Yakult Central Institute for Microbiological
Research and Department of Microbiology, 1796 Yaho, Kunitachi-shi,
Tokyo 186-8650, Japan
Received 22 September 1998/Returned for modification 22 October
1998/Accepted 2 December 1998
 |
INTRODUCTION |
Influenza is an acute viral
respiratory infection that results in high morbidity and significant
mortality in humans as well as in animals. The site of the virus entry
is the mucosa of the upper respiratory tract, and there is evidence
that influenza viruses can be spread by coughing and as a
small-particle aerosol directly into the lower respiratory tract
(28). An increase in specific antibody production at this
site is important for preventing infection in the upper and lower
respiratory tracts. In several studies, the degree of protection
against influenza virus infection was found to be correlated with the
levels of mucosal immunoglobulin A (IgA) in the respiratory tract and
serum IgG (4, 5, 14, 22, 23). Mucosal anti-influenza virus IgA inhibits viral attachment to epithelial cells in the mucosa, thereby preventing infection in the upper respiratory tract and stopping the spread of the infection. Furthermore, serum anti-influenza virus IgG prevents infection in the lower respiratory tract and protects the lungs against viral infection and thereby prevents death
from pneumonia (17). Subcutaneous vaccination with influenza virus, which is being performed at present, enhances the level of
anti-influenza virus IgG in serum and prevents infection by the virus
with the same surface hemagglutinin (HA) on the influenza virus virion
(12); however, this present method has some troublesome problems. If there is a safe oral adjuvant that enhances serum anti-influenza virus IgG and mucosal anti-influenza virus IgA, an oral
vaccine will become efficacius. Further, if there are foods that
enhance the immune response against influenza virus, these may be
functional foods that could prevent influenza virus infection.
In healthy breast-fed (but not formula-fed) infants, numerous
bifidobacteria inhabit the intestines (11). These bacteria are thought to play a role in the resistance to infection in humans and
animals (8, 16, 29). The intestines of adults have fewer of
these organisms (15), and some persons replace them with
yogurt and bifidobacteria cultures. We have found one strain of
Bifidobacterium (Bifidobacterium breve YIT4064)
that can induce large quantities of IgA among the many strains of
bifidobacteria isolated from human feces by the murine Peyer's patch
(PP) cell culture method (30). The organism enhances the
production of anti-influenza virus HA (31), antirotavirus,
and antipoliovirus antibodies (unpublished data) by PP cells in
response to the addition of HA, rotavirus, and poliovirus,
respectively. When the organism was administered orally to mice with
cholera toxin (CT), the amount of anti-CT IgA in feces and the levels
of anti-CT IgA production and proliferation in PP cells were
significantly greater than those after the administration of CT alone
or of CT and B. breve YIT4079, which did not induce IgA in
the in vitro PP cell culture (30). Furthermore, the level of
anti-rotavirus IgA in milk in mouse dams fed the organism orally with
rotavirus was significantly higher than that in dams immunized with
rotavirus only, and pups born to and nursed by dams fed the organism
and immunized orally with rotavirus were more strongly protected
against rotavirus-induced diarrhea than those born to and nursed by
dams immunized with rotavirus only (32).
In the present study, we investigated whether the oral administration
of B. breve YIT4064 augmented the level of anti-influenza virus IgG in serum and whether the antibody-enhanced mice were protected against influenza virus infection in the lower respiratory tract and were saved from death.
| |
MATERIALS AND METHODS |
Mice.
BALB/c female mice, 6 and 9 weeks old, were obtained
from Japan SLC, Inc. (Hamamatsu-shi, Japan) and used for the experiments.
Virus.
Influenza A/PR/8/34 (PR8, H1N1) virus was grown in
the allantoic sacs of 11-day-old chicken embryos at 34°C for 2 days
by the method of Takemoto et al. (20), with modifications.
