INFECTION AND IMMUNITY, July 2005, p. 3945–3953
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 7
Intranasal Administration of Recombinant Neisseria gonorrhoeae
Transferrin Binding Proteins A and B Conjugated to the
Cholera Toxin B Subunit Induces Systemic and
Vaginal Antibodies in Mice
Gregory A. Price,1Michael W. Russell,2and Cynthia Nau Cornelissen1*
Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth
University, Richmond, Virginia 23298-0678,1and Departments of Oral Biology and Microbiology and
Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo,
Buffalo, New York2
Received 21 December 2004/Returned for modification 2 February 2005/Accepted 19 February 2005
The transferrin binding proteins (TbpA and TbpB) comprise the gonococcal transferrin receptor and are
considered potential antigens for inclusion in a vaccine against Neisseria gonorrhoeae. Intranasal (IN) immu-
nization has shown promise in development of immunity against sexually transmitted disease pathogens, in
part due to the induction of antigen-specific genital tract immunoglobulin A (IgA) and IgG. Conjugation of
antigens to the highly immunogenic cholera toxin B subunit (Ctb) enhances antibody responses in the serum
and mucosal secretions following IN vaccination. In the current study, we characterized the anti-Tbp immune
responses following immunization of mice IN with recombinant transferrin binding proteins (rTbpA and
rTbpB) conjugated to rCtb. We found that both rTbpA-Ctb and rTbpB-Ctb conjugates administered IN
induced antibody responses in the serum and genital tract. IN immunization resulted in both IgA and IgG in
the genital tract; however, subcutaneous immunization mainly generated IgG. Surprisingly, rTbpA alone was
immunogenic and induced serum and mucosal antibody responses similar to those elicited against the
rTbpA-Ctb conjugate. Overall, rTbpB was much more immunogenic than rTbpA, generating serum IgG levels
that were greater than those elicited against rTbpA. Bactericidal assays conducted with sera collected from
mice immunized IN with TbpA and/or TbpB indicated that both antigens generated antibodies with bacteri-
cidal activity. Anti-TbpA antibodies were cross-bactericidal against heterologous gonococcal strains, whereas
TbpB-specific antibodies were less cross-reactive. By contrast, antibodies elicited via subcutaneous immuni-
zation were not cross-bactericidal against heterologous strains, indicating that IN vaccination could be the
preferred route for elicitation of biologically functional antibodies.
A 1995 World Health Organization report estimated that
there were 62.2 million cases of the sexually transmitted infec-
tion gonorrhea worldwide (19). This number is considered to
be an underestimate of the actual incidence, due in part to
inadequate reporting by physicians and clinics, as well as to the
prevalence of asymptomatic carriage (8, 40). One study found
that asymptomatic carriage in women can be as high as 55%
(17). Uncomplicated gonorrhea manifests as urethritis in men
and as endocervitis and/or urethritis in women. Serious down-
stream sequelae can afflict those individuals with asymptomatic
infection, since the infection can spread to the upper genital
tract. Ascension can result in epididimytis, salpingitis, ectopic
pregnancy, sterility, and disseminated gonococcal infection.
Antibiotics are the treatment of choice for gonorrhea, but the
increasing emergence of drug-resistant strains has made treat-
ment more difficult and expensive (25). Furthermore, it has
been shown that coinfection with Neisseria gonorrhoeae and
human immunodeficiency virus (HIV) can increase the risk of
transmission of HIV (10). These findings have made the need
for an effective vaccine more imperative. To date, attempts to
develop a gonococcal vaccine have been disappointing. Human
trials using partially lysed gonococci, purified pilin, or purified
porin all failed to confer protection upon natural exposure (4,
20, 45). These vaccine formulations, although immunogenic,
failed to protect, likely due in part to the intrinsic ability of the
gonococcus to undergo high-frequency phase and antigenic
variation of surface structures (28).
The gonococcal transferrin binding proteins, TbpA and
TbpB, have generated particular interest as vaccine antigens
because they are ubiquitously expressed among clinical iso-
lates, they exhibit low strain-to-strain variability, and they are
not subject to high-frequency antigenic or phase variation (11,
12, 29). Furthermore, their importance in gonococcal virulence
has been established in a human male challenge model of
infection (14). Subjects inoculated with a mutant strain of N.
gonorrhoeae that lacked the transferrin receptor showed no
signs or symptoms of urethritis, in contrast to subjects inocu-
lated with the parental strain (14). In spite of their expression
in vivo, we demonstrated that antibody responses to the trans-
ferrin binding proteins resulting from natural infections were
weak in the serum and nonexistent in vaginal washes and
seminal fluid (34). We postulate that the induction and sus-
tained production of an appropriate antibody response to one
or both Tbps in the genital tract could prevent colonization.
One of the shortcomings of parenteral immunization is its
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Medical College of Virginia Campus of Vir-
ginia Commonwealth University, Richmond, VA 23298-0678. Phone:
(804) 827-1754. Fax: (804) 828-9946. E-mail: firstname.lastname@example.org.
