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Infection and Immunity, February 2004, p. 1184-1187, Vol. 72, No. 2
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.2.1184-1187.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Intranasal Immunization of Mice with Group B Streptococcal Protein Rib and Cholera Toxin B Subunit Confers Protection against Lethal Infection
Charlotte Larsson,1* Jan Holmgren,2 Gunnar Lindahl,1 and Charlotta Bergquist2
Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund,1
Department of Medical Microbiology and Immunology, Göteborg University, Göteborg, Sweden2
Received 10 September 2003/
Returned for modification 9 October 2003/
Accepted 14 November 2003

ABSTRACT
Intranasal immunization of mice with Rib, a cell surface protein
of group B streptococcus (GBS), conjugated to or simply coadministered
with the recombinant cholera toxin B subunit, induces systemic
immunoglobulin G (IgG) and local IgA antibody responses and
confers protection against lethal GBS infection. These findings
have implications for the development of a human GBS vaccine.

INTRODUCTION
Several approaches have been tried in an attempt to design an
efficient vaccine against group B streptococcal (GBS) disease
(
7), and possible vaccine components include capsular polysaccharides
and cell surface proteins of GBS (
2,
8,
9,
12,
16,
17,
20,
27).
Mucosal immunization is an attractive strategy, since it is
noninvasive and has the potential to induce both systemic and
local immune responses (
3,
14,
22). Indeed, it should be advantageous
to use a GBS vaccine that not only induces protective immunoglobulin
G (IgG) antibodies that can be transplacentally transferred
to the fetus but also induces mucosal antibodies that prevent
genital colonization of the mother and thereby prevent transfer
of the bacteria to the fetus.
Some human and animal studies have focused on antibodies to GBS at the female genital mucosa (8-12, 24). Although, the role of these local antibodies in the pathogenesis of GBS infection is not clear, it seems reasonable to assume that they may to some extent protect against colonization with GBS (10). It has been reported that mucosal immunization with GBS polysaccharides or inactivated GBS bacteria induces systemic and local antibody responses in mice (8, 9, 12, 24). However, there is no previous report on mucosal immunization with purified GBS proteins.
Earlier studies have shown that parenterally administered cell surface proteins of GBS elicit a systemic IgG response and confer protection against experimental GBS infection (1, 6, 17, 18, 20). This made it of interest to examine if these proteins could also be used in a mucosal GBS vaccine. In the present study, Rib, a well-characterized GBS surface protein that is expressed by many strains causing invasive neonatal infection (17, 25, 26), was combined with recombinant cholera toxin B subunit (CTB) and administered intranasally (i.n.) to mice. The systemic and local IgG and IgA responses were examined. In addition, the protective capacity of this mucosal vaccination was evaluated by lethal intraperitoneal (i.p.) challenge with GBS.

Preparation of conjugate vaccine.
The Rib protein was isolated from the high-virulence type III
strain BM110 (
21,
25) by several purification steps and was
free of contaminating polysaccharides (
17,
25). Recombinant
CTB was purified from
Vibrio cholerae strain 358 (
19). The Rib
protein was conjugated to CTB using
N-succinimidyl 3-(2-pyridyldithio)propionate
(SPDP; Pharmacia AB, Uppsala, Sweden) as a bifunctional coupling
reagent. SPDP was added to both proteins according to the manufacturer's
instructions, using pH 8.5 for Rib and pH 7.5 for CTB. A similar
method has been used for coupling polysaccharides to CTB and
is described in detail elsewhere (
4,
5). A 10-fold molar excess
was used for Rib, and a 3-fold molar excess was used for CTB.
The 2-pyridyl disulfide-containing Rib was then reduced using
50 mM dithiothreitol, and the excess 2-pyridyl disulfide and
dithiothreitol was removed using a Sephadex G25 prepacked PD10
column (Pharmacia AB, Uppsala, Sweden). The two derivatized
proteins were mixed and allowed to react overnight at room temperature.
The conjugate was purified using a Superdex 200 10/30 column
connected to a BioLogic WorkStation (Bio-Rad). The fractions
containing the highest-molecular-weight material were collected,
pooled, and concentrated using an Amicon stirred concentration
cell (Amicon). The conjugate was tested by enzyme-linked immunosorbent
assay (ELISA), and both CTB and Rib were detected. The Lowry
protein determination assay was used to measure the total protein
content of the conjugate. However, the exact proportion of each
protein could not be determined. The conjugate was stored at
4°C with 0.005% Merthiolat (Kebo AB, Stockholm, Sweden)
as a preservative.

