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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 652-657, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.652-657.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Oral Administration of Influenza Vaccine in
Combination with the Adjuvants LT-K63 and LT-R72 Induces Potent Immune
Responses Comparable to or Stronger than Traditional
Intramuscular Immunization
John D.
Barackman,*
Gary
Ott,
Samuel
Pine, and
Derek T.
O'Hagan
Chiron Corporation, Emeryville, California
94608
Received 1 December 2000/Returned for modification 16 January
2001/Accepted 19 March 2001
 |
ABSTRACT |
Mucosal immunization strategies are actively being pursued in the
hopes of improving the efficacy of vaccines against the influenza
virus. Our group investigated the oral immunization of mice via
intragastric gavage with influenza hemagglutinin (HA) combined with
mutant Escherichia coli heat-labile enterotoxins K63
(LT-K63) and R72 (LT-R72). These oral immunizations resulted in potent
serum antibody and HA inhibition titers, in some cases stronger than
those obtained with traditional intramuscular administration, in
addition to HA-specific immunoglobulin A in the saliva and nasal
secretions. This study demonstrates that it may be possible to develop
effective oral influenza vaccines.
| |
TEXT |
Influenza is a serious human disease
exhibiting high mortality in vulnerable populations such as the very
young and the very old, as well as causing significant morbidity in the
general population (17). The social and economic costs
associated with yearly influenza outbreaks are high (7).
Formalin-inactivated whole-virus and split-virus vaccines administered
intramuscularly (i.m.) are commercially available to control the spread
and severity of influenza (15, 38). These prophylactic
vaccines, although important agents in controlling influenza, suffer
from a number of shortcomings that limit their efficacy and
acceptability. Notably, inactivated whole-virus and split-virus
vaccines are known to activate CD8+ cytotoxic T-lymphocyte
responses only sporadically, have poor cross-reactivity to antigenic
variants, and produce poor secretory immunoglobulin A (IgA) responses
(4, 7, 17, 24, 34, 36). In addition, injection site
reactogenicity and weak immune responses can be a problem in very young
children (18, 19). Significant efforts are currently being
pursued to improve the vaccines' efficacy and tolerability primarily
through the development of mucosally active influenza vaccines
(2, 7, 10, 33, 40). Oral immunization is considered by
many to be a highly desirable form of vaccination, although numerous
obstacles make oral immunization using subunit antigens a significant
challenge (3, 6, 11). Many approaches have been
investigated to develop viable orally active influenza vaccines
(3, 21, 29, 30). Mucosal adjuvants, primarily
Escherichia coli heat-labile enterotoxin (LT) and cholera
toxin (CT), are the most commonly employed vaccine enhancers (11,
12). Although potent mucosal adjuvants, LT and CT are toxic in
humans at doses useful for adjuvanticity due to their
ADP-ribosyltransferase activity (28). The nontoxic B
subunit of CT (CTB) has also been investigated; however, studies have
indicated that small amounts of the whole CT are required for
sufficient adjuvant potency, inhibiting the potential of CTB in humans
(44, 45, 46). Our group has investigated the mutant LT
toxins LT-K63 and LT-R72, which demonstrate extremely low (LT-R72) to
undetectable (LT-K63) levels of ADP-ribosyltransferase activity yet
maintain potent mucosal adjuvant activity, demonstrating that ADP-ribosyltransferase activity may not be linked to the adjuvant activity (2, 13, 16). In this study, the influenza
hemagglutinin (HA) antigens A/Beijing8-9/93 HA and A/Johannesburg/97 HA
were administered orally in mice with LT-K63 and LT-R72 and the results were compared to those obtained with i.m. immunization for induction of
serum antibody and mucosal IgA responses as well as serum HA inhibition
titers. Dosing studies were conducted to determine the optimum dose
levels of both antigen and adjuvant.
Vaccines used.
Purified monovalent A/Beijing8-9/93 (H3N2) and
A/Johannesburg/97 (H1N1) split-virus influenza antigens were provided
by Chiron Vaccines, Siena, Italy. Dosing was based on HA content as
assayed by single radial immunodiffusion as described previously
(25). LT-K63 and LT-R72 were prepared as described
previously (35). Wild-type LT (wtLT) was obtained from
Sigma (Escherichia coli heat-labile enterotoxin, lyophilized
powder; Sigma-Aldrich, St. Louis, Mo.). All immunogen preparations were
formulated in phosphate-buffered saline. Immunogens prepared for
intragastric gavage (i.g.) administration included 1.5% (wt/vol)
sodium bicarbonate.
