CLINICAL AND VACCINE IMMUNOLOGY, Jan. 2010, p.

7379
1556-6811/10/$12.00 doi:10.1128/CVI.00266-09
Copyright 2010, American Society for Microbiology. All Rights Reserved.

Vol. 17, No. 1

Physical and Chemical Characterization and Immunologic

Properties of Salmonella enterica Serovar Typhi Capsular
Polysaccharide-Diphtheria Toxoid Conjugates
Changfa Cui,1* Rodney Carbis,1 So Jung An,1 Hyun Jang,1 Cecil Czerkinsky,1
Shousun C. Szu,2 and John D. Clemens1
International Vaccine Institute, SNU Research Park, San 4-8 Bongcheon-7 Dong, Kwanak-Gu,
Seoul, Korea, 151-919,1 and Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, Maryland 208542
Received 18 June 2009/Returned for modification 14 September 2009/Accepted 21 October 2009

Typhoid fever remains a serious public health problem in developing countries, especially among young
children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed
vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever
for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A
(rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory
precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to
accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic
acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly
higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster
effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated
freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when
added to the infant combination vaccines.
and adults. However, neither vaccine is licensed for routine
immunization of infants (52).
The Vi capsular polysaccharide is both an essential virulence
factor and a protective antigen for S. Typhi (36, 38, 39). The
concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is
not suitable for routine immunization of infants and young
children because of its age-related immunogenicity and T-cell
independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae
(12, 20), Vi covalently bound with protein conferred T-cell
dependence and increased immunogenicity (4850). To date,
diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins
(CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella
pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi
polysaccharide in laboratory studies (16, 17, 32, 4850; personal communications). An improved method was developed
(24), utilizing adipic acid dihydrazide (ADH) as the linker and
Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as
the carrier. Clinical trials of Vi-rEPA conjugates conferred
89% protection in Vietnamese children 2 to 5 years old for 46
months (23, 26, 28). The level of serum IgG anti-Vi induced by
Vi-rEPA conjugates was correlated with prevention of typhoid
fever in these studies (7, 2123, 26, 28).
One limitation of using rEPA as the carrier protein is the
lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by
pharmaceutical companies in China and India were prepared

Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health
problem in Central Asia, Southeast Asia, Africa, and Latin
America (11, 52, 53). It was estimated that more than 21
million cases of typhoid fever and 200,000 deaths occurred in
2000 (10). The treatment of patients and management of
asymptomatic carriers are becoming more difficult due to the
worldwide emergence of multidrug-resistant (MDR) strains (2,
15, 29, 42, 43). Vaccination is considered the most promising
strategy for the control of typhoid fever in developing countries
(11, 19, 52, 53).
Typhoid fever in children younger than 5 years old has often
been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of
less than optimal culture media (35, 46). Several studies have
shown that the incidence of typhoid fever among children less
than 5 years old is similar to that in school age children and
young adults (14, 27, 34, 50, 51).
The 3 licensed typhoid vaccines have limited efficacy, and
none are suitable for young children under 5 years old. The use
of heat-inactivated whole-cell vaccine was suspended in many
countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were
introduced in the late 1980s; both vaccines are well accepted
and confer moderate protection (50 to 70%) in older children

* Corresponding author. Present address: 40 Boniface Drive, Rochester, NY 14620-3334. Phone: (585) 442-5451. Fax: (585) 563-4558.
E-mail: ccui@ivi.int.

Published ahead of print on 4 November 2009.

73

74

CUI ET AL.

(24, 48, 49). Modifications of conjugation procedures were

made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility
of stockpiling in disaster relief.
Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule
using alternating injections of polysaccharide and conjugate.
Priming or boosting effects of polysaccharide on its conjugate
vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no
consistent conclusion about various types of polysaccharides
studied (6, 9, 31, 40, 41). Here, we compared the immune
response of Vi polysaccharide injected before or after the
administration of Vi-DT with the responses of those receiving
2 injections of Vi-DT. We also investigated the dosage effect
for the purpose of better formulation.

