![Description of Inaba CHO-BSA immunogens (A) and timeline of immunization and serum sampling (B). Each treatment group consisted of five female BALB/c mice, aged 7 weeks at the start of the treatment regiment. Mice received injections of Inaba CHO-BSA neoglycoconjugate immunogen mixed 1:1 in RIBI adjuvant in 150 mM NaCl on days 0, 14, and 28. Immunogens were dosed at 10 μg per mouse per immunization based upon weight of CHO in each conjugate and administered via i.p. injection. Individual serum samples were collected on days 0 (preimmune control sample [PI]), 10 (primary sample), 17 (secondary sample), and 35 (tertiary sample) via the retro-orbital sinus and subsequently pooled by treatment group and stored at either 4 or −20°C until assessed.](https://www.researchgate.net/profile/Pavol-Kovac/publication/8495410/figure/fig2/AS:11431281220510579@1706523466554/Description-of-Inaba-CHO-BSA-immunogens-A-and-timeline-of-immunization-and-serum_Q320.jpg)
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INFECTION AND IMMUNITY, July 2004, p. 4090–4101 Vol. 72, No. 7
0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.7.4090–4101.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Synthetic Fragments of Vibrio cholerae O1 Inaba O-Specific
Polysaccharide Bound to a Protein Carrier Are Immunogenic in Mice
but Do Not Induce Protective Antibodies
Michael D. Meeks,
1
Rina Saksena,
2
Xingquan Ma,
2
Terri K. Wade,
1
Ronald K. Taylor,
1
Pavol Kova´cˇ,
2
and William F. Wade
1
*
Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire 03756,
1
and
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland 20892
2
Received 3 February 2004/Returned for modification 16 March 2004/Accepted 5 April 2004
Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the
correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and
Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody follow-
ing infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the
terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba.
The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal
hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide com-
ponent of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin
(BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody.
We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective.
Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and
hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate
(CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized
with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1)
were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba
antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked
immunosorbent assay but not enough to be protective in vivo.
Cholera, an enteric diarrheal disease caused by the gram-
negative bacterium Vibrio cholerae, continues to be a world-
wide health concern. V. cholerae lipopolysaccharide (LPS), a
critical component of the outer membrane that is required for
virulence, is a known target for immune responses following
infection or immunization. Antibodies specific for V. cholerae
LPS are correlated with protection against cholera (31, 32).
The importance of preexisting anti-LPS antibody was high-
lighted by a change in the susceptible population of a V. chol-
erae O139 outbreak, where disease was seen in adults that are
normally thought to have some immunity because of previous
exposure to cholera LPS antigens, but in this circumstance,
previous exposure did not cross-protect against the new LPS
antigens of O139 (2). Multiple serologic reagents (1, 12, 14-16,
29) have been developed against V. cholerae LPS and used to
define three O-antigen-associated B-cell epitopes (epitopes A,
B, and C). The A epitope is expressed equally well by V. cholerae
O1 serotypes Inaba and Ogawa LPS. Structurally, epitope A was
postulated to be either the perosamine residues or the N-tetronic
acid (N-3-deoxy-L-glycero-tetronic acid) side chain, elements com-
mon to both LPS serotypes (29). The serologic designation B is
found only in the Ogawa O-specific polysaccharide (O-SP) (29,
39). Several groups showed that anti-C reactivity was associated
with only the Inaba LPS, although some groups described anti-C
monoclonal antibody (MAb) reactivity to the Ogawa strain (1, 12,
14–16). The nature of the C epitope has not been experimentally
identified (29). The O-SP terminal sugar of V. cholerae LPS is now
known to differentiate Ogawa and Inaba serotypes. V. cholerae
O-SP consists of (132)-␣-linked 4-amino-4,6-dideoxy-D-mannose
(perosamine) whose amino group is acylated with 3-deoxy-L-glyc-
ero-tetronic acid (17, 21). In the Ogawa O-SP, the terminal sugar
is characterized by a 2-O-methyl group, while the terminal sugar
in the Inaba O-SP has a hydroxyl at the 2 position (17, 18, 40, 41).
V. cholerae serotypes can undergo serotype conversion in
both directions during epidemics or in areas where cholera is
endemic (9, 11). For example, the initial serotype in South
America in 1991 was 95% Inaba, whereas 1992 to 1995 saw
Ogawa as the predominant (90%) serotype (9). Others have
noted seroconversion in response to immune selective pressure
in vitro where anti-serotype-specific antibodies can select for
the nonreactive serotype (reviewed in reference 2). V. cholerae
O1 LPS induces protective immune responses in humans and
experimental animals (13, 19, 27, 35) and thus is an immuno-
gen of choice for cholera vaccine development. Therefore, it is
important to develop O-SP-based cholera vaccines that can
protect against Inaba as well as Ogawa serotypes. These vac-
cines could be based on the common A epitope or both the
unique B and C epitopes.
It has recently been reported that synthetic hexasaccharide-
* Corresponding author. Mailing address: Dartmouth Medical
School, Department of Microbiology and Immunology, 630 W. Bor-
well Bldg., Lebanon, NH 03756. Phone: (603) 650-6896. Fax: (603)
650-6223. E-mail: william.wade@dartmouth.edu.
4090
protein conjugate immunogens that mimic in part the terminus
of Ogawa LPS induced vibriocidal antibodies as well as pro-
tective antibodies, as measured by an infant mouse protection
assay (7). We reasoned that if antibodies specific for the ter-
minal sugar of Ogawa O-SP were protective, then antibodies to
the analogous structure in Inaba LPS would also be protective.
We now report that a series of conjugates made from Inaba di-,
tetra-, and hexasaccharide and bovine serum albumin (BSA)
are immunogenic in mice, inducing immunoglobulin M (IgM)
and the T-dependent IgG1 subclass. For the majority of the
conjugates, the length of oligosaccharide and the degree of
carbohydrate (CHO) substitution (CHO/BSA, mole/mole) did
not affect the serologic response in the tertiary sera. In contrast
to the protective antibody induced by the Ogawa O-SP protein
conjugates, the Inaba O-SP protein conjugates failed to induce
antibodies which were vibriocidal in vitro that were protective
in the infant mouse assay or that bound V. cholerae LPS in situ.