Immunization and sample collection.
Groups of 10 female BALB/c
mice (Charles River Labs, Wilmington, Mass.), 6 to 10 weeks old, were
i.m. or i.g. immunized at days 0, 21, and 35 using immunogen
preparations as described below. Mice were fasted 12 h prior to each
immunization to minimize the possibility of lectins (or other agents)
in the feed from inhibiting uptake of the orally delivered immunogens
(9). Immunizations were made either by i.m. injection (50 µl) into the posterior thigh muscle or by direct i.g. (200 µl) into
the stomach using a 20-gauge stainless steel feeding needle attached to
a 1-ml syringe. Animals were not anesthetized during immunizations.
Serum, saliva wash (SW), and nasal wash (NW) samples were collected
from individual animals 2 weeks after the final immunization (day 49)
using methods described previously (47).
Antibody ELISA.
Serum samples from individual animals were
assayed for total anti-HA Ig (IgG plus IgA plus IgM) titers by a
3,3',5,5'-tetramethylbenzidine-based colorimetric enzyme-linked
immunosorbent assay (ELISA) as previously described, with
A/Beijing8-9/93 or A/Johannesburg/97 as appropriate as coating antigen
(20). A490 was measured using a
standard ELISA reader. The titers represent reciprocal serum dilutions giving an A490 of 0.5 and were normalized to a
serum standard assayed in parallel. SW and NW samples from individual
animals were assayed for HA-specific IgA titers using a bioluminescence immunosorbent assay as previously described, with A/Beijing8-9/93 or
A/Johannesburg/97 as appropriate as coating antigen (47). The goat anti-mouse IgA biotin conjugate (EY Labs, San Mateo, Calif.)
used was presaturated with purified mouse IgG (Sigma Chemical Company,
St. Louis, Mo.) to reduce cross-reactivity. Quantitation was based on
the number of relative light units representing total luminescence
integrated over 3 s (arbitrary units). Titers represent log
dilution values linearly extrapolated from the log of the relative
light units to a cutoff value at least 2 standard deviations above mean background.
HI assay.
Serum samples pooled by group were assayed for
hemagglutination inhibition (HI) titer by the Viral and Rickettsial
Disease Laboratory (Department of Health Services, Berkeley, Calif.). The HI assay is based on the ability of sample sera to inhibit the
agglutination of goat erythrocytes in the presence of HA antigen. The
resulting titers are expressed as the reciprocal dilution required for
complete inhibition (22, 23).
Statistics.
Log anti-A/Beijing8-9/93 and
anti-A/Johannesburg/97 HA serum Ig, saliva IgA, and nasal IgA titers
from individual animals were analyzed for differences between test
groups using a Fisher least-significant-difference procedure using a
statistical significance of >5% (P 
0.05) as the
cutoff interval (1). Additionally, the resulting data were
graphically represented as mean titers ± standard errors (SE) in
the usual manner.
Effects of enterotoxin types and doses on antibody responses after
i.g. immunization.
A dose-ranging study was conducted to determine
the dose-response relationship for LT-K63 and LT-R72 for i.g.
immunization with A/Beijing8-9/93 HA. Groups of 10 mice were immunized
by the i.g. route with 20 µg of A/Beijing8-9/93 HA in combination
with three dose levels of wtLT (1, 10, and 25 µg), LT-K63 (1, 10, and 100 µg), and LT-R72 (1, 10, and 100 µg). Significant mortality in
the mice subjected to high doses of wtLT limited the upper dose of wtLT
to 25 µg. An unadjuvanted A/Beijing8-9/93 HA control group (HA only)
was immunized by the i.g. route at the highest dose level (20 µg) for
comparison purposes.
Serum antibody responses (Fig. 1) were
significantly higher in most cases in animals that received
A/Beijing8-9/93 HA in combination with either of the enterotoxins
tested than in animals that received unadjuvanted A/Beijing8-9/93 HA. A
dose response was not clearly demonstrated, although, in general,
groups that received A/Beijing8-9/93 HA in combination with LT-R72
showed serum antibody responses comparable to those of groups receiving
A/Beijing8-9/93 HA in combination with wtLT.