MATERIALS AND METHODS

Vi purification and characterization. Vi was extracted and purified from S.
Typhi strain Ty2 (ATCC 19430) as described previously (54). Briefly, the culture
supernatant was mixed with 0.2% hexadecyl-trimethyl-ammonium bromide
(Cetavlon; Sigma Life Science). The precipitate was washed with 0.1 M NaCl and
extracted with 1.0 M NaCl. The supernatant was brought to 25% ethanol and
centrifuged to remove nucleic acids and then was brought to 75% ethanol. The
precipitate was washed with 98% ethanol, freeze-dried, and extracted four times
with buffered phenol at 10C. The water phase of the extraction was precipitated
with 75% ethanol, dialyzed extensively against pyrogen-free deionized water, and
freeze-dried.
The antigenicity of Vi was confirmed by immunodiffusion reaction with burro
Vi hyperimmune serum (provided by NIH) (30). The molecular size of Vi was
estimated by the size exclusion chromatography (SEC) method in a 1.5- by 95-cm
Sepharose CL-4B column (GE Health). The O-acetyl content of Vi was determined by Hestrin assay, using acetylcholine chloride as a standard (50). The
nucleic acid content was determined with UV spectroscopy at a wavelength of
260 nm. The protein concentration was measured by the Coomassie protein assay
(Thermo Scientific), using bovine serum albumin (BSA) as a standard (5). The
gel-clotting method for the Limulus amebocyte lysate (LAL) test was used to
estimate the contamination of lipopolysaccharide (LPS) (Associates of Cape
Code Inc.).
Derivatization of DT. Bulk DT vaccines were provided by the Lanzhou Institute of Biological Products, Peoples Republic of China, for conjugates C1 and
C2 and by Shantha Biotech Inc., India, for conjugates C3, C4, and C5. DT was
concentrated by passing it through Vivaspin 6 centrifugal concentrators (10,000molecular-weight cutoff; Sartorius), and buffer was exchanged for saline in preparation for derivatization.
Ten mg/ml of DT was mixed with ADH (Sigma) at 35 mg/ml. 1-Ethyl-3(3dimethylaminopropyl) carbodiimide (EDAC) (Sigma-Aldrich Fine Chemicals)
was added as powder to a final concentration of 2 mg/ml. The pH was maintained
at 5.6 to 5.8 with 0.1 M HCl for 45 min at room temperature (24, 47). The
reaction mixture was passed through a Vivaspin 6 (MWCO 10K) 5 times to
remove EDAC and unbound ADH. The derivatized DT was designated DTAH.
The level of derivatization was measured by colorimetric reactions. The acid
hydrazide (AH) concentration was determined by reactions with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Fluka) using ADH as a standard (24, 44, 50), and
the protein concentration was measured by Coomassie protein assay. The degree
of derivatization was expressed as a percentage of AH by weight and the molar
ratio of AH to DT.
Conjugation. Vi (3 mg/ml) was dissolved in 70 mM MES (morpholineethanesulfonic acid) buffer (pH 5.5; Sigma Life Science) overnight. EDAC was added
as powder to a final concentration of 10 mM and mixed at room temperature for
5 min. DTAH solution was added dropwise to a final concentration of 3 mg/ml,
and the reaction proceeded at room temperature for 3 hours. The reaction
mixture was passed through Sephacryl S-1000 (2.5 by 95 cm; GE Healthcare) in
0.2 M NaCl. Fractions containing both Vi and DT, with a partition coefficient
(Kav) of 0.40, were pooled and designated Vi-DT, lots C1 to C5. Thimerosal
(Sigma Life Science) solution was added to a final concentration of 0.01%, and
the conjugates were stored at 4C (47, 50).

CLIN. VACCINE IMMUNOL.

