (Cross Reactive
Material, a nontoxic, single amino acid mutant of diphtheria toxin) in 0.5
ml of 0.9% saline used in vitro and in mouse immunizations was obtained
commercially. Hib-tetanus vaccine (ActHIB; Aventis Pasteur, Swiftwater
PA), which contained 10 ␮g of purified Hib PS conjugated to 24 ␮g of
inactivated tetanus toxoid in 0.5 ml of 0.9% saline, was also obtained
commercially. Experimental grade (not for vaccine use) unconjugated Hib
PS was supplied by Dr. R. Eby (Wyeth).
of 4 ⫻ 104 cells/well in triplicate. The cells were stimulated the following
day with the various dilutions of vaccines, carrier proteins, and controls,
and IL-8 release was measured in the supernatants using a commercially
available ELISA (DuoSet; R&D Systems, Minneapolis, MN). The experiment was repeated three times with similar results.
Murine dendritic cells were prepared by culturing bone marrow cells
from C57BL/6 mice or age- and sex-matched MyD88-deficient mice in
RPMI 1640 medium supplemented with 10% FCS, 10 ␮g/ml ciprofloxacin
(Mediatech, Herndon, VA), 10 ng/ml GM-CSF (PeproTech), and 50 ␮⌴
2-ME (Sigma-Aldrich) for 7 days. Dendritic cells were seeded into 96-well
plates (5 ⫻ 104 cells/well) 1 day before stimulation in triplicate. Cell culture
supernatants were removed and analyzed for cytokines by ELISA (R&D Systems). These experiments were repeated twice with similar results.
The Journal of Immunology
Detection of cytokine production by mouse splenocytes
WT and TLR2 KO mouse splenocytes obtained after four immunizations
(wk 0, 2, 4, and 6) with 83.5 ␮g of OMPC (seven WT and six KO mice)
were stimulated for 24 h with Hib-OMPC (5 ␮g/ml Hib PS, 83.5 ␮g/ml
OMPC), 83.5 ␮g/ml OMPC, 5 ␮g/ml unconjugated Hib PS, 5 ␮g/ml Hib
PS conjugated to CRM197 (HibTITER; Wyeth Lederle Laboratories), or 5
␮g/ml Hib PS conjugated to inactivated tetanus toxoid (ActHIB; Aventis
Pasteur, Swiftwater, PA). Supernatants were collected, and cytokine production by the stimulated cells was measured using ELISA, as previously
described, in duplicate wells (26, 27). IL-1, IL-2, IL-4, IL-5, IL-6, IFN-␥,
and TNF-␣ production was measured using OptEIA Mouse Cytokine Sets
(BD PharMingen, San Diego, CA), and IL-10 and IL-12 were measured
using Duo ELISA Development Sets (R&D Systems). Polystyrene microtiter
plates (MaxiSorp; Nunc) were coated with100 ␮l of the desired cytokinespecific capture Ab diluted according to the manufacturer’s protocol.
Statistics
Due to the skewness of the OMPC and Hib PS Ab levels, all analyses were
performed on the natural logarithmic (loge) scale. Measures of IgG, IgM,
and total Ig levels (absorbance) were analyzed and compared between the
KO and WT mice groups by a repeated measures analysis for unbalanced
data using maximum likelihood implemented with SAS Proc Mixed (version 8.2; SAS Institute, Cary, NJ). For each outcome, a model assuming
separate geometric means at each time point was fit to the data. Under this
model, a different geometric mean level is estimated at each time point
from wk 1 through wk 6 for each of the two groups. The covariance structure of the repeated measures was modeled using an unstructured model
that allows the variances and correlations of the levels to differ over time.
For each model, the baseline (wk 0) value was controlled for in the analysis
by including the term in the model. A test between the groups was performed at each week postbaseline, and comparison between the groups at
baseline was performed using the two-sample t test.
