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Infection and Immunity, October 1999, p. 5508-5513, Vol. 67, No. 10
0019-9567/99/$04.00+0
Outer Membrane Proteins as a Carrier for Detoxified
Lipooligosaccharide Conjugate Vaccines for Nontypeable
Haemophilus influenzae
Ting-Huai
Wu and
Xin-Xing
Gu*
Laboratory of Immunology, National Institute
on Deafness and Other Communication Disorders, Rockville, Maryland
20850
Received 18 May 1999/Returned for modification 22 June
1999/Accepted 22 July 1999
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ABSTRACT |
Nontypeable Haemophilus influenzae (NTHi) is a common
cause of otitis media and respiratory tract infections. Outer membrane proteins (OMP) and lipooligosaccharide (LOS) are major surface antigens
of NTHi and potential vaccine candidates. De-O-acylated LOS (dLOS) or
oligosaccharide (OS) was coupled to total OMP to form dLOS-OMP and
OS-OMP conjugates, while a dLOS-tetanus toxoid (TT) was synthesized for
comparison. These conjugates were evaluated in mice and rabbits for
immunogenicity. dLOS-OMP elicited a better boostable antibody response
against LOS than did dLOS-TT, while OS-OMP was not immunogenic.
Formulation of the conjugates with Ribi adjuvant significantly enhanced
the immunogenicity of dLOS-OMP and dLOS-TT but not that of OS-OMP. In
addition, rabbit antisera elicited by dLOS-OMP but not dLOS-TT (or OMP
alone) demonstrated bactericidal activity against 40% of the NTHi
strains tested. These results indicate that dLOS is a better derivative
of LOS than OS and that OMP is a good carrier for NTHi LOS-based
conjugate vaccines.
| |
TEXT |
Nontypeable Haemophilus
influenzae (NTHi) is a common cause of otitis media (OM) and
respiratory tract infections (6, 28). Unlike H. influenzae type b, for which a highly successful vaccine is
available, there is no vaccine against NTHi-induced diseases. Efforts
to develop NTHi vaccines have been focused on surface antigens such as
outer membrane proteins (OMP), pili/fimbriae, and lipooligosaccharide
(LOS) (3, 5, 9, 12, 27, 32). These antigens are believed to
play an important role in the interaction of the bacteria with the
hosts in vivo (23). OMP and LOS are two major surface
antigens that are considered to be potential vaccine candidates because
they induced bactericidal antibodies in humans (4, 8) and
conferred protection against experimental NTHi OM in animals (12,
20), although both antigens showed antigenic variation among NTHi
strains (26, 29).
Previously, two LOS-based protein conjugates were synthesized in our
laboratory (15). The conjugates elicited anti-LOS antibodies with bactericidal activity against homologous strains and a large percentage of heterologous strains and conferred protection against experimental NTHi OM in chinchillas. To further improve the
immunogenicity and biological activity of the LOS-based conjugate,
total OMP were selected as an alternative carrier to explore whether a
conjugate with two different surface components from NTHi would serve
as a vaccine candidate offering broader and better protection against NTHi infections than either LOS or OMP alone. To investigate the feasibility of such an approach, two different modified LOSs, de-O-acylated LOS (dLOS) and oligosaccharide (OS), were used to covalently couple to the OMP to form dLOS-OMP and OS-OMP. As a control,
dLOS-tetanus toxoid (TT) was also synthesized, and the immunological
properties of these conjugates were investigated in vitro and in animals.
Purification and characterization of OS, dLOS, and OMP from
NTHi.
The conditions for the growth of strain 9274 were described
previously (15). LOS was purified from strain 9274 by a
modified phenol-water extraction method (14). Two approaches
were used for the detoxification of the LOS which was hydrolyzed with
acetic acid to produce OS (36) and with hydrazine to produce
dLOS (16). The yield was approximately 50% for OS or 60%
for dLOS. The purity of dLOS and OS was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by
silver staining (33). There was no detectable LOS in 5 µg
of dLOS or OS loaded on the gels, indicating that the residual LOS in
dLOS or OS preparations was less than 1% compared with that of the LOS standard.