The allantoic fluid was removed and stored at 
80°C. The virus titer of allantoic fluid was expressed as the 50% egg-infecting doses (EID50) (27). Serial 10-fold dilutions of the
allantoic fluid were injected into five embryonated eggs, and the
presence of virus in the allantoic fluid of each egg was determined on
the basis of the hemagglutinating capacity 2 days after injection. The
virus titer, expressed as a magnification of dilution with EID50, was 109.2 EID50/ml. The
various dilutions of the allantoic fluid were used for oral
immunization and nasal infection. Oral immunization with various
concentrations of live PR8 via a stomach tube was performed in mice
that had been pretreated with an intramuscular injection of cimetidine
(Tagamet; Smith Kline & French Laboratories, Philadelphia, Pa.) (1.2 mg
per mouse) 1 h before (2) and with 3%
NaHCO3 via a stomach tube just before the oral
immunization. Nasal infection was performed by dropping 10 or 1 µl of
fluid containing various concentrations of PR8 into each nostril
(injection with 20 or 2 µl per mouse) after the mice were
anesthetized by an intraperitoneal injection of sodium amobarbital
(0.25 µg per mouse). Mice were infected in the upper and lower
respiratory tracts by dropping 10 and 1 µl of PR8 solution into each
nostril, respectively (21).
PR8 antigen.
The purified PR8 antigen was prepared from the
allantoic fluid that included PR8 grown in 11-day-old embryonated eggs.
The PR8 in the fluid was concentrated by centrifugation (174,500 × g, 4°C, 1 h) and then purified by zonal
centrifugation through a noncontinuous 30 and 60% sucrose gradient
(174,500 × g, 4°C, 50 min) and then a noncontinuous 35, 42, 52, and 60% sucrose gradient (174,500 × g, 4°C, 50 min). The virus preparation was treated with 0.01% formalin, stored at
80°C, and used as the antigen in an enzyme-linked immunosorbent
assay (ELISA) for antibody detection.
B. breve YIT4064.
B. breve YIT4064,
isolated from the feces of a healthy breast-fed infant, was treated
with heat and used in this study. B. breve YIT4064 was
incubated in prereduced modified PSYL broth medium at 37°C for
24 h. The composition of the modified PSYL medium (per liter) was
as follows: skim milk, 10 g; protease, 0.2 g; yeast extract,
10 g; CH3COONa, 3 g;
(NH4)2SO4, 3 g; lactose, 50 g; silicon, 0.1 ml. B. breve YIT4064 was harvested
by centrifugation at 7,000 × g for 10 min at 4°C,
washed three times with sterilized water, and allowed to autolyse by
incubation in sterilized water overnight at 15°C. B. breve
YIT4064 was heated at 100°C for 30 min and lyophilized. A 0.05%
(wt/wt) concentration of B. breve YIT4064 was added to MM-3
diet (Funabashi Farms, Funabashi-shi, Japan).
Protocol.
Six-week-old female BALB/c mice were given food
orally ad libitum (Fig. 1). Each group
consisted of 10 mice, except that group 8 consisted of 9 mice. Groups
2, 4, 6, 8, and 10 received food with 0.05% B. breve
YIT4064 added, and groups 1, 3, 5, 7, and 9 received food without the
organism. Groups 3 and 4, 5 and 6, and 7 and 8 were immunized via a
stomach tube with a single dose of 105.9 EID50s
of PR8 at 6, 8, and 10 weeks before nasal infection, respectively. Groups 9 and 10 were immunized via a stomach tube with two doses of
104.9 EID50 of PR8 at 6 and 2 weeks before
nasal infection. Groups 1 and 2 were not immunized before nasal
infection. These mice were monitored for symptoms and survival every
day after nasal infection. The symptoms monitored were erect hair and a
decrease in body weight. Nasal infection was performed by dropping 10 µl of fluid containing a virus concentration of 103.5
EID50/20 µl into each nostril. Blood was obtained from
retroorbital blood vessels just before the nasal injection of PR8, and
the serum was stored at 30°C until tested.
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FIG. 1.
