Download Immunomodulation of Tcell responses with Vibrio cholerae O135

RESEARCH ARTICLE
Immunomodulation of T-cell responses with Vibrio cholerae
O135 capsular polysaccharide and its protein conjugate, novel
cholera vaccine study models
Ema Paulovičová, Jana Korcová, Eva Machová & Slavomı́r Bystrický
IMMUNOLOGY & MEDICAL MICROBIOLOGY
Slovak Academy of Sciences, Institute of Chemistry, Centre of Excellence GLYCOMED, Bratislava, Slovakia
Correspondence: Ema Paulovičová, Slovak
Academy of Sciences, Institute of Chemistry,
Centre of Excellence GLYCOMED, Dubravská
cesta 9, 84538 Bratislava, Slovakia. Tel.: +42
125 941 0217; fax: +42 125 941 0222;
e-mail: [email protected]
Received 15 June 2011; revised 5 January
2012; accepted 16 March 2012.
Final version published online 4 May 2012.
DOI: 10.1111/j.1574-695X.2012.00957.x
Editor: Willem van Eden
Keywords
Vibrio cholerae; CPS; CPS-BSA; conjugate
vaccine; T cells; activation antigens.
Abstract
We studied T-cell immune responses to surface capsular polysaccharide (CPS)
of Vibrio cholerae O135 and its protein conjugate. CPS and CPS–bovine serum
albumin (BSA) activation and presentation are characterized with induced
alterations in expression and upregulation of membrane antigens CD25,
CD11b, CD16/32, MHCII and CD45 on blood- and spleen-derived T cells.
Expression of the early activation marker CD25 revealed efficient CPS-BSA
conjugate activation especially of CD4+CD3+ and CD8+ CD3+ cells. Specific
CPS-BSA-induced CD25+ T-cell subsets in blood were observed after the first
application, i.e. a 4.2-fold increase of CD4+CD25+ and 7.6-fold increase of
CD8+CD25+ vs. preimmune levels was determined. The upregulation of surface
antigens MHCII and CD45 involved in antigen presentation and cell activation
of CD3+ cells and their significant reciprocal correlation (R2 = 0.92) observed
only with CPS-BSA conjugate suggested efficient T-cell dependency and presentation. The pattern of accelerated T-cell activation and engagement of T cells
as antigen-presenting cells throughout CPS-BSA immunization contrary to CPS
alone was also confirmed in CD4+/CD8+/CD3+ splenic cells. The results
revealed different T-cell antigen presentation and activation following administration of CPS and CPS-BSA conjugates, as supported also by evaluation of
CD45, MHCII and CD25 expression on CD19+ B cells.
Introduction
The Gram-negative bacteria Vibrio cholerae O1 and O139
cause cholera accompanied by serious intestinal dysfunction and diarrhoea. Non-O1 non-O139 serotypes of
V. cholerae are increasingly being reported as the causal
agents of severe gastrointestinal disorders (Chatterjee
et al., 1998; Das & Gupta, 2005). And the isolation of
multidrug-resistant clinical isolates has focused the need
for a vaccine (Das & Gupta, 2005; Krishna et al., 2006).
The choice of prospective vaccine targets is linked to
immunodominant structures engaged in cholera pathogenesis (Kabir, 2005; Levine, 2010). The capsular polysaccharides (CPSs) of non-O1 vibrios, usually antigenically
and structurally very similar to O-polysaccharide, represent suitable vaccine components as important virulence
factors and protective antigens (Nesper et al., 2002; Jones,
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2005; Chaudhuri & Chatterjee, 2009). Serologic studies
on patients infected with V. cholerae serotype O139
revealed vibriocidal activity of sera mostly specific to CPS
(Kossaczka et al., 2000). Functional studies have suggested that CPS contributes to virulence by decreasing the
sensitivity to serum killing and by increasing adhesion to
intestinal epithelial cells (Johnson et al., 1992; Waldor
et al., 1994).
CPSs are classified as thymus-independent type-2 antigens. The conversion of originally T-cell-independent CPS
to T-dependent antigen is based on covalent coupling of
CPS to a T-cell-dependent protein carrier, capable of
immune cell activation through major histocompatibility
complex (MHC) class II-restricted CD4+ T cells followed
by more rapid and enhanced immune response on
re-exposure. The effective T-cell participation resulted in
enhanced immune responses and induction of immunoFEMS Immunol Med Microbiol 65 (2012) 422–430
Immunomodulation of T cell response with V. cholerae O135 CPS
logical memory accompanied by long-lasting protective
immunity even in infants and young children under
2 years, particularly susceptible to infection with encapsulated bacteria (Mosley et al., 1968; Rijkers & Mosier,
1985; Baker, 1992; Goldblatt, 2000; Weintraub, 2003).
In contrast to the contribution of B cells in anticholera
immunity and vaccination (Provenzano et al., 2006),
T-cell-mediated immunity, and the role of T cells and
their prominent activation membrane markers engaged
in cell interactions, activation and antigen-induced cell
stimulation in anti-V. cholerae defence remain largely
unknown. Knowledge of T-cell activation and participation in anticholera vaccine response is critical for developing vaccine strategies to control or prevent
infections.
We have previously revealed the novel structure of
V. cholerae O1 lipopolysaccharide-derived glycoconjugates
(Machová et al., 2002) and the immunological efficacy of
subcellular preparations of vibrios (Paulovičová et al.,
2006, 2010a, b; Korcová et al., 2010). Here we report the
results of in vivo studies on T-cell immunobiological
effectivity of V. cholerae O135 CPS and its conjugate, a
possible model structure for cholera vaccine.
