Immunogenicity of synthetic saccharide fragments of Vibrio
cholerae O1 (Ogawa and Inaba) bound to Exotoxin A
Terri K. Wade1, Rina Saksena2, Joseph Shiloach3, Pavol Kováč1 & William F. Wade2
1
Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH, USA; 2National Institutes of Health, NIDDK, Laboratory of
Medicinal Chemistry, Bethesda, MD, USA; and 3National Institutes of Health, NIDDK, Biotechnology Unit, Bethesda, MD, USA
Correspondence: William F. Wade,
Department of Microbiology and
Immunology, Dartmouth Medical School,
630W. Borwell Bldg., Lebanon, NH 03756,
USA. Tel.: 11 603 650 6896; fax: 11 603 650
6223; e-mail: william.wade@dartmouth.edu
Received 11 February 2006; revised 29 May
2006; accepted 9 July 2006.
First published online 29 September 2006.
DOI:10.1111/j.1574-695X.2006.00143.x
Editor: Artur Ulmer
Keywords
Vibrio cholerae ; Cholera vaccine; Ogawa O1
LPS; neoglycoconjugates; synthetic
oligosaccharide antigens.
Abstract
Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to
Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral
responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa
lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to
boost the vibriocidal response, but Inaba LPS did not prime for an enhanced
vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier,
and priming B cells with the LPS of either serotype, resulted in enhanced
vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished
response to the Inaba LPS. These studies demonstrate that the ‘functional’ B cell
epitopes on the LPS differ from those of the neoglycoconjugates and that the order
of immunization and the serotype of the boosting conjugate can influence the
epitope specificity and function of the antisera.
Introduction
Vibrio cholerae is a gram-negative bacterium that causes the
diarrheal disease cholera. A surface structure, the lipopolysaccharide (LPS) of V. cholerae (O1 serogroup), induces
protective humoral immune responses in humans and
experimental animals (Mosley, 1969; Qadri et al., 1999;
Chernyak et al., 2002; Meeks et al., 2004). Vibrio choleraespecific, anti-LPS antibodies are linked to protection against
cholera (Mosley, 1969). Development of LPS-based epitopes
for a cholera subunit vaccine is the focus of several groups
(Ariosa-Alvarez et al., 1998; Gupta et al., 1998). Inaba and
Ogawa are the two major serotypes of V. cholerae O1 that
cause either endemic or epidemic cholera (Manning et al.,
1994). Vibrio cholerae O-specific polysaccharide (O-SP)
consists of a polymer of (1 ! 2)-a-linked 4-amino-4, 6dideoxy-D-mannose (perosamine), the amino group of
which is acylated with 3-deoxy-L-glycero-tetronic acid
(Kenne et al., 1982; Hisatsune et al., 1993). The upstream
terminal sugar of the O-SP of V. cholerae LPS differentiates
the Ogawa and Inaba serotypes. A 2-O-methyl group (Pykett
& Preston, 1975; Ito et al., 1994; Liao et al., 2002; Villeneuve
et al., 2002) defines Ogawa LPS (sero-epitope B) while the
Inaba terminal sugar with a hydroxyl group at that position
is thought to define sero-epitope C (Manning et al., 1994;
FEMS Immunol Med Microbiol 48 (2006) 237–251
Liao et al., 2002; Villeneuve et al., 2002). Protective Ogawaspecific mAbs bind the O-SP upstream terminal sugar
(Wang et al., 1998; Chernyak et al., 2002). Other LPS
structures (core-O-SP junction) that are the same for Ogawa
and Inaba LPS can also induce protective antibody (Villeneuve et al., 1999).
We previously reported that neoglycoconjugates (hexasaccharide, carbohydrate-bovine serum albumin conjugate
– hereafter CHO-BSA) with carbohydrate components that
mimic the upstream elements of Ogawa LPS, induced
vibriocidal antisera (Chernyak et al., 2002). To approximate
a clinically relevant vaccine, we prepared conjugates composed of mono-, di- or hexasaccharide fragments of the OSP of V. cholerae O1, serotype Inaba and Ogawa as the B cell
epitopes and recombinant Pseudomonas aeruginosa exotoxin
A (rEPA). Compared to EPA (Pseudomonas aeruginosa
exotoxin A), rEPA has one of the glutamic acids in EPA
replaced with aspartic acid (Hickey & Mohn, 1996). rEPA
has been used previously as a carrier in conjugate vaccines
(Fattom et al., 1990, 2004; Schneerson et al., 2004). These
neoglycoconjugates were used in experiments to determine
if monosaccharides could boost a hexasaccharide-primed
host. Other studies addressed the efficacy of the hexasaccharide conjugates if the host was primed with LPS (Inaba
or Ogawa) prior to boosting with the neoglycoconjugates.
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238
T.K. Wade et al.
Finally, the efficacy of the conjugates was tested in a host
that had prior immunity to the carrier and B cells primed to
V. cholerae LPS.
priming with nonvibriocidal doses of purified LPS would
enhance the efficacy of a booster immunization (5 days
later) with the hexasaccharide of the contra serotype (Fig.
1b). Finally, schedule C was an attempt to replicate the
steady-state immune status of an individual with prior
exposure to the carrier and to V. cholerae LPS. Mice were
immunized twice with rEPA in RIBIs 2 weeks before
priming with a low dose of either Inaba or Ogawa LPS,
followed 5 days later with a booster containing the Ogawa
hexasaccharide conjugate (Fig. 1c). The Ogawa hexasaccharide conjugate was used to boost mice immunized with Inaba
LPS because we wanted to see if it could expand B cells
activated by the contra-serotype of LPS.
Blood collection via retro-orbital sinus/plexus was done
on days indicated in the individual time lines (Fig. 1). Sera
were designated either: prebleed (pb), primary (p), secondary (s), tertiary (t) or quaternary (q) on the graphs.
Resulting sera from individual mice was stored separately
at 4 1C or 20 1C until used. ELISA and the vibriocidal
assay assessed individual serum samples.