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FIG. 1.
Comparison of the effects of enterotoxin doses on
antigen-specific serum antibody responses after i.g. administration.
Shown are means ± SE of anti-A/Beijing8-9/93 HA antibody titers
in the sera of mice immunized with 20-µg doses of A/Beijing8-9/93 HA
antigen either alone (HA only) or in combination with wtLT, LT-K63, and
LT-R72 as indicated. Asterisks indicate groups whose values are
significantly greater than that of the HA only group (P 0.05).
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A clearer adjuvant dose response was found in the antigen-specific
saliva IgA responses (Fig.
2).
Significantly stronger saliva
IgA responses were demonstrated for all
but one of the LT-K63-
and LT-R72-adjuvanted groups than for animals
that received A/Beijing8-9/93
HA alone. Additionally, animals dosed
i.g. with 20 µg of A/Beijing8-9/93
HA in combination with 100 µg of
LT-R72 were found to have an
antigen-specific saliva IgA response
significantly higher (
P 0.05) than that of animals
dosed i.g. with either 10 or 25 µg
of wtLT.
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FIG. 2.
Comparison of the effects of enterotoxin doses on
antigen-specific SW IgA responses after i.g. administration. Shown are
means ± SE of anti-A/Beijing8-9/93 HA SW IgA antibody titers of
groups of mice immunized with 20-µg doses of A/Beijing8-9/93 HA
antigen either alone (HA only) or in combination with enterotoxins as
indicated. Asterisks indicate groups whose values are significantly
greater than that of the HA only group (P 0.05).
Double asterisks indicate a value that is significantly greater than
that of the groups immunized with 10 and 25 µg of wtLT (groups 3 and
4), in addition to that of the HA only group (P 0.05).
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Effects of A/Beijing8-9/93 HA dose on antibody responses at two
dose levels of LT-R72.
A second dose-ranging study was conducted
to determine the optimum dose of A/Beijing8-9/93 HA for i.g.
immunization when adjuvanted with LT-R72. Groups of 10 mice were
immunized by the i.g. route with three dose levels of A/Beijing8-9/93
HA (1, 5, and 20 µg) in combination with either 10 or 100 µg of
LT-R72. An unadjuvanted A/Beijing8-9/93 HA control group (HA only) was
immunized at the highest dose level (20 µg) for comparison purposes.
The antigen-specific serum antibody responses (Fig.
3) demonstrated a dose-response trend
with respect to the dose level of
A/Beijing8-9/93 and the dose level of
LT-R72 with which the animals
were immunized. Serum antibody responses
were significantly higher
(
P 0.05) in animals
immunized i.g. with 20-µg doses of A/Beijing8-9/93
HA in combination
with either 10 or 100 µg of LT-R72 than in the
unadjuvanted HA
control group. The group that received the highest
dose level tested
(20 µg of A/Beijing8-9/93 in combination with
100 µg of LT-R72) had
a significantly higher (
P 0.05) antigen-specific
serum antibody response than those of all other groups tested.
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FIG. 3.
Comparison of the effects of HA doses on
antigen-specific serum antibody responses after i.g. administration.
Shown are means ± SE of anti-A/Beijing8-9/93 HA antibody titers
in the sera of mice immunized either with 20 µg of HA administered
alone (HA only) or with 1-, 5-, or 20-µg doses of A/Beijing8-9/93 HA
in combination with either 10 or 100 µg of LT-R72 as indicated.
Asterisks indicate groups whose values are significantly different from
that of the HA only group (P 0.05). Double asterisks
indicate a value that is significantly greater than that of all other
groups shown (P 0.05).
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The antigen-specific saliva IgA responses (Fig.
4) matched the trend seen with the serum
antibody responses with the exception
of the group that received 1 µg
of A/Beijing8-9/93 in combination
with 10 µg of LT-R72 (group 12).
Animals that received either
5 or 20 µg of A/Beijing8-9/93 HA in
combination with 100 µg of
LT-R72 demonstrated a significantly higher
(
P 0.05) antigen-specific
saliva IgA response than
that of animals that received unadjuvanted
A/Beijing8-9/93 HA.
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FIG. 4.