Our specific objective was to compare the efficacy and
extent of the T-cell immune response induced by conjugated and unconjugated CPS, based on the observation of
different expression of principal surface differentiation
antigens engaged in cell activation.
Materials and methods
Cultivation of V. cholerae O135
Vibrio cholerae O135 was isolated from the river Váh near
Kolárovo, Slovakia, in 2000 and was serotyped at the
National Reference Center for Vibrionaceae in Komárno,
Slovakia. Serotype O135 represents the most abundant
serotype in the collection of V. cholerae strains isolated
from different aquatic biotypes in Slovakia during 1970–
2000. Bacteria were grown aerobically at 30 °C in medium
containing 10 g L 1 NaCl and 10 g L 1 of bacterial peptone (pH 8.6). Upon reaching the late logarithmic growth
phase, cultivation was stopped by centrifugation (20 min,
2711 g, 4 °C). Bacterial cells were suspended in distilled
water and killed by phenol. The suspension of the killed
cells was centrifuged (30 min, 3904 g, 4 °C). Vibrio cholerae biomass was stored at 20 °C.
Vibrio cholerae strain non-O1 NRC-66/171; serotype
O135; genotype NaG, OmpW+, OmpU , ToxR+, CT ,
ST , ZOT , ACE , HLY+ (unpublished data); chemotype a/II (according to the Heilberg–Smith–Goodner
scheme), which was arbitrarily selected, was used for
preparations of CPS and its conjugate.
FEMS Immunol Med Microbiol 65 (2012) 422–430
423
Preparation and characterization of CPS from
V. cholerae O135
Cell-wall-associated polysaccharides from V. cholerae
O135 were obtained by extraction of wet biomass with
90% phenol in water at 68 °C and detoxified by acid
hydrolysis (Hisatsune et al., 1985). Purification of polysaccharides was performed by size exclusion chromatography (Korcová et al., 2010). Two saccharide fractions were
obtained and CPS as the larger polysaccharide
(Mp ~ 197 000) was present in the first peak. The CPS of
V. cholerae O135 (Mw ~ 197 000 Da) contained < 1%
protein and < 2% nucleic acid.
Preparation and characterization of CPS–bovine
serum albumin (CPS-BSA) conjugate
CPS-BSA conjugate was prepared using adipic acid
dihydrazide (ADH) as a linker for binding of CPS to BSA
according to Machová et al. (2002). The content of free
hydrazide groups in CPS-ADH intermediate was evaluated
by using the trinitrobenzene sulfonic acid method (Fields,
1971). The conjugation of BSA to CPS-ADH was performed
through free hydrazide groups of CPS-ADH and carboxyl
groups of BSA activated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).
The CPS-BSA conjugate was purified by size exclusion
chromatography (SEC). The amount of protein was determined according to a modification of the method of Sedmak & Grossbergh (1977) using BSA as standard. The ratio
of saccharide/protein in the CPS-BSA conjugate was 10 : 1.
Potential endotoxic activity of CPS and the CPS-BSA conjugate was evaluated by a Limulus amoebocyte lysate test
(E-TOXATE Kit; Sigma). Neither CPS nor CPS-BSA exerted
pyrogenic activity (< 0.015 endotoxin unit mL 1).
Animals, experimental design and
immunisation
Female Balb/c mice aged 8–12 weeks (breeding facility
VELAZ, Prague, Czech Republic) were used. Animal experiments were conducted in accordance with the revised
Declaration of Helsinki, 1983, and follow the criteria for
the welfare of experimental animals, and with the ethical
guidelines issued by the Research Base of Slovak Medical
University, Institute of Preventive and Clinical Medicine
(Bratislava, Slovakia). Primary immunization and two
booster immunizations, (1) 200 lL CPS and (2) 200 lL
CPS- BSA conjugate, each with 4 lg polysaccharidic
moiety per dose, were administered subcutaneously (s.c) at
2-week intervals. Each experimental group comprised 10
animals. The primary–boost immunization protocol also
included a placebo group of saline-injected mice.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
E. Paulovičová et al.
424
Blood specimens were obtained by retro-orbital puncture. Preimmune blood samples were collected as negative
control before primary vaccination. Potassium EDTA
blood samples for immunocytometric evaluation were
collected 2 weeks after each immunization. Animals were
killed by cervical dislocation.
Spleens were carefully removed in an aseptic manner
into 10-mL Falcon tubes (Becton Dickinson) containing
sterile saline and splenic lymphocytes were carefully
washed out from spleens to Petri dishes with PBS (1 mL
per spleen). Washing was performed by centrifugation
(1500 g/5 min, 4 °C) and 50-lL aliquots of cell pellets of
freshly isolated mouse splenocytes were analysed by direct
labelling with monoclonal antibodies (see below).
Immunophenotyping
Peripheral blood and splenic cells were used for direct
staining and evaluation using a Beckman-Coulter FC 500
flow cytometer running under CXP software. For each
sample, fluorescence histograms of 10 000 cells were generated and analysed. Gates were set around lymphocytes
to exclude debris. The following panel of rat antimouse
fluorochrome-conjugated monoclonal antibodies was
used, i.e. fluorescein isothiocyanate-conjugated and phycoerythrin-conjugated: anti-CD3, anti-CD8, anti-CD4,
anti-CD25, anti-CD19, anti- CD45, anti-MHCII, antiCD11b and anti-CD16/32 (Antigenix America Inc.).