Protocol for feeding mice with B. breve
YIT4064:
B,
start;  , oral immunization with PR8;  , inoculation with PR8;
 , collection of sera;
- , observation.
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|
Hyperimmune serum.
Hyperimmune antiserum to PR8 was prepared
after four injections of PR8 (108.9 EID50) via
a stomach tube and one nasal injection of PR8 antigen (104.5 EID50) into BALB/c mice. By comparing
the A492 values against various dilutions of the
hyperimmune antiserum to PR8, we determined the dilution values that
best represented a straight line. At A492, 1 U
of anti-PR8 IgG was equivalent to a 104 dilution of the
hyperimmune antiserum to PR8.
Measurement of antibodies.
The antibody titers in serum were
measured by ELISA. The purified PR8 antigen (2 µg of protein/ml) was
coated onto the wells of a 96-well ELISA plate (Nunc, Roskilde,
Denmark) with carbonate buffer (pH 9.6). An experimental sample of
serum or a standard sample of hyperimmune antiserum against PR8 was
added to each well, and then the plate was incubated at 37°C for
1.5 h. Horseradish peroxidase-labeled goat anti-mouse IgG
(1:2,000; Organon Teknika-Cappel) was then added to the wells. The
wells were extensively washed between incubations. After an
o-phenylenediamine solution (0.4 mg/ml citrate buffer, pH
5.0) containing 0.02% H2O2 had been added to
each well, the plates were held at 37°C for 10 min. The reaction was
stopped by the addition of 2.5 M H2SO4, and the
A492 of the resulting solution was determined
with a Titertek Multiscan (Flow Laboratories, McLean, Va.).
Statistical analysis.
The statistical significance of the
difference between the groups fed B. breve YIT4064 and those
not fed the bacteria in the experiments was examined by means of
Fisher's exact test or the 
2 test.
| |
RESULTS |
Virus dose dependence of infection in mice after intranasal
challenge with PR8.
Nine-week-old mice were inoculated with 1 or
10 µl of a PR8 solution at various concentrations into each nostril
(inoculation with 2 or 20 µl of PR8 per mouse) and then monitored for
survival every day after inoculation. None of the mice given various
concentrations of PR8 in the upper respiratory tract, i.e., with a
2-µl solution of PR8 given per mouse, were infected, and therefore
these mice did not die (Fig. 2A).
Regarding infection of the lower respiratory tract (inoculation with 20 µl of PR8 per mouse), all mice inoculated with 103.5 to
105.5 EID50 of PR8 died between 7 and 10 days
after intranasal inoculation. Of the mice inoculated intranasally with
102.5 EID50 of PR8, 66.7% survived (Fig. 2B).
Therefore, 103.5 EID50, a lethal dose with
regards to infection of the lower respiratory tract (inoculation with
20 µl of PR8 per mouse), was used as the infectious dose to determine
the ability of B. breve YIT4064 to protect against influenza
virus infection.
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FIG. 2.
Kinetics of survival rate after inoculation with 1 (A)
or 10 (B) µl of a PR8 solution containing various doses into each
nostril. Mice were inoculated with 1 µl of a PR8 solution into each
nostril for infection of the upper respiratory tract (A) and with 10 µl of a PR8 solution into each nostril for infection of the lower
respiratory tract (B). Virus dose group symbols:  , 101.5
EID50 of PR8 (n = 6);  ,
102.5 EID50 of PR8 (n = 6);
 , 103.5 EID50 of PR8 (n = 6);  , 104.5 EID50 of PR8 (n = 6);  , 105.5 EID50 of PR8
(n = 6).
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Protection against influenza virus infection by oral immunization
with influenza virus.