MATERIALS AND METHODS
Animals. Six-week-old female BALB/c mice were purchased from the National
Cancer Institute (Bethesda, Md.). Pregnant, female CD-1 mice were purchased
from Charles River (Raleigh, N.C.) for the infant mouse protection studies. All
mice were housed under standard conditions in the Animal Resources Center
located at the Dartmouth-Hitchcock Medical Center, Lebanon, N.H.
Inaba CHO-BSA constructs. Immunogens 1a to 3c were prepared by linking
the chemically synthesized di-, tetra-, and hexasaccharide fragments of the O-SP
of V. cholerae O1 serotype Inaba to BSA by using squaric acid chemistry (20, 36,
38). The oligosaccharides were assembled in a stepwise manner (28) (Fig. 1)
from the monosaccharide glycosyl donor 4 and the monosaccharide glycosyl
acceptor 5. Note that in contrast to our previous syntheses of oligosaccharides
related to the O-SP of V. cholerae O1 from intermediates containing the azido
group at position 4, the building blocks 4 and 5 have the 4-(3-deoxy-L-glycero-
tetronic acid) side chain already in place. Also, the glycosyl acceptor 5 is a
glycoside of methyl 6-hydroxyhexanoate. Thus, the coupling of these intermedi-
ates afforded, after deprotection, haptens in the form of glycosides whose aglycon
made them amenable to conjugation to proteins. The advantage of the use of
fully equipped intermediates such as 4 and 5 is that the assembled oligosaccha-
rides have to undergo a lesser number of chemical manipulations. Briefly (Fig.
1A), condensation (23) of the glycosyl acceptor 5 and glycosyl donor 4, prepared
from the corresponding amine (7) and 2,4-O-benzylidene-3-deoxy-L-glycero-tetronic
acid (24, 28), gave the fully protected disaccharide 6. Partial deprotection by Zem-
ple´n deacetylation afforded alcohol 7, which was used as a glycosyl acceptor in the
next coupling with 4 to extend the oligosaccharide chain and obtain the trisaccharide
8. Complete deprotection of 7 was achieved by hydrogenolysis, which simultaneously
removed the benzyl and benzylidene protecting groups, to give the disaccharide
hapten 10. Repeating the sequence of reactions with 8 that effected the conversions
63738 and then repeating the sequence with the higher oligosaccharides provided
the linker-equipped tetrasaccharide (11) and the hexasaccharide hapten (12). Next
(Fig. 1B), haptens 10 through 12 were subjected to aminolysis to afford ethylenedi-
amine derivatives 13 through 15, which were treated with squaric acid diethyl ester.
The squaric acid monoesters 16 through 18, thus obtained, were treated with BSA to
give neoglycoconjugates 1a through 3c, whose molar hapten-BSA ratio was deter-
mined by surface-enhanced laser desorption ionization–time of flight (mass spec-
trometry) (6). The chemical composition of the synthetic components used to con-
struct the Inaba CHO-BSA immunogens were confirmed by nuclear magnetic
resonance and mass spectrometry after synthesis and then assembled to produce the
Inaba CHO-BSA.
Immunization and serum collection. Eight groups of five mice each were used
to test the immunogenicity of the Inaba O-SP epitope conjugates according to
the regimen shown in Fig. 2. Ten micrograms (based on carbohydrate weight) of
Inaba CHO-BSA conjugate resuspended in 150 mM NaCl and mixed 1:1 in RIBI
adjuvant (Sigma, St. Louis, Mo.) was injected intraperitoneally (i.p.) on days 0,
14, and 28. Blood collection via retro-orbital sinus or plexus was done on days 0,
10, 17, and 35, which represent preimmune, primary, secondary, and tertiary
sera, respectively (Fig. 3). Retro-orbital plexus bleeding yielded between 80 to
120 l of blood, which can provide 50% of that volume as serum after processing.
Resulting sera from individual mice within a group were pooled and stored at 4
or ⫺20°C until use.
BALB/c female mice used to generate control sera for protection and vibrio-
cidal-antibody assays were immunized with purified LPS from either V. cholerae
O1 Ogawa strain P1418 (a generous gift from S. Kondo, Josai University, Josai,
Japan) or Inaba strain 569B LPS (Sigma). Mice were immunized i.p. with 9 g
of Ogawa or 9 g of Inaba LPS on days 0, 15, 60, and 71. Tertiary and quaternary
sera were used as positive controls for these studies.
Serology. The presence of anti-O-SP Inaba-specific antibodies in individual
serum samples was measured by enzyme-linked immunosorbent assay (ELISA).
High-binding, flat-bottomed 96-well microtiter plates (Corning Life Sciences,
Acton, Mass.) were coated with 100 l of an Inaba solution (5 g of Inaba
LPS/ml) in 0.1 M carbonate-bicarbonate buffer (pH 9.5) per well and incubated
overnight at 4°C. Plates were washed three times by using a Molecular Devices
(Sunnyvale, Calif.) Skan plate washer with 250 l of phosphate-buffered saline
(PBS)–0.05% Tween 20 (Fisher Scientific, Pittsburgh, Pa.) per well. Nonspecific
binding was blocked by using 200 l of blocking buffer consisting of PBS–1.0%
fish gelatin (BioFX, Owings Mills, Md.)–1.0% normal goat serum (Jackson
ImmunoResearch, West Grove, Pa.)–0.05% Tween 20 per well for1hatroom
temperature. Plates were washed three times more, after which 50 l of anti-
serum (serially diluted twofold in blocking buffer) was added to each well and
incubated overnight at 4°C. The initial dilution of 1:25 was used for all sera for
the initial ELISA, and then a dilution of 1:100 was used for the tertiary serum
sample in a follow-up ELISA to determine an endpoint titer for these sera.