Materials and methods
Animals and immunization protocols
Six-week-old, female BALB/c mice from the National Cancer Institute (Bethesda, MD) were used as hosts for the
immunogenicity studies. Individual adult mice were immunized intraperitoneally (ip) with 10 mg (based on carbohydrate weight) of either the Ogawa or Inaba CHO- rEPA
conjugate (5 moles CHO/mole rEPA) suspended in
150 mM NaCl and mixed 1 : 1 with RIBIs adjuvant (Sigma,
St Louis, MO). Three immunization schedules (A–C) were
used in this study. Schedule A had four doses of immunogen. The first two doses were with the hexasaccharide
conjugates and the last two with the monosaccharide conjugate (Fig. 1a). Schedule B was developed to assess if
10 µg CHO
10 µg CHO
Ogawa Hexasaccharide or
Inaba Hexasaccharide
Ogawa Monosaccharide or
Inaba Monoasaccharide
(a)
Day 0
Sera
Collection PB
28
10
P
83
69
T
93
Q
10 µg CHO
Ogawa Hexasaccharide or
Inaba Hexasaccharide
2.5 µg Ogawa LPS or
2.5 µg Inaba LPS
(c)
59
S
(b)
Day 0
Sera
Collection
PB
38
5
14
10
35
P
S
rEPA
25 µg
Day -14
Sera
Collection PB
10 µg
0
2.5 µg Ogawa LPS
or 2.5 µg Inaba
LPS
14
10 µg Ogawa
Hexasaccharide
19
24
P
Fig. 1. Time lines for mouse immunizations and serum collection. (a) For schedule A, the first two inoculations were with either Ogawa or Inaba
hexasaccharide conjugates and the last two with the monosaccharide conjugate of the same serotype as the hexasaccharide. (b). Mice were primed
with 2.5 mg purified LPS from either Ogawa or Inaba and then boosted (5 and 14 days later) with the hexasaccharide of the contra-serotype. (c) Mice
were immunized twice with rEPA in RIBIs 2 weeks apart before priming with 2.5 mg of either Inaba or Ogawa LPS, followed 5 days later with a booster
of 10 mg of the Ogawa hexasaccharide conjugate. The day post priming for the immunizations and the blood collection are shown above and below the
time lines, respectively.
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FEMS Immunol Med Microbiol 48 (2006) 237–251
239
Anti-Ogawa O-SP antibodies
Ogawa CHO-rEPA constructs
condensation of the disaccharide glycosyl donor (1) with
tetrasaccharide glycosyl acceptor (2) and further conversions, as shown in Fig. 2a and b. The analogous Inaba
hexasaccharide (20), mono- and the disaccharides that
mimic the O-PS of Vibrio cholerae O:1, serotypes Inaba and
rEPA was prepared as described (Johansson et al., 1996). The
5-(methoxycarbonylpentyl) linker-equipped Ogawa hexasaccharide (19) was assembled (Saksena et al., 2005b) by
(a)
(b)
OMe
H C
OMe
O
H N
O
AcO
BnO
H C
H N
O
O
C
OH RO
OBn
O
H C
R O
OR
O
RO
BnO
H N
O
O
H C
C NH
R O
4
H
O
C NH
OR
O
O
O
RO
O
O
H C
C
NH
4
R O
BnO
O
COOCH
OR
R
13
R
R
COR
O
14
C Ac
Bn OCH
15
OH
Bn OCH
16
OH
OH OCH
17
OH
OH NHCH CH NH O
18
OH
OH NHCH CH HN
OC H
O
19
OH
O
O
OH
NHCH CH HN
NH
rEPA
4.9
Fig. 2. Schema for generation of neoglycoconjugate immunogens from the Ogawa hexasaccharide and rEPA carrier.
FEMS Immunol Med Microbiol 48 (2006) 237–251
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240
T.K. Wade et al.
OH
O
HO
H C
O
C NH
HO
O
OH
O
HO
H C
O
C NH
HO
OH
O
HO
O
O
H C
C
NH
4
O
O
HO
OH
20 O
CO NHCH CH HN
r EP A
NH
5 .4
O
HO
OH
O
H C
C
NH
O
O
HO
OH
21 O
C O NHCH CH HN
r EP A
NH
5
O
HO
OMe
O
H C
C
NH
O
O
HO
OH
22
CO N HCH CH HN
O
r EP A
NH
5 .3
OH
O
HO
H C
O
C NH
HO
OH
O
HO
O
O
H C
C
NH
O
O
HO
OH
23
O
CO NHC H CH HN
r EP A
NH
5
OMe
O
HO
H C
C NH
O
HO
OH
O
HO
H C
C
NH
O
O
O
O
HO
OH
24
O
CO NHC H CH HN
r EP A
NH
4.9
Ogawa used here (21–24), analogous to hexasaccharides 19
and 20, were from a stock of materials that were made and
characterized as reported previously (Gotoh & Ková, 1994;
Gotoh et al., 1994; Lei et al., 1995, 1996; Ogawa et al., 1996;
Saksena et al., 2005b). All linker-equipped saccharides were
converted to the squaric acid derivatives and conjugated to
rEPA as shown for the Ogawa hexasaccharide (Fig. 2b).
Briefly, an N-iodosuccinimide/silver trifluorosulfonatemediated reaction of glycosyl donor 1 (Johansson et al.,
1996) and glycosyl acceptor 2 (Peters & Bundle, 1989; Zhang
& Kovac, 1997) gave tetrasaccharide 3, which was sequen2006 Federation of European Microbiological Societies
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c
Fig. 2. Continued.
tially deacetylated ( ! 4) and methylated, to give glycosyl
donor 5. It was treated with alcohol 11 to obtain 5(methoxycarbonylpentyl) linker equipped hexasaccharide
12. The required compound 11 was obtained as follows.
Crystalline (Poirot et al., 2001) diacetate 6 was treated
(Saksena et al., 2005b) with methyl 6-hydroxyhexanoate in
the presence of boron trifluoride etherate and the 5-(methoxycarbonylpentyl) glycoside 7 formed was deacetylated
( ! 8). Reaction of 8 with thioglycoside 9 followed by
deacetylation of the formed 10 gave 11. To introduce the 3deoxy-L-glycerotetronic acid side chain, the hexaazide 12 was
FEMS Immunol Med Microbiol 48 (2006) 237–251
241
Anti-Ogawa O-SP antibodies
selectively reduced, and the corresponding hexamine 13 was
treated with 2-O-acetyl-4-O-benzyl-3-deoxy-L-glycerotetronic acid, to give the fully protected hexasaccharide 14. The
foregoing substance was subjected to two-step deprotection,
deacetylation ( ! 15) and hydrogenolytic debenzylation
( ! 16), and subsequent amidation with ethylenediamine
gave amine 17, whose reaction with squaric acid diethyl ester
at pH 7 gave the aquaric acid monoester 18. Treatment of
the latter with rEPA at pH 9 was monitored by surfaceenhanced laser desorption-ionization time-of-flight mass
spectrometry (SELDI-TOF MS) (Chernyak et al., 2001).