relatively poor ability to induce genital-tract-specific immuno-
globulin A (IgA) antibodies (5, 30). IgA is considered impor-
tant in protecting the genital tract from infection, as its pres-
ence is correlated with a protective role against chlamydia and
HIV (6, 7). Intranasal (IN) immunization, on the other hand,
has been more promising in terms of eliciting genital-tract
antigen-specific IgA and IgG in mice (18, 21, 47), primates
(42), and humans (3, 38). In addition, the genital-tract anti-
bodies generated as a function of IN immunization have been
demonstrated to be long lasting in mice (37, 47)
Cholera toxin B (Ctb), the nonenzymatic, nontoxic compo-
nent of cholera toxin, has been studied extensively for its ability
to augment antibody responses to coadministered and physi-
cally conjugated antigens following intranasal application (21,
23, 24, 48, 49). In this study, we evaluated the immunogenicity
of IN administered recombinant TbpA and TbpB, alone and in
combination with recombinant Ctb. We demonstrate that IN
immunization can elicit serum and vaginal antigen-specific an-
tibody responses. Furthermore, this route of immunization was
superior to subcutaneous immunization in the induction of
specific genital tract IgA. IN immunization generated antibod-
ies with greater serum bactericidal activity than did subcuta-
neous immunization. Importantly, this bactericidal activity was
detected against both homologous and heterologous gonococ-
(A preliminary account of these results was presented pre-
viously [G. A. Price, M. W. Russell, and C. N. Cornelissen,
14th Int. Pathog. Neisseria Conf., abstr. 188, 2004].)
MATERIALS AND METHODS
pUNCH412, was described previously (13). The tbpB expression plasmid,
pVCU711, was constructed by PCR amplification using a proofreading Taq
polymerase (Platinum Pfx; Invitrogen) of a previously described tbpB expression
plasmid, pVCU705 (34). The forward primer, oVCU240 (GGATCCTGTCTGG
GCGGAGGCGGCAGTTTCG), contained a BamHI site (shown in boldface)
and amplified the FA19 tbpB gene from the sequence that encodes amino acid 2
of the mature protein. The reverse primer, oVCU241 (CCCGGGTTATTTCAC
AAGCTTTTGGCGTTTCG), contained a SmaI site (shown in boldface) and the
stop codon of the FA19 tbpB gene. The PCR product was ligated into the
pQE-80L expression vector (QIAGEN). The resultant plasmid, pVCU711, en-
coded a recombinant TbpB in which the N-terminal six-histidine tag was fused to
amino acid 2 of the mature protein. The resulting protein lacked the amino-
terminal cysteine residue and was expressed under the control of the T5 pro-
moter. The ctb expression plasmid, pVCU710, was constructed by PCR ampli-
fication of the plasmid pCT?A1(21). The forward primer, oVCU238 (TGGCC
ACACCTCAAAATATTACTGATTTGTGTG) contained an MscI site (shown
in boldface) and amplified the mature ctb gene product. The reverse primer,
tained an XhoI site and amplified the 3? end of the ctb gene, including the stop
codon. The PCR product was ligated into the pET-22b(?) (Novagen) expression
vector. The resultant plasmid, pVCU710, contained the mature ctb gene product
fused with the Escherichia coli pelB leader sequence immediately upstream.
Gene expression was under the control of the T7 promoter. The expression hosts
for pVCU710 and pVCU711 were the E. coli strains BL21(DE3) (Novagen) and
TOP10 (Invitrogen), respectively.
Recombinant protein expression and purification. Recombinant proteins were
expressed in 1-liter cultures of Luria-Bertani broth containing 1% glucose and
500 ?g/ml of carbenicillin for recombinant TbpA (rTbpA) expression or 200
?g/ml of ampicillin for rTbpB and rCtb expression. When the cultures reached
an optical density at 600 nm of 0.4 to 0.6, they were induced with IPTG (iso-
propyl-?-D-thiogalactopyranoside). For rTbpA, prior to induction, cultures were
centrifuged for 15 min at 6,000 ? g to pellet the bacteria. The pellets were then
resuspended in fresh medium as described above with 0.5 mM IPTG and allowed
to express overnight at 27°C (?16 h). For rTbpB and rCtb expression, 0.5 mM
The tbpA expressionplasmid,
IPTG was added, and the cultures were allowed to express for 3 h at 30°C. After
induction, the cells were pelleted as described above and stored at ?80°C.
For rTbpA and rTbpB purification, the pellets were thawed on ice and resus-
pended in Tris buffer (100 mM Tris [pH 8.0] and 0.5 M NaCl). After the cells
were completely resuspended, Elugent (Calbiochem) was added to a final con-
centration of 2%. Protease inhibitors (Sigma), lysozyme, and DNase were added,
and the mixture was allowed to incubate overnight at 4°C. Solubilized prepara-
tions were centrifuged at 18,000 ? g for 30 min to remove insoluble material.
TbpA was purified using a transferrin affinity column (26). The rTbpA-trans-
ferrin column was washed with 20 bed volumes of 50 mM potassium phosphate
(pH 8.0)-0.5 M NaCl-0.05% lauryl maltoside (n-dodecyl-?-D-maltopyranoside;
Anatrace, Maumee, OH) and eluted with the above-mentioned buffer at pH 2.0.
The eluted proteins were immediately neutralized by the addition of 1 M potas-
sium phosphate, pH 8.0, and 0.05% lauryl maltoside. rTbpB was purified as
described previously (34). Ctb pellets were resuspended in 50 mM potassium
phosphate buffer, pH 6.8, and 100 ?g/ml lysozyme and placed at 30°C for 15 min.
Following the 15-min incubation, the cell pellets were subjected to sonication on
ice for 30 bursts repeated three times. Following centrifugation, the supernatants
were subjected to precipitation by ammonium sulfate, where Ctb precipitated at
60 to 80% saturation. The resulting precipitate was collected by centrifugation
and dissolved in 20 mM potassium phosphate buffer, pH 6.8. The dissolved
precipitate was dialyzed three times against a 1,000-fold excess of potassium
phosphate buffer. The dialyzed preparation was centrifuged to remove precipi-
tated material and then passed through a 0.45-?m-pore-size syringe filter. Ctb
was then purified by anion-exchange chromatography using an Econo-Pac High
S Cartridge (Bio-Rad) and gel filtration using a Superdex 200 column (Amer-
sham). Following purification, TbpB and Ctb were dialyzed four times against a
1,000-fold excess of phosphate-buffered saline (PBS), and TbpA was dialyzed
against PBS plus 0.05% lauryl maltoside.
