Journal of Medical Microbiology (2006), 55, 1441–1446 DOI 10.1099/jmm.0.46696-0
Synthesis and characterization of Pseudomonas
aeruginosa alginate–tetanus toxoid conjugate
Nasim Kashef,1 Qorban Behzadian-Nejad,1 Shahin Najar-Peerayeh,1
Kamran Mousavi-Hosseini,2 Mohammad Moazzeni3
and Gholamreza Esmaeeli Djavid4
Correspondence Department of Bacteriology1 and Department of Immunology3, School of Medical Sciences,
Nasim Kashef Tarbiat Modares University, Tehran, Iran
Iranian Blood Transfusion Organization, Research Center, Tehran, Iran
Academic Center for Education, Culture, and Research, Tehran, Iran
Chronic infection with Pseudomonas aeruginosa is the main proven perpetrator of lung function
decline and ultimate mortality in cystic ﬁbrosis (CF) patients. Mucoid strains of this bacterium
elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of
naıve animals elicits opsonic antibodies and leads to clearance of mucoid P. aeruginosa from the
lungs. Alginate was isolated from mucoid P. aeruginosa strain 8821M by repeated ethanol
precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve
immunogenicity, the puriﬁed antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide
(ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The
reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was
composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and
non-pyrogenic, and elicited high titres of alginate-speciﬁc IgG. Antisera raised against the
conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able
to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an
Received 24 April 2006 alginate-based vaccine has signiﬁcant potential to protect against chronic infection with mucoid P.
Accepted 13 July 2006 aeruginosa in the CF host.
INTRODUCTION Immunization with alginate antigen gives rise to antibodies
that have opsonic activity and lead to clearance of mucoid P.
The most common pathogen responsible for the morbidity
aeruginosa from the respiratory tract in mice and rats (Pier
and mortality seen in cystic ﬁbrosis (CF) patients is
et al., 1983, 1990, 1994).
Pseudomonas aeruginosa (Mathee et al., 1999; Koch &
Høiby, 1993). One of the clinically most important features One of the most effective modern technologies applied to
of infection by P. aeruginosa is the tendency of this active vaccination has been the conjugation of surface
bacterium to change to a mucoid phenotype, as a result carbohydrate capsular antigens to carrier proteins to increase
of the production of a polysaccharide known as alginate or their immunogenicity, particularly in young children
mucoid exopolysaccharide (MEP) (Doggett, 1966). In (Lakshman & Finn, 2002; Makela, 2003; Pelton et al.,
pathogenesis, this exopolysaccharide has potential roles as 2003). This converts polysaccharide from a T-cell-indepen-
a mechanism for bacterial adherence, as a barrier to dent to a T-cell-dependent antigen, and elicits a higher
phagocytosis and as a mechanism to neutralize oxygen and boostable immune response in animals (Sood et al.,
radicals (Govan & Deretic, 1996). Alginate also affects 1996). The applicability of this technology to the alginate
leukocyte functions, such as the oxidative burst and of P. aeruginosa has been investigated (Pier, 2005; Cryz
opsonization, and plays an immunomodulatory role via et al., 1991; Theilacker et al., 2003). Cryz et al. (1991)
induction of proinﬂammatory cytokines and suppression of used detoxiﬁed exotoxin A as a carrier protein, whereas
lymphocyte transformation (Pedersen, 1992). Theilacker et al. (2003) used keyhole-limpet haemocyanin
Abbreviations: ADH, adipic acid dihydrazide; CF, cystic ﬁbrosis; EDAC,
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; i.p., intraperitoneal(ly); In the present study, we describe the synthesis and
KLH, keyhole-limpet haemocyanin; MEP, mucoid exopolysaccharide; TT, characterization of alginate–tetanus toxoid (TT) conjugate
tetanus toxoid. using the native non-depolymerized polymer of alginate.