Comparison of the effects of HA doses on
antigen-specific SW IgA responses after i.g. administration. Shown are
means ± SE of anti-A/Beijing8-9/93 HA SW IgA antibody titers of
groups of mice immunized either with 20 µg of HA administered alone
(HA only) or with 1-, 5-, or 20-µg doses of A/Beijing8-9/93 HA in
combination with either 10 or 100 µg of LT-R72 as indicated.
Asterisks indicate groups whose values are significantly greater than
that of the HA only group (P 0.05).
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Comparison of i.g. and i.m. immunizations.
The serum antibody
responses of mice i.g. immunized with A/Johannesburg/97 HA either alone
or in combination with an LT were compared to those of mice immunized
with A/Johannesburg/97 HA by the i.m. route. Groups of 10 mice were
immunized by the i.g. route with 20 µg of A/Johannesburg/97 HA either
alone or in combination with two dose levels of wtLT (1 and 10 µg),
LT-K63 (10 and 100 µg), or LT-R72 (10 and 100 µg). A group
receiving 1 µg of A/Johannesburg/97 HA by the i.m. route was
included. The 1-µg HA i.m. dose level was chosen such that, based on
data from previous experiments, a strong, protective, immunogenic
response in mice would result (data not shown), and it was used here in
order to make comparisons with the i.g. responses.
Serum antigen-specific antibody responses (Fig.
5) for mice immunized i.g. with 20 µg
of A/Johannesburg/97 HA adjuvanted with
an LT were equivalent to or
higher than those for i.m. immunized
mice. Mice i.g. immunized with 20 µg of A/Johannesburg/97 HA either
unadjuvanted (HA only) or in
combination with 10 µg of LT-K63
showed serum antigen-specific
antibody responses significantly
lower (
P 0.05) than
those of mice immunized by the i.m. route;
nevertheless, i.g.
immunization in the presence of 10 µg of LT-K63
resulted in antibody
responses 1 log higher than those obtained
with i.g. immunization with
unadjuvanted A/Johannesburg/97 HA.
Mice i.g. immunized with 20 µg of
A/Johannesburg/97 HA in combination
with 100 µg of LT-R72 showed
serum antigen-specific antibody responses
that were significantly
(
P 0.05) higher than those found with
i.m.
immunization.
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FIG. 5.
Comparison of the effects of i.m. and i.g.
administrations of A/Johannesburg/97 HA on antigen-specific serum
antibody responses. Shown are means ± SE of
anti-A/Johannesburg/97 HA antibody titers in the sera of mice immunized
i.m. with 1 µg of A/Johannesburg/97 HA unadjuvanted (IM) or immunized
i.g. with 20-µg doses of A/Johannesburg/97 HA alone (HA Only) and in
combination with either 1 or 10 µg of wtLT, 10 or 100 µg of LT-K63,
or 10 or 100 µg of LT-R72 as indicated. Asterisks indicate groups
whose values are significantly different from that of the i.m.
immunized group (P 0.05).
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|
Serum HI titers (Fig.
6) for mice i.g.
immunized with 20 µg of A/Johannesburg/97 HA in combination with
either 10 µg of wtLT
or 100 µg of LT-R72 were comparable in potency
to those for i.m.
immunized mice. Mice that were i.g. immunized with 20 µg of A/Johannesburg/97
HA in combination with either 1 µg of wtLT,
10 µg of LT-R72, or
100 µg of LT-K63 showed modest HI titer levels.
Significant HI
titers were not demonstrated for mice i.g. immunized
with 20 µg
of A/Johannesburg/97 HA either alone or in combination
with 10
µg of LT-K63.
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FIG. 6.
Comparison of the effects of i.m. and i.g.
administrations of A/Johannesburg/97 HA on serum HI titers. The data
shown are for pooled sera from groups of mice immunized i.m. with 1 µg of A/Johannesburg/97 HA unadjuvanted (IM), or immunized i.g. with
20-µg doses of A/Johannesburg/97 HA alone (HA Only), and in
combination with either 1 or 10 µg of wtLT, 10 or 100 µg of LT-K63,
or 10 or 100 µg of LT-R72 as indicated.
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Antigen-specific NW IgA responses (Fig.
7) were found to be significant only in
those mice immunized i.g. with 20 µg of A/Johannesburg/97
HA in
combination with either wtLT, LT-K63, or LT-R72. Mice receiving
i.m.
immunization or immunized i.g. with 20 µg of A/Johannesburg/97
HA
alone did not show significant antigen-specific NW IgA responses.
