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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 247-250, Vol. 5, No. 2
Department of Medical Microbiology and
Immunology1 and
Department of
Infectious Diseases,2 Göteborg University,
Göteborg, Sweden
Received 7 July 1997/Returned for modification 6 October
1997/Accepted 15 December 1997
The possibility that a mucolytic drug, i.e., acetylcysteine, given
orally may enhance the gut mucosal or systemic immune response to an
oral B-subunit-whole-cell (B-WC) cholera vaccine was evaluated for 40 adult Swedish volunteers, and the kinetics of the immune responses were
monitored for responding volunteers. Two doses of vaccine induced
similar frequencies of immunoglobulin A (IgA) and IgG antitoxin
responses (80 to 90%) and vibriocidal titer increases (60 to 65%) in
serum irrespective of whether the vaccine was given alone or together
with 2 g of acetylcysteine. In feces the frequencies of IgA
antitoxin (67%) and antibacterial (33 to 40%) antibody responses were
also comparable in the two immunization groups. Six months after
vaccination, IgA and IgG antitoxin as well as vibriocidal antibody
titer increases in serum could still be detected in approximately 80%
of initially responding vaccinees. Significantly elevated fecal
antitoxin and antibacterial IgA antibody levels were found in,
respectively, 50 and 43% of those volunteers who initially had
responded to the vaccine. Determination of IgA antibodies in feces does
not seem to offer any advantages compared to determination in serum for
assessment of immune responses after immunization with inactivated
cholera vaccine.
Stimulation of the gut mucosal
immune system is most efficiently achieved by antigens applied directly
to the luminal surface of the small intestine (6). Studies
with animals have shown that a number of different compounds exert
adjuvant effects on the intestinal immune response when given orally
together with the antigen (8, 12). The most potent mucosal
adjuvant so far identified is cholera toxin, but its high toxicity
prevents its use in humans (7, 23). Several compounds have
been evaluated in humans with regard to possible adjuvant effects when
given together with various parenteral vaccines (11), but in
the case of oral vaccines the experience with adjuvants in humans is
still limited. Recently, a Lactobacillus strain was shown to
enhance the immune responses to a reassortant live oral rotavirus
vaccine in young children (14). The mucosal adjuvant effect
was explained as facilitation of antigen transport to underlying
lymphoid cells in the intestine.
Mucolytic substances have so far not been evaluated with regard to
their possible adjuvant effects in mucosal immunizations. The proposed
mode of action for such agents would be to enhance the antigen uptake
in the small intestine by affecting the mucus layer. Acetylcysteine is
a mucolytic agent that has been extensively used in humans for
treatment of chronic obstructive pulmonary disease (3). When
administered topically, it exerts rapid mucolytic activity by splitting
the disulfide bonds in mucus molecules (9, 29). Cystic
fibrosis patients with "meconium ileus equivalent" have been
treated with daily doses up to 18 g, and the toxicity has been low
(9).
The aim of the present study was to examine whether acetylcysteine had
any adjuvant effect on the gut mucosal and systemic immune responses to
an oral cholera vaccine, as well as to evaluate whether determination
of specific IgA antibodies in feces could be a reliable method for
assessing and monitoring the kinetics of the intestinal immune
responses in humans after immunization. The cholera vaccine, consisting
of a combination of the purified B subunit of cholera toxin (CTB) and
heat- and formalin-killed O1 whole cells (B-WC), has in large field
trials been shown to be completely safe and to confer protection
against cholera for at least 3 years (5, 28). The antitoxin
and antibacterial antibody responses in feces as well as in sera of
Swedish volunteers given two doses of the oral B-WC vaccine together
with acetylcysteine were compared with the responses found in
volunteers receiving the vaccine alone.
Recombinant B-WC cholera vaccine.
The oral recombinant B-WC
cholera vaccine was produced by the Swedish National Bacteriological
Laboratory (Stockholm, Sweden) as previously described (17).