46696 G 2006 SGM Printed in Great Britain 1441
N. Kashef and others
METHODS Pyrogenicity determination. New Zealand White rabbits
(2–2?5 kg each), three in each group, were used. Alginate–TT anti-
Puriﬁcation of alginate. Alginate was puriﬁed from mucoid P. gen was administered intravenously at 1 ml per kg rabbit body
aeruginosa strain 8821M donated by Dr Parviz Owlia (Faculty of weight. Rectal temperatures were recorded at 15 min intervals for
Medicine, Shahed University, Tehran, Iran), as previously described 3 h after challenge.
(Kashef et al., 2005). Brieﬂy, 4 l of modiﬁed Mian’s medium was
inoculated with the test strain and incubated at 37 uC for 72 h. Toxicity test. The lethal effect of alginate–TT conjugate was evalu-
Cultures were stirred with a magnetic bar for 3 to 5 h, and bacterial ated in ﬁve female mice (weight 22 g) and two guinea pigs (weight
cells were removed by centrifugation for 1 h at 17 700 g at 4 uC. 250 g). One human dose (10 mg ml21) of conjugate was admini-
Crude alginate was precipitated from the supernatant by the addi- stered intraperitoneally (i.p.). Animals were observed for 5 days
tion of cold absolute ethanol to a ﬁnal concentration of 80 % (v/v). post-challenge.
The precipitate was collected by centrifugation at 3000 g for 30 min,
washed twice in 80 % (v/v) ethanol and once in 96 % (v/v) ethanol, Stability test. Alginate–TT conjugate was placed at 37 uC for
dialysed against distilled water for 48 h, and lyophilized. Freeze- 1 week, and rerun over the same size-exclusion chromatography
dried crude alginate was redissolved at 2 mg ml21 in PBS, pH 7?2, column. The proﬁles of the protein and polysaccharide were deter-
supplemented with 5 mM MgCl2 and 1 mM CaCl2, and DNase I mined to check stability.
and RNase A were added (each at 100 mg ml21). After overnight Immunization of mice. Female BALB/c mice, 6–8 weeks old, were
incubation at 37 uC, proteinase K was added (100 mg ml21) for 4 h injected i.p. in groups of ﬁve on days 0, 14 and 28 with either puri-
with incubation at 56 uC. Chromatography was performed with a ﬁed alginate or alginate–TT conjugate suspended in PBS. Blood was
Pharmacia XK 16 column of Sephacryl S-400 (1?6630 cm) equili- obtained from the orbital sinus on days 14, 28 and 42. All immuni-
brated in PBS, pH 8?6. Eluted fractions were assayed for uronic acid zations were done without adjuvant. Alginate-speciﬁc IgG was deter-
content, and positive fractions eluting near the void volume of the mined by ELISA.
column were pooled, concentrated, passed through a 0?45 mm pore-
size ﬁlter and stored at 4 uC. ELISA. ELISAs were performed as follows. Microtitre plates were
coated with alginate derived from P. aeruginosa strain 8821M
Chemical analysis of alginate. The puriﬁed antigen was analysed (6 mg ml21 in PBS, pH 7?4), and kept overnight at 4 uC. Between
for uronic acid content by the carbazole-borate assay (Knutson & incubation steps, plates were washed three times with PBS contain-
Jeanes, 1986) with sodium alginate as the standard, for protein ing 0?05 % Tween 20 (PBS-Tw). Individual mouse sera were diluted
by the Bradford assay (Bradford, 1976) with BSA as the standard, in the blocking buffer (1/50) and assayed in triplicate. Incubation
for nucleic acid by A260, and for LPS (endotoxin) by the was performed for 1?5 h at 37 uC. Bound antibodies were allowed to
Limulus amoebocyte lysate assay with Escherichia coli endotoxin as react with a horseradish peroxidase-conjugated goat anti-mouse IgG
the standard. diluted 1 : 8000 as secondary antibody, for 1?5 h at 37 uC. o-
Phenylenediamine dihydrochloride (Sigma; 0?4 mg ml21 in 0?2 M
Protein. TT was obtained from Razi Vaccine and Serum Research
Na2HPO4, 0?1 M citric acid, pH 5) and 10 ml H2O2 was used as sub-
Institute of Iran. TT preparations were concentrated by ultraﬁltration
strate. After 15 min incubation in the dark, the reaction was stopped
with a molecular-size cutoff of 100 000 Da. Chromatography was
by the addition of 0?05 ml H2SO4 (20 %), and A492 was measured.