Results
OMPC and Hib-OMPC, but not other Hib glycoconjugate,
vaccines engage human TLR2 expressed on HEK cells
We first compared the effect of OMPC and other glycoconjugates
on HEK-TLR2 transfected cells with control cells stably expressing the pcDNA3 plasmid (empty vector). The cells were incubated
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FIGURE 1. Hib-OMPC and OMPC engage human TLR2. HEK cells stably transfected with TLR2-YFP and MD-2 or with
empty vector pcDNA3 were incubated with
increasing concentrations of heat-killed Listeria monocytogenes (HKLM) or three different Hib conjugate vaccines (A). Cell supernatants were analyzed for IL-8 secretion
by ELISA in triplicate wells after 16 h of
stimulation. B, The stimulatory activity of
the nonconjugated molecules (OMPC or Hib
PS) was compared with that of porin prepared from N. meningitidis. These experiments were repeated three times with similar
results, and the data shown are from one representative experiment.
2433
2434
H. influenzae TYPE B OMPC GLYCOCONJUGATE VACCINE ENGAGES TLR2
OMPC and Hib-OMPC stimulate TNF release from murine
bone marrow-derived dendritic cells that is MyD88 dependent
We tested the ability of murine bone marrow-derived dendritic
cells to respond to the different vaccine preparations as Ag-processing cells may be some of the first cells to interact with glycoconjugate vaccines after immunization. To assess the necessity of
the TLR adapter molecule MyD88 for dendritic cell stimulation by
OMPC we also included dendritic cells prepared from MyD88⫺/⫺
mice in these experiments. Both OMPC and Hib-OPMC robustly
stimulated murine DCs to release TNF-␣ in a MyD88-dependent
fashion (Fig. 3A). As seen before with the LPS-sensitive HEKTLR4/MD-2 cell line (Fig. 2), Hib PS and Hib-tetanus vaccine led
to TNF-␣ production at higher doses. However, these stimulatory
activities could be completely abrogated by addition of the LPS
antagonist B1287, indicating that the activity seen is probably due
to LPS contamination of these preparations (Fig. 3B). From these
data we conclude that murine cells are stimulated by OMPC in a
similar manner as human cells, and that the responses are dependent on the TLR adapter molecule MyD88.
The ability to make Hib capsular polysaccharide Abs after HibOMPC immunization is reduced in TLR2 KO mice
WT and TLR2 KO mice were immunized with three doses of
Hib-OMP vaccine in a dose previously found to yield optimal Hib
PS Ab levels in the WT mice and in a regimen designed to mimic
the multiple doses administered to human infants and were bled
weekly to measure serum anti-Hib PS Ab levels. WT mice had
significantly higher IgM anti-Hib PS Ab levels 6 wk after the first
immunization with Hib-OMPC than TLR2 KO mice ( p ⫽ 0.038;
Fig. 4A). In addition, WT mice had significantly more IgG anti-Hib
PS Abs at wk 4, 5, and 6 after the first immunization ( p ⫽ 0.012,
0.025, and 0.012, respectively; Fig. 4B). Finally, total anti-Hib PS
Ab Ig levels at wk 4, 5, and 6 after the first immunization in WT
mice were also higher compared with those in TLR2 KO mice
( p ⫽ 0.011, 0.012, and 0.003, respectively; Fig. 4C). No detectable IgA anti-Hib PS Abs were found in either WT or TLR2 KO
mice, and control mice injected with PBS did not make Hib PS
Abs (data not shown).
Abs against the carrier protein OMPC are also reduced in
TLR2 KO mice
Serum OMPC Ab levels after WT and TLR2 KO mouse immunization with three doses of Hib-OMPC were also measured by
ELISA in a subset of WT and KO mice. WT mice had significantly
higher IgM OMPC Ab levels at wk 1, 4, and 5 postimmunization
( p ⫽ 0.002, 0.047, and 0.009, respectively; Fig. 5A). WT mice had
significantly higher IgG OMPC Ab levels wk 1 postimmunization
( p ⫽ 0.001; Fig. 5B), but IgG levels were similar in both groups
after wk 1. Finally, total Ig levels of OMPC Abs were also higher
in WT mice wk 2 postimmunization ( p ⫽ 0.012; Fig. 5C). Control
mice injected with PBS did not make OMPC Abs (data not
shown).