The reaction was terminated when a molar carbohydrate–
protein ratio 5 was reached, as indicated by SELDI-TOF
MS, to afford conjugate 19.
Serology
The presence of anti-O-SP Ogawa or Inaba antibody was
measured by ELISA as described in detail previously (Chernyak et al., 2002; Meeks et al., 2004; Saksena et al., 2005a).
The ELISA test antigen was purified Ogawa LPS P1418 or
Inaba LPS (Sigma, St Louis, MO). Endpoint titers for ELISA
were defined as the reciprocal of the antibody dilution for
the last well in a column with a positive optical density for
each sample after subtracting twice the average optical
density background. The disaccharide-BSA was used as an
ELISA test antigen at 0.5 mg well1(Saksena et al., 2005a).
Vibriocidal microtiter assay
The microtiter vibriocidal test developed by Fournier’s
group (Boutonnier et al., 2003; Meeks et al., 2004; Saksena
et al., 2005a) was used. This assay is based on metabolically
active bacteria metabolizing a substrate to produce a violet
color in the well, which indicates the presence of live vibrios.
Inhibition of bacterial growth (end-point titer) is reported
as the reciprocal of the antibody dilution for the negative
well containing the lowest concentration of antibody. A titer
of o 1 : 40 is considered not vibriocidal. The negative
control ( cont.) included bacteria and complement only,
while the positive control (1cont.) was quaternary sera
from mice immunized four times with either purified
Ogawa or Inaba LPS.
Results
Rationale for studies
We reported that synthetic B cell epitopes, which mimic the
terminal sugars of Ogawa or Inaba LPS, are immunogenic
(Chernyak et al., 2002). Ogawa immunogens, especially the
terminal hexasaccharide, efficiently induce vibriocidal and
protective antibody, whereas the Inaba-based immunogens
did not induce protective responses (Chernyak et al., 2002;
FEMS Immunol Med Microbiol 48 (2006) 237–251
Meeks et al., 2004; Saksena et al., 2005a, 2006). Bovine
serum albumin (BSA) was the carrier component of the
neoglycoconjugates for our early studies. Because BSA is not
an appropriate carrier for human vaccines, we tested the
nontoxic rEPA for its suitability as a carrier.
Anti-LPS (IgM) titers induced by hexasaccharide
conjugates can be boosted by monosaccharide
conjugates
We assessed the immunogenicity and efficacy of the rEPAbased conjugates for mice given two doses of hexassacharide
followed by two doses of the monosaccharide conjugates
(Fig. 1a). We chose this approach because two doses of
Ogawa hexasaccharide were effective at inducing vibriocidal
serum antibody, and Ogawa monosaccharides were found to
be able to enhance the humoral response of mice primed
with low doses of LPS (Saksena et al., 2006). This experimental design will provide information that has implications for the vaccination protocol and also the production of
the vaccine. If monosaccharides can boost the anti-LPS
antibody response as efficiently as hexasaccharide conjugates, then six times more doses of vaccine will be available
from the same production level of carbohydrate.
Four immunizations (schedule A) induced comparable if
not higher anti-LPS (IgM) end-point titers (Fig. 3a) in the
tertiary and quaternary sera than three immunizations with
hexasaccharide immunogens (data not shown). The fourdose immunization protocol resulted in anti-LPS IgM endpoint titers in quaternary sera that were similar in magnitude to those induced by four immunizations with purified
LPS (Fig. 3a and b, 1cont.). The kinetics of the anti-Inaba
and anti-Ogawa responses to the neoglycoconjugates was
similar, but the anti-LPS IgM titers were higher by a log for
mice immunized with Ogawa conjugates. The antibodies
induced, regardless of serotype, were cross-reactive to the
LPS that was heterologous to the immunizing LPS. An antiLPS IgG1 response was apparent in tertiary and quaternary
sera of mice immunized with Ogawa-based conjugates and
in the quaternary sera of mice immunized the Inaba-based
conjugates (Fig. 3c).
Vibriocidal titers in response to immunization
with hexasaccharide and monosaccharide
conjugates
Immunization schedule A resulted in vibriocidal titers in the
secondary sera for the mice immunized with the Ogawa
conjugates (Fig. 3d). It is noteworthy that the quaternary
sera of mice immunized with Ogawa conjugates using rEPA
were highly vibriocidal, with the majority of the end-point
titers in the range of 1 : 10 000. The serum from mice
immunized with the Ogawa conjugate was specific for
Ogawa LPS and did not kill Inaba bacteria (Fig. 3e). Neither
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242
T.K. Wade et al.
co
nt
.
Q
S
PB
Ogawa hexa
Ogawa mono
+
Q
S
PB
.
nt
co
−
vibriocidal titers
Inaba hexa
Inaba mono
Inaba bacteria
(e)
P
P
PB
P
PB
P
PB
P
PB
P
PB
Inaba LPS on plate
(c)
Ogawa bacteria
P
P
PB
P
(d)
PB
P
PB
P
PB
P
PB
PB
Ogawa LPS on plate
(b)
PB
IgM end point titers
(a)
Inaba bacteria
1.0 µg
Inaba LPS
2.5 µg
5.0 µg
Ogawa LPS
1.0 µg
2.5 µg
.
nt
co
Ogawa hexa
Ogawa mono
Q
S
PB
Q
PB
S
Inaba hexa
Inaba mono
+
−
co
nt
.
Vibriocidal titers
Ogawa bacteria
5.0 µg
Fig 3. Humoral and vibriocidal response of mice immunized with Ogawa or Inaba hexasaccharides twice before boosting with two doses of the
monosaccharides of the same serotype. (a) The individual end-point titers of anti-Ogawa LPS IgM are shown for prebleed (PB, open squares), primary (P,
open triangles), secondary (S, closed inverted triangles), tertiary (T, open diamonds) and quaternary (Q, closed triangles) sera. The bar represents the
arithmetic mean of the data. The1cont. is quaternary sera (Q, closed circles) from mice immunized with purified Ogawa LPS, four times every 14 days.