Following incubation with primary sera, plates were washed three times, and 50
l of horseradish peroxidase-labeled goat anti-mouse IgM (-chain-specific) or
anti-IgG1 (␥1-specific) (Southern Biotechnology Associates, Birmingham, Ala.)
detector antibodies (diluted 1:6,000) was added to each well and incubated at
room temperature for1hinthedark. Plates were washed three times and then
developed with 100 lofO-phenylenediamine dihydrochloride (OPD) peroxi-
dase substrate (Sigma) per well for 5 min at room temperature. OPD peroxidase
substrate was prepared by diluting 10-mg tablets into 0.05 M phosphate-citrate
buffer, pH 5.0 (Sigma), to a final concentration of 0.4 mg/ml. Fresh 30% H
2
O
2
was added to the OPD substrate solution immediately before use to a final
concentration of 0.02%. The reaction was stopped with an equal volume of 3 M
HCl. Optical densities (OD) were read at 490 nm (OD
490
) by using a Dynex
Technologies MRX microtiter plate reader (Thermo Labsystems, Helsinki, Fin-
land) with Dynex Revelation 3.04 software.
We compared the tertiary serum sample titers, as they were the source of sera for
the functional assays. Endpoint titers for ELISA were defined as the reciprocal of
the antibody dilution for the last well in a column with a positive OD for each sample
after subtracting the background. Background values were determined with preim-
munization sera. Preimmunization serum samples for each treatment group were
analyzed on multiple 96-well plates. The OD values of the preimmunization sera
were averaged and then doubled. This value was subtracted from the OD of all the
wells containing the titration of the pooled serum samples.
Vibriocidal antibody. (i) Spread plate method. Titers of vibriocidal antibody
against V. cholerae (classical Ogawa strain O395, classical Inaba strain 569B, and
El Tor Inaba strain N16961) were assessed in vitro (10, 22). Bacteria were grown
in Luria-Bertani (LB) broth at 37°C for 18 h. The culture was centrifuged for 10
min, resuspended into an equal volume of PBS plus 0.1% peptone, and diluted
1:1.0 ⫻10
4
in PBS. Pooled preimmune and tertiary sera from each treatment
group were diluted in 50 l of ice-cold PBS containing 20% guinea pig comple-
ment (Sigma) with the dilutions of 1:(1.0 ⫻10
2
), 1:(1.0 ⫻10
3
), 1:(1.0 ⫻10
4
),
1:(5.0 ⫻10
4
), and 1:(1.0 ⫻10
5
) and kept in an ice-water bath until needed.
Bacteria (3.2 ⫻10
4
CFU) were mixed with diluted antiserum (1:1), incubated for
1 h on a platform shaker at 37°C (125 rpm), and then returned to the ice-water
bath. Each sample (100-l total volume) was then spread on LB agar plates and
allowed to dry at room temperature before overnight incubation at 37°C. CFU
were recorded for each plate. Inhibition of bacterial growth (endpoint titer) was
considered significant if 50% or more of the bacteria were killed compared to
CFU from plates containing preimmune serum and complement.
(ii) Microtiter method. The recently developed microtiter test protocol (5) was
generously provided by Fournier’s group (Pasteur Institute, Paris, France). V.
cholerae O1 El Tor Inaba strain N16961 was inoculated into 2.0 ml of alkaline
peptone water (1.0% peptone and 1.0% NaCl [pH 8.6]) and grown overnight at
37°C. The culture was transferred to a prewarmed nutrient agar plate and
incubated for 90 min at 37°C. Five milliliters of cold PBS was applied to the plate
and swirled gently to resuspend the bacteria and then was transferred to a 15-ml
conical tube. The OD
600
of the bacterial suspension was adjusted to 0.80 with
PBS to approximate the bacteria to 10
9
CFU/ml. Seven volumes of cold PBS, 2
volumes of guinea pig complement, and 1 volume of bacterial suspension were
mixed in a chilled tube and kept on ice for 20 min. Fifty microliters of heat-
inactivated mouse serum from the various treatment groups was placed in a
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4091
FIG. 1. Schema for generation of Inaba neoglycoconjugate immunogens and Inaba CHO-BSA.
4092 MEEKS ET AL. INFECT.IMMUN.
round-bottomed sterile microtiter plate with a lid and serially diluted 1:2 in PBS.
Twenty-five microliters of the complement-treated bacteria was added to each
well and covered and incubated for1hat37°C. One hundred fifty microliters of
LB broth was then added to each well, and the plate was incubated uncovered in
a humidified chamber for2hat37°C. An aqueous solution containing 1 volume
of 1.0% neotetrazolium chloride (ICN Biomedicals, Irvine, Calif.) and 9 volumes
of 2.7% sodium succinate (ICN Biomedicals) was made. Twenty-five microliters
of this solution was added to each well and incubated uncovered for 15 min at
room temperature before the OD
570
was recorded. The plate was then placed in
a humidified chamber at 4°C overnight, and the optical density was recorded
again the next day. A violet color in the well indicated the presence of live vibrios.
Inhibition of bacterial growth (endpoint titer) was reported as the reciprocal of
the antibody dilution for the negative well containing the lowest concentration of
antibody for each sample tested.
Infant mouse challenge. The suckling mouse challenge model for cholera was
used for assessing the protective quality of anti-O-SP Inaba-specific antibodies in
vivo (33, 37). Cultures of V. cholerae (El Tor Inaba strain N16961) were grown for
16 h in LB broth, pH 6.5, at 30°C. Twenty-five microliters of bacterial suspension,
representing 25 to 44 50% lethal doses (LD
50
) (44 LD
50
were used to test the
positive control sera), was combined with 25 l of either preimmune sera (negative
control), sera from BALB/c mice previously immunized with Inaba LPS (positive
control), or tertiary anti-O-SP Inaba-specific sera immediately before administration
intragastrically to 4- to 5-day-old CD-1 mice. Challenged mice were kept at 30°C and
monitored every 4 h starting 24 h postchallenge.