performed with a Sephacryl S-200 (Pharmacia XK 16) column. The
concentrated TT preparations were applied to the column, which was Opsonophagocytosis assay. Opsonophagocytic killing was deter-
equilibrated with 0?2 M NaCl (ﬂow rate 30 ml h21). Optical densi- mined by using 100 ml heat-inactivated mouse serum diluted 1 : 10,
ties of eluted fractions were measured by spectrophotometry at 100 ml mouse macrophages at 16107 ml21, 100 ml 4 % fresh infant
280 nm. The peak corresponding to a molecular mass of 150 000 Da rabbit serum as a complement source, and 100 ml mucoid P. aerugi-
was pooled and concentrated. The ﬁnal material was passed through nosa 8821M at 16107 ml21. These components were mixed in ster-
a 0?45 mm pore-size ﬁlter and stored at 4 uC. ile microfuge tubes. Control tubes, from which antibody,
complement or macrophages were omitted, and 100 ml RPMI
Derivatization and conjugation of alginate. The alginate was medium/fetal calf serum was substituted, were run with each assay.
derivatized as follows. Alginate (10 mg) and adipic acid dihydrazide For all assays involving mouse sera, pooled serum from members of
(ADH) (0?5 M ﬁnal concentration) were dissolved in 5 ml 0?05 M the respective immunization groups was used. The tubes were held
PBS buffer, pH 7?4, and the pH was adjusted to 5?0 by adding at 37 uC for 90 min with gentle shaking and a 10 ml sample was
0?3 M HCl. After stirring at 4 uC for 4 h, 1-ethyl-3-(3-dimethylami- removed, diluted in saline, and plated for bacterial counts. The
nopropyl)-carbodiimide (EDAC) was added (0?2 M ﬁnal concentra- plates were incubated overnight at 37 uC, and mucoid colonies were
tion), and the reaction mixture was stirred at 4 uC for 18 h while the counted. The percentage kill was calculated as follows:
pH was maintained between 4?9 and 5?1. The reaction mixture was
dialysed exhaustively against distilled H2O at 4 uC. Percentage kill=[12(c.f.u. of immune serum at 90 min/c.f.u. of
preimmune serum at 90 min)]6100.
A total of 10 mg TT was added to the adipic hydrazide (AH) derivative
of alginate, and coupling was done with 0?1 M EDAC for 1 h at room Active protection. Mice were divided into three groups, A–C, each
temperature and 24 h at 4 uC. The reaction mixture was passed through containing six mice. Groups A and B were immunized i.p. three
a Sepharose CL-4B column with PBS, pH 7?4, used as running buffer times (on days 0, 7 and 14) with 4 mg MEP–TT and MEP (in 0?1 ml
(ﬂow rate 30 ml h21). Void-volume fractions that assayed positive for PBS, pH 7?4), respectively. Group C contained six unimmunized
both protein and uronic acid were designated polysaccharide–protein control mice. Two weeks after the last immunization, mice were
conjugate and were pooled, passed through a 0?45 mm pore-size ﬁlter challenged i.p. with 36108 c.f.u. (46 LD50) of the heterologous
and stored at 4 uC. strain of mucoid P. aeruginosa suspended in sterile PBS, pH 7?4; the
inoculum was given in a volume of 1 ml. Mice were observed for
Chemical analysis of alginate–TT conjugate. The amounts of 7 days, and mortality was recorded.