As appropriate, isotype antibody-negative controls
anti-IgG1 and anti-IgG2a were used. The conjugated
monoclonal antibodies (5 lL) and whole-blood samples
(in K2EDTA) or splenic cells (50 lL) were added to
5-mL sterile Falcon tubes (Becton Dickinson) and
incubated for 30 min in the dark at 4 °C. Lysis of
erythrocytes was carried out with 250 lL of Optilyse C
lysing solution (Immunotech, A Beckman Coulter
Company). Lysis was stopped after 30 min of incubation
in the dark at 4 °C with PBS, pH 7.2. Finally, the
samples were evaluated by a single- and/or dual-colour
flow cytometric assay.
Computational statistical analyses
Experimental results were calculated as means ± SD. The
normality of distribution was evaluated according to
Shapiro–Wilk’s test at the 0.05 level of significance. Statistical comparison between experimental groups was performed using one-way ANOVA and post-hoc Bonferroni’s
and Tukey’s tests. The results were significant if the
difference between the analysed groups equalled or exceeded
the 95% confidence level (P < 0.05). Statistics were performed with ORIGIN 7.5 PRO software (OriginLab Corp.,
Northampton, MA). Pearson’s correlation coefficient (r)
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
was used to compare the strength of the relationship
between immunological parameters.
Results
Analysis of peripheral and splenic CD3+CD4+
and CD3+CD8+ T cells
The induced changes of T-cell immunophenotype following immunization with CPS and CPS-BSA were assessed
by immunocytometric measurements of blood- and
spleen-derived T-cell populations. The frequency of
CD3+CD4+ and CD3+CD8+ T cells was established
throughout the immunization period (Table 1, Fig. 1).
Although there were slight changes of proportions of both
T-cell subsets induced with CPS and CPS-BSA administration, significant expression of the CD4+ portion on
splenic T cells was observed after the second boost with
both formulas (P < 0.05). A significant increase of
CD3+CD4+/CD3+CD8+ T-cell ratio (immunoregulatory
index) (40.2%, P < 0.01) in peripheral blood was detected
after the primary vaccination with CPS-BSA and
remained significant after the two boosters (P < 0.05), in
contrast to the 18.5% (non-significant) increase after the
first administration, followed by a subsequent decline
observed in the CPS-treated group (Fig. 1). A final induction of the immunoregulatory index with CPS-BSA was
more evident in peripheral blood than in spleen (1.3
times higher). By contrast, administration of CPS resulted
in a 1.03-fold higher index in the spleen population compared with blood values.
CD25
To characterize the corresponding early activation of
CD3+CD4+ and CD3+CD8+ cells, the expression of CD25
(IL-2Ra) was measured (Figs 2 and 3). Increased CD25
expression on CD4+ blood-derived T cells (second booster, 5.96-fold, P < 0.001) and CD8+ blood-derived T cells
(primary vaccination, 9.53-fold, P < 0.001) in comparison
with preimmune levels was related to CPS immunization.
The initially enhanced CD25+ expression on CD8+ cells
(P < 0.001) decreased slightly, but remained significant
(P < 0.01). The frequency of CD25+ expression on CD4+
cells was significantly boosted throughout the whole
immunization period, with maximal intensity following
primary vaccination and following the second booster
injection (P < 0.001). Expression of CD25 on both T-cell
subtypes was much greater than that observed in the
placebo group. The frequency of CD25+ cells among the
T cells in spleen after the second booster was four times
higher in comparison with baseline and 2.6 times higher
than in the placebo group. The enhanced CD8+CD25+
FEMS Immunol Med Microbiol 65 (2012) 422–430
Immunomodulation of T cell response with V. cholerae O135 CPS
425
CPS-BSA
CPS
Spleen
2.5
2.0
CD3+CD4+/CD3+CD8+
1.5
1.0
0.5
0.0
4
Preimmune
3rd
**
Blood
Control
*
*
3
2
1
0
1st
Preimmune
2nd
3rd
Control
Immunization
Fig. 1. Changes in CD3+CD4+/CD3+CD8+ ratio throughout immunization.
The expression of CD4+ and CD8+ cell populations was measured on
CD3+ cells of all lymphocytes. The experimental data are expressed as
geometric means of double measurements ± SD. Levels of significance:
**0.001 < P < 0.01, *0.01 < P < 0.05. Differences were considered
significant at 0.01 < P < 0.05.
response was more pronounced in spleen (3.6 times
higher than in blood) compared with the CD4+CD25+
response (ratio spleen/blood = 1.4). The specific CPSBSA-induced CD25+ T cells in blood were observed after
the first application of formula: a 4.2-fold increase of
CD4+CD25+, P < 0.001 vs. preimmune level and 7.6-fold
increase of CD8+CD25+, P < 0.01 vs. preimmune level
(Fig. 1). The spleen/blood ratio of the CD4+CD25+ subpopulation following the second CPS boost was 1.38 and
for CD8+CD25+ was 13.78.
CD11b
The CPS-BSA immunization was associated with profound changes in the expression of lymphocyte adhesive
molecule CD11b following immune activation (Fig. 3).