In situ analysis of anti-LPS antibody binding. Live V. cholerae O1 El Tor
Inaba bacteria were used to assess the binding ability of anti-O-SP Inaba-specific
antibodies in situ. Cultures of V. cholerae strain N16961 were grown for 16 h in
standard LB medium at 37°C. One hundred microliters of bacterial suspension,
representing approximately 10
9
CFU, was pelleted in a microcentrifuge and
washed in PBS (pH 7.2) three times to remove all culture medium. The bacteria
were then resuspended in an equal volume of tertiary anti-O-SP Inaba-specificor
anti-Inaba whole-LPS serum which was diluted 1:10 in PBS. The resulting mix-
ture was incubated at room temperature for 2 h followed by overnight incubation
at 4°C. Samples were then washed three times with PBS (pH 7.2) to remove
unbound antibodies. Pelleted bacteria were resuspended in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis protein sample buffer containing
2-mercaptoethanol. Two micrograms of mouse IgM was used as a positive con-
trol for the IgM heavy chain. Each sample was boiled for 5 min and then
centrifuged for 5 min to remove precipitates. Twenty microliters of each sample
was loaded onto a 12% Tris-HCl polyacrylamide gel (Bio-Rad Laboratories, Inc.,
Hercules, Calif.) and electrophoresed for 45 min at 150 V. The samples were
then transferred to a nitrocellulose membrane by using a semidry transfer cell
(Bio-Rad Laboratories, Inc.) at 23 V for 30 min. The membrane was blocked at
room temperature for 2 h in PBS, 0.05% Tween 20, and 5.0% nonfat dry milk.
Horseradish peroxidase-conjugated goat anti-mouse IgM antibody (Southern Bio-
technology Associates) was added at a dilution of 1:5,000 and allowed to incubate at
room temperature for another 2 h. The membrane was washed for 5 min in PBS with
6 buffer changes and then exposed to enhanced chemiluminescent Western blot
detection reagent (Amersham Biosciences, Piscataway, N.J.) for 1 h. Data were
recorded with Kodak BioMax MR scientific imaging film. The developed film was
scanned with Adobe Photoshop 7.0 by using a UMax PowerLook 1120 overhead
flatbed scanner at 1,200 dpi and converted into JPEG format.
Statistical analyses. The ELISA titers of the tertiary anti-Inaba IgM and
anti-Inaba IgG1 were compared for significant differences by using an estab-
lished parameter that requires a fourfold or greater difference between endpoint
titers of pooled individual sera for significance (42). The Prism GraphPad pro-
gram was used to evaluate the statistical significance of the cross-reactivity
analysis and the infant mouse protection assay data. The analysis of significance
for the cross-reactivity curves was based on assessing the null hypothesis that the
slopes of the lines (anti-Inaba versus anti-Ogawa) are not different. A Pvalue of
less than 0.050 indicates that the slopes are significantly different. If the slopes
are statistically the same, a second test (with a Pvalue of ⬎0.050 indicating
significance) determines whether the lines are identical or parallel. The analysis
of significance for the protection data was based on the log rank test, which is
equivalent to the Mantel-Haneszel test. The null hypothesis that was tested was
that the survival curves are identical in the overall population; i.e., treatment did
not change survival.
FIG. 2. Description of Inaba CHO-BSA immunogens (A) and timeline of immunization and serum sampling (B). Each treatment group
consisted of five female BALB/c mice, aged 7 weeks at the start of the treatment regiment. Mice received injections of Inaba CHO-BSA
neoglycoconjugate immunogen mixed 1:1 in RIBI adjuvant in 150 mM NaCl on days 0, 14, and 28. Immunogens were dosed at 10 g per mouse
per immunization based upon weight of CHO in each conjugate and administered via i.p. injection. Individual serum samples were collected on
days 0 (preimmune control sample [PI]), 10 (primary sample), 17 (secondary sample), and 35 (tertiary sample) via the retro-orbital sinus and
subsequently pooled by treatment group and stored at either 4 or ⫺20°C until assessed.
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4093
RESULTS
Inaba CHO-BSA conjugates. It was recently reported that
conjugates of the hexasaccharide fragment of the O-SP of V.
cholerae (Ogawa) and BSA were both immunogenic and able
to induce protective antibody responses in mice (7). To deter-
mine if a related structural epitope on the Inaba O-SP was a
target for B-cell responses, we synthesized Inaba CHO-BSA
conjugates that varied in the length (di-, tetra-, and hexasac-
charide) of the O-SP fragment (Fig. 1B). Note that the linker
(spacer) described here, which provides a connection between
FIG. 3. V. cholerae Inaba LPS-specific IgM (A) and IgG1 (B) responses following immunization with synthetic Inaba CHO-BSA neoglyco-
conjugates. For a description of Inaba constructs, see Materials and Methods. Horizontal dashed lines indicate the starting dilution (1:25) of
antiserum used in the ELISA. Serum samples were collected as described in the legend to Fig. 2.
4094 MEEKS ET AL. INFECT.IMMUN.
the antigen and the carrier protein in the Inaba CHO-BSA
conjugates, differs from the one used previously in the similar
Ogawa hexasaccharide-BSA constructs (7). With the Ogawa
hexasaccharide (7), the amino group required by the squaric
acid chemistry of conjugation was generated in the spacer-
equipped hexasaccharide by hydrazinolysis of the methyl ester
with hydrazine. In the present study, the amino group was
introduced by aminolysis of the methyl ester with ethylenedi-
FIG. 4. Cross-reactivity of tertiary Inaba CHO-BSA antisera to V. cholerae Ogawa and Inaba LPS reported as the OD
490
. The preimmune
serum control sample is represented by the closed squares. Pooled tertiary sera reacted against Inaba LPS (open circles) and against Ogawa LPS
(closed inverted triangles). Initial serum dilutions were 1:100 for the ELISA for IgM (A) and IgG1 (B). A comparison of the slopes of the lines
for the anti-Inaba and anti-Ogawa responses indicated that within any particular group, the IgM ELISA and the IgG1 ELISA results did not differ.
Only the lines for the IgG1 ELISA describing groups 2 (P⫽0.043), 3 (P⫽0.032), and 6 (P⫽0.001) were not identical but parallel; for all other
comparisons, the lines were identical.
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4095
amine (Fig. 1B). Consequently, the linker used for the Inaba
conjugates is longer than the linker used in Ogawa conjugates
by two methylene groups (7). Preliminary data (data not
shown) and the data presented here indicate that the change in
linker length does not substantially affect the immunogenicity
of the CHO-BSA conjugates.