We investigated whether influenza virus
infection was prevented in mice immunized with various doses of PR8
orally. Nine-week-old mice were immunized via a stomach tube with a
single dose of 105.9 to 108.9 EID50
of PR8 at 2 weeks before nasal inoculation or with two doses of
104.9 EID50 of PR8 at 6 and 2 weeks before
nasal inoculation of PR8 (103.5 EID50) and were
monitored for survival for 14 days after nasal inoculation. As shown in
Table 1, 100% of the mice immunized orally with 108.9 EID50 of PR8 showed
enhancement of the level of anti-PR8 IgG in serum just before and
survival after nasal inoculation of PR8, and 25.0% of the mice
immunized orally with 105.9 EID50 of PR8 showed
enhancement of the level of anti-PR8 IgG just before and survival after
nasal inoculation of PR8. Therefore, immunization with
108.9 and 105.9 EID50 of PR8
protected mice against infection perfectly and partially, respectively.
All the mice immunized orally with two doses of 104.9
EID50 of PR8 died. Therefore, correlation was observed
among the oral immunization dose of PR8, augmentation of anti-PR8 IgG production in serum, and survival rate with regards to PR8 infection. Therefore, we studied, by using immunization doses of 105.9
and 104.9 EID50 of PR8, whether B. breve YIT4064 enhanced the level of anti-PR8 IgG in serum and the
survival rate after virus infection.
In the next study, we observed differences in the serum IgG titers and
survival rates for the period between oral immunization and nasal
inoculation. Nine-week-old mice were immunized with a single dose of
105.9 EID50 of PR8 or with two doses of
104.9 EID50 of PR8 at various numbers of weeks
or at 6 and 2 weeks before nasal inoculation of PR8, respectively.
After nasal inoculation, the mice were monitored for survival for 14 days. As shown in Fig. 3, the number of
mice with enhanced anti-PR8 IgG in serum was increased at 2, 4, and 6 weeks after oral immunization with 105.9 EID50
of PR8, but mice with the antibody-enhanced serum were not observed at
8 or 10 weeks after oral immunization with 105.9
EID50 of PR8 or after immunization with two doses of
104.9 EID50 of PR8 (Fig. 3A). Mice infected
intranasally at 6 weeks after oral immunization with 105.9
EID50 of PR8 showed the highest survival rate. The survival
rate in mice infected at 8 and 10 weeks after immunization with
105.9 EID50 of PR8 was decreased and was the
same as that in nonimmunized mice. Mice immunized with two doses of
104.9 EID50 of PR8 did not show an increase in
survival rate but showed the same survival rate as that of nonimmunized
mice, too (Fig. 3B). The decrease in the survival rate was thought to
depend on a decrease or absence of anti-PR8 IgG in serum. Therefore, we investigated whether B. breve YIT4064 augmented antibody
production in serum and enhanced the survival rate in mice infected in
the lower respiratory tract when the effectiveness of the immunization was decreased for long periods between immunization and infection (8 and 10 weeks) or when the immunization was not effective because a
small amount of the immunogen (two doses of 104.9
EID50 of PR8) was used.
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FIG. 3.
Differences in percentages of anti-PR8 IgG production in
serum (A) and survival rates (B) based on the period between oral
immunization and nasal infection or on the quantity of immunogen.
Nine-week-old female BALB/c mice were immunized with a single dose of
105.9 EID50 and with two doses of
104.9 EID50 of PR8 at various weeks and at 6 and 2 weeks before nasal inoculation (103.5
EID50 of PR8), respectively. The anti-PR8 IgG titer in
serum was measured just before nasal inoculation of PR8. The survival
rates were observed for 14 days after nasal inoculation of PR8.
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Protection against PR8-induced influenza virus infection by oral
administration of B. breve YIT4064.