Immunizations and antibody responses.
Five groups with four 10-week-old C3H/HeN female mice (Charles
River, Sulzfeld, Germany) in each group were immunized i.n.
with either of the following vaccines: protein Rib conjugated
to CTB (Rib-CTB) (8 µg), Rib mixed with CTB (Rib+CTB)
(4 µg plus 4 µg), Rib alone (Rib) (4 µg),
CTB alone (CTB) (4 µg), or phosphate-buffered saline (PBS)
three times 2 weeks apart. One group with four mice received
5 µg of Rib subcutaneously (s.c.) and 4 weeks later a
booster of 2.5 µg s.c. The antigens were diluted in PBS
to a volume of 25 µl for i.n. administration and 150 µl
for s.c. administration, respectively. The mice were lightly
anesthetized with methoxyflurane (Methofane) (Mallincrodt Veterinary,
Mundelein, Ill.) during immunizations. Sera and genital tissue
extracts were obtained from the immunized mice. Two weeks after
the last vaccine dose, mice were anesthetized and bled to death
from the subclavian vein. The mice were then extensively perfused
with 0.1% heparin-PBS through the heart until all blood was
removed. The ovaries, fallopian tubes, uterus, and vagina were
taken out and then frozen at -20°C. For analyses of antibody
contents, the organs were extracted by 2% (wt/vol) saponin-PBS,
1 µl/mg of organ, and allowed to thaw overnight. After
centrifugation, the supernatants were stored at -20°C (
5).
The titers of IgG and IgA to protein Rib in serum and in genital extracts were determined by ELISA. The wells of microtiter plates (Nunc, Roskilde, Denmark) were coated by incubation overnight at room temperature with 100 µl of a solution of Rib in PBS (0.6 µg/ml). The plates were blocked with 0.1% bovine serum albumin-PBS by incubation for 30 min at 37°C and then washed once with PBS. Samples and controls were added in threefold serial dilutions. As a reference and a positive control, a serum pool from mice immunized with Rib-CTB, Rib+CTB i.n., and Rib s.c. was used. To detect specific antibodies, the plates were washed three times with PBS containing 0.05% Tween (PBST), and horseradish peroxidase-conjugated goat anti-mouse
chains (Jackson Immunoresearch Laboratories, Westgrove, Pa.) diluted 1:3,000 in 0.1% bovine serum albumin-PBST or goat anti-mouse IgA (Southern Biotechnology Inc., Birmingham, Ala.) diluted 1:1,000 in 0.1% bovine serum albumin-PBST was added to the wells. The plates were incubated for 90 min at room temperature. After three washes with PBST, the plates were developed using o-phenylenediamine (Sigma) and H2O2. ELISA titers are given as the reciprocal dilution giving an absorbance of 0.4 above the background level in a microplate reader (Labsystems Multiscan Plus, Helsinki, Finland) at 450 nm.
Intranasal immunization of mice with Rib-CTB or Rib+CTB elicited significant levels of serum IgG to protein Rib (Table 1). In contrast, the IgG response induced by i.n. immunization with Rib alone was approximately 100 times lower (Table 1). In agreement with previous studies (17, 18), s.c. immunization with protein Rib elicited a systemic Rib-specific IgG response (Table 1).
Rib-specific IgA antibodies were found in serum and in genital
tissue extracts from mice i.n. immunized with Rib-CTB. Low levels
of IgA were also detected in serum from mice that received Rib+CTB
(Table
2). In contrast, Rib given i.n. without CTB did not result
in any measurable IgA response. The method used in this study
for measuring local antibodies in mucosal tissue has previously
been evaluated regarding the proportion of serum antibodies
from blood retained in the tissue extracts. The proportion of
serum IgA antibodies in genital tissue extracts in that control
experiment was approximately 2% (
13). We compared the titers
of IgA in the genital tissue extracts after i.n. immunization
with Rib-CTB with the corresponding titers of IgA in serum,
and we found that most of the IgA antibodies in the genital
tissue appeared to be locally produced, since the titer of IgA
exceeded 2% of the titer in serum (Table
2). Low levels of Rib-specific
IgG were detected in the genital tissue extracts from mice i.n.
immunized with Rib-CTB or Rib+CTB or s.c. immunized with Rib
alone (data not shown); however, most of these IgG antibodies
were considered to be transuded from serum rather than locally
produced, according to the observations made in the previous
control experiment referred to above (
13).
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TABLE 2. Titers of Rib-specific IgA in serum and genital tissue extract after immunization with different vaccine compositionsa
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Protection against lethal GBS infection.
Groups of 15 mice were vaccinated with Rib-CTB i.n., Rib+CTB
i.n., Rib s.c., or PBS i.n., as described above. Two weeks after
the booster mice were challenged i.p. with a