(b) The individual end-point titers of anti-Inaba LPS IgM are shown. The symbols are as in a. The 1cont. is hyperimmune anti-Inaba LPS sera. (c) The
individual end-point titers of anti-Ogawa LPS IgG1 are shown. The symbols and 1cont. are as a. ELISA endpoint titers of 100 are considered negative
( cont.). (d) The vibriocidal titers of PB (open circles), secondary (S, closed circles), and quaternary (Q, inverted closed triangles) antisera against Ogawa
bacteria. The cont. is bacteria and complement without sera; the 1cont. is quaternary hyperimmune Ogawa LPS antisera. (e) The vibriocidal titers and
symbols as in d. but against Inaba bacteria and the 1cont. is anti-Inaba LPS hyperimmune sera (open circle).
Inaba conjugate induced vibriocidal antibody (Fig. 3e),
consistent with a previous report (Meeks et al., 2004). We
performed bacterial adsorption experiments using quaternary antisera from mice immunized with either Inaba LPS or
Inaba conjugates. Under incubation conditions used in the
vibriocidal assay (3 h at 37 1C), antibodies from anti-Inaba
LPS and anti-Inaba conjugate sera both bound V. cholerae
LPS in situ equally well (data not shown), yet only the anti2006 Federation of European Microbiological Societies
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Inaba LPS sera was vibriocidal (Fig. 3e). Antibodies in
Ogawa LPS antisera bound as well as antibodies in antisera
made to Ogawa conjugates; both antisera were vibriocidal.
These results suggest that the concentration and affinity of
the antibodies (IgM or IgG1) in the Inaba antisera are similar
to those in the Ogawa antisera and certainly at a level that
could be functional. The reason for nonvibriocidal Inaba
response is not known but likely involves antibody epitopes.
FEMS Immunol Med Microbiol 48 (2006) 237–251
243
Anti-Ogawa O-SP antibodies
Priming with native LPS and enhancement of
vibriocidal response by contra-serotype
conjugate
This experiment (immunization schedule B) was designed
to determine whether priming with purified LPS would
enhance the immunogenicity and efficacy of a booster with
contra-serotype neoglycoconjugates. A low dose (2.5 mg) of
either Inaba or Ogawa LPS was delivered intraperitoneally
(ip) on day zero, followed 5 and 14 days later by an ip
inoculation of the contra-serotype hexasaccharide neoglycoconjugate (Fig. 1b). This immunization protocol was motivated by the fact that people living in areas of endemic
cholera can be exposed to either serotype of V. cholerae and
it is, therefore, important to know if that preexisting
immunity to either serotype can influence the response to
the neoglycoconjugates.
None of the doses of Ogawa LPS used to prime induced
measurable anti-LPS IgM titers against Ogawa or Inaba LPS
(Fig. 4a). Purified Inaba LPS induced low titers of anti-IgM
antibody at all doses tested (Fig. 4b). Surprisingly, a vibriocidal response was present for mice that had no or low antiLPS IgM titers (Fig. 4c). The Inaba vibriocidal response was
higher than that of the anti-Ogawa vibriocidal response,
which was less than 1 : 100 (compared to 1/1000 for mice
immunized with Inaba LPS).
Having established the control responses of mice immunized with LPS alone, we examined sera from mice that were
primed with LPS and boosted with a conjugate of the
contra-serotype. Regardless of the serotype of the priming
LPS, mice responded with anti-LPS IgM antibodies at
days 10 and 35 (Fig. 4d and e). Day 35 IgM titers were not
statistically different from those of day 10, although the
trend was for the mean end-point titer to be lower.
In general, the mice did not respond to the primeboost immunization protocol with measurable IgG1 titers
(Fig. 4f).
Reactivity of antisera to Ogawa or Inaba
disaccharide epitope
Mice immunized once with either the Ogawa or Inaba
hexasaccharide conjugates produced significant amounts of
IgM antibody reactive with Inaba and Ogawa disaccharide
conjugates at day 10 (Fig. 4g and h). The average of
antidisaccharide IgM end-point titers was 1/10 000, similar
to the antidisaccharide response of mice hyperimmune
(1cont.) to either Inaba or Ogawa LPS. Mice immunized
with 2.5 mg of purified LPS did not have serum antibody that
reacts with disaccharide conjugates (Fig. 4i and j). When
mice were immunized four times with purified LPS, the
response to the homologous (immunogen) disaccharide was
always 10-fold higher than the response to the heterologous
disaccharide, suggesting that anti-LPS sera has both serotype-specific and cross-reactive antibodies (Fig. 4i and j).
Fig. 4. Humoral and vibriocidal response of mice primed with purified LPS and boosted with a hexasaccharide of the contra-serotype. (a) ELISA titers of
anti-Ogawa LPS (IgM) of mice immunized intraperitoneally with different doses of Inaba or Ogawa LPS. Prebleed titers (open squares) and titers for the
primary (P, open triangles) sera are shown. The horizontal bar represents the mean of the particular data set. (b) ELISA titers of anti-Inaba LPS (IgM) for
mice immunized with varying doses of either Inaba or Ogawa LPS. ELISA end-point titers of 100 are considered negative (-cont.). (c) The PB sera of mice
immunized against Inaba (left-hand panel) or Ogawa (right-hand panel) LPS were not vibriocidal (not shown). The vibriocidal titers against Inaba (open
circles) and Ogawa (closed circles) for individual P serum samples are shown. Vibriocidal titers of less than 1 : 40 are considered negative. (d) Mice were
immunized with 2.5 mg of either Inaba or Ogawa LPS followed by a booster 5–14 days later of a hexasaccharide conjugate of the contra-serotype to the
priming LPS. The individual end-point titers of anti-Ogawa LPS IgM are shown for prebleed (PB, open squares), primary (P, open triangle), and secondary
(S, closed inverted triangles) sera. The horizontal bar represents the arithmetic mean of the data. The 1cont. is quaternary (Q, closed circles) sera from
mice immunized with purified Ogawa LPS, four times every 14 days. (e) The individual end-point titers of anti-Inaba LPS IgM are shown. The symbols are
as in d. The 1cont. is hyperimmune anti-Inaba LPS sera. (f) The individual end-point titers of anti-Ogawa LPS IgG1 are shown. The symbols and 1cont.