We studied the
protection against influenza virus infection by oral administration of
B. breve YIT4064 according to the protocol shown in Fig. 1.
Figure 4 shows the time courses of the
accumulated symptom rate (percentage of mice showing erect hair and/or
decrease in body weight) in mice that were nonimmunized (Fig. 4A) and
in mice that were immunized with 105.9 EID50 of
PR8 at 6, 8, and 10 weeks (Fig. 4B to D) and with two doses of
104.9 EID50 of PR8 (Fig. 4E) before nasal
inoculation of PR8 (103.5 EID50). In the mice
immunized with 105.9 EID50 of PR8 at 10 weeks
(Fig. 4D) and with two doses of 104.9 EID50 of
PR8 (Fig. 4E) before nasal inoculation of PR8, oral administration of
B. breve YIT4064 significantly decreased the accumulated
symptom rate of influenza virus infection. In the nonimmunized mice
(Fig. 4A) and in the mice immunized with 105.9
EID50 of PR8 at 6 and 8 weeks before nasal inoculation of
PR8 (Fig. 4B and C), the accumulated symptom rate of influenza virus infection did not decrease upon oral administration of B. breve YIT4064. As shown in Fig. 5,
in the mice immunized with 105.9 EID50 of PR8
at 10 weeks (Fig. 5D) and with two doses of 104.9
EID50 of PR8 (Fig. 5E) before nasal inoculation of PR8
(103.5 EID50), the survival rate of mice fed
B. breve YIT4064 (groups 8 and 10) was significantly greater
than that of those not fed the organism (groups 7 and 9). In the
nonimmunized mice (Fig. 5A) and in the mice immunized with
105.9 EID50 of PR8 at 6 and 8 weeks before
nasal inoculation of PR8 (Fig. 5B and C), the survival rate of mice fed
B. breve YIT4064 was nearly the same as that of those not
fed B. breve YIT4064. Thus, mice fed B. breve
YIT4064 and immunized with PR8 were better protected than mice
immunized only with PR8, when the efficiency of immunization was
decreased for long periods between immunization and infection (8 and 10 weeks), and the effect of immunization was not recognized when a small
amount of the immunogen (two doses of 104.9
EID50 of PR8) was used. Our next experiment sought to
determine whether the protection against influenza virus infection by
oral administration of B. breve YIT4064 was due to an
increase in serum antibodies.
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FIG. 4.
Protection against morbidity due to influenza virus
infection in mice administered B. breve YIT4064 orally. Mice
were not immunized (A) (groups 1 and 2), were immunized with a single
dose of 105.9 EID50 of PR8 at 6 (B) (groups 3 and 4), 8 (C) (groups 5 and 6), and 10 (D) (groups 7 and 8) weeks, or
were immunized with two doses of 104.9 EID50 of
PR8 at 6 and 2 weeks (E) (groups 9 and 10) before nasal inoculation
(103.5 EID50 of PR8). Mice were not fed ()
or were fed () B. breve YIT4064 throughout these
experiments. After nasal inoculation of PR8, the mice were observed for
accumulated symptom rate for 14 to 21 days.
 and
,
P < 0.02 and P < 0.05, respectively,
versus accumulated symptom rate in groups 9 and 7, respectively, by
Fisher's exact test.
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FIG. 5.
Protection against mortality due to influenza virus
infection in mice administered B. breve YIT4064 orally. Oral
immunization and nasal inoculation in each group were performed in the
same manner as described in the legend to Fig. 4. After nasal injection
of PR8, the mice were observed for survival for 14 to 21 days.
,
P < 0.05 versus survival rate in group 7 or 9, by
Fisher's exact test.
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Antibody titers in sera of mice.
We measured serum antibodies
to determine whether oral administration of B. breve YIT4064
augmented anti-PR8 IgG production in serum, which plays a role in the
prevention of infection in the lower respiratory tract and mortality.
As shown in Table 2, the number of mice
with an enhanced level of anti-PR8 IgG in serum increased significantly
upon oral administration of B. breve YIT4064 in mice
immunized with 105.9 EID50 of PR8 at 10 weeks
(groups 7 and 8) or with two doses of 104.9
EID50 of PR8 (groups 9 and 10) before nasal inoculation of
PR8. But, it did not increase upon oral administration of B. breve YIT4064 in nonimmunized mice (groups 1 and 2) or in mice
immunized with 105.9 EID50 of PR8 at 6 and 8 weeks before nasal inoculation of PR8 (groups 3 to 6).