Splenocytes from TLR2 KO mice make significantly less IL-6
and TNF-␣ than WT mice after immunization with OMPC
FIGURE 2. Hib-OMPC and OMPC stimulatory activity is TLR2 specific. Stable transfectants of HEK cells expressing TLR2-YFP (left panel)
or TLR4-YFP (right panel) together with MD-2 were incubated with LPS
(100 ng/ml), OMPC (40 ␮g/ml), Hib PS (40 ␮g/ml), or the three conjugate
vaccines (1/10, v/v) alone or together with the LPS antagonist B1287 (10
␮g/ml). After 16-h incubation, IL-8 release into cellular supernatants was
measured by ELISA in triplicate wells. The experiment was repeated twice
with similar results, and the data shown are from one representative
experiment.
A subset of WT and TLR2 KO mice receiving immunization
with OMPC were sacrificed, and their splenocytes were stimulated
with OMPC, Hib-OMPC, and a variety of other glycoconjugate
vaccines to determine the cytokines produced after exposure to
OMPC. Splenocytes obtained from OMPC-immunized TLR2 KO
mice made significantly less IL-6 and TNF-␣ than WT mice after
stimulation with OMPC ex vivo (Fig. 6, A and B, respectively).
There were no differences in the production of IL-1, IL-2, IL-4,
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with the three different glycoconjugate vaccines, Hib-OMPC,
Hib-tetanus, and Hib-CRM197, and as a positive control with
heat-killed Listeria monocytogenes, a potent TLR2-stimulating
bacteria. Hib-OMPC induced vigorous production of IL-8 by the
TLR2-transfected HEK cells as did heat-killed Listeria monocytogenes (Fig. 1A). Neither preparation was able to elicit an IL-8
response in the control cells. Hib-CRM197 and Hib-tetanus vaccines did not engage TLR2 (Fig. 1A). We then used the single
components of the conjugate vaccine Hib-OMPC to decipher
which part of the conjugate was responsible for the TLR2 activation. As a further positive control and to compare the TLR2 activity, we used a porin preparation derived from Neisseria meningitidis, which is known to strongly engage TLR2 (17). The TLR2stimulating activity could be attributed to the protein (OMPC) part
of the conjugate vaccine, as unconjugated Hib PS did not significantly engage TLR2 and was found to be equally potent to the
TLR2-stimulating activity of neisserial porin (Fig. 1B).
We next tested whether the different conjugate vaccine preparations had TLR4/MD-2 stimulatory activity. Due to the ubiquitous presence and stability of bacterial LPS, contamination of the
reagents and/or reaction mixtures was possible. We thus used the
LPS antagonist compound B1287 to block the LPS activity present
in the various vaccine preparations or ligands. The compound
B1287 did not significantly influence the TLR2-stimulating activity of either OMPC or the Hib-OMPC conjugate vaccine (Fig. 2).
By contrast, TLR4/MD2-expressing HEK cells responded vigorously to LPS, and the addition of the compound B1287 completely
abrogated the LPS stimulatory activity. HEK-TLR4/MD-2 cells
also responded to the polysaccharide portion of the vaccines (experimental grade Hib PS) and, to a much lesser extent, to the
conjugate vaccines, Hib-OMPC and Hib-tetanus. As these activities were completely abrogated by the addition of B1287, it seems
likely that the cells detected minor LPS contaminations present in
the different preparations.
The Journal of Immunology
2435
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FIGURE 3. Murine bone-marrow derived dendritic cells are activated by OMPC and Hib-OMPC and produce TNF-␣ in a MyD88-dependent fashion.