are as d. The immunization schedule to identify the groups is shown below the graphs. (g) The IgM titers of prebleed (PB, open squares) and primary (P,
open triangles) sera to Ogawa disaccharides of mice immunized once with Ogawa or Inaba hexasaccharide are shown. The 1cont. are either hyper
immune Ogawa LPS antisera (closed circles) or hyper immune Inaba LPS antisera (open circles). h. The prebleed (PB open squares) and primary (P, open
triangles) end-point titers of Inaba disaccharides of mice immunized once with Inaba hexasaccharide or Ogawa hexasaccharide are shown. Symbols are
as in panel g. (i) The IgM titers of prebleed (PB, open squares) and primary (P, open triangle) sera to Ogawa disaccharides of mice immunized with Ogawa
or Inaba LPS are shown. The symbols are as in g. (j) The IgM titers of prebleed (PB, open squares) and primary (P, open triangles) sera to Inaba
disaccharides of mice immunized with Ogawa or Inaba LPS are shown. The symbols are as in g. k. The IgM titers of prebleed (PB, open squares) and
primary (P, open triangles) sera against Ogawa disaccharides of mice immunized according to the prime-boost strategy. (l) The IgM titers of prebleed (PB,
open squares) and primary (P, open triangles) sera against Inaba disaccharides of mice immunized according to the prime-boost strategy. m. The serum
vibriocidal response against Ogawa bacteria of mice primed to Ogawa or Inaba LPS before boosting with the contra-serotype hexasaccharide. The open
circles represent prebleed (PB) responses while the closed circles represent the vibriocidal titers in primary (P) sera and the closed triangles represent the
vibriocidal titers in secondary (S) sera. Horizontal bars are the mean of the individual data sets. The cont. represents the vibriocidal activity of bacteria
and complement only. The 1cont. (closed circles) represent the vibriocidal capacity of hyperimmune Ogawa LPS antisera. (n) The vibriocidal activity
against Inaba bacteria of PB, P, or S sera of mice immunized according to the prime-boost strategy. The symbols are as in m, except for the 1cont. (open
circles) which is the vibriocidal activity of anti-Inaba LPS hyperimmune sera. The immunizing protocol is shown below the x-axis.
FEMS Immunol Med Microbiol 48 (2006) 237–251
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244
T.K. Wade et al.
(b)
+
co
nt
S
.
Ogawa LPS on plate
P
PB
IgM end point titers
P
P
B
P
P
PB
P
PB
P
PB
Inaba LPS on plate
(e)
1.0 µg
Inaba LPS
2.5 µg
5.0 µg
Ogawa LPS
1.0 µg
2.5 µg
t.
co
n
S
P
Inaba LPS
Ogawa
+
Prime:
Ogawa LPS
Boost:
Inaba
(CHO-rEPA)
PB
S
PB
Vibriocidal titers
Ogawa bacteria
Ogawa LPS on plate
(f)
P
P
P
Inaba bacteria
IgG1 end point titers
(c)
B
P
P
PB
P
PB
P
PB
P
PB
PB
+
co
nt
.
S
P
PB
S
PB
Inaba LPS on plate
P
P
(d)
Ogawa LPS on plate
PB
PB
IgM end point titers
(a)
P
Serum vibriocidal titers were apparent at days 10 and 35 for
mice primed with one LPS serotype and boosted with the
contra-serotype neoglycoconjugate (Fig. 4m and n). Immu-
PB
Serum vibriocidal titers of mice primed with LPS
and boosted with neoglycoconjugates of the
contra-serotype
nization of mice with conjugates alone does not induce
vibriocidal titers after 5 days (data not shown). Immunization with only purified LPS induced low vibriocidal titers (1/
100) in 25% of the mice immunized with Ogawa LPS
whereas 60% of the mice immunized with 2.5 mg of Inaba
LPS had an average end-point titer of 1/1000. When mice
were primed with a low dose of Ogawa LPS, Inaba hexasaccharide conjugates enhanced ( 4 1/1000) the Ogawa-specific
vibriocidal response (Fig. 4m). The day 10 sera vibriocidal
end-point titers were only about a log lower than the
vibriocidal titers of mice hyperimmune (four doses) to
Ogawa LPS (1cont.). The ability of the Ogawa hexasaccharide to enhance the Ogawa vibriocidal response of Inaba LPS-
S
Mice primed with 2.5 mg of purified LPS and boosted
with the neoglycoconjugates of the contra-serotype to the
priming LPS had comparable primary end-point titers to
the disaccharide epitope as mice immunized with neoglycoconjugates alone (compare Fig. 4k and l and Fig. 4g and h).
5.0 µg
Fig. 4.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Immunol Med Microbiol 48 (2006) 237–251
245
Anti-Ogawa O-SP antibodies
(m)
co
nt
.
Ogawa bacteria
(n)
.(
nt
co
+
.
co
nt
S
Inaba LPS
Ogawa
+
Prime:
Ogawa LPS
Boost:
Inaba
(CHO-rEPA)
P
PB
S
P
Inaba Di-on plate
PB
.
nt
co
+
Inaba LPS
Ogawa
I)
)
.(
nt
+
+
(l)
S
O
P
Inaba LPS
co
co
+
co
nt
.
P
+
P
PB
I)
nt
.(
nt
co
+
P
PB
S
Prime:
Ogawa LPS
Boost:
Inaba
(CHO-rEPA)
Inaba Di-on plate
Ogawa LPS
Ogawa Di-on plate
P
PB
(k)
.(
O
)
P
PB
PB
P
Inaba LPS
Ogawa hexa
(j)
Ogawa Di-on plate
Ogawa LPS
PB
Inaba hexa
PB
Ogawa hexa
(i)
P
co
nt
.
+
P
+
Inaba hexa
Inaba Di-on plate
PB
(h)
co
nt
.
PB
Ogawa Di-on plate
P
PB
IgM anti-Disacchairde end point titers
(g)
Prime:
Ogawa LPS
Boost:
Inaba
(CHO-rEPA)
Inaba LPS
Ogawa
t.
co
n
P
S
+
Prime:
Ogawa LPS
Boost:
Inaba
(CHO-rEPA)
PB
S
P
PB
.
nt
co
co
n
t.