Serologic response to Inaba CHO-BSA conjugates. Eight
groups of BALB/c mice were immunized three times i.p. with
10 g (based on the CHO weight) of the various Inaba BSA-
CHO constructs (Fig. 2A). Serum was collected over a 35-day
period as shown in the immunization and serum collection
schema (Fig. 2B). Purified Inaba LPS was used to assess
pooled serum from individual mice of the various groups for
Inaba-specific IgM and IgG1 antibodies by ELISA. The IgM
anti-Inaba response can be detected in the primary sera, which
were collected 10 days after the primary immunization (Fig. 2B
and 3A). Most groups of mice showed increased levels of
IgM-specific Inaba antibody in serum over the next 25 days,
with the exception of groups 2 and 8, whose levels of antibody
in serum did not increase after the first immunization. In con-
trast, while the IgG1 response was universal at day 35 after
initiation of the immunization schedule, only select groups of
mice had IgG1-specific Inaba antibody at day 17, a point at
which mice were immunized twice with the Inaba CHO-BSA
conjugate (Fig. 3B). Groups 2, 3, 4, and 6, which were immu-
nized with disaccharide (15.8 mol of CHO/mol of BSA [group
2]), tetrasaccharide (4.5 mol of CHO/mol of BSA [group 3]
and 11.6 CHO/mol of BSA [group 4]), and hexasaccharide (2.9
mol CHO/mol of BSA [group 6]) conjugates, had different
kinetics of accumulation of IgG1 specific for Inaba O-SP in
serum. There was, however, no correlation with length of sac-
charides or level of substitution for these earlier responses.
With the exception of group 8, the tertiary ELISA titers for
either Inaba-specific IgM or Inaba-specific IgG1 in serum were
not considered different among the groups, as the endpoint
titers did not differ by fourfold or more (42). The responses to
Inaba CHO-BSA conjugates in serum were similar in magni-
tude for some of the groups immunized with Ogawa CHO-
BSA conjugates (7).
Cross-reactivity of Inaba CHO-BSA conjugate antisera with
Ogawa LPS. We assessed the cross-reactivity of pooled tertiary
sera from the groups immunized with the various Inaba CHO-
BSA conjugates against Ogawa LPS. In general, the anti-Inaba
CHO-BSA sera reacted equivalently with Ogawa and Inaba
LPS in an ELISA (Fig. 4) that assessed either IgM or IgG1
antibodies. Sera induced by the Inaba CHO-BSA disaccharides
(groups 1 and 2), as well as those induced by the low-level
substitution tetrasaccharide (group 3) and hexasaccharide
(group 6) conjugates were more reactive to the Ogawa LPS
epitopes. A comparison of the slopes of the lines for the anti-
Inaba and anti-Ogawa responses was performed. The slopes of
the lines within any particular group for the IgM ELISA were
the same, as were those for the IgG1 ELISA. Further analysis
to determine if the lines were parallel or identical revealed that
only the curves for the IgG1 ELISA for groups 2 (P⫽0.043),
3(P⫽0.032), and 6 (P⫽0.001) were not identical yet were
parallel; the lines for all other groups were identical. The
results for group 1, mice immunized with the disaccharide (6.9
mol of CHO/mol of BSA), had a Pvalue of 0.071 and thus were
not considered significant. With the exception of group 4, mice
immunized with the Inaba CHO-BSA tetrasaccharide (11.6
mol of CHO/mol of BSA), these groups (2, 3, and 6) were also
the groups that had faster accumulations of anti-Inaba IgG1.
Vibriocidal activity of antisera specific for Inaba. A well-
accepted assay for assessing the functional significance of V.
cholerae anti-LPS antibodies is the in vitro vibriocidal-antibody
assay that measures complement-mediated killing. Using an
agar plate-based complement fixation assay, we tested pooled
sera from mice immunized with the various Inaba CHO-BSA
conjugates to determine if immunization induced vibriocidal
antibody (Table 1). Control antisera generated to either Inaba
or Ogawa whole-LPS induced vibriocidal antibody of high titer
(ⱖ50,000). This result was in sharp contrast to the results
obtained with sera from tertiary bleeds of mice immunized
TABLE 1. Antivibriocidal activity of pooled sera of mice immunized with Inaba CHO-BSA
a
Inaba CHO-BSA conjugate groups
(mol of CHO per mol of BSA)
Vibriocidal titers of pooled sera determined by
f
:
Spread plate method for strain: Microtiter method for
strain N16961 (Inaba)
e
569B (Inaba)
b
O395 (Ogawa)
c
N16961 (Inaba)
d
Preimmune
sera
Tertiary
sera
Preimmune
sera
Tertiary
sera
Preimmune
sera
Tertiary
sera
Preimmune
sera
Tertiary
sera
Group 1, disaccharide (6.9) ⬍100 ⬍100 ⬍100 ⬍100 ⬍20 ⬍20 ⬍50 ⬍50
Group 2, disaccharide (15.8) ⬍100 ⬍100 ⬍100 ⬍100 ⬍20 ⬍20 ⬍50 ⬍50
Group 3, tetrasaccharide (4.5) ND ND ND ND ⬍20 ⬍20 ⬍50 ⬍50
Group 4, tetrasaccharide (11.6) ND ND ND ND ⬍20 ⬍20 ⬍50 ⬍50
Group 5, tetrasaccharide (14.6) ⬍100 ⬍100 ⬍100 1,000 ⬍20 ⬍20 ⬍50 ⬍50
Group 6, hexasaccharide (2.9) ⬍100 ⬍100 ⬍100 100 ⬍20 ⬍20 ⬍50 ⬍50
Group 7, hexasaccharide (6.6) ND ND ND ND ⬍20 ⬍20 ⬍50 ⬍50
Group 8, hexasaccharide (19.0) ⬍100 ⬍100 ⬍100 ⬍100 ⬍20 ⬍20 ⬍50 ⬍50
Anti-Inaba LPS control ⬍100 ⱖ50,000 ⬍100 ⱖ50,000 ND ⬎8,000 ND ⱖ100,000
Anti-Ogawa LPS control ⬍100 ⬍100 ⬍100 ⱖ100,000 ND ND ND ND
a
Synthetic Inaba LPS epitope.
b
Input, 7.15 ⫻10
3
CFU/sample.
c
Input, 8.9 ⫻10
3
CFU/sample.
d
Input, 4.5 ⫻10
3
CFU/sample.
e
Input, 9.3 ⫻10
5
CFU/sample.
f
ND, not determined.