The level of expression increased significantly with CD4+
T-cell activation in blood, with a peak value after the first
boost (4.5-fold increase vs. pre-immune level and 1.3-fold
increase vs. placebo, P < 0.001). The impact of BSA
immunization was also apparent on splenic T-cell activation. The CD4+CD11b+ T-cell population was boosted
2.2-fold greater compared with preimmune levels and
1.9-fold vs. placebo group (P < 0.01). The CD4+CD11b+
T-cell population was more accelerated in spleen: 3.9folds greater vs. the same population in blood.
A steady upregulation of CD11b on CD4+ T cells in
blood associated with activation was observed also with
CPS administration (Fig. 2), although to a lesser extent
than with CPS-BSA (2.1-fold increase vs. baseline, second
booster, P < 0.01). Expression of CD11b on the CD4+
T-cell subset in spleen was less significant (second booster, P < 0.05). The expression of surface antigen CD11b
was 1.5 times greater than the preimmune level and 1.37
times vs. placebo group.
CD45
Total T-cell stimulation by CPS and CPS-BSA was evaluated based on expression of activation molecule CD45
(B220) on CD3+ T cells in blood and spleen (Figs 2 and 3).
The kinetics of this population in blood increased with
cell activation following CPS-BSA administration, with a
peak response 4.14-fold higher than baseline and 1.97fold higher vs. placebo after the second booster
(P < 0.01). The final booster increased expression of
CD45+ 3.48 times compared with primary vaccination.
The boosted expression pattern was also evaluated in the
splenic CD3+ T-cell population: 2.1 times vs. preimmune
group and 1.85 times vs. placebo, P < 0.05. Only moderate activation was noted after administration of CPS, with
peak value in peripheral blood CD3+ T cells after the first
Table 1. Influence of CPS and CPS-BSA immunization on the distribution of CD3+CD4+ and CD3+CD8+ T cells given as the per cent of all
peripheral blood and splenic lymphocytes
CPS
CPS-BSA
+
+
+
CD3 CD4 cells
Preimmune
Immunization
1st
2nd
3rd
Control
Spleen
70.40 ± 1.3
63 ± 1.4
±
±
±
±
1.1
1.8
1.5
1.1
CD3+CD4+cells
CD3 CD8 cells
Blood
74.14
74.07
72.25
69.70
+
–
–
73 ± 0.95*
65 ± 0.89
Blood
CD3+CD8+cells
Spleen
Blood
Spleen
20.3 ± 2.1
27.3 ± 0.89
70.40 ± 1.3
63 ± 1.4
±
±
±
±
–
–
30.1 ± 0.72
27.9 ± 0.54
76.64
75.47
75.58
69.70
25.35
24.63
27.73
24.3
0.9
1.0
1.1
0.57
±
±
±
±
0.6
1.4
0.9
1.1
–
–
75 ± 0.65*
65 ± 0.89
Blood
Spleen
20.3 ± 2.1
27.3 ± 0.89
±
±
±
±
–
–
29.9 ± 0.72
27.9 ± 0.54
21.94
24.24
22.03
24.3
1.31
1.83
0.57
0.57
Comparisons of vaccinated groups with preimmune reference values were performed by ANOVA. The experimental data are expressed as geometric
means of double measurements ± SD.
Level of significance: *0.01 < P < 0.05. Differences were considered significant at 0.01 < P < 0.05.
FEMS Immunol Med Microbiol 65 (2012) 422–430
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
E. Paulovičová et al.
%
426
8
7
6
5
4
3
2
1
0
6
**
Spleen
*
*
*
Pre-immune
Blood
5
*
Control
3rd
MHCII
**
**
***
3
** **
2
*
1
0
**
1st
Pre-immune
**
*
*
*
*
Control
2nd
3rd
Immunization
%
%
Fig. 2. Patterns of expression of CPS-induced T-cell surface markers.
The expression of CD11b, MHCII, CD45 and CD25 antigens was
measured either on CD3+, CD4+ or CD8+ cells of all lymphocytes. The
experimental data are expressed as geometric means of double
measurements ± SD. Levels of significance: ***0.000 < P < 0.001;
**0.001 < P < 0.01, *0.01 < P < 0.05. Differences were considered
significant at 0.01 < P < 0.05.
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Spleen
CD4/CD11b
CD3/MHCII
CD8/CD25
CD4/CD25
CD3/CD45
***
**
**
*
*
Pre-immune
*
3rd
Pre-immune
4.0 Blood
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
***
***
*
booster (2.1 times vs. pre-immune levels and 1.02 times
vs. placebo group, P < 0.05). Surface expression of CD45
in the splenic CD3+ T-cell population after the second
boost was lower (1.8 times vs. pre-immune level,
P < 0.05) in comparison with a peak value in blood after
the first injection. The second boost revealed more accelerated expression of CD45 (1.8 times vs. preimmune
level, P < 0.05) in splenic lymphocytes in comparison
with that observed in peripheral blood lymphocytes (1.3
times vs. pre-immune level, P < 0.05).
***
***
4
%
CD4/CD11b
CD3/MHCII
CD8/CD25
CD4/CD25
CD3/CD45
**
**
**
**
*
*
1st
**
*
2nd
*
3rd
**
*
*
Control
Immunization
Fig. 3. Patterns of expression of CPS-BSA-induced T-cell surface markers.
The expression of CD11b, MHCII, CD45 and CD25 antigens was measured
either on CD3+, CD4+ or CD8+ cells of all lymphocytes. The experimental
data are expressed as geometric means of double measurements ± SD.