Tbp-Ctb conjugate preparation. TbpA (1 mg in 1 ml PBS plus 0.05% lauryl
maltoside), TbpB (2 mg in 1 ml PBS), and Ctb (2 mg in 1 ml PBS) were treated
with 5 ?l of a 20 mM stock solution of SPDP [N-succinimidyl 3-(2-pyridyldithio)
proprionate; Pierce] in dimethyl sulfoxide for 1 h at room temperature. Each
protein was dialyzed against the corresponding initial buffers to remove free
SPDP. To 1 ml of derivatized TbpA or TbpB, 0.5 ml of acetate buffer (100 mM
sodium acetate, 100 mM NaCl, and 0.05% lauryl maltoside for TbpA only)
containing 12 mg of dithiothreitol was added, and the mixture was incubated for
30 min at room temperature. The reduced proteins were passed through a
desalting column (Pierce), and protein concentrations were determined by bicin-
choninic acid assay (Pierce). Equimolar amounts of derivatized Ctb were added
to the reduced proteins and allowed to incubate overnight at 4°C. For TbpA
conjugation, the derivatized Ctb was diluted to half by the addition of PBS plus
0.1% lauryl maltoside in order to keep the detergent concentration at 0.05%.
Conjugated proteins were separated from unconjugated proteins by size exclu-
sion chromatography using a Superdex 200 column (Amersham).
GM1ganglioside ELISA. Purified conjugates were analyzed for the presence of
Ctb and TbpA or TbpB using the GM1ganglioside enzyme-linked immunosor-
bent assay (ELISA). ELISA plates (Nunc) were coated with 0.05 ml GM1gan-
glioside (Sigma) diluted at 2 ?g/ml in methanol. Following evaporation of the
methanol, the plates were blocked with 0.2 ml of PBS plus 1% skim milk for 1 h
at 37°C. The test samples were diluted at 1/100 in PBS or PBS plus 0.05% lauryl
maltoside for the TbpA conjugate and applied to each well in 0.1-ml volumes.
The plate was then incubated at 30°C for 1 h. The plates were washed three times
with PBS to remove unbound material, and bound conjugates were probed for
1 h at room temperature with 0.05 ml of either anti-TbpA, anti-TbpB, or anti-CT
(Sigma) rabbit serum diluted in PBS plus 1% skim milk. The plates were again
washed as described above and probed with 0.05 ml of alkaline phosphatase-
conjugated goat anti-rabbit IgG (Bio-Rad) for 1 h at room temperature. The
plates were washed again and developed with 0.05 ml of p-nitrophenylphosphate
substrate (Sigma) diluted in carbonate buffer (0.05 M sodium carbonate, 1 mM
MgCl2, pH 9.8). After sufficient color developed, the optical density of each well
was measured at 405 nm and compared to those of blank and control wells.
Immunizations and sample collection. Female BALB/c mice, 7 to 8 weeks old,
were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). The mice were
housed in microisolator cages and were under the care and supervision of the
Division of Animal Resources. The protocols were approved by the Virginia
Commonwealth University Institutional Animal Care and Use Committee. At
the start of the experiment, the mice were approximately 10 weeks old. Groups
of five mice were immunized either intranasally or subcutaneously with Tbp-Ctb
conjugate(s) or Tbps with or without Ctb as an adjuvant (Table 1 shows immu-
nization details). All groups were immunized three times at 10-day intervals. Sera
and vaginal secretions were collected on days 0, 17, 28, 35, and 65. Sera were
obtained from tail vein blood samples and stored at ?20°C. Vaginal secretions
3946 PRICE ET AL.INFECT. IMMUN.
were obtained by pipetting 0.05 ml of PBS in and out of the vaginal vault three
times. This procedure was repeated twice, and the vaginal washes were pooled.
The protease inhibitor phenylmethylsulfonyl fluoride (Sigma) was added to each
wash sample at a concentration of 1 mM following collection. The vaginal washes
were kept at ?80°C until use.
ELISAs. Serum and vaginal washes were assayed for total and specific anti-
bodies as described previously (34). For antibodies specific to Ctb, plates were
first coated with 0.1 ml of GM1ganglioside as described above. All capture
antibodies and alkaline phosphatase-conjugated goat anti-mouse isotype-specific
antibodies were purchased from Southern Biotechnology Associates (Birming-
ham, AL). The standard curve was generated using a mouse reference serum
Serum bactericidal assays. Mouse sera were pooled by group and heat inac-
tivated at 56°C for 30 min. Gonococcal strains were plated from freezer stocks
directly onto plates containing GC medium base (Difco) plus Kellogg’s supple-
ment I (25a) and 5 ?M desferal to induce iron stress. For strains FA19 and
FA1090, plates were allowed to incubate at 37°C and 5% CO2for approximately
24 h, at which time they were passed again as described above. Following the
second passage, the plates were allowed to incubate for 16 to 18 h. Isolated
colonies were picked from the plate and suspended in prewarmed 37°C Gey’s
balanced salt solution (Sigma) containing 0.1% gelatin and 5 ?M desferal
(GBSS?G?D). The optical density of the inoculum at 600 nm was monitored
until it reached 0.20 (0.23 for strain MS11), and then it was serially diluted to
10?5in prewarmed GBSS?G?D. Immediately following dilution, 80 ?l of the
diluted cell suspension was added to a prewarmed 96-well microtiter plate con-
taining 10 ?l of the appropriate serum samples diluted in GBSS?G?D. The
plate was incubated at 37°C and 5% CO2for 15 min, and then 10 ?l of normal
human pooled serum (Quidel Corp.) was added and the plate was again incu-
bated as described above for 45 min. After incubation, viable gonococci were
detected by plating them onto plates containing GC medium base plus Kellogg’s
supplement I and 12.5 ?M ferric nitrate. The plates were incubated for approx-
imately 24 h as described above, after which colonies were enumerated. The
bactericidal titer was determined as the lowest dilution that gave ?50% killing
compared to control sera at the same dilution. Strain MS11 was plated only once
directly from the freezer stock and allowed to grow for 16 to 18 h. Because of its
moderate sensitivity to human serum, bactericidal activity against strain MS11
was tested in 5% human serum with incubation for 15 min.