The content of Vi in conjugates was determined by Hestrin assay, using Vi as
a standard (24). The DT content was determined by Coomassie protein assay.
Stability tests. The accelerated method was applied for assay of stability (16,
18). Conjugate C3 was stored at 4C, 25C, and 37C for 5 weeks or subjected to
20 cycles of freeze-thaw at 20C. High-pressure liquid chromatography
(HPLC) with an Ohpak SB-806 M HQ column (Shodex, Japan) was used to
estimate the molecular sizes of the conjugates. Dextrans 110, 500, and 2000
(Pharmacia) were used as references.
Immunization. Outbred female mice (CD1; Charles River Laboratories) were
used for all experiments unless otherwise specified. Hyperimmune Vi sera were
raised in 8-week-old mice by multiple subcutaneous (s.c.) injections of formalininactivated S. Typhi (1, 30). DT hyperimmune sera were raised in 8-week-old
mice by multiple s.c. injections of DT with incomplete Freund adjuvant (Pierce
Biotechnology), as described previously (24).
For immunogenicity evaluation, 6-week-old mice (10/group) were injected
twice s.c. at 4-week intervals with 100 l of the immunogen in saline containing
2.5 g of Vi as a conjugate or as a polysaccharide. Blood samples were collected
through eye bleeding 2 weeks after each injection.
Immunogenicity of Vi-DT C1 was compared with that of Vi-rEPA (lot 13106),
a clinical lot prepared at NIH. Following the NIH research protocol, 5- to
6-week-old female mice (Swiss Webster; 10/group) were injected s.c. twice 2
weeks apart with conjugates containing 2.5 g of Vi per injection. The mice were
exsanguinated 7 days after each injection. The negative controls were mice
injected with 100 l saline.
Vi as a priming or boosting vaccine. The effect of Vi on the immunogenicity
of Vi conjugate was evaluated in five groups of mice (6 weeks old; 10/group) by
injecting Vi polysaccharide s.c. at the first or the second dose and was compared
with those receiving 2 doses of conjugate C5. Vi and C5 were injected into mice
in various sequences (two injections 4 weeks apart): Vi/C5, C5/Vi, and C5/C5.
Serum samples were collected via eye bleeding 2 weeks after each injection. The
control groups were those injected with C5 once or Vi alone. Serum samples
were taken at 2 and 6 weeks after the injection.
Dosage study. Three groups of mice (6 weeks old; 10/group) were injected s.c.
with various amounts of C4 containing 2.5 g, 1.25 g, and 0.5 g of Vi,
respectively. Mice were boosted with the same dosage 4 weeks later. Blood
samples were taken 2 weeks after each injection via eye bleeding.
Serology. Double immunodiffusion was performed in 1% agarose in saline.
Serum IgG Vi and DT antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) using Nunc Maxisorp plates as described previously
(13, 48). ELISA data were processed with Program ELISA for Windows (Centers for Disease Control and Prevention) (33). IgG anti-Vi and IgG anti-DT
levels were expressed in ELISA units (EU) using hyperimmune sera as references. International Vaccine Institute (IVI) anti-Vi hyperimmune serum was
calibrated with NIH reference serum (100 EU) and assigned a value of 82 EU
(4750). IVI anti-DT hyperimmune serum was assigned 100 EU.
The results were tabulated as the geometric mean (GM) and 95% confidence
interval (CI).
Statistical evaluation. Antibody levels were expressed as the GM) of EU. Sera
with antibodies below detectable levels were assigned 0.02 EU (one-half of the
lowest detectable level) for the purpose of GM calculation. Statistical significance was calculated using a two-tailed Students t test with a confidence level of
95% (P 0.05).

RESULTS
Vi polysaccharide. The purified Vi met WHO requirements
(in parentheses) for polysaccharide typhoid vaccine: the Oacetyl content was 2.56 mmol/g of Vi (2.0 mmol/g of Vi), and
57.6% of Vi (50%) was eluted from a Sepharose CL-4B
column before the Kav reached 0.25. The purity of the polysaccharide was as follows: nucleic acid content 2.0%
(2.0%), protein content 0.25% (1.0%), and an endotoxin
level 0.3 EU per g of Vi (54).
Characterization of DTAH. The level of derivatization of
DTAH was between 2.9% and 3.5% AH/DT (wt/wt), corresponding to an AH/DT molar ratio of 10 to 12 (Table 1). Based
on the 14% SDS-PAGE pattern, the molecular weight of
DTAH was similar to that of the native DT (data not shown).

VOL. 17, 2010

S. TYPHI Vi-DT CONJUGATE VACCINES

75

TABLE 1. Chemical compositions of S. Typhi Vi-DT conjugates

AH/DT
Conjugate

%
(wt/wt)

Molar
ratiob

C1
C2
C3
C4
C5

3.5
3.1
3.2
2.9
3.4

12
11
11
10
12

Vi concn
(g/ml)

Yielda
(Vi, %)

Vi/DT
ratio
(wt/wt)

Vi repeating
units/DTb

58.05
53.83
47.00
50.77
56.04

42
48
c
44
52

0.80
1.06
0.87
0.91
1.09

198
263
216
226
270

Yield was based on the recovery of Vi.