−
−
+
S
P
PB
S
P
PB
co
nt
.
vibriocidal titers
Inaba bacteria
Inaba LPS
Ogawa
Fig. 4. Continued.
FEMS Immunol Med Microbiol 48 (2006) 237–251
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
246
primed mice was less apparent, with titers of 1/100 being the
average, when Ogawa bacteria were the target (Fig. 4m).
These titers were lower by a log than the vibriocidal titer
induced by 2.5 mg of Inaba LPS alone (Fig. 4b). The use of
Ogawa LPS to prime mice for a booster with Inaba hexasaccharides was ineffective at inducing a vibriocidal response, as
measured against Inaba bacteria (Fig. 4n). Priming mice with
Inaba LPS and boosting with the Ogawa hexasaccharide
resulted in low vibriocidal titers (1/100) against Inabaexpressing bacteria, which contrasts with the 10-fold higher
vibriocidal titers (1/1000) in sera for mice inoculated with
2.5 mg of Inaba LPS. Changing the structure or context of the
LPS-based B cell epitopes available during the course of the
immunization affected the vibriocidal response.
Response of mice preimmune to the rEPA,
primed with either Inaba or Ogawa LPS, and
boosted with Ogawa hexasaccharide conjugate
This experiment was designed (Fig. 1c) to test the efficacy of
the neoglycoconjugates in a circumstance that may represent
the immune status of human vaccinees: the presence of
memory helper T cells specific for the carrier protein and
memory/activated B cells specific for various LPS epitopes.
Mice immunized twice with rEPA in an adjuvant before LPS
priming and then immunized with rEPA associated with the
conjugate at day 5 after the LPS priming, had anti-rEPA
titers greater than 1/3.2 106 compared to lower serum
titers (1/50 000–100 000) of mice that only received rEPA in
the form of the conjugate at day 5 (data not shown).
Regardless of the LPS serotype used to prime, mice made
good anti-LPS IgM responses at day 10 (Fig. 5a and b). On
average, the end-point titers in the primary sera were higher
in this scenario than for mice immunized according to the
schedule in Fig. 1b, which involved priming with LPS but
not with the carrier. The anti-LPS IgM end-point titers in
the primary sera of mice primed to carrier and LPS before
boosting were less than fivefold different from titers of sera
from mice hyperimmunized to purified Ogawa or Inaba
LPS. As did sera from mice from the other immunization
protocols, sera from mice immunized according to schedule
C bound both serotypes of LPS.
T.K. Wade et al.
The prior exposure to the rEPA resulted in higher endpoint titers to the Ogawa and Inaba disaccharides at day 10
compared to mice primed with LPS but not the carrier (Fig.
5c and d). The anti-disaccharide titers were similar to serum
titers of mice given four doses of Ogawa or Inaba LPS. The
prime boost strategy did not enhance the response to
disaccharide of a particular serotype.
Vibriocidal responses of mice primed to carrier
and LPS before immunization with Ogawa
neoglycoconjugates
Ogawa hexasaccharide neoglycoconjugates are superior at
inducing vibriocidal antibodies compared to the Inaba
hexasaccharide conjugates (Chernyak et al., 2002; Meeks
et al., 2004). We wanted to know if Ogawa hexasaccharides
could enhance the vibriocidal responses of mice primed
with either serotype type of LPS. Priming mice with rEPA
and LPS and later immunized with Ogawa hexasaccharide
conjugates induced vibriocidal titers that were cross-reactive
with V. cholerae LPS (Fig. 5e and f). The serum vibriocidal
response (against Ogawa bacteria) of mice primed with
Inaba LPS and boosted with Ogawa hexasaccharide conjugate was on average 1/1000 10 days past LPS priming. The
vibriocidal antibodies (against Ogawa bacteria) induced by
the homologous system, Ogawa LPS priming and a booster
with Ogawa conjugate, were on average 1/2000, but the
response was less variable (Fig. 5e). The mean anti-Inaba
vibriocidal titer of mice primed to carrier and Inaba LPS and
boosted with Ogawa hexasaccharide was 1 : 200 (Fig. 5f).
Mice primed with rEPA and Ogawa LPS and boosted with
Ogawa hexasaccharide conjugates had a slightly lower (1/
100) vibriocidal titer to Inaba-LPS expressing bacteria.
Discussion
Cholera subunit vaccines based on V. cholerae LPS B cell
epitopes without the attending Toll receptor 4 agonist
component of LPS are being developed (Gupta et al., 1998;
Chernyak et al., 2002; Meeks et al., 2004). Ogawa-based
neoglycoconjugates (synthetic hexasaccharide variously
linked to BSA) induce vibriocidal and protective antibody
(Chernyak et al., 2002). Human clinical trials require
Fig. 5. Humoral response and vibriocidal of mice primed with rEPA and LPS (Inaba or Ogawa) before being boosted with Ogawa hexasaccharides. (a)
The individual end-point titers of anti-Ogawa LPS IgM are shown for prebleed (PB, open squares) and primary (P, open triangle). The horizontal bar
represents the arithmetic mean of the data. The 1cont. is quaternary (Q, closed circles) sera from mice immunized with purified Ogawa LPS, four times
every 14 days. (b) The individual end-point titers of anti-Inaba LPS IgM are shown. The symbols are as in a. The 1cont. is hyperimmune anti-Inaba LPS
sera. ELISA endpoint titers of 100 are considered negative (-cont.). (c) The prebleed (PB, open squares) and primary (P, open triangles) IgM titers against
Ogawa disaccharides of mice immunized according to the prime-boost strategy shown in schedule C. (d) The prebleed (PB, open squares) and primary
(P, open triangles) sera IgM titers against Inaba disaccharides of mice immunized according to the prime-boost shown in schedule C. (e) The vibriocidal
titers of prebleed (PB, open squares) and tertiary (T, closed circles) antisera against Ogawa bacteria. The -cont. is bacteria and complement without sera;
the 1cont. is hyperimmune Ogawa LPS antisera. Vibriocidal titers of less than 1 : 40 are considered negative. (f) The vibriocidal titers as in panel e. but
against Inaba bacteria. The open circles for the 1cont. is anti-Inaba LPS hyperimmune sera. The immunizing protocol are shown below the x-axis.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Immunol Med Microbiol 48 (2006) 237–251
247
Anti-Ogawa O-SP antibodies
neoglycoconjugates with a medically acceptable carrier
protein such as rEPA which has been used for other O-SP
conjugate vaccines (Pavliakova et al., 1999). Synthetic Oga-
.
co
+
.
nt
co
I)
)
.(
co
nt
nt
co
rEPA
Ogawa
Ogawa
+
rEPA
Inaba
Ogawa
+
.(
O
P
PB
P
.(
nt
co
+
Carrier prime:
LPS prime:
Boost:
Inaba bacteria
(f)
Ogawa bacteria
Carrier prime:
LPS prime:
Boost:
rEPA
Inaba
Ogawa
rEPA
Ogawa
Ogawa
.