Murine bone marrow-derived dendritic cells from C57BL/6 or MyD88⫺/⫺ mice were incubated with increasing doses of the indicated ligands for 16 h, and
cellular stimulation was assessed by measuring TNF-␣ release in triplicate wells containing cell supernatants (A). The LPS antagonist compound B1287
was used in B to decipher the contribution of LPS contamination in some of the reagents used in A. Bone marrow-derived dendritic cells were stimulated
as described in A (highest doses), and B1287 LPS-inhibitor (10 ␮g/ml) was added where indicated. This experiment was performed twice with similar
results, and the data shown are from one representative experiment.
IL-5, IL-10, or IFN-␥ production by the stimulated splenocytes
from TLR2 KO or WT mice (data not shown).
Discussion
Glycoconjugate vaccines consisting of the capsular PS of Hib have
been effective in greatly reducing invasive infections caused by
this pathogen. Unfortunately, most of the available glycoconjugate
vaccines require three or four doses to elicit long-lasting immunity
in small infants most susceptible to infection. Interestingly, one
glycoconjugate vaccine that uses a carrier protein derived from the
OMPC of N. meningitidis is often capable of inducing protective
levels of anti-capsular Hib Abs after only one dose. We set out to
determine whether this carrier protein interacted with the immune
system in ways other than providing carrier protein-derived T cell
help, as has been hypothesized to be the major contribution of
carrier proteins to enhanced PS-specific Ab responses. We found
that OMPC, either unconjugated or chemically linked to Hib PS,
vigorously and specifically engaged TLR2 in cell lines that stably
express human TLR2 and produce IL-8 after TLR2 engagement.
The OMPC was specific for TLR2 and was not contaminated with
LPS, as cell lines stably transfected with TLR4 and MD-2 (but not
TLR2) were not stimulated by OMPC or Hib-OMPC. In addition,
2436
H. influenzae TYPE B OMPC GLYCOCONJUGATE VACCINE ENGAGES TLR2
the engagement of TLR2 was MyD88 dependent, as MyD88⫺/⫺
cells could not be stimulated by OMPC. Finally, Hib conjugated to
two other carrier proteins, tetanus toxoid and CRM197, a nontoxic
diphtheria toxin mutant, as well as unconjugated Hib PS did not
engage TLR2. These vaccines did slightly engage TLR4, but only
due to minor LPS contamination, as the addition of the LPS antagonist B1287 abrogated TLR4 engagement. The unconjugated
Hib PS engaged TLR4 as expected, as this was an experimental lot
of PS that was not vaccine grade and contained traces of LPS.
The OMPC used in the Hib-OMPC vaccine consists of the
OMPC of N. meningitidis that is randomly cross-linked to the Hib
capsular polysaccharide. Previous studies have demonstrated that
the PorB porin protein from the outer membrane of N. meningitidis
stimulates B cells and up-regulates surface expression of B7-2 via
engagement with TLR2 in a MyD88-dependent fashion (16, 17).
OMPC contains various outer membrane proteins, including porins; it thus seems likely that the TLR2 engagement mediated by
Hib-OMPC vaccine is due to the presence of porins, as one of the
carrier protein components linked to the Hib capsular PS.
As the Hib-OMPC glycoconjugate vaccine is known to be immunogenic after fewer doses than other Hib glycoconjugate vac-
FIGURE 5. TLR2 KO mice make less OMPC Abs after immunization
with Hib-OMPC glycoconjugate vaccine compared with WT mice. Serum
OMPC Ab levels after WT and TLR2 KO mouse immunization with three
doses of Hib-OMPC were also measured by ELISA. WT mice had significantly higher IgM OMPC Ab levels at wk 1, 4, and 5 postimmunization
(five WT and nine KO mice; p ⫽ 0.002, 0.047, and 0.009, respectively; A).
WT mice had significantly higher IgG OMPC Ab levels at wk 1 postimmunization (p ⫽ 0.001; B) and higher total Ig levels at wk 2 postimmunization (p ⫽ 0.012; C).
cines in infants, we wanted to determine whether the presence of
the TLR2 ligand was crucial to the enhanced immunogenicity in an
animal model. TLR2 KO mice made significantly less IgM and
IgG anti-Hib PS Abs after immunization with Hib-OMPC than
WT mice. These data suggest that much of the enhanced immunogenicity of the Hib-OMPC vaccine may be related to the presence of TLR2 ligands and that the use of such ligands may be a
useful method to improve the immunogenicity of bacterial PS and
glycoconjugates (28).