Relationship between the level of anti-PR8 IgG in serum just before
nasal infection and the survival of mice after nasal infection.
As
shown in Fig. 6, all mice in which the
level of anti-PR8 IgG in serum was enhanced were protected against
influenza virus infection in the lower respiratory tract and death.
Anti-PR8 IgG was not detected in the mice that died in any group. One
or two mice survived in all groups, and anti-PR8 IgG was not detected in the serum of these mice. We thought that since the activity of
nonspecific immune cells and NK cells, etc., differed among the lots of
mice, the sensitivity to infection with different lethal doses of PR8
might differ among the lots of mice and noninfected mice might appear
in some lots of mice.
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FIG. 6.
Relationship between level of anti-PR8 IgG in serum and
survival of mice after infection. The anti-PR8 IgG titers in sera of
mice from various groups were measured just before nasal inoculation of
PR8 (103.5 EID50). The anti-PR8 IgG titers in
dead and surviving mice are shown individually.
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Therefore, when the efficiency of immunization was decreased for long
periods between immunization and infection or was not recognized due to
the use of a small quantity of the immunogen, the oral administration
of B. breve YIT4064 enhanced antigen-specific IgG production
in serum and prevented influenza virus infection in the lower
respiratory tract.
| |
DISCUSSION |
All mice injected with 103.5 to 105.5
EID50 of PR8 in the lower respiratory tract died. But, for
infection of the upper respiratory tract (inoculation with 2 µl of
PR8 per mouse), 100% of the mice survived even with inoculation of
105.5 EID50 of PR8 (Fig. 2). It was thought
that if PR8 was injected directly into the lower respiratory tract, a
small amount of PR8 (103.5 EID50) proliferated
sufficiently in the lungs to kill all mice. Serum anti-influenza virus
IgG is important in preventing pneumonia and in protecting mice from
death after lower respiratory infection, although it is not always
effective in preventing infection in the upper respiratory tract
(1, 17). We found that oral vaccination with PR8 produced
anti-PR8 IgG in serum, prevented infection in the lower respiratory
tract, and protected mice from death. The observations in our present
study resemble the results of Chen and Quinnan (3); i.e.,
intragastric administration of inactivated influenza virus vaccine
induced a predominance of HA-specific IgG in serum. The level of
anti-PR8 IgG in serum and the survival rate of mice after nasal
inoculation of PR8 depended on the dose of PR8 given orally (Table 1).
In the next study, we observed the relation between the decrease in the
serum IgG titer just before nasal inoculation of PR8 and the increase
in the infection rate after nasal inoculation of PR8. The anti-PR8 IgG
level in serum at 2, 4, and 6 weeks after oral immunization and the
survival rate of mice after infection increased at the same time and
peaked at 6 weeks after immunization (Fig. 3). These results are
similar to those of Murphy et al. (13), who reported that
after inoculation of influenza virus, the IgM and IgA antibody levels
peaked at 2 weeks and then began to decrease, whereas IgG antibodies,
first detectable during the same period, continued to increase in titer
until the maximal level was reached at 4 to 7 weeks.
The oral administration of B. breve YIT4064 with the oral
injection of rotavirus augmented anti-rotavirus IgA antibodies in milk
secreted from the mammary glands and prevented rotavirus-induced diarrhea in pups (32). Anti-influenza virus IgG in serum
prevented influenza virus infection in the lower respiratory tract and
protected mice from death (Table 1). We examined whether the oral
administration of B. breve YIT4064 augmented anti-PR8 IgG
production in serum and prevented influenza virus infection in the
lower respiratory tract. When the efficiency of immunization was
decreased for long periods after oral immunization (8 and 10 weeks),
and when immunization was not recognized due to the small amount of the
immunogen (two doses of 104.9 EID50 of PR8),
the oral administration of B. breve YIT4064 augmented anti-PR8 IgG production in serum and prevented influenza virus infection (Table 2; Fig. 4 and 5).