Each dose of vaccine contained 1 mg of B subunit purified from the
fermentation medium of a Vibrio cholerae O1 strain from
which cholera toxin had been deleted and which harbored a recombinant
plasmid that provides high-level production of CTB (26). The
WC component consisted of 1011 killed O1 vibrios
representing three different cholera strains belonging to Inaba and
Ogawa serotypes and to classical and El Tor biotypes (13,
17).
Assessment of B-WC activity after exposure to
acetylcysteine.
Initial experiments were undertaken to assess the
antigenic contents of the antitoxin and antibacterial components of the B-WC vaccine before and after exposure to different concentrations of
acetylcysteine. One vaccine dose (3 ml) was mixed with 100 ml of a 4%
sodium bicarbonate-1.5% citric acid buffer solution in order to
protect the B-subunit component from stomach acidity (4).
After 10-ml aliquots of the vaccine-buffer solution were dispensed into
flasks, a 200 mg ml
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Kinetics of Local and Systemic Immune Responses to an Oral
Cholera Vaccine Given Alone or Together with Acetylcysteine
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 acetylcysteine solution
(Acetylcystein Tika; Tika Läkemedel AB, Lund, Sweden) was added
at final concentrations of 1, 2, and 5%. The vaccine-buffer solution
alone was used as a control. The B-subunit activity in each aliquot was
determined by a GM1-enzyme-linked immunosorbent assay (ELISA)
(30), and the WC component activity was determined by an
inhibition ELISA method (21), in which V. cholerae O1 lipopolysaccharide (LPS) (3 µg ml1)
was used as the solid-phase antigen and an immunoglobulin M (IgM) mouse
monoclonal antibody against V. cholerae O1 LPS (8:4) (0.25 µg ml1) was used for inhibition.
Study design. Forty healthy adult Swedish persons (26 females and 14 males), aged 23 to 53 years, who had not been vaccinated against cholera previously, volunteered to participate in the study, which was approved by the Research Ethical Committee at the Medical Faculty, Göteborg University. Each volunteer received two oral doses of the recombinant B-WC cholera vaccine, with an interval of 2 weeks. The vaccine was administered in 100 ml of a 4% sodium bicarbonate-1.5% citric acid buffer solution (4). The buffer ingredients were delivered as two effervescent tablets, each consisting of 2,000 mg of sodium bicarbonate, 750 mg of citric acid, and 1,025 mg of lactose (ACO; ACO AB, Stockholm, Sweden) (18). Half of the volunteers were given the B-WC vaccine in buffer alone, and half were given the vaccine in buffer with 2 g of acetylcysteine (Tika Läkemedel AB). The trial was performed in a randomized open manner, since the taste of the acetylcysteine could not be concealed from the volunteers.
Serum and fecal specimens were collected immediately before the first immunization (day 0) and then 9 days after the second vaccine dose. In some instances, serum and fecal specimens were also obtained 180 days (range, 150 to 210 days) after the first immunization (Table 1). Sera were obtained by venous puncture and stored in aliquots at
20°C. Fecal samples were collected and
frozen at 70°C within 2 to 3 h after collection. Fecal
extracts were prepared by mixing 6 g of homogenized feces with 24 ml of a buffer solution containing enzyme inhibitors as described
elsewhere (2, 10). The fecal extracts were stored in
aliquots at 70°C. All analyses were performed within 2 months after
collection.
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Antibody determinations. Serum antitoxin antibody responses of the IgA and IgG classes were determined by the GM1-ELISA method as previously described (31). Levels of antibacterial antibodies in serum were determined by a microtiter vibriocidal assay (24). Threefold (GM1-ELISA) or twofold (vibriocidal test) serial dilutions of pre- and postvaccination specimens were tested in duplicate. The antibody titer ascribed to each sample was the mean from duplicate determinations. A twofold or greater (antitoxin) or a fourfold or greater (vibriocidal) increase in endpoint titer in postvaccination specimens compared to prevaccination specimens was used to signify seroconversion at a P value of <0.05 (15, 18). Confidence intervals (CI) were calculated by using the t distribution in a two-tailed fashion.
Levels of total IgA in fecal specimens were determined by a modified microplate ELISA method (32). Specimens with very low IgA concentrations,
20 µg ml1, or with a
greater-than-10-fold difference in total IgA content between pre- and
postvaccination samples were excluded from further analyses, since our
previous studies have shown that antibody titrations of such specimens
give unreliable results (2).