No
significant differences were seen in the antigen-specific IgA
responses
as a consequence of enterotoxin dose level or type.
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FIG. 7.
Comparison of the effects of i.m. and i.g.
administrations of A/Johannesburg/97 HA on antigen-specific NW IgA
antibody responses. Shown are means ± SE of
anti-A/Johannesburg/97 HA NW IgA antibody titers of mice immunized i.m.
with 1 µg of A/Johannesburg/97 HA unadjuvanted (IM) or immunized i.g.
with 20-µg doses of A/Johannesburg/97 HA alone (HA Only) and in
combination with either 1 or 10 µg of wtLT, 10 or 100 µg of LT-K63,
or 10 or 100 µg of LT-R72 as indicated. Asterisks indicate groups
whose values are significantly greater than that of the i.m. immunized
group (P 0.05).
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Current commercial influenza vaccines are shown to induce in healthy
adult humans serum antibody responses that are protective
against viral
challenge, but this protective immunity tends to
be variable in potency
and is relatively short lived, particularly
in the elderly and infant
populations (
7,
15,
24,
34,
38). Mucosal immunization
strategies have been extensively investigated
as a means to improve the
efficacy and duration of influenza vaccination
by providing a broader
immune response than that afforded by i.m.
immunization (
14,
31,
32,
33,
42). Like intranasal immunization,
oral immunization has
been shown to induce strong secretory IgA
responses, improve protective
cellular immune responses, and result
in significant serum antibody
responses as well (
5,
14,
26,
29,
31,
32,
43). The
secretory IgA responses for oral
immunization have been shown for human
subjects to be strongest
in the urogenital and rectal tracts, and when
compared to intranasal
immunization, oral immunization has
resulted in somewhat muted
upper respiratory, nasopharyngeal, and
salivary secretory IgA
responses (
39). These relatively
weak upper respiratory IgA
responses would seem to be a problem
with respect to achieving
effective protection against viral challenge
against viruses whose
primary mode of entry is via the upper
respiratory tract (such
as influenza). Other studies, however, have
shown that there are
sufficient local secretory IgA responses, and more
importantly,
there is evidence of antigen-primed B- and T-cell
migration to
the upper respiratory sites to induce potent protective
immunity
(
26,
43). Furthermore, oral immunization has been
shown to
promote memory B-cell maintenance in the bone marrow, a factor
that may be important in the development of the persistence of
immunity
against viral challenge (
5). However, to obtain strong
immune responses from many antigens, a potent mucosal adjuvant,
usually
an enterotoxin, must be coadministered (
9).
Studies have shown that immune responses to orally immunized antigens
were significantly stronger if the antigen by itself
had mucosal
binding properties or could be made to have mucosal
binding properties
by chemically coupling to agents with mucoadhesive,
lectin, or
receptor-binding properties (
8,
9,
21,
30).
CTB has been
used for these purposes with some success (
8,
9).
Influenza HA binds neuraminic acid-rich glycoproteins,
while LT-R72 and
LT-K63 bind GM
1 ganglioside, as well as galactose
containing glycoproteins and lipopolysaccharides, all of which
ligands
are found ubiquitously in the gut (
27,
37,
41).
Our group
has found that antigens that do not have any mucoadhesive
or
gut-associated binding properties have minimal immunogenicity
when
delivered orally in mice, other than at very high dose levels,
either
in the absence of LTs or as mixtures of soluble antigen
with soluble LT
(unpublished data). With influenza antigens, however,
our group and
others have shown that modest immune responses occur
when reasonable
dose levels are delivered orally but substantial
and broad immune
responses result when the antigens are adjuvanted
with LT or CT
(
26).
Our group has demonstrated here that potent antigen-specific serum
antibody titers that are comparable to or stronger than
i.m.
immunization, as well as modest salivary and nasal IgA responses,
can
be induced in mice with influenza HA antigens using i.g. immunization
by adjuvanting with mutant LTs that demonstrate significantly
reduced
(LT-R72) and unmeasurable (LT-K63) levels of ADP-ribosyltransferase
activity (
16). The dose level of influenza HA antigen (20 µg)
and mutant LTs (10 to 100 µg) found necessary for the strongest
immune responses may be relatively high. Further formulation efforts
are under way in our group to improve the efficiency of oral
immunization
and, if possible, lower the dose
requirements.
| |
ACKNOWLEDGMENTS |
We acknowledge the contributions made by Rino Rappuoli and
Mariagrazia Pizza for advice and technical support for LT-K63 and LT-R72, Mildred Ugozzoli for developing the luminometer-based ELISA,
Russell Reeve for help with statistical analyses, and Diana Atchley for
her skills in animal husbandry.