+
co
nt
P
PB
P
PB
.
nt
co
−
co
nt
.
+
P
PB
P
PB
−
co
nt
.
vibriocidal titers
(e)
PB
I)
)
O
.(
nt
co
rEPA
Ogawa
Ogawa
+
rEPA
Inaba
Ogawa
P
PB
P
PB
Carrier prime:
LPS prime:
Boost:
Inaba Di- on plate
(d)
Ogawa Di- on plate
(c)
nt
P
rEPA
Ogawa
Ogawa
+
rEPA
Inaba
Ogawa
P
P
PB
PB
Inaba LPS on plate
(b)
Carrier prime:
LPS prime:
Boost:
IgM endpoint titers
PB
P
Ogawa LPS on plate
PB
IgM end pointtiters
(a)
wa or Inaba saccharide fragments conjugated to rEPA were
immunogenic and induced vibriocidal antibodies. The
rEPA-based conjugates were not as readily soluble nor did
Carrier prime: rEPA
LPS prime:
Inaba
Boost:
Ogawa
rEPA
Ogawa
Ogawa
Fig. 5.
FEMS Immunol Med Microbiol 48 (2006) 237–251
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
248
they easily induce an IgG response compared to the BSAbased conjugates (Chernyak et al., 2002; Meeks et al., 2004).
The utility of rEPA as a carrier for synthetic V. cholerae
carbohydrate epitopes is questionable.
Serotypes, immunodominance and epitope
Structurally, Ogawa and Inaba V. cholerae LPS are almost
identical, differing only at the upstream terminal sugar
where an O-2 methyl group or a hydroxyl at that position
defines Ogawa and Inaba, respectively (Hisatsune et al.,
1993; Ito et al., 1994; Liao et al., 2002; Villeneuve et al.,
2002). Inaba LPS is thought to be a superior immunogen to
Ogawa LPS (Longini et al., 2002). The data presented herein
supports that notion. The immunologic explanation for this
is unknown and at variance with the protective efficacy of
the LPS-based Ogawa and Inaba hexasaccharide conjugates
we have developed. Ogawa hexasaccharide immunogens are
superior to their Inaba counterparts, as the latter only rarely
induce vibriocidal antibody (Chernyak et al., 2002; Meeks
et al., 2004).
The structure of V. cholerae LPS and that of the corresponding neoglycoconjugates differ. The O-SP component
of LPS has more monomeric units (12–15 vs. 6) which are
linked to the core saccharides. The longer LPS with its
additional sugars (perosamine and core residues) could
contain an additional protective B cell epitope (Liao et al.,
2002; Chatterjee & Chaudhuri, 2003). Another difference
between the two immunogens is the ability to induce the
innate immune response. Native LPS contains lipid A, a
Toll-4 receptor (TLR-4) agonist which can enhance human
and murine B cell activation leading to enhanced immunoglobulin responses (Ogata et al., 2000). The neoglycoconjugates do not contain lipid A, and thus attending
inflammatory signals required to enhance immunogenicity
are derived from RIBIs adjuvant that contains monophosphoryl lipid, a lipid A look alike which was engineered to
remove the phosphate group from the reducing end sugar
and the ester-linked fatty acidy at the 3-positon to obviate
most of the toxic aspects of LPS’s lipid A (Baldridge &
Crane, 1999).
Do these differences contribute to the different efficacy of
LPS and the neoglycoconjugates at inducing vibriocidal
antibody? The analysis of the geometric mean end-point
titers (anti-LPS IgM and antidisaccharide IgM) and the
corresponding vibriocidal capacity of the sera (day 10) from
mice immunized according to the different protocols is
instructive. Mice immunized with 2.5 mg of Inaba LPS had
low titer anti-LPS responses, did not have a measurable antidisaccharide response, but did have mid-level vibriocidal
titers. Low doses of Inaba LPS, and to some extent of Ogawa
LPS also, can induce vibriocidal antibody that does not bind
the terminal disaccharides of either LPS serotype. This is
2006 Federation of European Microbiological Societies
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c
T.K. Wade et al.
consistent with a LPS B cell epitope (common epitope,
designated as C), other than the two terminal sugars, being
more effective at inducing early protective responses than
the terminal LPS sugars. The C eptitope was postulated to be
the tetronic acid residue of the perosamines (Manning et al.,
1994).
Mice immunized with either Inaba or Ogawa hexasaccharide neoglycoconjugates had similar if not higher antiLPS IgM titers than the LPS-immunized mice (348–606 vs.
200) and significant levels of antidisaccharide antibody
(5572–11 143 end-point titers that bound both LPS serotypes. However, these sera were not vibriocidal, suggesting
that antibodies to terminal disaccharides of V. cholerae LPS
are not strictly correlated with a protective response. Others
have reported that a vibriocidal, anti-Ogawa mAb (S-20-4;
IgG1) binds the terminal sugars (mono- and disaccharide)
of Ogawa LPS (Wang et al., 1998; Villeneuve et al., 2002). S20-4 was generated from a hyperimmune mouse and is very
specific for Ogawa LPS, reacting with Inaba monglycosides
with about 800-fold less affinity (Wang et al., 1998; Liao
et al., 2002). We speculate that the early IgM (10 day)
response to the disaccharides we report, which has not
undergone isotype switching and thus is likely not to be
somaticly mutated, is the reason these anti-disaccharide
antibodies were not protective.