IgA antibody responses to cholera toxin in fecal extracts were studied
by the GM1-ELISA (31), and antibacterial antibodies were
studied by an ELISA in which the WC component of the B-WC vaccine
(2.5 × 107 bacteria ml1) or
V. cholerae O1 LPS (purified from a classical Inaba
strain, 569B; 3 µg ml1) was used as the solid-phase
antigen. Threefold (antitoxin) or twofold (antibacterial) serial
dilutions of pre- and postvaccination specimens were tested side by
side. The specific IgA antitoxin and antibacterial activities in fecal
specimens were determined by dividing the IgA ELISA antibody titer by
the total IgA concentration (in micrograms per milliliter) of the
sample. Based on previous calculations, a greater-than-twofold increase
in IgA antibody titer/total IgA observed in postimmunization specimens
compared to preimmunization specimens was chosen to signify
seroconversion (1).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Antibody responses in serum.
Prior to immunization, the IgA
and IgG antitoxin and vibriocidal antibody titers in serum were
comparable in volunteers receiving B-WC vaccine alone and those
receiving it together with acetylcysteine. Two doses of vaccine induced
similar frequencies of significant IgA and IgG antitoxin titer
increases in the two immunization groups, and the magnitudes of the
responses were also comparable (Table
2). Thirteen (65%) of the volunteers
receiving the acetylcysteine-vaccine mixture responded with
significant increases in vibriocidal titer; the corresponding figure
for persons receiving vaccine alone was 60% (Table 2). The magnitude
of the vibriocidal response was higher in volunteers given vaccine
alone than in those receiving the acetylcysteine-vaccine mixture, but
the difference was not statistically significant (Student's
t test, unpaired, two-tailed). In both groups the titer
increases tended to be higher in volunteers with low preimmune levels
of vibriocidal antibodies (<1:40) than in those with high
prevaccination titers (
1:40).
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Antibody responses in feces.
Fecal extracts from five (25%)
of the volunteers given B-WC vaccine together with acetylcysteine and
from eight (40%) of those receiving vaccine alone contained 20 µg
of total IgA per ml or showed a greater-than-10-fold difference in
total IgA between pre- and postvaccination specimens and were therefore
excluded from the analyses.
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Kinetics of immune responses. Serum and fecal specimens were obtained approximately 180 days after the first immunization from most of those volunteers who had initially responded to B-WC vaccine in feces (data from the two immunization groups were pooled). In total, the kinetics of the antitoxin immune responses were monitored for 18 volunteers and the kinetics of the antibacterial responses were monitored for 7 individuals.
In serum, elevated IgA and/or IgG antitoxin levels in relation to preimmune titers could still be found 180 days after immunization in 13 of the 18 volunteers (Fig. 1). A substantial decline in the magnitude of the IgA antitoxin titer increases among responders, from 21-fold (95% CI, 11.5 to 40) to 3.5-fold (95% CI, 1.6 to 7.4), was noted at follow-up, whereas the magnitude of the IgG antitoxin titer increases among responders had decreased from 8.1- to 4.1-fold. Also in feces, significantly elevated CTB-specific IgA antibody levels could be demonstrated 180 days after vaccination in 9 (50%) of 18 volunteers (Fig. 1), although the magnitude of the titer increases among responders had decreased from 7.1- to 4.2-fold.
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) and approximately 180 (150 to
210) days (
) after the first immunization. The numbers of volunteers
responding are indicated above the bars.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study does not support the hypothesis that acetylcysteine may promote the immunogenicity of the oral B-WC cholera vaccine in the intestines of Swedish volunteers, as both the frequencies and magnitudes of antitoxin and antibacterial antibody responses in feces and serum were comparable in volunteers receiving vaccine alone and in those receiving vaccine together with 2 g of acetylcysteine. A possible explanation for this inability of acetylcysteine to enhance the immune responses to the vaccine may be that the mucus layer does not prevent the uptake of the vaccine by the antigen-presenting cells. Alternatively, the concentration of the mucolytic agent reaching the small intestine could have been too low to exert any substantial topical action (25), even though the amount of acetylcysteine given was 10 times higher than the normal dose used in patients with chronic bronchitis.