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FOOTNOTES |
*
Corresponding author. Mailing address: Chiron
Corporation, 4560 Horton St., Bldg. M-2, Emeryville, CA 94608. Phone:
(510) 923-8194. Fax: (510) 923-4116. E-mail:
john_barackman{at}cc.chiron.com.
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REFERENCES |
| 1.
|
Andrews, H. P.,
R. D. Snee, and M. H. Sarner.
1980.
Graphical display of means.
Am. Statistician
34:195-199[CrossRef].
|
| 2.
|
Barackman, J. D.,
G. Ott, and D. T. O'Hagan.
1999.
Intranasal immunization of mice with influenza vaccine in combination with the adjuvant LT-R72 induces potent mucosal and serum immunity which is stronger than that with traditional intramuscular immunization.
Infect. Immun.
67:4276-4279[Abstract/Free Full Text].
|
| 3.
|
Barackman, J. D.,
M. Singh,
M. Ugozzoli,
G. S. Ott, and D. T. O'Hagan.
1998.
Oral immunization with poly(lactide-co-glycolide) microparticles containing an entrapped recombinant glycoprotein (gD2) from herpes simplex type 2 virus.
S. T. P. Pharma Sci.
8:41-46.
|
| 4.
|
Bender, B. S.,
M. P. Johnson, and P. A. Small.
1991.
Influenza in senescent mice: impaired cytotoxic T-lymphocyte activity is correlated with prolonged infection.
Immunology
72:514-519[Medline].
|
| 5.
|
Benedetti, R.,
P. Lev,
E. Massoch, and J. Fló.
1998.
Long-term antibodies after an oral immunization with cholera toxin are synthesized in the bone marrow and may play a role in the regulation of memory B-cell maintenance at systemic and mucosal sites.
Res. Immunol.
149:107-118[CrossRef][Medline].
|
| 6.
|
Challacombe, S. J.,
D. Rahman,
H. Jeffery,
S. S. Davis, and D. T. O'Hagan.
1992.
Enhanced secretory IgA and systemic IgG antibody responses after oral immunization with biodegradable microparticles containing antigen.
Immunology
76:164-168[Medline].
|
| 7.
|
Clements, M. L., and I. Stephens.
1997.
New and improved vaccines against influenza, p. 545-570.
In
M. M. Levine, G. C. Woodrow, J. B. Kaper, and G. S. Cobon (ed.), New generation vaccines, 2nd ed. Marcel Dekker, Inc, New York, N.Y.
|
| 8.
|
Czerkinsky, C.,
M. W. Russell,
N. Lycke,
M. Lindblad, and J. Holmgren.
1989.
Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues.
Infect. Immun.
57:1072-1077[Abstract/Free Full Text].
|
| 9.
|
De Aizpurua, H. J., and G. J. Russell-Jones.
1988.
Oral vaccination: identification of classes of proteins that provoke an immune response upon oral feeding.
J. Exp. Med.
167:440-451[Abstract/Free Full Text].
|
| 10.
|
De Haan, A.,
H. J. Geerligs,
J. P. Huchshorn,
G. J. M. Van Scharrenburg,
A. M. Palache, and J. Wilschut.
1995.
Mucosal immunoadjuvant activity of liposomes: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with an influenza subunit vaccine and coadministered liposomes.
Vaccine
13:155-162[CrossRef][Medline].
|
| 11.
| Dickingson, B. L., and J. D. Clements.
Use of Escherichia coli heat-labile enterotoxin as an oral
adjuvant, p. 73-87. In H. Kiyono, P. L. Ogra, and
J. R. McGhee (ed.), Mucosal vaccines. Academic Press, New York,
N.Y.
|
| 12.
|
Elson, C. O.
1996.
Cholera toxin as a mucosal adjuvant, p. 59-72.
In
H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, New York, N.Y.
|
| 13.
|
Freytag, L. C., and J. D. Clements.
1999.
Bacterial toxins as mucosal adjuvants.