Molar ratio calculated based on the following: DT, Mr 62,000; Vi repeating unit, Mr 250; ADH, Mr 174.
c
The accidental loss of some material prevented accurate determination of the
yield.
b

Double immunodiffusion with DT antiserum showed a line of

identity between DT and DTAH (Fig. 1).
Chemical compositions of Vi-DT conjugates (Table 1). The
Sephacryl S1000 gel filtration profiles of all five lots of the
conjugate reaction mixtures were similar and were exemplified
by C2 in Fig. 2. One major peak was eluted from the void
volume (Kav 0) and trailed through the rest of the column.
Only the fractions eluted before the Kav 0.4 were pooled as
the final conjugate C2. Since C2 was eluted before Vi and
DTAH, this indicated C2 had a higher molecular weight than
the individual components. The size distribution was further
confirmed by the HPLC analysis (Fig. 3, left). All five conjugates showed similar profiles, with a single major peak at 6.06
min (range, 5.04 to 7.18 min), ahead of both Vi and DT.
The conjugates failed to enter the 14% SDS-polyacrylamide
gel, and no free protein was detected (data not shown).
The Vi/DT ratios of the conjugates ranged from 0.80 to 1.09
by weight, equivalent to 198 to 270 Vi repeating units per DT
molecule. Based on the recovery of Vi, the yield was between
42% and 52% (Table 1).
The conjugates formed a line of identity with Vi when reacting with anti-Vi serum in immunodiffusion (data not
shown). In contrast, none of the conjugates formed visible
precipitation when reacting with anti-DT in immunodiffusion.
Stability test. Conjugate C3 underwent repeated freezethaw cycles 20 times and showed no visible change in solubility
and immunogenicity (data not shown). The HPLC profile of
the treated C3 was identical to that of C3 kept at 4C. In
contrast, C3 stored at 25C or 37C for 5 weeks showed obvious
size reduction, evident from the appearance of trailing in the
HPLC profile. No dissociation of C3 was observed when it was
stored at 20C for the same length of time (Fig. 3, right).

FIG. 1. Double immunodiffusion in 1% agarose. DTAH showed a

line of identity to DT against DT antiserum.

FIG. 2. Gel filtration profiles of Vi, DT, and Vi-DT conjugate (exemplified by C2) through a 2.5- by 95-cm column of Sephacryl S-1000
in 0.2 M NaCl (pH 7.4). Fractions (15 ml/fraction) in which Kav 0.40
were collected as conjugates. Detector wavelength, 280 nm (milliabsorbance unit).

Immunogenicity (Table 2). Vi elicited 0.39 EU (GM) of

serum IgG Vi antibodies. All Vi-DT conjugates elicited levels
of IgG anti-Vi antibodies in mice significantly higher than
those elicited by Vi alone: anti-Vi levels were about 16 and 42
times higher than those in the Vi groups after the first and
second injections, respectively (P 0.001). The IgG Vi antibody levels increased from 6.5 to 11.1 EU after the first injection and to 16.9 to 18.7 EU after the second injection (1st
versus 2nd injections, P 0.05 for all conjugates except C4,
P 0.19). The GM levels of IgG Vi antibodies after the 1st
and 2nd injections were similar among the 5 conjugates (P
0.083 and P 0.69, respectively).
One injection of the conjugates in mice did not elicit detectable IgG anti-DT antibodies. After the second injection, anti-DT antibodies were detected in all groups, with C3 having
the highest level. Since dosages of DT varied in different
groups, no statistical analysis was performed.
Dosage effect (Table 3). As observed in humans injected with
Vi-rEPA, the serum IgG Vi antibody response was related to
the dosage of Vi-DT: the higher the dosage, the higher the
level of antibodies after each of the two injections (7). The
group of mice that received 2.5 g elicited levels of antibodies
significantly higher than in those that received 0.5 g after
each of the two injections (P 0.001). There was a difference
between the groups that received 2.5 g and those that received 1.25 g after the first injection (P 0.05), but there was
no difference following the second injection (P 0.3). IgG
anti-Vi levels were significantly higher in the 1.25-g group
than in the 0.5-g group after either of the two injections (P
0.002 and P 0.003, respectively).
The effects of Vi polysaccharide on the immunogenicity of
Vi-DT (Table 4). After one injection, as observed before,
groups that received C5 had significantly higher anti-Vi levels
than those that received Vi (11.32, 12.95, and 13.20 versus 0.35
and 0.20; P 0.001) (4750). After 2 injections, the C5/C5
group had significantly higher levels of anti-Vi than those injected with either Vi/C5 or C5/Vi (16.88 versus 6. 31 and 5.81;
P 0.001). However, IgG anti-Vi levels were comparable in
the C5 and Vi/C5 groups 2 weeks after C5 injection (11.32
versus 5.81; P 0.12), indicating that previous immunization