protein and uronic acid present in the conjugate were quantiﬁed by
the Folin–Lowry assay with BSA as the standard (Lowry et al., Statistical analysis. The statistical analysis was performed using
1951), and the carbazole-borate assay with sodium alginate as the SPSS version 11.5 (SPSS). Differences in the mean ELISA absorbance
standard, respectively. and the mean percentage of opsonic killing were compared by analysis
1442 Journal of Medical Microbiology 55
P. aeruginosa alginate–tetanus toxoid conjugate
of variance (ANOVA) by using a post hoc multiple comparison as conformational epitopes of bacterial polysaccharides are
(Bonferroni correction) test. The chi square test was used to analyse often stabilized by polymer length, which can be destroyed
the survival data from the protection experiment. P¡0?05 was con-
by depolymerization (Watson et al., 1992). Another alginate
conjugate vaccine was synthesized and evaluated by
Theilacker et al. (2003), who bound thiolated alginate to
RESULTS AND DISCUSSION KLH by using succinimidyl-4-(N-maleimidomethyl)cyclo-
hexane-1-carboxylate (SMCC) as a linker. Using this
Characterization of alginate technology, they were able to construct a water-soluble
conjugate of native, large-molecular-weight alginate. A
The puriﬁcation of the alginate produced by P. aeruginosa
potential disadvantage of the chosen carrier protein and
should ensure removal or inactivation, or both, of other
conjugation chemistry is that neither KLH nor the cross-
immunogenic or biologically active substances, such as
linker SMCC have yet been used in bacterial polysaccharide
proteins, toxins and LPS. At the same time, puriﬁcation
conjugate vaccines injected into humans.
should not result in any signiﬁcant change in the structure
and properties of the alginate. We used TT as the carrier protein because in practice, only a
On a Sephacryl S-400 column, sodium alginate eluted at a handful of proteins of bacterial origin, such as TT and
lower volume than puriﬁed alginate, indicating a somewhat diphtheria toxoid, have been used for the preparation of the
larger molecular size. Chemical analysis showed that the conjugate vaccines that have been licensed for human use or
puriﬁed antigen contained 91?6 % (w/w) uronic acid, are currently under development (Sood et al., 1996). The TT
<7?6 % protein, <0?0061 % LPS, and 0?7 % nucleic acid. molecule (Mr 150 000) is a more effective carrier protein
than diphtheria toxoid (Mr 58 000), perhaps owing to its size
Only the large-size polymer fractions of alginate were (Watson et al., 1992). However, its use as a universal carrier
collected, because it has been shown that only the highest- may prove to be undesirable, as its frequent use may
molecular-size polymers of alginate are able to induce overload the immune system with large doses of this protein
opsonic antibodies in mice with pre-existing levels of non- in combined vaccines, and therefore result in a higher
opsonic antibodies (Pedersen & Kharazmi, 1990). Non- frequency of adverse reactions due to pre-existing anti-
opsonic antibodies are frequently seen in healthy individuals bodies in targeted populations (Peters et al., 1991; Barington
as well as in most patients with CF, even before the onset of et al., 1994). Overall, diversifying our carrier-protein pool
detectable infection (Pedersen & Kharazmi, 1990). may prove to be crucial for the development of human
Efﬁciency of coupling reaction
Our initial attempts to conjugate native, non-depolymer-
The conjugation of capsular polysaccharide (CP) or other ized alginate to TT were not successful. We found that ADH
bacterial polysaccharide-based vaccines to a carrier protein coupling via EDAC had to be repeated once or twice to
is a well-established approach to increase the immunogeni- obtain sufﬁcient ADH bound to the alginate, or alternatively
city of the former (Fattom et al., 1995). Because of the very that the reaction time of ADH, alginate and EDAC needed to
large molecular mass of alginate, conjugating it to carrier be increased.
proteins to produce immunogenic vaccines has proven
difﬁcult. Cryz et al. (1991) constructed a conjugate vaccine In preliminary studies, the concentration of the reactants
of depolymerized alginate with exotoxin A of P. aeruginosa. during the coupling reaction, and the reaction time,
This approach, however, has some potential disadvantages, inﬂuenced the yield of conjugate. The coupling reactions
Fig. 1. Elution proﬁle of alginate–TT conju-
gate from a Sepharose CL-4B column. Solid
line, A525; dotted line, A720.