Levels of significance: ***0.000 < P < 0.001; **0.001 < P < 0.01,
*0.01 < P < 0.05. Differences were considered significant at 0.01 <
P < 0.05.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Expression of MHCII on CD3+ T cells associated with
activation and antigen presentation resembles the kinetic
pattern of CD45 T-cell expression (Figs 2 and 3). The
changes of T-cell expression of MHCII surface antigen
throughout the immunization were more significant with
CPS-BSA conjugate formula compared with CPS, especially after the second booster (7.5-fold increase vs. placebo and 102.7% increase in comparison with primary
vaccination); the increase observed with CPS after the
second booster was slightly lower (6.5-fold increase vs.
placebo and 62.5% increase vs. primary vaccination). The
expression of MHCII on splenic CD3+ T cells was
induced to a greater extent with CPS-BSA (2.05 times vs.
preimmune levels, P < 0.05) than with CPS (1.5 times vs.
preimmune levels, P > 0.05, n.s.) immunizations.
CD16/CD32
Expression of the low-affinity Fcc II and III receptors
(CD32 and CD16) resembles that of CD45 and MHCII
antigens, especially with the CPS-BSA formula (Fig. 4).
With CPS-BSA conjugate, expression was increased as a
result of immunisation (131.6% increase after the second
booster vs. primary vaccination, 3.66-fold greater in comparison with preimmune levels). By contrast, expression
of Fcc receptors differed following administration of CPS
formula; expression of low-affinity Fcc II and III receptors declined after booster injections. The peak value was
observed following primary vaccination (twofold vs. preimmune and 1.71-fold vs. placebo). The most significant
increase of Fcc receptors in splenic CD3+ T cells was
revealed with CPS-BSA immunization after the second
boost (3.3 times vs. placebo, P < 0.001). Expression of
Fcc receptors induced with CPS administration was significantly lower (2.6 times vs. placebo, P < 0.05).
Discussion
The immunobiological efficacy of CPS and CPS-BSA on
T-cell responses and, in particular, on upregulation of
FEMS Immunol Med Microbiol 65 (2012) 422–430
Immunomodulation of T cell response with V. cholerae O135 CPS
5
***
Spleen
CPS-BSA
CPS
CD3+/CD16+/CD32+ cells (%)
4
*
3
2
1
0
2,5
3rd
Pre-immune
2,0
Control
**
***
Blood
*
1,5
1,0
0,5
0,0
Pre-immune
1st
2nd
immunization
3rd
Control
Fig. 4. Induced changes of T-cell expression of Fcc II and III receptors
during immunization. Expression of the CD16+/CD32+ cell population
was measured on CD3+ cells of all lymphocytes. The experimental
data are expressed as geometric means of double measurements ±
SD. Levels of significance: ***0.000 < P < 0.001; **0.001 <
P < 0.01, *0.01 < P < 0.05. Differences were considered significant
at 0.01 < P < 0.05.
early and late activation surface antigens CD25, MHCII,
CD11b, CD16/32 and CD45 expression was followed up
on blood- and spleen-derived T cells. While induced
changes in counts of immune effector cells reflect the
extent of immunization, expression of surface antigens
involved in cell activation and antigen presentation illustrates the intensity of antigen specificity. The spleen, a
major peripheral lymphoid organ, containing 25% of all
white blood cells, mainly lymphocytes, is important in
immune responses to all antigens, especially when
encountering an immunogenic moiety for the first time,
irrespective of the mode of presentation. Splenic lymphocytes are characterized predominantly by B-phenotypic
features; the T-cell population represents approximately
35% of all lymphocytes (Hudson & Hay, 1989). The
influence of immunization with CPS and CPS-BSA on
the frequency of T-cell subsets was more profound in the
splenic T-cell population and subtypes in comparison
with blood (Table 1). The trend towards higher average
percentages was more apparent on CD3+CD4+ cells after
the second boost with CPS-BSA (19% increase in spleen,
7.4% in blood) vs. the second boost with CPS (15.8%
increase in spleen and only 2.3% in blood). The data
indicated that antigen-driven changes of phenotype of
splenic T cells are manifested on blood T cells to a lesser
extent.
The prevalence of the CD3+CD4+ T-cell population
corresponds to a central role of the T-helper lymphocyte
subset in regulating the cell-mediated immune response
to bacteria. Moreover, this population reflected the signifFEMS Immunol Med Microbiol 65 (2012) 422–430
427
icant antigen-derived activation, as evidenced by expression of early activation antigen CD25 (Figs 2 and 3).
CD25, a type I transmembrane protein, represents the
alpha chain of the trimeric interleukin-2 receptor,
expressed after triggering of the T-cell receptor. Approximately 10% of peripheral CD4+ cells and less than 1% of
CD8+ cells in healthy, naive adult mice express the CD25
molecule (Sakaguchi et al., 1995). The present results on
upregulation of CD25 expression on CD4+ blood T cells
(P < 0.001) and CD8+ blood T cells (P < 0.01) even after
the first immunization demonstrate strong CPS-BSA-driven activation of T lymphocytes in vivo. Splenic cells also
resembled this pattern. In comparison, when treated with
CPS, the extent of CD4+CD25+ and CD8+CD25+ splenic
cells was significantly lower (P < 0.01 and P < 0.05,
respectively; Fig. 2). The expression of CD25 as a functional marker of T-cell activation occurs on activated T
cells within 2–24 h after stimulation by cytokines and
persists for only a few days after diminution of stimulating antigens (Robb et al., 1981; Poulton et al., 1988).