Statistics. Analysis of variance for multiple group comparisons was performed
using the Tukey-Kramer multiple-comparison test or the Kruskal-Wallis multi-
ple-comparison Z value test where appropriate. A P value of ? 0.05 was con-
sidered significant. These comparisons were performed on logarithmically trans-
formed data. Group data are therefore presented as geometric means ?/?
standard deviation, after back transformation of the logarithmic means ? stan-
Serum antibody responses against TbpA and TbpB. The
serum antibody responses were measured over time using a
quantitative ELISA, with which we measured antibody levels
following each immunization. Sera were collected at days 0, 17,
28, 35, and 65. All day zero sera were assayed and found to be
negative for antibodies specific to all antigens tested. The an-
tibody responses to TbpA and TbpB following vaccination
were strikingly different and were dependent on antigen prep-
aration and route of immunization. For TbpA, the highest
antibody responses were seen in the subcutaneously immu-
nized group, in which antibodies to TbpA peaked on day 35
and remained high through day 65 (Fig. 1A). The groups
receiving TbpA conjugated to Ctb and TbpA alone generated
the next-highest responses through day 28. Interestingly, the
presence of Ctb in admixtures with TbpA appeared to delay
the immune response against TbpA. However, by day 65,
TbpA levels were similar for all IN immunized groups (Fig.
Unlike TbpA, conjugation of TbpB to Ctb significantly en-
hanced antibody titers compared to the groups where Ctb was
admixed with TbpB (all comparisons, P ? 0.05, days 17 to 65)
(Fig. 1B). Another important difference between TbpA and
TbpB was that TbpB was poorly immunogenic when adminis-
tered by itself (Fig. 1B) whereas TbpA alone was as immuno-
genic as the conjugated form (Fig. 1A). IN immunization with
TbpB conjugate or with TbpB and TbpA conjugates together
did not result in antibody levels that were significantly different
from those elicited in the subcutaneously immunized group
(Fig. 1B). In terms of antibody response, the groups immu-
nized with both Tbps did not differ significantly from the group
immunized with only one antigen on any day tested (Fig. 1A
and B). Thus, although each antigen individually elicited dis-
tinct antibody responses, the presence of a coadministered
antigen did not adversely affect antibody levels generated by
IN vaccination. Antibody responses to Ctb were robust in all
groups tested (Fig. 1C). Not surprisingly, the subcutaneously
immunized group elicited the highest Ctb antibody titers, ex-
cept on day 65 (Fig. 1C).
We were also able to detect serum IgA antibody responses
specific for TbpA, TbpB, and Ctb (Fig. 2). Serum IgA levels
against TbpA were transient and not measurable until day 28
and were completely undetectable in all groups by day 65 (Fig.
2A). The low IgA levels detected against TbpA were probably
reflective of the antigen’s lower overall immunogenicity, as
shown by lower serum IgG titers against TbpA than against
TbpB (Fig. 1A and B). Serum IgA responses to TbpB were
much higher than those measured against TbpA, with the
highest detected serum IgA antibody responses found in the
groups immunized with the TbpB-Ctb conjugates (Fig. 2B).
The levels measured in the conjugate groups were not signif-
TABLE 1. Immunization groups
Group (immunization route)ImmunogenAmt administereda(?g)
A ? Ctb (IN)
B ? Ctb (IN)
A ? Ctb ? B ? Ctb (IN)
A ? Ctb (IN)
B ? Ctb (IN)
A ? B ? Ctb (IN)
A only (IN)
B only (IN)
S.c.A ? B ? Ctb (s.c.b)
Ctb ? TbpA conjugate
Ctb ? TbpB conjugate
Ctb ? TbpA ? Ctb ? TbpB conjugates
Ctb ? TbpA admixed
Ctb ? TbpB admixed
Ctb ? TbpA ? TbpB admixed
Ctb ? TbpA ? TbpB admixed
20 ? 20
10 ? 10
10 ? 10
10 ? 10 ? 10
10 ? 10 ? 10
aGroups of mice (n ? 5) were immunized three times at 10-day intervals.
bOne group was immunized subcutaneously (s.c.) with an admixture of TbpA, TbpB, and Ctb.