Curr. Top. Microbiol. Immunol.
236:215-236[Medline].
|
| 14.
|
Gallichan, W. S., and K. L. Rosenthal.
1996.
Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization.
J. Exp. Med.
184:1879-1890[Abstract/Free Full Text].
|
| 15.
|
Ghendon, Y.
1989.
The immune response of humans to live and inactivated influenza vaccines.
Adv. Exp. Med. Biol.
257:37-45[Medline].
|
| 16.
|
Giuliani, M. M.,
G. Del Giudice,
V. Giannelli,
G. Dougan,
G. Douce,
R. Rappuoli, and M. Pizza.
1998.
Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knock-out of ADP-ribosyltransferase activity.
J. Exp. Med.
187:1123-1132[Abstract/Free Full Text].
|
| 17.
|
Glezen, P. W.
1982.
Serious morbidity and mortality associated with influenza epidemics.
Epidemiol. Rev.
4:25-44[Free Full Text].
|
| 18.
|
Groothuis, J. R.,
M. V. Levin, and M. J. Levin.
1994.
Safety and immunogenicity of a purified haemagglutinin antigen in very young high-risk children.
Vaccine
12:139-141[CrossRef][Medline].
|
| 19.
|
Groothuis, J. R.,
M. J. Levin,
G. P. Rabalais,
G. Meiklejohn, and B. A. Lauer.
1991.
Immunization of high-risk infants younger than 18 months of age with split-product influenza vaccine.
Pediatrics
87:823-828[Abstract/Free Full Text].
|
| 20.
|
Harlow, E., and D. Lane (ed.).
1988.
Antibodies: a laboratory manual, p. 553-612.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 21.
|
Harokopakis, E.,
G. Hajishengallis, and S. M. Michalek.
1998.
Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system.
Infect. Immun.
66:4299-4304[Abstract/Free Full Text].
|
| 22.
|
Hierholzer, J. C., and M. T. Suggs.
1969.
Standardized viral hemagglutination and hemagglutination-inhibition tests. I. Standardization of erythrocyte suspensions.
Appl. Microbiol.
18:816-823[Medline].
|
| 23.
|
Hierholzer, J. C.,
M. T. Suggs, and E. C. Hall.
1969.
Standardized viral hemagglutination and hemagglutination-inhibition tests. II. Description and statistical evaluation.
Appl. Microbiol.
18:824-833[Medline].
|
| 24.
|
Hoskins, T. W.
1979.
Assessment of inactivated influenza-A vaccine after three outbreaks of influenza A at Christ's Hospital.
Lancet
i:33-35.
|
| 25.
|
Johannsen, R.,
H. Moser,
J. Hinz,
H. J. Freisen, and H. Gruschkau.
1985.
Quantitation of haemagglutinin of influenza tween-ether split vaccines by immunodiffusion.
Vaccine
3:235-240[CrossRef][Medline].
|
| 26.
|
Katz, J. M.,
X. Lu,
S. A. Young, and J. C. Galphin.
1997.
Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine.
J. Infect. Dis.
175:352-363[Medline].
|
| 27.
|
Kuziemko, G. M.,
M. Stroh, and R. C. Stevens.
1996.
Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance.
Biochemistry
35:6375-6384[CrossRef][Medline].
|
| 28.
|
Lyche, N.,
T. Tsuji, and J. Holmgren.
1992.
The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.
Eur. J. Immunol.
22:2277-2281[Medline].
|
| 29.
|
Meitin, C. A.,
B. S. Bender, and P. A. Small, Jr.
1994.
Enteric immunization of mice against influenza with recombinant vaccinia.
Proc. Natl. Acad. Sci. USA
91:11187-11191[Abstract/Free Full Text].
|
| 30.
|
Neutra, M. R., and J. Kraekenbuhl.
1996.
Antigen uptake by M cells for effective mucosal vaccines, p. 41-55.
In
H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, New York, N.Y.
|
| 31.
|
Novak, M.,
M. Yamamoto,
K. Fujihashi,
Z. Moldoveanu,
H. Kiyono,
J. R. McGhee, and J. Mesechy.
1995.
Ig-secreting and interferon- -producing cells in mice mucosally immunized with influenza virus.
Adv. Exp. Med. Biol.
371B:1587-1590.
|
| 32.
|
Ogra, P. L.
1996.