Evidently, CPS-BSA is a strong inducer especially of
CD3+CD4+ T cells even after the first dose, in contrast to
CPS, where the most profound expression of CD25
antigen was reached only after the repeating boosters,
suggesting the different activation and presentation mode
and B-cell engagement in mutual interactions.
Additionally, to further characterize CD3+CD4+ T-cell
activation, we measured CPS- and CPS-BSA-induced
upregulation of CD11b, the a-chain of the integrin b2
Mac-1, also known as complement receptor 3. CD11b
surface receptor is known to be upregulated in association
with activation and expansion of T-cell clones (Wagner
et al., 2001; Wagner & Hansch, 2006). The increased
prevalence of CD4+CD11b+ cells in peripheral blood and
their recruitment to the infected site in acute bacterial
infection have been reported (Wagner et al., 2008;
Kotsougiani et al., 2010). CD4+CD11b+ T cells were
upregulated by CPS and CPS-BSA vaccination to lesser
degree than CD4+CD25+ T cells. The induction of CD11b
observed in blood T cells was 4.6 times lower after primary vaccination than CD25 upregulation, in contrast to
splenic cells (1.6 times lower). Evidently, CD25 seems to
be more sensitive than CD11b in the context of CPS and
CPS-BSA immunization.
T-cell activation (CD3+ T cells), as a result of CPS and
CPS-BSA immunizations, was characterized according to
expression of MHCII, CD45, R/B220 and CD16/CD32
antigens, also reflecting antigen presentation. MHCII
molecules, crucial surface antigens in initiation of the
immune response, are found on specialized cells: professional antigen-presenting cells. The immunocompetent
cell upregulation of MHCII HLA-DR antigens signifies
their involvement in the induction and regulation of
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
E. Paulovičová et al.
428
cellular immune responses through antigen presentation
(Germain, 1994). Notably, T lymphocytes expressed
MHCII molecules following activation. Simultaneous triggering of CD3 and MHCII antigens leads to an increase
in CD3-mediated T-blast proliferation. Thus, MHCII
molecules on activated T cells represent antigen-presentation molecules, affecting the activity of bystander T
lymphocytes and also capable of providing signals modulating T-cell functions (Barnaba et al., 1994; Pichler &
Wyss-Coray, 1994; Mannie & Walker, 2001). Accelerated
MHCII expression on the CD3+ population, especially
after the second booster injection, was observed with CPS
(62.5% vs. preimmune level) and was more pronounced
with CPS-BSA (87.5% vs. preimmune baseline). Antigendriven MHCII expression on splenic CD3+ T-cell population shows a similar pattern after the second boost:
55.5% (CPS) and 105.55% (CPS-BSA) vs. preimmune
MHCII values. A steady increase of CD3+ MHCII+ T cells
was observed throughout CPS-BSA immunization, with
102.7% increased expression after the second boost as
compared with primary immunization; the resulting
boost with CPS represents only a 1.6% increase. Thus,
CPS-BSA conjugate is evidently presented in a different
manner to CPS.
To evaluate early CD3+ T-cell activation triggered by
CPS and CPS-BSA, expression of the CD45R antigen was
measured. T lymphocytes express multiple forms of leucocyte-common antigen CD45, a type 1 transmembrane
molecule. The various isoforms expressed differentially on
T cells are involved in different stages of development
and activation. Activated murine T cells express the
B-cell-specific CD45R isoform, B220 (Watanabe & Akaike,
1994). The almost 1.4-fold higher expression (P < 0.01)
of CD45R/B220 on CD3+ T cells in blood was triggered
by administration of CPS-BSA formulation in comparison
with CPS (< 1%). Although this antigen is assumed to be
an early activation marker, the frequency of CD25+ and
CD11b+ correlates more closely with the initial phases of
immunization, particularly with CPS-BSA (Figs 2 and 3).
This observation was also evidenced by comparison of
CD45/B220 T-cell expression with the frequency of MHCII
(late activation antigen) on CD3+ T cells.
Regression analysis suggested a strong association
between subsets CD3+CD45+ and CD3+MHCII+ (R2 =
0.92) as a response to CPS-BSA immunization. By contrast,
the CD3+MHCII+ subset was not strongly associated with
the CD3+CD45+ T-cell subset (R2 = 0.09071, n.s.) after
CPS formulation. Evidently, BSA conjugation altered the
T-cell-independent character of CPS toward effective T-cell
dependency. This was confirmed also by the kinetics of
expression of CD45, MHCII and CD25 on blood-derived
CD19+ B cells, with an increasing trend following CPS
immunisation and the highest increase after the second
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
boost [CD19+CD45+ cells (1.14 times vs. placebo),
CD19+MHCII+ cells (1.04 times vs. placebo) and
CD19+CD25+ cells (3.4 times vs. placebo)], suggesting the
clear B-cell activation by CPS. By contrast, the decreasing
tendency of CD19+ B-cell expression of these surface antigens was observed in particular after CPS-BSA immunisation, most notably after the second boost in comparison
with primary vaccination [CD19+CD45+ cells (5.19%
decrease), CD19+MHCII+ cells (14.6% decrease) and
CD19+CD25+ cells (43.1% decrease)]. Results on CD19+
B-cell and CD3+ T-cell activation revealed the pro-B-cell
character of CPS and pro-T-cell character of CPS-BSA, the
different activation and presentation pattern with pure CPS
and its conjugate.