VOL. 73, 2005 INTRANASAL VACCINATION WITH Tbps3947
icantly different from one another but were different from the
only other groups with measurable serum IgA against TbpB,
namely, the group immunized with TbpA plus TbpB plus Ctb
and the subcutaneously immunized group (P ? 0.05; days 28 to
65) (Fig. 2B). The subcutaneously immunized animals had the
highest serum IgG antibody titers against TbpB; however, the
serum IgA titers elicited by this route were detectable only on
day 65 (Fig. 2B). Serum IgA titers to Ctb (Fig. 2C) initially
were highest in the animals immunized with the TbpA-Ctb
conjugate, followed by the other two conjugate groups. By day
FIG. 1. Serum IgG levels specific for TbpA, TbpB, and Ctb. (A) Se-
rum IgG levels specific for TbpA detected at days 17, 28, 35, and 65.
(B) Serum IgG levels specific for TbpB detected at the same time
points. (C) Serum IgG levels specific for Ctb detected at the same time
points. Results are expressed as the geometric mean of antibody titers
?/? standard deviation. For all immunization groups, n ? 5.
FIG. 2. Serum IgA levels specific for TbpB and Ctb. (A) Serum IgA
levels specific for TbpA detected at days 17, 28, 35, and 65. (B) Serum
IgA levels specific for TbpB detected at the same time points. (C) Se-
rum IgA levels specific for Ctb detected at the same time points.
Results are expressed as the geometric mean of antibody titers ?/?
standard deviation. For all immunization groups, n ? 5.
3948PRICE ET AL.INFECT. IMMUN.
65, all IN immunized groups had similar levels of Ctb-specific
IgA antibody. Interestingly, on days 17 and 28, we were able to
measure serum IgA to Ctb in the subcutaneously immunized
group, but by day 35, Ctb-specific serum IgA was undetectable
and remained so on day 65 (Fig. 2C).
Vaginal antibody responses to TbpA and TbpB. The relative
immunogenicities of TbpA and TbpB were also reflected in the
detectable antibody levels measured in the vaginal washes.
Vaginal-wash antibodies to TbpA detected on day 28 (7 days
after the final immunization) (Table 2) were not as robust as
those detected against TbpB (Table 3). For TbpA-specific IgA
(Table 2), on day 28 the highest response measured was gen-
erated by the group of animals immunized with the TbpA-Ctb
conjugate: TbpA-specific IgA represented 1% of the total IgA
antibody detected. This level, however, was only significantly
different from the group immunized with both Tbp conjugates
(P ? 0.05) among the groups in which we were able to measure
TbpA-specific IgA. Furthermore, only in the IN immunized
groups were we able to detect TbpA-specific IgA. Interestingly,
TbpA-specific IgA responses declined on day 35 in all groups
with measurable IgA; however, these levels had returned to
similar or slightly higher levels by day 65 (Table 2). Although
the group receiving both TbpA and TbpB conjugates had in-
creased antibody levels by day 65, they were still significantly
lower than those of the groups immunized with TbpA-Ctb and
with both Tbps admixed with Ctb (P ? 0.05) (Table 2). TbpA-
specific IgG levels were undetectable on day 28. We were
unable to measure vaginal IgG until days 35 and 65 (Table 2).
For the most part, vaginal IgG antibody levels specific for
TbpA were lower and more sporadic than vaginal IgG mea-
sured against TbpB (Table 3).
In contrast to antibody levels measured against TbpA,
TbpB-specific IgA and IgG levels were robust as early as day 28
(Table 3). Vaginal IgA levels specific for TbpB were highest in
groups immunized with the Ctb conjugates and were statisti-
cally different from those of the other IN immunized and
subcutaneous groups (P ? 0.05 day 28), and they remained
significantly different through day 65 (P ? 0.05). The day 28
TbpB-specific IgG responses were also robust, with the highest
levels measured in the IN immunization groups immunized
with the Ctb conjugates and in the subcutaneously immunized
group. In the IN groups immunized with admixed Ctb, we were
unable to detect TbpB-specific IgG on day 28; however, levels
increased on subsequent days (Table 3), consistent with serum
IgG increases (Fig. 1B). Though TbpB-specific levels of IgA
and IgG were initially robust in the groups immunized with the
TbpB-Ctb conjugate, they were in decline by day 65 (Table 3).
Vaginal antibody responses to Ctb were also robust and
were generally higher than those responses measured against
TbpA or TbpB (data not shown). This is presumably reflective
TABLE 2. Vaginal antibody levels specific for TbpA detected at days 28, 35, and 65a
Day 35Day 65
IgAIgG IgA IgG IgAIgG
TbpA ? Ctb
TbpA ? Ctb ? TbpB ? Ctb
TbpA ? Ctb
TbpA ? TbpB ? Ctb
S.c.hTbpA ? TbpB ? Ctb
1.0 ?/? 1.8
0.4 ?/? 6.3e
0.5 ? 4.1e
0.2 ?/? 1.7
0.2 ?/? 3.8
0.2 ? 2.0
0.6 ?/? 10.6d
2.8 ?/? 3.8
0.9 ?/? 1.4
0.3 ?/? 2.3
1.5 ?/? 3.1
0.6 ?/? 10.8e
0.6 ?/? 1.7f
0.5 ?/? 1.3e
0.3 ?/? 2.7f
0.2 ?/? 4.4g
1.2 ?/? 1.3
aData are expressed as the geometric mean of the percentage of total corresponding antibody isotype concentrations ?/? standard deviation.
bDay 28 is 7 days after final immunization.
cOnly one mouse had detectable TbpA-specific antibodies.
dOnly two mice had detectable TbpA-specific antibodies.
eOnly three mice had detectable TbpA-specific antibodies.
fn ? 4; one mouse removed due to very low total IgG.
gn ? 3; two mice removed due to very low total IgG.