4096 MEEKS ET AL. INFECT.IMMUN.
with Inaba CHO-BSA immunogens. The latter sera were uni-
formly negative in two different assessments of the plate vibrio-
cidal-antibody assay. In general, for the vibriocidal-antibody
assay, there was no cross-reaction of anti-Inaba sera with
Ogawa LPS, with the exception of two groups that had low titer
responses (2.9 mol of hexasaccharide, 1:100; 14.6 mol of tet-
rasaccharide, 1:1,000). This was in contrast to the anti-Inaba
whole-LPS sera, which bound purified LPS from either sero-
type of V. cholerae O1.
Because the plate vibriocidal-antibody assay uses large vol-
umes of reagents and requires a large input of bacteria for the
enumeration of colonies, it is difficult to test lower dilutions of
antiserum or to vary the number of target bacteria. To confirm
the results, we used a microtiter test (5) developed by Fourni-
er’s group (Pasteur Institute). This test measures the metabolic
activity of the bacteria following treatment with antisera and
complement. As with the plate method, the vibriocidal activity
of the anti-Inaba CHO-BSA antiserum was negative (Table 1).
Similar to its effectiveness in the plate vibriocidal-antibody
assay, positive-control anti-Inaba LPS serum made to whole
Inaba LPS was effective in inhibiting bacterial growth and thus
metabolic activity as measured by the microtiter assay.
FIG. 5. (Top) Percent survival of neonatal mice following oral challenge with live V. cholerae. Four- to five-day-old CD-1 neonatal mice were
orally administered by gavage 25 ⫻10
6
CFU of virulent (25 LD
50
)V. cholerae O1 El Tor Inaba strain N16961, which was cultured overnight in
LB medium at 30°C for 16 h and then mixed 1:1 with tertiary antisera or preimmune antisera. The untreated group did not receive challenge.
Tertiary and preimmune antisera were diluted into normal mouse sera for a final dilution of 1:5. Eight mice were used per treatment group. The
data from the preimmune group are a collective result from three randomly chosen preimmune sera which were individually evaluated. Mice were
kept at 30°C and monitored every 4 h starting 24 h after oral challenge until termination of the experiment at 48 h. Groups 1 and 2 showed a
potential for protection, and the tests for these groups were repeated in the analysis with 44 LD
50
of bacteria and found not to be protective (data
not shown). (Bottom) The tabulated values show the results of a log rank comparison test for significance between the survival curves. Multiple
comparisons were made. The top row of the table defines the vaccine modality used to generate serum for a particular Inaba CHO-BSA conjugate.
The columns under the individual vaccine modality headings show the Pvalues for the various comparisons to the groups listed in the first column.
The second to the last and the last row show the Pvalue generated from the comparison of the survival curve for mice treated with tertiary sera
of a particular vaccine modality to the survival curves of either mice treated with preimmune sera or untreated mice, respectively. The Pvalues
shown in boldface type are less than 0.050, which is considered significant.
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4097
Anticolonization capacity of antisera specific for Inaba. An-
other means of assessing the functional activity of antisera
directed against V. cholerae surface antigens is the infant
mouse protection assay. Similar to the results with the vibrio-
cidal-antibody assays, the antisera from the various groups did
not provide protection to infant mice (Fig. 5). A log rank
analysis of the survival curves did not reveal any significant
difference (Pof ⬍0.050 is significant) in survival of mice
treated with either preimmune sera or tertiary sera of mice
immunized with the various Inaba CHO-BSA conjugates.
There was a hint of protection in groups 1 and 2, namely, mice
immunized with the disaccharide Inaba CHO-BSA, but re-
peated analysis of those tertiary sera compared to the corre-
sponding prebleed sera did not support this contention (data
not shown).
In situ LPS binding. The lack of vibriocidal or protective
capacity of the anti-Inaba sera raised against the Inaba CHO-
BSA conjugates is interesting given the relatively high titers of
anti-Inaba LPS IgM and IgG1 present in the tertiary sera. In
addition, similar and even lower IgM-IgG ELISA titers in-
duced in response to Ogawa CHO-BSA conjugates were pro-
tective for mice (7). Sera from mice immunized i.p. with whole,
purified Inaba LPS have ELISA (secondary [IgM]) titers sim-
ilar to those measured by using pooled sera from mice immu-
nized with the Inaba CHO-BSA conjugates (data not shown).
This result suggests similar concentrations and/or affinities of
Inaba-specific antibodies in the antisera regardless of the im-
munogen used. Perhaps the LPS concentration used in the
ELISA is not reflective of the LPS environment (density and
spatial distribution) of in situ LPS on V. cholerae bacteria.
We tested whether the inability of the sera elicited by the
Inaba CHO-BSA conjugates to protect resulted from the lack
of binding LPS due to in situ considerations or from binding
LPS being in a position that was not protective. We used
anti-whole-Inaba LPS and anti-Inaba CHO-BSA sera at dilu-
tions similar to those used for the protection assay, with
amounts of bacteria that were the same as that of the challenge
dose for the infant mouse protection assay. After an overnight
incubation with sera, bacterial pellets were washed extensively,
and the binding capacities of the two sera were compared by
assessing the bacterium-bound IgM in a Western blot (Fig. 6).
The tertiary sera from the various groups of mice immunized
with the Inaba CHO-BSA conjugates did not bind to the bac-
teria at detectable levels. The quaternary and tertiary anti-
whole-LPS sera had a titer that was higher (log
10
) than that of
the secondary sera. Similarly, the quaternary and tertiary sera
derived from mice immunized with Inaba LPS efficiently asso-
ciated with LPS in situ. The secondary anti-Inaba whole-LPS
sera had an ELISA titer similar to that of the sera of mice
immunized with the Inaba CHO-BSA; it also did not effectively
FIG. 6. Adsorption of anti-O-SP Inaba-specific and anti-Inaba whole-LPS antibodies with V. cholerae O1 El Tor Inaba bacteria. Bacteria (10
9
CFU) were incubated with 1:10 dilutions of antisera overnight, washed to remove unbound antibody, and then run on sodium dodecyl sulfate-
polyacrylamide gel electrophoresis buffer followed by transfer to nitrocellulose membranes. The nitrocellulose membranes were probed with
horseradish peroxidase-labeled goat anti-mouse IgM. (A) Adsorbed IgM from anti-whole-LPS Inaba quaternary antiserum and the positive-control
mouse IgM samples are in the two center lanes, flanked on either side by anti-Inaba CHO-BSA tertiary antiserum. (B) Adsorbed Ig assessed by
using anti-whole-LPS Inaba preimmune, primary, secondary, tertiary, and quaternary antiserum samples is shown with the positive IgM control.