CPS and CPS-BSA immunization triggered expression
of low-affinity IgG receptors Fcc RII (CD32) and Fcc RIII
(CD16) on CD3+ T cells, highlighting the participation of
CPS-BSA on activation and antigen presentation of blood
and splenic T cells (P < 0.01 and P < 0.001, respectively)
(Fig. 4). IgG Fc receptors are expressed on cells of haematopoietic lineages where they regulate and mediate
multiple physiological functions in host defence. The relationship between expression of low-affinity Fcc II and III
receptors and T-lineage development and antigen-responsiveness has been investigated (Engelhardt et al., 1995; de
Andres et al., 1999).
Our observations indicate the immunobiological effectiveness of V. cholerae O135-derived CPS-BSA conjugate
with respect to T-cell-mediated immune responses. The
effectiveness of originally thymus-independent CPS was
efficiently improved by conjugating the polysaccharidic
moiety, as revealed by the high level of expression of
CPS-BSA-induced T-cell surface markers characteristic for
early and late T-cell activation, mainly CD25, CD11b and
CD45. Moreover, our experimental data on MHCII
expression pointed to efficient CPS-BSA T-cell presentation.
The results of this pilot study suggest that both
V. cholerae O135 CPS and CPS-BSA conjugate might be
beneficial as prospective candidates for construction of
subcellular vaccines by taking advantage of the effective
T-cell engagement with respect to the different activation
and presentation modes essential for pharmacokinetics
and pharmacodynamics.
Acknowledgements
This contribution is the result of the project Centre of
Excellence for Glycomics, ITMS 26240120031, supported
by the Research & Development Operational Programme
funded by ERDF. This work was supported by the Slovak
Research and Development Agency under contract no.
APVV-0032-06, by the Grant Agency of Slovak Academy
FEMS Immunol Med Microbiol 65 (2012) 422–430
Immunomodulation of T cell response with V. cholerae O135 CPS
of Sciences VEGA no. 2/0040/10 and by the Centre of
Excellence Glycomed, CE SAS Glycomed. We thank Professor Milan Buc (Department of Immunology, Comenius
University School of Medicine, Bratislava, Slovakia) for
his valuable comments and suggestions.
References
Baker PJ (1992) T cell regulation of the antibody response to
bacterial polysaccharide antigens: an examination of some
general characteristics and their implications. J Infect Dis
165: S44–S48.
Barnaba V, Watts C, deBoer M, Lane P & Lanzavecchia A
(1994) Professional presentation of antigen by activated
human T-cells. Eur J Immunol 24: 71–75.
Chatterjee S, Mondal AK, Begum NA, Roychoudhury S & Das
J (1998) Ordered cloned DNA map of the genome of Vibrio
cholerae 569B and localization of genetic markers. J Bacteriol
180: 901–908.
Chaudhuri K & Chatterjee SN (2009) Cholera Toxins.
Springer-Verlag, Berlin.
Das S & Gupta S (2005) Diversity of Vibrio cholerae strains
isolated in Delhi, India, during 1992–2000. J Health Popul
Nutr 23: 44–51.
de Andres B, Hagen M, Sandor M, Verbeek S, Rokhlin O &
Lynch RG (1999) A regulatory role for Fcc receptors
(CD16 and CD32) in hematopoiesis. Immunol Lett 68:
109–113.
Engelhardt W, Matzke J & Schmidt RE (1995) Activationdependent expression of low affinity IgG receptors Fc
gamma RII(CD32) and Fc gamma RIII(CD16) in
subpopulations of human T lymphocytes. Immunobiology
192: 297–320.
Fields R (1971) The measurement of amino groups in protein
and peptides. Biochem J 124: 581–590.
Germain RN (1994) MHC-dependent antigen processing and
peptide presentation: providing ligands for T-lymphocyte
activation. Cell 76: 287–299.
Goldblatt D (2000) Conjugate vaccines. Clin Exp Immunol 119:
1–3.
Hisatsune K, Yamamoto F & Kondo S (1985) The acid
hydrolysis. Advances in Research on Cholera and Related
Diarrhoeas (Kuwahara S & Pierce NF, eds), pp. 17–24. KTK
Scientific Publishers, Tokyo.
Hudson L & Hay FC (1989) Practical Immunology, 3rd edn.
Blackwell Scientific Publications, Oxford.
Johnson JA, Panigrahi P & Morris JG (1992) Non-O1 Vibrio
cholerae NRT36S produces a polysaccharide capsule that
determines colony morphology, serum resistance, and
virulence in mice. Infect Immun 60: 864–869.
Jones C (2005) Vaccines based on the cell surface
carbohydrates of pathogenic bacteria. An Acad Bras Cienc
77: 293–324.
Kabir S (2005) Cholera vaccines the current status and
problems. Rev Med Microbiol, 16: 101–116.
FEMS Immunol Med Microbiol 65 (2012) 422–430
429
Korcová J, Machová E, Farkaš P & Bystrický S (2010)
Immunomodulative properties of conjugates composed of
detoxified lipopolysaccharide and capsular polysaccharide of
Vibrio cholerae O135 bound to BSA-protein carrier. Biologia
65: 768–775.