Mucosal immunoprophylaxis: an introductory overview, p. 3-14.
In
H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, New York, N.Y.
|
| 33.
|
Oh, Y.,
K. Ohta,
H. Kuno-Sakai,
R. Kim, and M. Kimura.
1998.
Local and systemic influenza haemagglutinin-specific antibody responses following aerosol and subcutaneous administration of inactivated split influenza vaccine.
Vaccine
10:506-511.
|
| 34.
|
Patriarca, P. A.,
J. A. Weber,
R. A. Parker,
W. N. Hall,
A. P. Kendal,
M. S. Bregman, and L. B. Schonberger.
1985.
Efficacy of influenza vaccine in nursing homes: reduction in illness and complications during an influenza A (H3N2) epidemic.
JAMA
253:1136-1139[Abstract/Free Full Text].
|
| 35.
|
Pizza, M.,
M. Domenighini,
W. Hol,
V. Giannelli,
M. R. Fontana,
M. M. Giuliani,
C. Magagnoli,
S. Peppoloni,
R. Manetti, and R. Rappuoli.
1994.
Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.
Mol. Microbiol.
14:51-60[CrossRef][Medline].
|
| 36.
|
Powers, D. C.
1993.
Influenza A virus-specific cytotoxic T-lymphocyte activity declines with advancing age.
J. Am. Geriatr. Soc.
41:1-5[Medline].
|
| 37.
|
Pritchett, T. J.,
R. Brossmer,
U. Rose, and J. C. Paulson.
1987.
Recognition of monovalent sialosides by influenza virus H3 hemagglutinin.
Virology
160:502-506[CrossRef][Medline].
|
| 38.
|
Riddiough, M. A.,
J. E. Sisk, and J. C. Bell.
1983.
Influenza vaccination: cost-effectiveness and public policy.
JAMA
249:3189-3195[Abstract/Free Full Text].
|
| 39.
|
Rudin, A.,
E. Johansson,
C. Bergquist, and J. Holmgren.
1998.
Differential kinetics and distribution of antibodies in serum and nasal and vaginal secretions after nasal and oral vaccination of humans.
Infect. Immun.
66:3390-3396[Abstract/Free Full Text].
|
| 40.
|
Santiago, N.,
S. Haas, and R. A. Baughman.
1995.
Vehicles for oral immunization, p. 413-438.
In
F. M. Powell, and M. J. Newman (ed.), Vaccine design: the subunit and adjuvant approach. Plenum Press, New York, N.Y.
|
| 41.
|
Spangler, B. D.
1992.
Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.
Microbiol. Rev.
56:622-647[Abstract/Free Full Text].
|
| 42.
|
Staats, H. F., and J. R. McGhee.
1996.
Application of basic principles of mucosal immunity to vaccine development, p. 17-39.
In
H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, New York, N.Y.
|
| 43.
|
Takase, H.,
Y. Murakami,
A. Endo, and T. Ikeuchi.
1996.
Antibody responses and protection in mice immunized orally against influenza virus.
Vaccine
14:1651-1656[CrossRef][Medline].
|
| 44.
|
Tamura, S.,
H. Funato,
Y. Hiroabayashi,
Y. Suzuki,
T. Nagamine,
C. Aizawa, and T. Kurata.
1991.
Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.
Eur. J. Immunol.
21:1337-1344[Medline].
|
| 45.
|
Tamura, S.,
Y. Ito,
H. Asanuma,
Y. Hirabayashi,
Y. Suzuki,
T. Nagamine,
C. Aizawa, and T. Kurata.
1992.
Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit.
J. Immunol.
149:981-988[Abstract].
|
| 46.
|
Tamura, S.,
H. Asanuma,
T. Tomita,
K. Komase,
K. Kawahara,
H. Danbara,
N. Hattori,
K. Watanabe,
Y. Suzuki,
T. Nagamine,
C. Aizawa,
A. Oya, and T. Kurata.
1994.
Escherichia coli heat-labile enterotoxin B subunit supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine.
Vaccine
12:1083-1089[CrossRef][Medline].
|
| 47.
|
Ugozzoli, M.,
D. T. O'Hagan, and G. S. Ott.
1998.
Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses.
Immunology
93:563-571[CrossRef][Medline].
|
Clinical and Diagnostic Laboratory Immunology, May 2001, p. 652-657, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.652-657.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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