TABLE 3. Vaginal antibody levels specific for TbpB detected at days 28, 35, and 65a
Day 35Day 65
IgAIgGIgA IgG IgAIgG
TbpB ? Ctb
TbpA ? Ctb ? TbpB ? Ctb
TbpB ? Ctb
TbpA ? TbpB ? Ctb
S.c.gTbpA ? TbpB ? Ctb
12.2 ?/? 2.9
8.2 ?/? 3.6
32.5 ?/? 1.4f
20.2 ?/? 5.5
20.5 ?/? 1.8
6.4 ?/? 2.7
2.6 ?/? 2.9
15.9 ?/? 1.5f
20.4 ?/? 1.5
1.8 ?/? 2.9e
0.5 ?/? 1.3c,f
12.9 ?/? 1.5
3.5 ?/? 1.7
1.9 ?/? 2.9
2.0 ?/? 3.8d
9.7 ?/? 4.0e
1.6 ?/? 2.7c
15.8 ?/? 1.5
aData are expressed as the geometric mean of the percentage of total corresponding antibody isotype concentrations ? standard deviation.
bDay 28 is 7 days after final immunization.
cOnly one mouse had detectable TbpB-specific antibodies.
dOnly two mice had detectable TbpB-specific antibodies.
eOnly three mice had detectable TbpA-specific antibodies.
fn ? 4; one mouse removed due to very low total IgG.
VOL. 73, 2005INTRANASAL VACCINATION WITH Tbps3949
of this antigen’s higher immunogenicity and is consistent with
the higher serum IgG levels shown in Fig. 1. The groups im-
munized with the Ctb conjugates, as opposed to the admix-
tures, generally induced the highest Ctb-specific antibody re-
sponses. Interestingly, the subcutaneously immunized group
had high levels of Ctb-specific IgA, whereas subcutaneous im-
munization with the Tbps resulted in IgA levels that were
almost zero (Tables 2 and 3 and data not shown).
Serum bactericidal activity. In order to determine whether
serum antibodies had bactericidal activity, we performed in
vitro serum bactericidal assays using pooled mouse serum from
day 35 with human serum as a complement source. The data
demonstrate that those animals immunized with both Tbp-Ctb
conjugates had the greatest bactericidal activity against both
homologous and heterologous strains tested (Table 4). The
sera from the group immunized with the TbpA-Ctb conjugate
was more effective at killing the homologous strain (FA19) and
one heterologous strain (MS11) than were the TbpB-Ctb sera.
This outcome is interesting, considering the significantly lower
serum antibody titers generated against TbpA in comparison
to those generated against TbpB (Fig. 1A and B). The subcu-
taneously immunized group had the highest TbpA and TbpB
antibody titers on day 35 (Fig. 1A and B); however, these sera
were the least bactericidal against the homologous strain of all
the groups tested. Furthermore, sera from this group were the
only ones that failed to show bactericidal activity against any of
the heterologous strains tested.
IgG subclass analysis. We performed IgG subclass analysis
on selected serum samples in an effort to gain insight into why
some serum pools performed better than others in bactericidal
assays. It had been shown previously that mouse IgG2a is the
most efficient IgG subclass in activating complement, while
IgG1 is poor and may be inhibitory (16, 27, 44). We found that
animals immunized with the TbpA-Ctb conjugate had higher
IgG2a antibody responses, and hence a lower IgG1/IgG2a ratio
(Fig. 3A). Those animals immunized with the TbpB-Ctb con-
jugate produced significantly more IgG1 than IgG2a antibod-
ies and had a very high IgG1/IgG2a ratio (Fig. 3B). Interest-
ingly, in the animals immunized simultaneously with both Ctb
conjugates, the presence of TbpA and TbpB in the same an-
tigen preparation influenced the IgG1-to-IgG2a ratio of anti-
bodies elicited against the individual antigens. The presence of
TbpA increased the level of IgG2a compared to TbpB,
whereas the presence of TbpB resulted in increased produc-
tion of IgG1 and decreased levels of IgG2a against TbpA.
Contrary to expectations, the subcutaneously immunized ani-
mals had low IgG1/IgG2a ratios against both TbpA and TbpB;
however, as demonstrated above, sera from this group per-
formed the most poorly in terms of bactericidal activity.
Previous studies have shown IN immunization to be an ef-
fective means for the induction of serum and mucosal antigen-
specific antibodies (21, 23, 24, 48, 49). The prolonged induction
of genital tract antigen-specific antibodies following IN vacci-
nation has highlighted this route of immunization as an attrac-
tive potential method for preventing sexually transmitted in-
fections (37, 47). We explored this possibility by immunizing
mice IN with recombinant transferrin binding protein A and/or
B in conjunction with the mucosal adjuvant cholera toxin B.
We demonstrated that IN immunization with these antigens is
FIG. 3. IgG1 and IgG2a subtype analysis. (A) IgG1 and IgG2a
antibody levels specific for TbpA detected in sera collected at day 35.
(B) IgG1 and IgG2a antibody levels specific for TbpB detected in sera
collected at day 35. The bars represent the geometric mean ?/?
standard deviation. The values above the bars represent the IgG1/
IgG2a ratios of the corresponding immunization groups, indicated
below each graph. For all groups, n ? 5.
TABLE 4. Serum bactericidal activities of sera collected at day 35
Serum bactericidal titerafor strain:
TbpA ? Ctb ? TbpB
TbpA ? Ctb
TbpB ? Ctb
S.c.dTbpA ? TbpB ?