Kossaczka Z, Shiloh J, Johnsson V, Taylor DN, Finkelstein RA,
Robbins JB & Szu SC (2000) Vibrio cholerae O139 conjugate
vaccines: synthesis and immunogenicity of Vibrio cholerae
O139 capsular polysaccharide conjugates with recombinant
diphtheria toxin mutant in mice. Infect Immun 68: 5037–
5043.
Kotsougiani D, Pioch MB, Prior V, Heppert G, Hansch M &
Wagner C (2010) Activation of T lymphocytes in response
to persistent bacterial infection: induction of CD11b and of
toll-like receptors on T cells. Int J Inflam, Article 1: 526740–
526750.
Krishna BVS, Patil AB & Chandrasekhar MR (2006)
Fluoroquinolone-resistant Vibrio cholerae isolated during
cholera outbreak in India. Trans R Soc Trop Med Hyg 100:
224–226.
Levine MM (2010) Immunogenicity and efficacy of oral
vaccines in developing countries: lessons from live cholera
vaccines. BMC Biol 8: 129.
Machová E, Bystrický S, Gáliková A & Kogan G (2002)
Preparation of a subcellular conjugate with the
lipopolysaccharide from Vibrio cholerae 01 using b-D-glucan
as matrix. Eur J Med Chem 37: 681–687.
Mannie MD & Walker MR (2001) Feedback activation of Tcell antigen-presenting cells during interactions with T-cell
responders. J Leucoc Biol 70: 252–260.
Mosley WH, Feely JC & Pittman M (1968) The
interrelationships of serological responses in humans, and
the active mouse protection test to cholera vaccine
effectiveness. International Symposium on Enterobacterial
Vaccines. Series on Immunobiological Standards (Regamey
RH, Stanic M & Ungar J, eds), pp. 185–196. Karger, Basel.
Nesper J, Schild S, Lauriano CM, Kraiss A, Klose KE & Reidl J
(2002) Role of Vibrio cholerae O139 surface polysaccharides
in intestinal colonization. Infect Immun 70: 5990–5996.
Paulovičová E, Machová E, Hoštacká A & Bystrický S (2006)
Immunological properties of complex conjugates based on
Vibrio cholerae O1 Ogawa lipopolysaccharide antigen. Clin
Exp Immunol 144: 521–527.
Paulovičová E, Kováčová E & Bystrický S (2010a) Vibrio
cholerae O1 Ogawa detoxified lipopolysaccharide structures
as inducers of cytokines and oxidative species in
macrophages. J Med Microbiol 59: 158–164.
Paulovičová E, Korcová J, Farkaš P & Bystrický S (2010b)
Immunological efficacy of glycoconjugates derived from
Vibrio cholerae O1 serotype Ogawa detoxified LPS in mice. J
Med Microbiol 59: 1440–1448.
Pichler WJ & Wyss-Coray T (1994) T cells as antigenpresenting cells. Immunol Today, 15: 312–315.
Poulton TA, Gallagher A, Potts RC & Beck JS (1988) Changes
in activation markers and cell membrane receptors on
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
430
human peripheral blood T lymphocytes during cell cycle
progression after PHA stimulation. Immunology 64: 419–425.
Provenzano D, Kováč P & Wade WF (2006) The ABCs
(Antibody, B-cells and Carbohydrate epitopes) in cholera
immunity: considerations for an improved vaccine.
Microbiol Immunol 50: 899–927.
Rijkers GT & Mosier DE (1985) Pneumococcal polysaccharides
induce antibody formation by human B lymphocytes in
vitro. J Immunol 135: 1–4.
Robb RJ, Munck A & Smith KA (1981) T cell growth factor
receptors: quantification, specificity, and biological
relevance. J Exp Med 154: 1455–1474.
Sakaguchi S, Sakaguchi N, Asano M, Itoh M & Toda M (1995)
Immunologic self-tolerance maintained by activated T cells
expressing IL-2 receptor alpha-chains (CD25). Breakdown
of a single mechanism of self-tolerance causes various
autoimmune diseases. J Immunol 155: 1151–1164.
Sedmak JJ & Grossbergh SE (1977) A rapid, sensitive and
versatile assay for protein using CBB G-250. Anal Biochem
79: 538–544.
Wagner C & Hansch GM (2006) Receptors for complement
C3 on T-lymphocytes: relics of evolution or functional
molecules? Mol Immunol 43: 22–30.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
E. Paulovičová et al.
Wagner C, Hansch GM, Stegmaier S, Denefleh B, Hug F &
Schoels M (2001) The complement receptor 3, CR3
(CD11b/CD18), on T lymphocytes: activation-dependent
upregulation and regulatory function. Eur J Immunol 31:
1173–1180.
Wagner C, Kotsougiani D, Pioch M, Prior B, Wentzensen A &
Hansch GM (2008) T lymphocytes in acute bacterial
infection: increased prevalence of CD11b+ cells in the
peripheral blood and recruitment to the infected site.
Immunology 125: 503–509.
Waldor MK, Colwell R & Mekalanos JJ (1994) The Vibrio
cholerae O139 serogroup antigen includes an O-antigen
capsule and lipopolysaccharide virulence determinants.
P Natl Acad Sci USA 91: 11388–11392.
Watanabe Y & Akaike T (1994) Activation signal induces the
expression of B-cell specific CD 45R epitope (6B2) on
murine T-cells. Scand J Immunol 39: 419–425.
Weintraub A (2003) Immunology of bacterial polysaccharide
antigens. Carbohydr Res 338: 2539–2547.
FEMS Immunol Med Microbiol 65 (2012) 422–430