The number of surface-IgG-positive cells was very low in PP
(6), and anti-HA IgG was not produced upon the addition of HA antigen to a PP cell culture (31). Therefore, it was not thought that after PR8 was taken up by M cells in PP, anti-PR8 IgG was
produced in PP. The mechanism underlying the appearance of anti-PR8 IgG
in serum after oral vaccination of PR8 was thought to be that the HA of
PR8 might bind to sialic acid (13) on epithelial cells in
the lamina propria of the intestine and be absorbed by the epithelial
cells and then act as an immunogen and produce anti-PR8 IgG in serum.
Umesaki and Setoyama have shown that substances with the ability to
bind to intestinal epithelial cells are absorbed into the lamina
propria, are strong immunogens, and produce antigen-specific IgG in
serum (26). The mechanism underlying the enhancement of
anti-PR8 IgG upon oral administration of B. breve YIT4064 is not clear. The adjuvant activity of B. breve YIT4064 was not
observed when B. breve YIT4064 was administered orally and
HA antigen was injected nasally (unpublished data), being found only
when B. breve YIT4064 was given orally with an antigen
(30, 32). B. breve YIT4064 administered orally
was taken up by M cells in PP and was scarcely taken up by epithelial
cells on the lamina propria of the intestine (19).
Therefore, it was assumed that the PR8 antigen was absorbed by
epithelial cells in the intestine and that B. breve YIT4064
was taken up by M cells in PP encountered in mesenteric lymph nodes or
another immune tissue and then anti-PR8 IgG in serum was produced in an
amount greater than that upon oral injection of PR8 only. Furthermore,
it is presumed that B. breve YIT4064 fed orally induces the
production of some cytokines in epithelial cells on the lamina propria
and accelerates the immune response and antibody production in response
to absorbed PR8 at that site.
The attractive attributes of an oral vaccine include the simplicity of
administration: no need for sterile syringes, needles, or trained
personnel, less stringent requirements for the preparation of orally
delivered antigens than for injectable ones, and fewer problems with
the storage of dry, lyophilized oral vaccines than with liquid
injectable vaccines. However, because antigens administered orally are
inactivated by gastric acid and digestive enzymes, it is difficult for
them to be taken up and processed by PP. Therefore, the immune response
after oral administration of an antigen is weaker than that after
systemic immunization. The immunogenicity of vaccines is augmented by
the administration of potent adjuvants. A number of promising new
adjuvants, such as immunostimulating complexes, CT subunit B,
proteoliposomes, 6-o-acylmuramyldipeptide, a lipid A
analogue, and others, enhance the immune responses of animals and
humans to vaccines (7, 10, 18, 23-25). However, adjuvants
that stimulate mucosal immunity have some undesirable side effects
after oral administration. Because B. breve YIT4064 has been
isolated from the intestines of healthy breast-fed infants and shows
weak antigenicity (30), it is safe as an adjuvant for oral
vaccination. Furthermore, B. breve YIT4064 may be used as
the host in the development of a new oral vaccine which involves rearrangement of a gene. If it is possible to transfer various antigenic genes of a pathogen into B. breve YIT4064 and for
the bacteria to inhabit the intestine, one may obtain a new hopeful oral vaccine. We are now studying whether the oral administration of
B. breve YIT4064 enhances the secretory anti-PR8 IgA level in nasal and bronchoalveolar washings and prevents the growth of
influenza virus in the upper respiratory tract. In preliminary experiment, oral administration of B. breve YIT4064 enhanced
antigen-specific IgA against an influenza virus HA vaccine administered
orally in a nasal wash (unpublished data). Since B. breve
YIT4064 exhibits a protective effect against influenza virus infection,
it can be used as a probiotic for protection against infection and is a
candidate for production as a kind of "physiologic function food"
that was recently defined in Japan (9).
We thank S. Tamura and T. Kurata (National Institute of Health,
Tokyo, Japan) and Y. Suzuki (Kitasato Institute, Saitama, Japan) for
help with the study and M. Watanuki for helpful discussions.
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Abstract
Introduction