A published method (7) was used for the purification of OMP
with modifications. Briefly, strain 12 was grown (15),
suspended in 0.1 M Tris buffer (pH 8.5) containing 0.2 mM EDTA (TE),
sheared with a blender for 10 min, sonicated with a Labsonic 1000 (B. Braun Biotech Inc., Allentown, Pa.) under conditions of circle for
0.3 s and output 100 for 10 min, and then centrifuged at
120,000 × g at 4°C for 3 h. The resulting
pellets were dissolved in TE buffer, incubated at 37°C for 10 min,
and purified by a Sephadex G-50 column (1.6 by 85 cm) eluted with 0.02 M Tris buffer (pH 8.5) containing 2 mM EDTA, 1% Na-deoxycholate, and
0.01% NaN3. A peak around the void volume was pooled and
designated total OMP (or OMP). The yield of OMP preparation was 0.1 to
0.3% of the wet cell mass. The protein profile of OMP by SDS-PAGE is
typical for gram-negative bacteria composed of approximately 20 proteins, 4 to 6 of which are major components (Fig.
1) (24). A major band with an
apparent molecular mass of 37 kDa corresponding to P2 or porin
(17) accounts for approximately 65% by density. The
residual LOS in OMP was 1.4% (wt/wt) by SDS-PAGE and silver staining
analysis.
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FIG. 1.
Coomassie blue-stained 12% gel of OMP from NTHi strain
12 after SDS-PAGE. Lane 1, molecular weight markers (in thousands);
lane 2, OMP (22 µg). Arrow indicates a molecular mass of 37 kDa.
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Synthesis and characterization of conjugates.
A method
(13) for synthesizing OS-TT was tried first, but OMP was
precipitated under acid conditions. Therefore, a method described by
Verheul et al. (35) was adopted with modifications. Briefly,
OS or dLOS (10 mg/ml) was coupled to cystamine by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and sulfo-NHS at pH 4.8. The modified OS or dLOS was incubated in 0.1 M
Na2HPO4 buffer (pH 8.1) containing 0.2 M
dithiothreitol to generate free SH groups. After the bromoacetylation
of OMP or TT with N-succinimidyl bromoacetae, the resulting
OMP or TT was further conjugated to OS-SH or dLOS-SH in a ratio of 1 to
5 (wt/wt). After each reaction, three conjugates were synthesized and
referred to as OS-OMP, dLOS-OMP, or dLOS-TT.
The compositions and yields of the conjugates were analyzed (Table
1). The weight ratios of OS or dLOS to
OMP and dLOS to
TT were 0.12, 0.58, and 1.6, respectively. The yields
calculated
from the modified OS or dLOS were 0.6, 2.1, and 12.6% for
OS-OMP,
dLOS-OMP, and dLOS-TT. The yield for OMP was approximately
50%.
To determine the antigenicity of the conjugates in vitro, a
double
immunodiffusion assay was used (Fig.
2). dLOS-OMP, dLOS-TT, and
LOS formed
identical precipitation lines with two monoclonal antibodies
against
LOS (
34), indicating that the conjugates retained the
epitopes on LOS. OS and OS-OMP did not form precipitation lines.
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FIG. 2.
Double immunodiffusion assay. The central wells contain
mouse monoclonal antibodies against 9274 LOS (34). (A)
6245B4; (B) 6347C11. Wells: 1, LOS (1 mg/ml); 2, dLOS (1 mg/ml); 3, dLOS-TT (0.29 mg/ml) (carbohydrate content); 4, TT (0.37 mg/ml); 5, dLOS-OMP (carbohydrate content) (0.132 mg/ml); 6, OMP (0.228 mg/ml).
Each well was loaded with a 10-µl sample.
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Immunization and antibody production.
Female mice (NIH/Swiss),
5-week-old (10 mice per group), were subcutaneously injected with 5 µg of each conjugate (carbohydrate) alone or with Ribi-700 adjuvant.
The mixtures of 5 µg of dLOS and 8 µg of OMP (or 5 µg of TT),
designated dLOS + OMP or dLOS + TT, were used as controls.