We next determined whether the reduced immunogenicity of the
PS component of Hib-OMPC in TLR2 KO mice might be related
to differences in the induction of cytokines after exposure to
OMPC. Interestingly, splenocytes obtained from OMPC-immunized WT mice made significantly more IL-6 after ex vivo stimulation with OMPC than splenocytes from OMPC-immunized KO
mice. IL-6 was originally described as the B cell differentiation
factor, which encourages terminal differentiation and maturation of
Ab-producing plasma cells (29). In addition, IL-6 is known to
induce IL-4 production and promote the differentiation of Th2
CD4⫹ T cells, the T cells that appear to be most important to the
enhanced immunogenicity of bacterial glycoconjugate vaccines
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FIGURE 4. TLR2 knockout mice make significantly less Hib capsular
polysaccharide Abs after immunization with Hib-OMPC glycoconjugate
vaccine compared with WT mice. TLR2 KO and WT mice immunized
three times with Hib-OMPC were bled weekly, and Hib PS Ab levels were
measured via ELISA. WT mice had significantly higher IgM Hib PS Ab
levels 6 wk after the first immunization with Hib-OMPC than TLR2 KO
mice (seven WT and 13 KO mice; p ⫽ 0.038; A) and more IgG Hib PS Abs
wk 4, 5, and 6 wk after the first immunization (p ⫽ 0.012, 0.025, and 0.012,
respectively; B). Finally, total anti-Hib PS Ab Ig levels at wk 4, 5, and 6
after the first immunization in WT mice were also significantly higher than
those in TLR2 KO mice (p ⫽ 0.011, 0.012, and 0.003, respectively; C).
The Journal of Immunology
(26, 27, 30). Thus, our data suggest that at least one method by
which stimulation of TLR2 by the OMPC carrier protein yields
superior anti-PS Ab titers after fewer doses than other glycoconjugate vaccines may be due in part to induction of IL-6 and subsequent enhanced differentiation of PS-specific B cells as well as
carrier protein-specific CD4⫹ T cells. As direct administration of
IL-6 results in toxicity in animal models, stimulation of TLR2 via
OMPC or other agonists may represent an effective adjuvant for
the improved immunogenicity of glycoconjugate vaccines. Others
have also found that TLR2 agonists elicit Th2-type cytokines in
mice (31). These data, however, differ somewhat from a previous
report in which bacterial lipopeptides that engaged TLR2 induced
production of Th1 cytokines such as IFN-␥. However, these experiments were performed in human PBMC not mouse models, did
not include the variety of cells found in the mouse splenocytes, and
utilized different TLR2 ligands (32). Production of TNF-␣ also
was increased in OMPC-immunized WT mouse splenocytes compared with TLR2 KO mouse splenocytes. These data seem logical
given the vigorous induction of TNF-␣ we observed in the bone
marrow-derived dendritic cells also obtained from the WT mice.
As TNF-␣ is a comitogen for B and T cells and induces MHC
expression, it seems possible that the enhanced Hib-PS-specific
immunogenicity of Hib-OMPC glycoconjugate may also be related to induction of TNF-␣ after TLR2 engagement.