Corresponding IgM ELISA and vibriocidal titers (previously described in the legend to Fig. 3 and in Table 1, respectively) are shown.
4098 MEEKS ET AL. INFECT.IMMUN.
associate with LPS in situ. However, in contrast to the anti-
Inaba CHO-BSA sera, the secondary, anti-whole-Inaba LPS
sera were vibriocidal. These results suggest that the antibodies
induced by the Inaba CHO-BSA conjugates do not bind with
enough affinity or specificity to native LPS when expressed on
the bacterial surface. Alternatively, the anti-whole-Inaba LPS
sera may be specific for more epitopes other than the Inaba
terminal sugar which enhances the association with LPS in situ.
DISCUSSION
The search for a universally effective cholera vaccine is on-
going. Subunit vaccines have the potential to combine multiple
defined targets for optimal immunogenicity. A consensus com-
ponent for any cholera vaccine is LPS or its derivatives. Anti-V.
cholerae LPS antibodies correlate with protection against vir-
ulent V. cholerae challenge of vaccinated volunteers and those
infected naturally (19, 27, 32, 35). It is important to develop a
V. cholerae LPS-based vaccine that can induce neutralizing
antibodies to both Inaba and Ogawa serotypes, as both sero-
types can initiate infection. Individuals need to be immune to
both serotypes for optimal protection, as immune pressure can
drive serotype conversion (9, 11). Currently, there are two
well-defined antibody targets on V. cholerae LPS associated
with the Inaba and Ogawa serotypes. Several protective MAbs
that bind the terminal sugar of Ogawa (S-20-4), and are thus
Ogawa specific (39, 40), have been described, while an epitope
found on both Inaba and Ogawa LPS recognized by MAb
I-24-2 is defined by the core and O-SP regions of V. cholerae
LPS, suggesting an epitope found at the boundary of the core
and O-SP (39). Inaba-specific MAbs, while reported, are not
presently in general use for experimental manipulation (12,
14-16, 29).
The two O1 serotypes of V. cholerae associated with endemic
and epidemic cholera, Ogawa and Inaba, are defined by the
composition of the terminal sugar of their respective O-SP.
The Inaba O-SP differs from that of Ogawa in that the terminal
perosamine moiety has a hydroxyl group, rather than a me-
thoxyl group, at the 2 position. The methoxyl group has been
shown to be critical for protective anti-Ogawa MAb binding
(40, 41). To test whether the terminal sugar of Inaba LPS was
also a protective B-cell epitope, we constructed Inaba CHO-
BSA conjugates similar to the Ogawa hexasaccharide conju-
gates described previously (7). The solution binding studies
with Ogawa and Inaba oligosaccharides by Wang and col-
leagues (41) did not reveal observable binding of the Inaba
oligosaccharides by anti-Ogawa-specific MAbs, suggesting sub-
stantially different epitopes based on Inaba and Ogawa struc-
tural differences. Thus, anti-O1 serotype-specific sera would be
predicted to bind unique structural elements which should
provide protection unless the Inaba LPS is differently distrib-
uted or expressed in situ compared to Ogawa LPS.
The studies we report herein tested the hypothesis that In-
aba CHO-BSA conjugates would induce protective immunity.
Analogous to the results reported for the Ogawa CHO-BSA
conjugates (7), the Inaba conjugates induced antibody re-
sponses in serum that were of similar magnitude or higher than
those of some of the anti-Ogawa CHO-BSA conjugates but, in
contrast, failed to induce antivibriocidal antibodies or antibod-
ies that were protective in the infant mouse assay. A new
finding for synthetic V. cholerae LPS antigens is that anti-Inaba
CHO-BSA responses were not modulated by the length of the
saccharide attached to the carrier. This finding is consistent
with the data for synthetic Streptococcus pneumoniae conju-
gates, whereby the length of the saccharides did not affect the
magnitude of the humoral immune responses (3), but is dif-
ferent from results of immunization studies involving synthetic
Shigella dysenteriae oligosaccharides, which showed that the
difference in the length of the antigen, as well as antigen-
carrier ratio, affected the magnitude of the murine immune
response (34).
The only structural difference between the Ogawa and Inaba
serotypes places the serotype-defining epitope at the terminal
sugar of O-SP (17, 18, 40, 41). This difference, and the exis-
tence of MAbs specific for either Inaba or Ogawa LPS (1, 12,
14–16, 29), supports the notion that unique, serologically de-
fined epitopes exist for the Inaba and Ogawa serotypes. The
Inaba MAbs were developed for serologic typing; we are un-
aware of any protective efficacy that might have been reported
(12, 14–16, 29). The anti-Inaba MAbs were screened by ELISA
with purified LPS used as the test antigen. Some anti-Inaba
MAbs were found to bind LPS in situ but were not tested for
protective efficacy. Multiple, anti-Ogawa MAbs have been
made and, importantly, some have been reported to be pro-
tective (7, 12, 14–16, 29, 39). One MAb, S-20-4, was cocrystal-
lized with the terminal Ogawa monosaccharide or disaccharide
(40). Recently, the analysis of the cross-binding of S-20-4 to the
terminal monosaccharide of the Inaba O-SP was reevaluated
(25). The difference of a methoxyl group versus a hydroxyl
group at position 2 on the terminal perosamine moiety de-
creased the affinity of S-20-4 binding to Inaba synthetic termi-
nal monosaccharides 840-fold compared to that of the Ogawa
terminal O-SP residue. Liao and colleagues (25) postulated
that for the Inaba terminal monosaccharide, the loss of the O-2
methyl group and its electron-donating effect might alter the
negativity of the 3-OH group. Previously, the 3-OH group had
been shown to play an important role in hydrogen bonding in
the crystal structure of the S-20-4/Ogawa saccharide complex
(40, 41).