The mice were given three injections at 14-day intervals and bled 14 days after the first injection and 7 days after the second and third
injections. Female New Zealand White rabbits weighing 2 to 3 kg (two
per group) were given a total of two subcutaneous and intramuscular
injections at 28-day intervals with 50 µg of each conjugate alone or
with the adjuvant and were bled before and 14 days after each
injection. The mixtures of 50 µg of dLOS and 80 µg of OMP or 50 µg of TT were used as controls. Antibody levels in serum were
detected by an enzyme-linked immunosorbent assay (ELISA) and expressed
as ELISA units (15) or titers (OMP).
In mice, the levels of anti-LOS antibodies elicited by OS-OMP did not
increase after three injections (Table
2). However,
dLOS-OMP and dLOS-TT
elicited significant amounts of LOS antibodies,
especially for
immunoglobulin G (IgG). There was a 139-fold increase
of anti-LOS IgG
elicited by dLOS-OMP and a 21-fold increase of
anti-LOS IgG elicited by
dLOS-TT after three injections. dLOS-OMP
elicited higher levels of LOS
antibodies than dLOS-TT (
P < 0.01).
Formulation of the
conjugates with Ribi adjuvant enhanced the
antibody response in both
dLOS-OMP and dLOS-TT but not in the
OS-OMP group. Since OS-OMP was not
immunogenic in mice, only the
other two conjugates were further tested
for their immunogenicity
in rabbits (Table
3). dLOS-OMP elicited a 115-fold increase
of
anti-LOS IgG, while dLOS-TT elicited a 27-fold increase after
two
injections. Formulation of both conjugates with Ribi adjuvant
further
enhanced the antibody response in rabbits.
All conjugates elicited significant anti-protein IgG with booster
responses in mice (Table
4) and rabbits
(Table
5) after
two or three injections.
Formulation of the conjugates with Ribi
adjuvant enhanced their IgG
antibody responses. Nonconjugated
OMP and TT elicited higher IgG
antibody levels than those of the
conjugates in rabbits but not in
mice.
Bactericidal activity (15).
Mouse antisera elicited by the
conjugates were not bactericidal to strain 9274. Only one of four
rabbit antisera to dLOS-TT showed a low level of bactericidal activity
against strain 9274 at a titer of 1:2. However, all four rabbit
antisera to dLOS-OMP showed bactericidal activities against strain 9274 at titers of 1:2 to 1:64. Table 6 shows a
comparison between bactericidal activities and levels of antibodies to
LOS and OMP among six rabbit antisera elicited by dLOS-OMP or the
mixture of dLOS and OMP. A linear regression analysis showed a
correlation between LOS IgG (not IgM) levels and bactericidal titers
against strain 9274 (r = 0.98, P < 0.001) or
strain 12 (r = 0.81, P = 0.048) and no correlation
between OMP IgG or IgM levels and bactericidal titers against strain
9274 or strain 12 (P > 0.3) among the rabbit antisera.
The bactericidal activities of the rabbit antisera (no. 2, 4, and 6)
were further assayed with 20 strains from the United
States and Japan
(Table
7). The rabbit antisera elicited
by dLOS-OMP
alone or with adjuvant demonstrated bactericidal activities
to
8 of 20 strains (40%), while the antiserum elicited by the mixture
of dLOS and OMP or OMP with Ribi adjuvant showed bactericidal
activities to 1 of 20 strains (5%) and 3 of 20 strains (15%),
respectively.
Summary.
The aim of this study was to investigate the effects
of OMP as an alternative carrier of immunological properties of
LOS-based conjugates. By the method of Verheul et al. (35),
in which the reaction pH ranged from 6 to 8, dLOS and OS were coupled
to OMP without precipitation. Although the yields for both saccharides were low, more dLOS than OS was bound to OMP. Immunological analysis revealed that OS-OMP was not immunogenic in mice even with Ribi adjuvant, consistent with the observation that NTHi
OS-CRM197 (a nontoxic mutant of diphtheria toxin)
synthesized by reductive amination was a poor immunogen in mice
(10). However, a meningococcal OS-TT conjugate was
immunogenic in both mice and rabbits (13). The reason for
the poor immunogenicity of the NTHi OS-based conjugates in mice is
unclear. Besides the low ratio of OS to OMP (Table 1), one possible
interpretation is that the removal of the whole lipid A portion may
result in the relaxation of the conformation of the LOS molecule as
well as the loss of its adjuvant effect (18).