N. Kashef and others
Serum antibody response to native and
Puriﬁed alginate failed to induce a signiﬁcant rise in IgG
antibodies to alginate antigen (Fig. 2). The ﬁrst immuniza-
tion with alginate alone elicited low levels of IgG antibody,
but this did not increase signiﬁcantly after second and third
immunizations (P=0?214 and P=0?133, respectively). The
ﬁrst immunization with conjugate also induced low levels of
IgG. No signiﬁcant rise in alginate-speciﬁc IgG was observed
14 days after the second vaccine dose (P=0?963), but a
third immunization boosted antibody levels signiﬁcantly
(P<0?0001). As shown in Table 1, immunization with the
alginate–TT conjugate also induced IgG against the carrier
protein. The second immunization with conjugate induced
Fig. 2. Induction of anti-alginate IgG in BALB/c mice over a high levels of TT-speciﬁc IgG compared to the ﬁrst
period of 42 days after the beginning of immunization with
immunization (P=0?007), but no signiﬁcant rise was
either alginate–TT conjugate or alginate alone. Bars represent
observed 14 days after the third immunization (P=0?10).
the mean ELISA absorbance, and error bars indicate the range.
Native alginate therefore acted as a typical T-cell-indepen-
White bars, day 14; grey bars, day 28; black bars, day 42.
dent antigen, whereas a T-cell-dependent response was
observed with the conjugate.
Induction of IgG against the components of the alginate–TT
were not effective at ADH and EDAC concentrations lower conjugate over a period of 42 days is shown in Fig. 3. Mice
than 0?5 and 0?2 M, respectively, and optimal results were immunized with conjugate not only elicited IgG against the
obtained with concentrations of TT and alginate of two components of the conjugate, but also induced
10 mg ml21. There was a progressive increase in the signiﬁcant levels of IgG antibodies to the complete vaccine.
efﬁciency of the coupling reaction with time (data not A considerable rise in alginate–TT-speciﬁc IgG was observed
shown). after the second and third vaccine dose (P<0?0001).
Characterization of conjugate Opsonic activity of mouse sera against mucoid
Gel ﬁltration of the alginate–TT conjugate on a Sepharose
CL-4B column yielded a large peak at the void volume High levels of opsonic antibodies to alginate correlate with
consisting of both protein and polysaccharide, one protein clearance of mucoid P. aeruginosa from the lung in rodent
peak at higher elution volumes, and two minor peaks models of chronic infection (Pier et al., 1990). We therefore
positive for uronic acid (Fig. 1). Since native alginate elutes compared the potential of native and conjugated alginate to
from this resin at higher volumes, only fractions eluting at induce opsonic antibodies speciﬁc for alginate. The
and just past the void volume were presumed to be free of experiment was repeated three times and the results are
non-conjugated polysaccharide, and these represented shown in Fig. 4. In the opsonophagocytic killing assay,
alginate–TT conjugate containing 74?6 % protein and antisera from mice immunized with the conjugate mediated
25?4 % uronate. There were no overt signs of toxicity or phagocytic killing (86?3 %) against the vaccine strain on day
pyrogenicity after i.p. or intravenous administration 42, but antiserum from mice primed with native alginate
(respectively) of the conjugate vaccine to animals. The was less efﬁcient in mediating phagocytic killing (68?5 %).
elution proﬁle of the conjugate did not alter on storage at There was no phagocytic killing when antibody, comple-
37 uC. ment or macrophages were omitted.
Table 1. Antibody responses of mice immunized with alginate–TT conjugate or alginate alone
BALB/c mice were immunized with two candidate vaccines, as indicated, i.p. in PBS. Values show the mean±SEM of A492.
Immunogen Target antigen Isotype Day 14 Day 28 Day 42
Alginate–TT conjugate Alginate–TT IgG 0?357±0?03 0?770±0?14 0?888±0?05
Alginate IgG 0?226±0?07 0?248±0?10 0?725±0?30
TT IgG 0?368±0?04 0?628±0?08 0?798±0?04
Alginate Alginate IgG 0?268±0?04 0?349±0?17 0?258±0?04
1444 Journal of Medical Microbiology 55
P. aeruginosa alginate–tetanus toxoid conjugate
Fig. 3. Induction of anti-TT, anti-alginate
and anti-conjugate IgG in BALB/c mice over
a period of 42 days after immunization with
alginate–TT conjugate. Bars represent the
mean of ELISA absorbance, and error bars
indicate the range. White bars, day 14; grey
bars, day 28; black bars, day 42.