Perhaps similar to anti-Ogawa MAbs (7), the remodeling of
the antigen binding pocket of anti-Inaba might be critical for
optimal binding to the Inaba terminal sugar, especially for in
situ LPS. Somatic mutations in the anti-Inaba Fab, in VDJ/VJ
sequences (antigen binding domains of the IgG heavy and light
chains, respectively), or within the Ig fold framework region
sequences that alter the shape of the binding pocket are driven
by multiple immunizations with reagents that drive B-cell pro-
liferation, such as LPS. The Ig variable segment (V heavy/light)
family member(s) that is initially selected for binding Inaba
terminal sugars might be different than those that bind Ogawa,
as the Inaba antibody Fabs need to accommodate the smaller
and electrochemically different Inaba terminal sugar. Anti-
idiotype sera (a measure of V-segment uniqueness) specific for
the anti-Inaba MAb idiotype did not bind anti-Ogawa (epitope
B) MAb, suggesting that the MAbs specific for Inaba and
Ogawa do use different variable Ig gene segments to obtain
serotype specificity (12). We speculate that because of the
limitations imposed by the molecular signature of the Inaba
epitope, the anti-Inaba antibodies induced by the Inaba CHO-
BSA conjugates are of lower affinity than are anti-Ogawa an-
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4099
tibodies induced by Ogawa CHO-BSA conjugates or antibod-
ies induced by native Inaba LPS. This is consistent with the
studies of Ghosh and Campbell, who reported that three dif-
ferent Ogawa MAbs could effectively compete for antigen with
three different anti-Inaba MAbs but that anti-Inaba MAbs
could not compete with anti-Ogawa MAbs (12). Multiple In-
aba-specific MAbs (C6, A18, 11A, 14B1, and 14C3) were
readily measured by ELISA (12, 14–16, 29), which used solid-
phase binding to purified LPS, a physico-chemical state of the
antigen which enhances binding because of the enhanced po-
tential (monogamous bivalency) for one of the Fabs of IgG to
be bound at any time (4). However, similar to our results, A18
MAb was not reactive when tested against LPS in situ by using
a whole-cell ELISA (1). The anti-Inaba antibodies raised
against the Inaba CHO-BSA conjugates did not functionally
bind LPS in situ, as evidenced by the vibriocidal-antibody as-
say, Western blot analysis, and the infant mouse protection
assay.
A potential complicating issue for protective antibodies is
that of the IgG subclass, as subclass-defining structures or
sequences can affect anti-carbohydrate binding (8, 30). The
early studies to generate anti-Inaba MAb used native LPS as
the immunogens, and thus, IgM and the IgG3 subclasses were
dominant among the MAbs (12, 14–16, 29). We found low
levels of anti-Inaba O-SP IgG3 (W. F. Wade, unpublished
data) but high levels of IgG1 in response to Inaba CHO-BSA
conjugates.
The data presented herein raise the question as to whether
protective, anti-Inaba O-SP-specific antibodies can be easily
generated. Our results are seemingly in conflict with some
epidemiological evidence that supports the perspective that
Inaba LPS is a better immunogen than Ogawa LPS (reference
26 and references therein). The vibriocidal-antibody data we
present suggest that the Inaba LPS is a better immunogen than
Ogawa LPS, as antisera to Inaba LPS cross-reacts more readily
with Ogawa LPS in the vibriocidal-antibody assays (Table 1).
Caution must be employed, however, when the results of the
vibriocidal-antibody assays we report here are compared to the
epidemiologic data and inferences that Inaba is a superior
immunogen. The systems that lead to the different data sets are
quite diverse with respect to the complexity (whole cell versus
purified LPS) of immunogens used and the potential for pre-
existing immunity to a particular serotype or other undefined
antigens (reference 26 and references therein). The Inaba con-
jugates we investigated do not have the same number of
epitopes as that of native V. cholerae LPS; thus, it is inappro-
priate to compare them for efficacy. The common core O-SP
epitope is missing from the Inaba CHO-BSA conjugate; this
region has been previously shown to be a protective epitope
(39). The lack of this epitope may be the reason that the
anti-Inaba conjugate serum is less effective than anti-Inaba
whole-LPS serum. An alternate explanation we favor, as dis-
cussed above, is that native LPS has an advantage that can
more efficiently select and drive the proliferation of B cells that
express an antibody with a specificity that is protective or can
be driven to somatically mutate to a protective antibody.
Others have reported that anti-Inaba polyclonal antibodies
induced to purified, detoxified LPS conjugated to cholera toxin
are vibriocidal (13). The ELISA titers of these IgG or IgM
antibodies from BALB/c mice were substantially lower than
those we report in response to immunization with Inaba CHO-
BSA conjugates. However, the sera of whole detoxified LPS
conjugates were vibriocidal against both Inaba and Ogawa
LPS-expressing bacteria, suggesting that a shared epitope was
the target for in vitro killing. The B-cell epitope(s) for the
antisera described by Gupta and coworkers (13) was not de-
fined, but reactivity was adsorbed by LPS, detoxified LPS, or
O-SP.
The failure of the Inaba CHO-BSA conjugates to induce
protective antibodies could be due to the fact that the terminal
sugars of Inaba LPS are not protective epitopes or that the
means by which the Inaba CHO-BSA was delivered have not
been optimized. Future studies are needed to conclusively de-
termine if fragments of the Inaba O-SP can be used to induce
protective antibody, regardless of the vaccine format in which
they are presented. A panel of anti-Inaba-specific MAbs
should be generated so that the affinity and concentrations can
be carefully controlled and compared to the protective capacity
of an anti-Ogawa-specific MAb panel. If the majority of the
Inaba MAbs should be found to have, on average, lower affinity
for their targets and do not afford in vivo protection, then the
hope for the terminal fragments of the Inaba O-SP as a vaccine
antigen would be diminished. However, if a proportion of the
MAbs that are anti-Inaba terminal sugar specific or even cross-
reactive with the Ogawa epitope but that are protective can be
found, it will motivate the search for immunization schemas or
alterations in the Inaba CHO structure and/or conjugate ar-
chitecture to maximally induce the B cells that express the
protective antibody (25).
ACKNOWLEDGMENTS
This work was supported by NIH grants to R.K.T. (AI25096) and
W.F.W. (AI47373) and by intramural NIH support to P.K.
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Editor: J. D. Clements
VOL. 72, 2004 ANTI-INABA O-SP ANTIBODIES 4101
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