90% lethal dose
of log-phase bacteria (BM110) diluted in 0.5 ml of Todd-Hewitt
broth. Deaths were recorded daily for 7 days (
17). Vaccine efficacy
(or protective efficacy) was calculated by the following formula:
(mortality in nonvaccinated - mortality in vaccinated)/(mortality
in nonvaccinated). Statistical significance was estimated by
Fisher's exact test and confidence intervals (CI). The confidence
level was set at 0.95. A
P value of less than 0.05 was considered
statistically significant.
Immunization with CTB-Rib or CTB+Rib provided protection against lethal infection with the GBS type III strain BM110 expressing the Rib protein (Fig. 1). Ten of fifteen mice vaccinated with Rib-CTB i.n. and 12 of 15 mice vaccinated with Rib+CTB survived a challenge with a lethal i.p. dose of GBS. The protective efficacy for Rib-CTB was 55% (CI, 37 to 73; P = 0.03) and 73% (CI, 57 to 89; P = 0.005) for Rib+CTB. In agreement with previous reports (17, 18), s.c. vaccination with the Rib protein protected against lethal GBS infection. We observed, however, that even though the amount of the Rib protein used for s.c. immunization was reduced and was given without adjuvant, it still induced sufficient immunity to confer complete protection against lethal GBS infection (P = 0.00002) (Fig. 1).

Conclusions.
Intranasal immunization of mice with the Rib protein and CTB
seems to induce systemic and local antibody responses and to
confer protective immunity against GBS infection. The titers
of IgG induced by immunization with Rib+CTB and Rib-CTB i.n.
were higher than the titers induced by Rib s.c. (Table
1). Yet,
the protective efficacy for Rib given s.c. was higher than for
Rib+CTB and Rib-CTB given i.n. (Fig.
1). However, s.c. injection
and mucosal application represent presentation of the vaccine
antigen (Rib) to the immune system in two different ways. In
addition, the mucosal route included an adjuvant (CTB). These
differences may have resulted in vaccine-induced antibodies
with different avidities for the Rib protein. It seems possible
that the conjugation process may be further optimized, resulting
in a more immunogenic preparation.
We observed a wide range of antibody responses in some mouse groups (Tables 1 and 2). This variable antibody response was concordant with the individual response patterns for antibodies in both serum and genital tissue. However, since the number of mice in each group was small, it is difficult to draw general conclusions about the levels of antibody response to the different vaccines. The local antibody levels in the female genital tract vary with the stage of the estrous cycle, and it has been shown that pretreatment of mice with progesterone before mucosal immunizations increases the number of antibody-secreting cells in the genital tract not only in response to local vaginal immunization but also in response to i.n. immunization (13). Hence, the individual variation and the level of the antibody response might have been different in the present experiment had the mice been pretreated with hormones.
A relevant question is how the antibody response would be in humans. Although only vaccination studies with humans will give the full answer to this question, some encouraging observations have been published. In one study rhesus monkeys (Macacca mulatta) were immunized i.n. with a streptococcal protein antigen (AgI/II) either chemically conjugated to or mixed with CTB, which resulted in both systemic and local IgG and IgA responses to the vaccine antigens (23). Since these nonhuman primates are phylogenetically close to humans, it seems possible that humans would respond similarly to i.n. immunization with vaccines composed of bacterial protein and CTB. Further, i.n. immunization with CTB in humans has stimulated strong systemic as well as respiratory and vaginal mucosal antibody responses (3, 14, 22). Moreover, recent studies of naturally acquired antibodies to GBS cell surface proteins in different human populations have indicated that antibodies to GBS cell surface proteins are prevalent (10, 15), indicating that these protein antigens indeed are immunogenic also in humans.
The majority of clinically important GBS strains express either of the two proteins Rib or
(17, 25). Previously, we have shown that it is possible to include these two GBS cell surface proteins in a vaccine and elicit protective immunity in mice without any sign of immunogenic competition between the vaccine antigens (18). We conclude that i.n. immunization with a vaccine containing cell surface proteins from GBS strains that are prevalent among pregnant women may be an alternative strategy in the ongoing efforts to design an efficient vaccine against neonatal GBS disease.

ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Research
Council (Medicine), the Meningitis Research Foundation (United
Kingdom), Sida-SAREC's programme for research on AIDS and related
sexually transmitted infections, the "Förenade Liv" Mutual
Group Life Insurance Company, the Swedish Society for Medical
Research, the Royal Physiographic Society in Lund, and the trusts
of Jerring, Kock, and Österlund.
We thank Margaretha Stålhammar-Carlemalm for her help and for purified protein Rib, and we thank Gun Wallerström for laboratory assistance.

FOOTNOTES
* Corresponding author. Mailing address: Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, S-22362 Lund, Sweden. Phone: 46 46 173243. Fax: 46 46 135936. E-mail:
charlotte.larsson{at}mmb.lu.se.

Editor: D. L. Burns

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Infection and Immunity, February 2004, p. 1184-1187, Vol. 72, No. 2
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.2.1184-1187.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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