Although IgG titres determined by ELISA are useful to Active protection
screen sera for serologic responses, the levels of opsonic
Three immunizations of mice with 4?0 mg conjugate per
antibodies are the best predictor of protective efﬁcacy in
dose showed signiﬁcant protection (P<0?01) against
animal models (Pier et al., 1990), and are associated with the
intreperitoneal challenge with 46 LD50 of wild-type
resistance of CF patients to chronic mucoid P. aeruginosa
mucoid P. aeruginosa. This challenge dose killed 6/6 of
infection (Pier et al., 1983). In the opsonophagocytic killing
mice that were uninoculated, 1/6 of mice immunized with
assay, alginate conjugated to TT was superior to the native
conjugate and 3/6 of mice immunized with alginate alone.
polysaccharide in its ability to induce opsonic antibodies in
There was no signiﬁcant difference in the rates of survival of
mice. In contrast to the result found here, Theilacker et al.
mice immunized with alginate and those of uninoculated
(2003) did not report the induction of opsonic antibodies in
mice after their immunization with native alginate. A
possible explanation for these conﬂicting results may be the A key feature of any vaccine is its ability to protect against
different composition of the alginate preparations used in infection with strains of the organism heterologous to the
the two studies: the alginate antigens differed in Kd, the ratio one from which the vaccine was derived. For alginate, with
of mannuronic acid to guluronic acid, and acetate content its considerable variation in the ratio of mannuronic to
(Garner et al., 1990). guluronic acid and degree of O-acetylation, it is crucial to
establish that vaccination with a single preparation induces
antibodies reactive against heterologous mucoid P. aerugi-
nosa strains (Sherbrock-Cox et al., 1984). Our data suggest
that immunization of mice with the alginate–TT conjugate
results in an increased LD50 after heterologous-type
challenge, and IgG induced by the conjugate was cross-
reactive to the heterologous mucoid strain. In contrast to
our results, in another study, cross-reactive IgG was virtually
absent after immunization of rats with an alginate–exotoxin
A conjugate (Johansen et al., 1994, 1995). These data suggest
that extensive depolymerization and/or de-O-acetylation
may lead to the loss of epitopes shared between heterologous
In conclusion, conjugation of alginate to TT utilizing ADH
coupling via EDAC yielded an alginate-based conjugate rich
in protein and uronate, which was non-toxic, non-
Fig. 4. Opsonophagocytic killing of P. aeruginosa strain pyrogenic, and elicited high titres of alginate-speciﬁc IgG.
8821M by serum from BALB/c mice after immunization with Antisera raised against the conjugate had high opsonic
either alginate–TT conjugate or native alginate. Bars represent activity against the vaccine strain. The alginate conjugate was
the mean percentage killing of three replicates relative to the also able to protect mice against a lethal dose of mucoid P.
respective preimmune serum, and error bars represent SEM. aeruginosa. These data indicate that an alginate-based
White bars, day 14; grey bars, day 28; black bars, day 42. vaccine has signiﬁcant potential to protect against chronic
*P<0?01; **P<0?001. infection with mucoid strains of P. aeruginosa in the CF host.
N. Kashef and others
ACKNOWLEDGEMENTS Koch, C. & Høiby, N. (1993). Pathogenesis of cystic ﬁbrosis. Lancet
Special thanks to Dr Gerald B. Pier (Professor of Medicine, Lakshman, R. & Finn, A. (2002). Meningococcal serogroup C
Microbiology and Molecular Genetics, Harvard Medical School) for conjugate vaccine. Expert Opin Biol Ther 2, 87–96.
his invaluable guidance. Support was provided by the Department of
Lowry, O. H., Rousebrough, N. J., Farr, A. L. & Randall, R. J. (1951).
Bacteriology, School of Medical Sciences, Tarbiat Modares University.
The technical expertise was donated by the Iranian Blood Transfusion Protein measurement with Folin phenol reagent. J Biol Chem 193,
Organization, Research Center. The experiments comply with the 265–275.
current laws of the country in which they were performed. Makela, P. H. (2003). Conjugate vaccines – a breakthrough in
vaccine development. Southeast Asian J Trop Med Public Health 34,
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