800 (84 ? 5.7) 200 (64 ? 1.4) 400 (60 ? 0.7)
400 (83 ? 4.2)
200 (64 ? 1.4)
100 (70 ? 1.4)
200 (64 ? 1.4)
50 (56 ? 4.9)
400 (71 ? 9.9)NDc
aData are represented as the lowest reciprocal dilution that gave ?50%
killing. The average percent killing determined from duplicate assays ? standard
deviation is shown in parentheses.
bAssays conducted at 1/25 dilution were performed only once, and lower
dilutions were not tested.
cND, not determined.
3950PRICE ET AL.INFECT. IMMUN.
an effective means of eliciting specific serum and vaginal anti-
Tbp antibodies. However, each Tbp antigen behaved differ-
ently in regard to overall immunogenicity.
TbpB was the more immunogenic of the two proteins. Large
differences in immunogenicity between the antigens were ap-
parent regardless of the route of immunization. IN immuniza-
tion elicited the highest anti-TbpB titers when TbpB was con-
jugated to Ctb. Admixing TbpB with Ctb improved the
immunogenicity of TbpB over control groups; however, in gen-
eral, differences between admixed groups and those in which
TbpB was conjugated to Ctb were statistically significant. TbpB
was poorly immunogenic if administered alone in the absence
of the Ctb adjuvant. By contrast, maximal TbpA-specific serum
antibody responses following IN immunization were not de-
pendent on the presence of Ctb. Mice immunized IN with
TbpA alone elicited serum antibody titers similar to those
generated by the group immunized with the TbpA-Ctb conju-
gate. This may have been the result of the inclusion of the
nonionic detergent lauryl maltoside in the TbpA antigen prep-
arations. Lauryl maltoside has been shown to act as an absorp-
tion enhancer in the nasal cavity (1, 32). This may have allowed
better absorption of TbpA, as high-molecular-weight proteins
are usually poorly absorbed in the nasal cavity without enhanc-
ers (36). On the other hand, the relatively poor immunogenic-
ity of TbpB administered alone was likely due to the solubility
of the TbpB used in this study. Native TbpB is a lipoprotein
and is anchored to the bacterial outer membrane via a lipid
tail, and it contains no predicted transmembrane segments. To
simplify recombinant protein expression and purification, we
expressed TbpB in E. coli without the amino-terminal cysteine,
where lipidation normally occurs. Because of its overall hydro-
philicity, it is likely that overexpressed, lipid-free TbpB would
have been excluded from detergent micelles (22). The en-
hanced immunomodulatory effects with TbpB conjugated to
Ctb, therefore, are likely due in part to binding of Ctb to GM1
ganglioside on nasal mucosa cells, which is thought to enhance
antigen uptake and presentation to the immune system.
Interestingly, Ctb admixed with TbpA delayed the genera-
tion of antibodies against TbpA, as shown by the statistically
significant differences in antibody titers measured on days 17
and 28. However, this effect was abrogated by day 65, at which
time there were no significant differences in the levels of
TbpA-specific antibody titers against TbpA among any of the
IN immunized groups. The concurrent IN immunization with
both TbpA and TbpB did not have a negative effect on levels
of antibodies to either antigen compared to groups in which
each antigen was administered alone; however, the IgG sub-
class distribution was influenced by the presence of either
antigen. These alterations in IgG subclass distribution, how-
ever, did not appear to be deleterious, as the bactericidal
activity of pooled sera from the group immunized with both
TbpA and TbpB was superior to those of sera from animals
immunized with a single antigen. This demonstrates that the
two antigens can be administered simultaneously without neg-
atively influencing antibody levels or serum bactericidal activ-
Similar to the situation with specific antibody levels in the
serum, vaginal antibody responses to TbpB were generally
much higher than those elicited against TbpA. The robust
genital tract TbpB-specific antibody responses measured were
also dependent on conjugation to Ctb, whereas this was not the
case with TbpA. Immunization with TbpA elicited mostly IgA,
while measurable IgG responses were low and sporadic. The
low levels of TbpA-specific vaginal IgG were not surprising, as
it is thought that most vaginal IgG originates from serum
transudation (41). Although TbpA IgA levels were low, they
remained mostly steady through day 65, except for a transient
decrease on day 35. It is possible that this decrease could have
resulted from the mouse estrus cycle, as levels of genital tract
immunoglobulins fluctuate during the cycle (37). The levels of
TbpB-specific IgA and IgG, though initially robust, decreased
significantly during the course of the study. This decline in
antibody levels over time is not uncommon. Wu et al. followed
the genital tract antibody levels in IN immunized mice for a
1-year period (47). They demonstrated that by 4 months
postimmunization, antibody levels had decreased extensively
from their initial analysis but appeared to level out throughout
the course of 1 year (47). The aim of the current study was not
to characterize the duration of anti-Tbp immune responses,
but future studies will address the longevity of the antibody
response following IN immunization and whether these im-
mune responses are protective.
We performed serum bactericidal assays as a correlate for
the induction of protective antibody responses. We detected
serum bactericidal activity against the homologous gonococcal
strain (FA19) and two heterologous strains (FA1090 and
MS11) using human serum as a complement source. We found
that all IN immunization groups yielded sera with greater bac-
tericidal activity than the subcutaneously immunized group.