Interestingly, the TLR2 KO mice also made less IgM and IgG
OMPC Abs than WT mice, although these differences were much
less pronounced than those observed with Hib PS Abs. These data
are consistent with a recent report showing decreased outer surface
protein A Abs made in TLR2 KO mice after immunization with
outer surface protein A from Borrelia burgdorferi, in which the
response to a T cell-dependent protein Ag was blunted in the KO
mice. Macrophages from TLR2 KO mice were also found to produce less IL-6 than those from WT mice after immunization. Cooperation between engagement of TLR1 and TLR2 was also observed, as was altered surface expression of TLR1 in low
responder humans (33). TLR2 receptor activation is believed to
occur after association with other TLRs, such as TLR1 and TLR6,
and other recent experiments in mice with targeted lesions in
TLR1 or TLR6 revealed that coengagement of TLR2 with TLR1
or TLR6 results in the recognition of subtle differences in the molecular structure of the ligand. For example, TLR1/TLR2 cooperatively recognize synthetic triacylated lipoproteins, and TLR2/
TLR6 coengagement is needed for the recognition of synthetic
diacylated lipoproteins (34, 35). As the HEK cells we used are
known to express TLR1 and TLR6, we assume that coengagement
occurred as part of the response we observed to TLR2. The role of
TLR2 interactions with other TLR in the OMPC-mediated enhancement of Hib PS Ab levels remains to be investigated.
Dendritic cells presumably initiate T cell help that is necessary
for the development of the enhanced immunogenicity and memory
responses observed after immunization with glycoconjugate vaccines via uptake of the vaccine and processing and presentation of
carrier protein-derived peptides to specific T cells in the context of
MHC II molecules. We therefore were interested to determine
whether Hib-OMPC directly stimulated dendritic cells. We found
that Hib-OMPC and OMPC vigorously induced production of
TNF-␣ by bone marrow-derived dendritic cells in a MyD88-dependent fashion. As TNF-␣ is known to induce MHC expression,
it seems possible that TLR2 engagement by the glycoconjugate in
these cells may yield improved Ag processing efficiency of the
carrier protein, resulting in enhanced carrier protein-specific T cell
help. Others have also found that use of an Ag that directly ligates
APC TLR2 improved the efficiency of Ag presentation and subsequent CD4⫹ T cell responses (36). Furthermore, dendritic cell
activation leads to up-regulation of costimulatory molecules,
which also enhances the activation of peptide-specific T cells. It
will be important to determine whether the OMPC component of
the glycoconjugate vaccine yields enhanced carrier protein-specific CD4⫹ T cell stimulation that represents another mechanism
for the PS-specific immunogenicity of this vaccine after fewer
doses than other Hib glycoconjugate vaccines. As OMPC contains
multiple proteins, isolation of each component may be required in
future experiments to confirm which protein mediates TLR2 engagement and/or enhanced CD4⫹ T cell stimulation.
We conclude that Hib-OMPC glycoconjugate vaccine engages
human TLR2, which enhances the immunogenicity of Hib PS. The
mechanism of enhanced PS-specific immunogenicity of the HibOMPC may be related to increased induction of IL-6 and TNF-␣.
As a glycoconjugate vaccine containing this carrier protein is already in extensive use as a licensed vaccine for the prevention of
Hib disease in infants, further use of TLR2 agonists to enhance
vaccine immunogenicity in humans may be readily feasible. These
data may be useful in the design of second-generation glycoconjugate vaccines that require fewer doses to achieve protective Ab
levels against the capsular PS of bacterial pathogens.
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FIGURE 6. TLR2 KO mice make significantly less IL-6 and TNF-␣
after immunization with OPMC compared with WT mice. TLR2 KO and
WT mice were immunized three times with OPMC. Then their spleens
were removed, splenocytes were stimulated ex vivo with OMPC and other
Ags, and cytokine levels in cell supernatants were measured by ELISA.
Splenocytes obtained from postimmunization TLR2 KO mice made significantly less IL-6 (seven WT and six KO mice; p ⬍ 0.04; A) and TNF-␣
(p ⬍ 0.01; B) after stimulation with OMPC than splenocytes from postimmunization WT mice.
2437
2438
H. influenzae TYPE B OMPC GLYCOCONJUGATE VACCINE ENGAGES TLR2
Acknowledgments
We thank Dr. H. Lester Kirchner for assistance with statistical evaluations,
and Kristen Halmen, Rhonda Kimmel, and Sheryl Petersen for technical
assistance.
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