LPS priming before boosting with
neoglycoconjugates
If mice are primed with purified LPS of one serotype and
boosted with a hexasaccharide neoglycoconjugate of the
contra-serotype, there is no effect on the magnitude of the
early anti-LPS (IgM) response. Unexpectedly, the antidisaccharide response is lower compared to mice immunized
with neoglycoconjugates alone, yet vibriocidal titers are
more evident from mice immunization with the primeboost schedule compared to mice immunized with LPS or
neoglycoconjugates alone. These results suggest that priming with Ogawa LPS expands B cells that the Inaba hexasaccharide can also activate. We do not know if the
terminal sugars or the tetronic acid epitope is the target for
the booster effect. The ability of the humoral response to
LPS priming can be manipulated by the epitope composition of the booster immunogen, as is evident for mice
primed with Inaba LPS and boosted with Ogawa hexasaccharide. Mice only inoculated with Inaba LPS respond at
day 10 with a geometric mean vibriocidal titer of 420. If
mice are boosted at day 5 with Ogawa neoglycoconjugates,
the titer in day 10 sera is reduced to 80.
The sera of mice primed with rEPA and LPS (Ogawa or
Inaba) before being boosted with the neoglycoconjugate had
higher anti-LPS IgM and anti-disaccharide geometric mean
titers compared to the sera of mice immunized by protocol
FEMS Immunol Med Microbiol 48 (2006) 237–251
249
Anti-Ogawa O-SP antibodies
A or B. It is reasonable to expect that higher anti-LPS and
anti-disaccharide titers would correlate with enhanced vibriocidal titers. However, mice immunized according to
protocol C had vibriocidal titers within the range of those
titers resulting from the other immunization schedules. As
in the other prime boost immunization schedule, boosting
with Ogawa oligosaccharide-based immunogens was synergistic for the vibriocidal response, whereas boosting InabaLPS-primed mice with the Ogawa hexasaccharide resulted in
a reduced anti-Inaba LPS response.
Serotype-specific and common structures of
LPS: effect on immunogenicity and protective
response
What explains the lack of an additive response to prime
boost immunization if the immunogens are not identical
LPS serotype structures? We do not know the answer to this
question, but hypothesize that the immunodominance of
the B cell epitopes (B and C) in different LPS serotypes is
linked to the effect. The structure of the Inaba epitope may
provide a weaker epitope for the anti-Inaba antibody
repertoire than that of the anti-Ogawa antibody, and thus
the terminal sugars of Inaba are not as immunogenic as
those of Ogawa. This idea is supported by the work of Liao
and colleagues. (Liao et al., 2002), who suggested that the
loss of the methyl group at O-2 influences the polarity of the
Inaba epitope, making not only the resulting 2-OH more
polar, compared to 2-OMe, but also the oxygen of the
neighboring 3-OH less negative due to the lack of the
electron-donating effect of the methyl group at the 2
position. Compared to Ogawa LPS, the Inaba terminal sugar
may not be immunodominant if presented in the context of
LPS, and the B cell response is focused on the C antigen,
which is why Inaba LPS can induce cross-reactive protective
antibody following immunization (Villeneuve et al., 1999).
In individuals exposed to or immunized with V. cholerae
LPS, the initial B cell response is directed against the LPS
immunodominant epitope, which differs based on the
serotype. Ogawa LPS and a booster with the corresponding
serotype conjugate would focus on the terminal sugars. The
cross-reaction of anti-Ogawa antibodies to the terminal
Inaba LPS sugar would allow for an additive response. Inaba
LPS priming followed by a booster with Ogawa conjugates
would be the least likely to be effective. The Inaba LPS
presents the common epitope in the priming event. The
immunodominant Ogawa epitope in an Ogawa conjugate
booster would be presented in the context of B cells
activated and expanded to the C antigen and, thus, it would
represent an initial immunization rather than booster.
Immune pressure results in serotype conversion, especially
from Ogawa to Inaba. It is hypothesized that a gene product,
rfbT a methyl transferase, provides the Ogawa serotype
FEMS Immunol Med Microbiol 48 (2006) 237–251
structure. The loss of mutation of this gene results in the
Inaba serotype becoming the dominant serotype in the
individual or the community. We do not think that immune
pressure has selected a different serotype in our studies as
there is no active infection. However, our data support the
immunodominance of Ogawa over Inaba if the immunogen
is in the form of neoglycoconjugates. These data, along with
the immune-mediated serotype conversion, highlight the
complexity of the anti-LPS response in cholera.
The fact that individuals can have similar levels of antiLPS antibody (ELISA) and have different levels of protection
or not be protected at all, shows there is much to be learned
about the epitope specificity to the V. cholerae anti-LPS
response. We need to know how to manipulate LPS-based
immunogens to achieve maximal protection. Synthetic LPSbased neoglycoconjugates may be useful for this as they can
be generated to present restricted B cell epitopes, perhaps
the cross-reactive C epitope. In order to manipulate the
anti-LPS response, we need to determine the specificities
(LPS structures bound by antibody) and the affinities of the
antibodies induced by the neoglycoconjugates and by purified LPS. We also need to determine if there is a hierarchy to
the LPS-immunogen epitopes. Depending on the LPS
serotype, do some epitopes stimulate B cells more effectively
and thus optimize early production of protective antibody?
The information needs to be evaluated in the context of
prior exposure to V. cholerae LPS of individuals living in
cholera-endemic areas.
Conclusion
The results we report show that synthetic neoglycoconjugates can enhance the serum and vibriocidal response of
mice primed with purified LPS. The novel finding that
Ogawa neoglycoconjugates can boost Inaba-primed mice
while Inaba neoglycoconjugates can not boost Ogawaprimed mice highlights the issue of immunodominant LPS
epitopes that we propose, based on experimental evidence,
differ between Ogawa and Inaba LPS. Neoglycoconjugates
are being developed for use as boosters for cholera vaccine
responses that have waned. The neoglycoconjugates are an
attractive alternative because they can be delivered parenterally without the attending inflammation of LPS, and they
have a carrier component to enhance B cell memory and
antibody-isotype switching. Parenteral immunization obviates the problems of immune interference that have been
reported for boosting using the existing oral cholera vaccines.
Acknowledgements
This work was supported by an NIH grant to WFW (AI
47373) and by intramural NIH support to PK.
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c
250
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