76

CUI ET AL.

CLIN. VACCINE IMMUNOL.

FIG. 3. HPLC profiles of Vi-DTs using refractive index detectors (milliabsorbance units). The column was a Shodex OHpak SB-806 HQ (8 by
300 mm), the eluent was 0.2 N saline (pH 7.2), and the elution speed was 1 ml/minute. (Left) All five conjugates showed similar profiles, with a
single major peak at 6.06 min (spanning from 5.04 to 7.18 min, ahead of both Vi and DT). (Right) Conjugate C3 underwent repeated freeze-thaw
cycles 20 times and showed a profile identical to that of C3 kept at 4C. In contrast, C3 stored at 25C or 37C for 5 weeks showed obvious size
reduction. No dissociation of C3 was observed when it was stored at 20C for the same period.

with Vi did not impair or enhance the immune response to

Vi-DT.
There was a slight increase in the IgG anti-Vi level in the
C5/Vi group compared with those that received C5 only, but
the difference was not statistically significant (6.31 versus 2.66;
P 0.05).
Immunogenicity comparison between Vi-rEPA and Vi-DT
(Table 5). The effects of different carriers on immunogenicity
were examined by comparing conjugates C1 and Vi-rEPA in
mice. The polysaccharide/protein molar ratios were similar in
C1 and Vi-rEPA. One week after each of the two injections,
the IgG anti-Vi level induced by Vi-rEPA was higher than that
elicited by C1, but the difference was not statistically significant
(43.99 versus 35.01; P 0.58). Both conjugates induced levels
of IgG anti-Vi statistically higher than those elicited by Vi (P
0.001) (Table 5). The slightly higher response in C1 (35.01 EU)
than those observed in immunogenicity evaluation (18.74 EU)

(Table 2) could be due to the different strains of mice used in

the studies.
DISCUSSION
In this study, Vi was conjugated to DT, a licensed vaccine for
routine infant immunization. Physical and chemical characterization of all 5 lots of Vi-DT showed similar polysaccharide/
protein ratios, yields, and antigenicities. Similar levels of serum
IgG Vi antibodies elicited by all Vi-DT conjugates that were
significantly higher than those elicited by Vi alone were also
observed. These levels were also comparable to those elicited
by Vi-rEPA, a clinical lot prepared at the National Institutes of
Health. These findings demonstrated that the synthesis of Vi
conjugates could be reproduced with another carrier protein in
a different laboratory.
Because dosage-related immunogenicity has been shown for

TABLE 2. Immunogenicity evaluation in mice for S. Typhi Vi-DT conjugatesa

GM of IgG (95% CI) 2 wk after indicated injections
Conjugate

C1
C2
C3
C4
C5
Vi
DT
Saline

Anti-Vi IgG

Anti-DT IgGc

1st

2nd

1st

2nd

8.95 (4.6717.20)
6.45 (3.9510.53)
9.34 (5.5315.79)
11.09 (7.0617.41)
10.24 (6.7315.56)
0.39 (0.240.64)
NDd
0.02 (0.020.02)

18.74 (13.2826.42)
16.85 (10.0928.14)
17.39 (13.6222.20)
17.38 (9.5731.55)
18.05 (12.1426.82)
0.40 (0.151.07)
ND
ND

0.02 (0.020.02)
0.02 (0.020.03)
0.02 (0.020.02)
0.03 (0.020.07)
0.02 (0.020.03)
ND
0.02 (0.020.02)
0.02 (0.020.02)