The immunogenicities of dLOS-OMP and dLOS-TT were compared in mice and
rabbits. Both conjugates induced boostable IgG antibody
responses
against LOS, and the antibody levels elicited by dLOS-OMP
were
considerably higher than those of dLOS-TT. A possible explanation
for
the fact that OMP is a better carrier is its mitogenic activity
for
lymphocytes (
22) and stimulatory effect on macrophages
(
1).
OMP may have adjuvant effects, since it is a part of a
gram-negative
bacterial cell wall including a minor amount of LOS,
which has
a variety of biological activities (
19), such as
an effective
immunomodulating agent and adjuvant (
18,
25).
To investigate the protective capacity of the conjugate-induced
antisera, a bactericidal assay was performed with NTHi clinical
strains. Rabbit antisera but not mouse antisera showed bactericidal
activity, consistent with our previous observations with meningococcal
LOS-derived OS-TT and NTHi dLOS-protein conjugates (
13,
15).
However, with
Moraxella catarrhalis dLOS-protein conjugates,
20
to 45% of mouse antisera showed bactericidal activity
(
11).
Many factors may result in the lack of bactericidal
antibodies
generated by the conjugates in mice. These factors include
bacterial
strains and species, sources of complement, antibody levels
and
affinities, and immunization routes. The bactericidal levels
elicited
by dLOS-OMP varied among rabbit antisera from each group but
correlated
with the levels of anti-LOS IgG and not the levels of
anti-OMP.
Two selected antisera elicited by dLOS-OMP also demonstrated
bactericidal
activities against 8 of 20 NTHi strains, while the
antisera elicited
by the mixture of dLOS + OMP or OMP showed
bactericidal activities
in 1 and 3 of 20 strains, respectively. These
data indicate that
LOS is the major target of bactericidal antibody and
that OMP
and dLOS have synergetic effects on the dLOS-OMP conjugate. In
addition, the adjuvant could enhance the level of bactericidal
activity
but could not expand the spectrum for dLOS-OMP.
In this study, dLOS-TT exhibited very little bactericidal activity and
was not as immunogenic as dLOS-OMP. In contrast, a
previously
synthesized dLOS-TT (ADH) was very immunogenic (
15),
and the
rabbit antisera demonstrated bactericidal activities against
to 45 to
75% of the 20 strains described above, although the levels
of
bactericidal activities were lower than those of the rabbit
sera
elicited by dLOS-OMP (data not shown). The immunogenic differences
between dLOS-TT and dLOS-TT (ADH) probably resulted from differences
in
two conjugation methods, different ratios of dLOS to TT (1.6
in dLOS-TT
and 0.5 to 1 in dLOS-TT [ADH]), different coupling
groups for TT
(dLOS was coupled to amino groups of TT by the present
method instead
of carboxyl groups in dLOS-TT [ADH]), and different
distances between
dLOS and TT (approximately 15 Å at dLOS-TT and
20 Å at dLOS-TT
[ADH]). It has been reported that, besides the
size of the
carbohydrates and the choice of the carrier protein
(
2,
21,
30,
31), the ratios of carbohydrate to protein,
the use of spacers,
the distance between carbohydrates and carrier
proteins, and the
methods of coupling have significant effects
on the immunogenicity of
conjugate vaccines. In conclusion, OMP
is a good and alternative
carrier for NTHi or other conjugate
vaccines.
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ACKNOWLEDGMENTS |
We thank M. A. Apicella, S. J. Barenkamp, H. Faden, and
G. Mogi for providing strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: NIDCD, NIH, 5 Research Court, 2A31, Rockville, MD 20850. Phone: (301) 402-2581. Fax: (301) 402-4200. E-mail: guxx{at}nidcd.nih.gov.
Editor:
D. L. Burns
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