The group immunized IN with both TbpA and TbpB gave the
highest serum bactericidal titers and was the only pool of sera
that contained bactericidal antibodies reactive against all three
strains tested. Surprisingly, the group immunized IN with
TbpA elicited the second-highest bactericidal titers, in spite of
the fact that serum IgG levels were approximately 20-fold
lower than TbpB titers at that time point. This suggests that
TbpA may be the more ideal target in the development of a
vaccine. Studies have shown that TbpA is the more conserved
of the two proteins (11, 12), which may be why TbpA elicited
antibodies with more cross-bactericidal activity. Furthermore,
in a meningococcal-vaccine study, mice immunized with TbpA
or TbpA and TbpB were completely protected following lethal
challenge, but the group immunized with TbpB only was not
The obvious discrepancies between antibody titers and se-
rum bactericidal activities suggested that qualitative rather
than quantitative differences existed among antibody prepara-
tions, which prompted us to perform IgG subclass analysis. We
found that those animals immunized IN with the TbpA-Ctb
conjugate elicited higher levels of IgG2a than did those ani-
mals immunized with the TbpB-Ctb conjugate. In mice, the
IgG2a isotype is the most efficient activator of complement,
while the IgG1 isotype is the poorest complement activator
(16, 27, 44). Thus, the lower IgG1/IgG2a ratio detected could
in part explain the enhanced bactericidal activity observed with
the TbpA antiserum. The results of the IgG subclass analysis
do not, however, explain why the subcutaneously immunized
group, immunized with both Tbps, differed so dramatically
from its IN immunized counterpart in terms of bactericidal
activity. The IgG1/IgG2a ratios against TbpA for both IN and
VOL. 73, 2005INTRANASAL VACCINATION WITH Tbps 3951
subcutaneously immunized groups were similar (0.5 and 0.8)
(Fig. 3). By contrast, the IgG1/IgG2a ratio against TbpB in the
subcutaneously immunized group was nearly three times lower
than that of the IN group (6.0 and 17.9) (Fig. 3). In spite of
this, the bactericidal activity of the subcutaneously immunized
group was comparatively poor. These results suggest that an-
tigens delivered by IN immunization may better retain a native
conformation than those delivered by subcutaneous immuni-
zation. Bactericidal activity is associated with high-avidity an-
tibodies, elicitation of which correlates with the ability to keep
protein antigens in native conformation (9). Furthermore, vac-
cine studies using meningococcal PorA demonstrated that
PorA is immunogenic when administered via subcutaneous
immunization in conjunction with a variety of adjuvants; how-
ever, only mice immunized with PorA contained in outer mem-
brane vesicles or liposomes generated antibodies with bacteri-
cidal activity (2, 9). This suggests that antigens delivered
subcutaneously may be subject to misfolding or possibly pro-
teolysis unless they are protected in a membrane. By contrast,
the current study indicates that protein degradation or mis-
folding, resulting in nonnative presentation, may not be as
problematic if the antigens are delivered intranasally.
Whether bactericidal activity is an important mediator of
immunity in the genital tract is a matter of speculation. Though
complement lytic activity has been demonstrated in human
cervical mucus (35), complement levels are highly variable
among individuals and are influenced by hormonal cycles (43).
Furthermore, IgA levels are high in the female genital tract,
and IgA has been shown to be inhibitory to IgG complement
activation (39). Therefore, bactericidal activity in the female
genital tract may not be an important mediator of protection.
Mucosal IgA has been shown to be important in the protection
of the mucosal surfaces from invading bacteria, viruses, and
toxins (39). Furthermore, studies have shown enhanced pro-
tective abilities of polymeric IgA compared to IgG in passive
protective studies in mice (39). The precise roles of the differ-
ent antibody isotypes in protection of the genital tract remain
to be elucidated. However, studies performed with mice have
shown that protection against Chlamydia trachomatis genital
infection is attributable to IgA. Cui et al. showed that, follow-
ing immunization and subsequent Chlamydia challenge, clear-
ance of cervical chlamydial antigen correlated with increases in
cervical IgA, but not in IgG (15). Furthermore, Pal et al.
showed that a monoclonal IgA antibody against the chlamydial
major outer membrane protein could confer passive protection
in mice (31). Finally, in humans, levels of IgA in vaginal se-
cretions and the amount of C. trachomatis isolated from the
cervix are inversely correlated (7). In a recent study, IN im-
munization of mice with gonococcal outer membrane prepa-
rations resulted in strong serum bactericidal activity, and it
decreased the gonococcal vaginal colonization of estradiol-
treated mice (33). This study also showed that antigen-specific
IgA titers were 8- to 16-fold higher than IgG titers in the mice
with reduced vaginal colonization (33). These studies suggest
that IgA may be more important than IgG for protection
against sexually transmitted bacterial infections and highlight
the importance of inducing genital IgA following vaccination.
In conclusion, we have demonstrated induction of both se-
rum and vaginal antibodies following IN immunization with
TbpA, TbpB, or both. TbpA was poorly immunogenic in com-
parison to TbpB in both serum and vaginal antibody responses;
however, the bactericidal activities of TbpA-specific sera were
much greater. This, combined with the greater degree of TbpA
sequence conservation among gonococcal strains, suggests that
TbpA may be the more efficacious vaccine target. Future stud-
ies will be aimed at enhancement of TbpA immunogenicity in
an effort to elicit higher serum and vaginal antibody titers and
at determining whether Tbp-specific antibodies in the genital
tract can confer protection.
We gratefully acknowledge Heather Strange for excellent technical
assistance and Chris Elkins and Heather Masri for advice on bacteri-
cidal assay methodology.
This work was supported by NIH grant AI47141 to C.N.C. and NIH
grants AI46561 and DE06746 to M.W.R. G.A.P was supported by a
Training in Molecular Pathogenesis grant (T32 AI07617) from the
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Editor: J. T. Barbieri
VOL. 73, 2005 INTRANASAL VACCINATION WITH Tbps 3953