0.20 (0.070.62)
1.89 (0.734.95)
0.04 (0.020.12)
0.73 (0.281.87)
0.63 (0.142.81)
ND
0.44 (0.210.95)
ND

a
Female mice (6 weeks old; 10/group) were injected s.c. twice, 4 weeks apart, with 2.5 g of Vi as a conjugate or Vi alone per injection. Serum samples were taken
2 weeks after each injection. IgG anti-Vi is expressed as EU with respect to reference Vi and DT sera.
b
All conjugates versus the Vi group (for all injections), P 0.001; within each conjugate group, 1st versus 2nd injection, C1, C2, C3, and C5, P 0.05; C4, P 0.19.
There was no statistical difference among conjugates after the 1st and 2nd injections (P 0.083 and P 0.69, respectively).
c
Conjugates induced anti-DT responses in mice similar to those with DT. Dosages of DT varied in different groups, so no statistical analysis was performed.
d
ND, not done.

VOL. 17, 2010

S. TYPHI Vi-DT CONJUGATE VACCINES

TABLE 3. Comparison of immune responses in mice injected with

various dosages of Vi-DT C4a
Dose

GM of IgG anti-Vi (95% CI), 2 wk after

indicated injectionsb
1st

Vi-DT C4
2.5 g
1.25 g
0.5 g
Saline

24.04 (11.07.52.19)
17.65 (8.8935.04)
4.33 (1.835.88)

0.02 (0.020.02)

NDc

a
Female mice (6 weeks old; 10/group) were injected s.c. twice, 4 weeks apart,
with Vi-DT conjugate C4 at various dosages (2.5 g, 1.25 g, and 0.5 g of Vi
per injection as Vi-DT). Serum samples were collected 2 weeks after each
injection. IgG anti-Vi was assayed by ELISA.
b
2.5 g versus 1.25 g, 13.24 versus 8.81, P 0.05 and 24.04 versus 17.65, P
0.3; 2.5 g versus 0.5 g, 13.24 versus 4.36 and 24.04 versus 4.33, P 0.001; 1.25
g versus 0.5 g, 8.81 versus 4.36, P 0.002 and 17.65 versus 4.33, P 0.003.
c
ND, not done.

other polysaccharide conjugates, such as Haemophilus influenzae type b and S. pneumoniae, here we tested various dosages
of Vi-DT in mice (7, 8, 23, 4850). Within the range of dosages
studied here, the higher the dosage, the higher the level of
anti-Vi elicited. This dosage dependence was also consistent
with results observed in the clinical study of Vi-rEPA in 2- to
5-year-old children (7, 26).
The Vi-DT conjugates did not form a line of precipitation
with the anti-DT serum in immunodiffusion. This could be due
to the distinct physical conformations of the two molecules: the
compact globular conformation of DT as opposed to the long
unstructured form of Vi (Vi molecular mass, 1,000 kDa). It
is probable that Vi exerted shielding effects on the antigenic
sites of DT. However, the immunogenicity of DT was retained,
as evidenced by the induction of anti-DT in mice.
In our study, two different sources of DT were used to
prepare Vi-DT, and there was no significant difference among
the conjugates prepared. Utilizing DT as a carrier protein has
the following advantages: (i) both DT and Vi are licensed
vaccines, and the safety concerns are minimal; (ii) both vaccines are already produced locally in many developing coun-

TABLE 4. Effects of Vi upon immunogenicity of Vi-DT conjugatea

Conjugate

C5
C5/Vi
C5/C5
Vi/C5
Vi
Saline

TABLE 5. Immunogenicity comparison of Vi-DT C1 and clinical

lot of Vi-rEPA (lot 13106)a
Conjugate

2nd

13.24 (9.3618.73)
8.81 (6.8411.35)
4.36 (2.125.11)

GM of anti-Vi IgG (95% CI), 2 wk after

indicated injectionsb
1st

2ndc

11.32 (6.3120.34)d
12.95 (8.3120.16)
13.20 (9.3518.64)
0.35 (0.190.66)
0.20 (0.060.60)
0.02 (0.020.02)