The most reliable method for assessing intestinal immune responses in humans after oral immunization with enteric vaccines has been to determine specific IgA antibodies in intestinal lavage fluid (15). However, the lavage procedure is laborious and cannot be used for evaluations of immune responses in larger immunization groups, e.g., in children and under field conditions. Recently, intestinal antibacterial IgA antibody responses to an oral live auxotrophic Shigella flexneri vaccine, detected in feces, were described (22). Determination of antitoxin as well as antibacterial IgA antibody responses in stool specimens has also been shown to be a reliable proxy measure for intestinal lavage in Swedish volunteers given an oral inactivated enterotoxigenic Escherichia coli vaccine (33). In the present study, significant antitoxin and antibacterial IgA antibody responses in feces were seen in, respectively, 67 and 37% of the volunteers given two doses of B-WC vaccine. The frequencies and magnitudes of the fecal IgA antibody responses were somewhat lower than those recently observed in intestinal lavage fluid after oral immunization with a B-WC cholera vaccine (20).
When the lavage procedure is used to assess intestinal immune responses
after vaccination, approximately 10% of the volunteers have to be
excluded from analyses due to very low levels of total IgA in their
lavage fluids (1, 2). Analyses of fecal specimens are
associated with similar disadvantages. In the present study, 12.5% of
the volunteers had to be excluded due to very low fecal IgA
concentrations (20 mg ml1). Since determination of
fecal IgA immune responses has some advantages compared to analyses of
intestinal lavage fluid, e.g., in the ease of collecting and handling
the specimens, we consider measurement of fecal immune responses
feasible in situations where intestinal lavage is difficult to perform.
The kinetics of intestinal immune responses after oral immunization with B-WC cholera vaccine have been monitored in intestinal lavage fluid only up to 28 days after a second vaccine dose (15, 32). At that time, significant IgA antitoxin responses could still be detected in lavage fluid from two-thirds of the volunteers who initially had responded to the vaccine 7 to 9 days after immunization. Antibacterial IgA titers also remained elevated in most vaccinees. In the present study, we followed the kinetics of the intestinal immune responses for approximately 6 months after vaccination. Fecal antitoxin and antibacterial IgA antibody responses could still be detected in 50 and 43% of initially responding volunteers, respectively, although the magnitudes of the responses were lower than shortly after immunization.
The antitoxin IgA and IgG antibody titer increases and vibriocidal responses in serum are consistent with previous serological findings for Swedish volunteers who have been immunized with B-WC cholera vaccine (18, 20). The observation that individuals with high preimmune levels of vibriocidal antibodies had lower vibriocidal responses than those with low prevaccination titers is in accordance with findings for North American volunteers receiving recombinant B-WC cholera vaccine (27).
In earlier studies the kinetics of immune responses after oral cholera vaccination have been followed up to 5 years by measuring serum IgA and IgG antitoxin and vibriocidal antibodies (16, 19), since these antibodies have been shown to be useful indirect measures of the gut mucosal immune responses (15). Our data showed that 81% of initially responding volunteers still had significantly increased titers of IgA and IgG antitoxin in serum after 6 months, which is in accordance with previous findings for adult Swedish volunteers (16, 19). Vibriocidal antibody titer increases could still be found in most volunteers at follow-up, but due to the small number of subjects followed (five persons), no conclusions could be drawn. Earlier studies have shown a more rapid decrease in vibriocidal titers (16, 19). Six months after vaccination, sustained antitoxin and vibriocidal antibody levels were more often observed in serum than in feces.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Swedish Medical Research Council (16X-09089) and the Swedish Agency for Research Cooperation with Developing Countries (SAREC).
We are grateful to Kerstin Andersson, Ingela Ahlstedt, and Ingrid Högberg for skillful technical assistance and to Lena Widerström for collecting the specimens.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. Phone: 46 (31) 60 46 81. Fax: 46 (31) 82 69 76.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 247-250, Vol. 5, No. 2
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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