2.66 (1.126.34)e
6.31 (3.2012.46)e
16.88 (14.5519.60)
5.81 (2.8311.94)d
ND
ND

a
Female mice (6 weeks old; 10/group) were injected s.c. twice, 4 weeks apart,
with 2.5 g of Vi alone or as a conjugate per injection. Serum samples were
collected 2 weeks after each injection. For group C5 and group Vi, blood samples
were collected 2 and 6 weeks after the injection.
b
All groups that received 1 or 2 doses of C5 showed significantly higher levels
of IgG anti-Vi than the Vi group (P 0.001).
c
C5/C5 versus C5/Vi and Vi/C5, 16.88 versus 6.31 and 5.81, P 0.001. ND, not
done.
d
C5 versus Vi/C5, 11.32 versus 5.81, P 0.12.
e
C5 versus C5/Vi, 2.66 versus 6.31, P 0.05.

77

GM of anti-Vi IgG (95% CI), 1 wk after

indicated injections
1st

Vi-DT C1
Vi-rEPA
Vic
Saline

2nd
d

ND
0.64 (0.261.56)
0.37 (0.141.03)
0.02 (0.020.02)

35.01 (15.3479.91)b
43.99 (30.0964.32)b
ND
ND

a
Female mice (5 to 6 weeks old; 10/group) were injected s.c. twice, 2 weeks
apart, with 2.5 g of Vi as conjugates or Vi alone per injection. The mice were
exsanguinated 7 days after each injection. The sera were assayed for IgG anti-Vi
by ELISA, using the NIH hyperimmune serum as a reference (100 EU).
b
35.01 versus 43.99, P 0.58.
c
35.01 and 43.99 versus 0.37, P 0.001.
d
ND, not done.

tries, so no new facilities would be required for raw material

preparation, and the cost of production could be lower; (iii)
anti-DT elicited by Vi-DT bears clinical advantages; and (iv)
not introducing a new carrier protein, such as rEPA, will potentially simplify Vi conjugate licensing. Finally, the production of DT is economical due to its unusually high yield from
fermentation.
Recently, a locally produced Vi conjugate using TT as a
carrier protein was licensed in India (45). Several other manufacturers in developing countries have proceeded to perform
clinic trials and are in the process of licensing Vi conjugates
(pesonal communications).
We evaluated the effects of priming and/or boosting of Vi
polysaccharide on conjugates by alternate immunization. Our
results showed that injecting conjugates twice induced the
highest level of anti-Vi; replacing either injection with Vi substantially lowered the level of antibody elicited. However,
priming with Vi did not weaken or enhance the immune response to Vi-DT. Other reasons for such trials were to explore
alternative immunization schedules and formulations. There
were several attempts to study the immunological memory
induced by polysaccharide on the conjugate, or vice versa (3, 4,
6, 9, 31, 40, 41). For certain types of pneumococcus, there were
enhancements of immune response in groups primed with the
conjugate vaccines and benefits to the subsequent injection of
polysaccharide (31, 40). This phenomenon was not obvious in
the cases of meningococcal or Haemophilus influenzae type b
conjugates, where the best immune response still came from
using the conjugate vaccine for the full course of immunization
(3, 4, 6, 9, 41).
Vi-DT and Vi-rEPA elicited similar levels of anti-Vi IgG in
mice. Vi-rEPA was demonstrated to be safe and efficacious in
2- to 5-year-old children in an area of high endemicity for
typhoid fever (23, 26). Since the mouse has been shown to be
a reliable animal model for evaluation of the immune responses of polysaccharide-protein conjugates, we predict that
Vi-DT is as immunogenic and protective as Vi-rEPA. We plan
to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.
ACKNOWLEDGMENTS
We thank Jin Kyung Park of IVI for performing statistical analysis;
Steven Hunt of NIH for skillful assistance; and the Lanzhou Institute

78

CUI ET AL.

CLIN. VACCINE IMMUNOL.

of Bioproducts, Peoples Republic of China, and Shantha Biotech Inc.,

India, for providing diphtheria toxoid. We are grateful to John B.
Robbins and Rachel Schneerson of NIH for their support in technology transfer, scientific discussion, and writing of the paper. We also
thank the members of the Scientific Advisory Group (SAG) of IVI for
constructive comments and suggestions for the project.
The typhoid conjugate vaccine project is supported by the governments of Korea, Kuwait, and Sweden and by the Disease of the Most
Impoverished Program (DOMI). The DOMI program is funded by the
Bill and Melinda Gates Foundation.
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