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Department of Pathology, National Institute of Infectious Diseases,
Yakult Central Institute for Microbiological Research, and
Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Research Center for Biologicals, Kitasato Institute, Saitama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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20 and 0.3 times those in wild-type mice, respectively). The
degree of the reduction of protection or cross-protection was
moderately reversed by the low but non-negligible level of nasal IgA,
transudates from the accumulated serum IgA. These results, together
with the absence of the IgA-dependent cross-protection in the lower RT
and the unaltered level of nasal or serum IgG in wild-type and pIgR-KO
mice, confirm that the actively secreted IgA plays an important role in
cross-protection against variant virus infection in the upper RT, which
cannot be substituted by serum IgG. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The direct biological role of S-IgA in protection against virus infection has also been demonstrated in a number of studies (3, 4, 20, 21, 22, 23, 24, 25). Anti-influenza S-IgA purified from the RTs of mice immunized with HA molecules protected nonimmune mice from the virus infection, when administered i.n. (21, 22). Anti-HA-specific monoclonal polymeric (p)IgA injected i.v. was transported more efficiently into the nasal surface than were monomeric IgA or IgG1 (23). Treatment of mice, which were immunized with the live virus, with anti-IgA, but not anti-IgG and anti-IgM, abrogated the protection (24). In addition, monoclonal pIgA and S-IgA are several times more effective than monoclonal monomeric IgA in terms of hemagglutination inhibition (HI) and virus neutralization (NT) activities (25). These results suggest that S-IgA is a major mediator of nasal immunity and has inherently greater antiviral activity due to its polymeric nature.
It has been proposed that S-IgA Ab is a dimeric IgA (dIgA) that is produced by IgA-producing B cells in the lamina propria and secreted across the mucosal epithelial cells to the mucosal surface. Thus, dIgA binds to a pIgR on the basolateral surface and is transported to the apical side of the epithelial cells, where the secretory component (SC), the extracellular region of pIgR cleaved by a specific protease, remains bound to the dIgA, resulting in the formation of S-IgA (26). The involvement of pIgR in the transepithelial transport of dIgA has been verified by many investigators (27, 28, 29), and data showing that pIgR binds to dIgA via the J chain have also been reported (30). Recently, to further define the physiological roles of the J chain, IgA or pIgR, KO mice with a disruption of each gene, have been generated (31, 32, 33, 34, 35).
Mbawuike et al. (33) demonstrated that in IgA knockout (KO) mice, i.n. and i.p. immunizations with a CTB*-combined vaccine provided protection against a lethal dose of homologous viruses, in parallel with the induction of both mucosal and serum anti-HA IgG and IgM Abs, suggesting that IgA is not required for preventing influenza virus infection. The IgA-KO mouse seems to be an ideal model for individuals with selective IgA deficiency whose IgG and IgM levels are either normal or elevated (36, 37, 38). Thus, the finding that IgA is not necessary for mucosal protection in IgA-KO mice challenges the notion that S-IgA is responsible for protection against virus infection in the RT. However, in the previous experiments, the important role of S-IgA Abs in providing cross-protection against heterologous virus infection in the upper RT was not examined. The ability of S-IgA to provide cross-protection can be determined by analyzing Ab and virus titers in the nasal wash of mice immunized i.n. with a CTB*-combined vaccine and challenged by a RT infection with heterologous viruses (14).
Shimada et al. (34) have established a mouse strain lacking exon 2 of the pIgR gene and demonstrated that in pIgR-KO mice both intestinal and hepatic transcytoses of dIgA were severely blocked, resulting in the massive accumulation of dIgA in the serum; this suggests that dIgA is mainly transported by pIgR on the epithelial cells of the intestine and hepatocytes. Johansen et al. (35) have also established a mouse strain lacking exon 3 of the pIgR gene and demonstrated similar results. Under these circumstances, the superior ability of S-IgA over IgG in preventing influenza in the upper RT remains to be clarified in vaccine-immunized pIgR-KO mice.
In this study, the protective roles of S-IgA against influenza were examined in pIgR-KO mice immunized i.n. with different CTB*-combined inactivated vaccines and challenged by a RT infection with the virus. In addition, to delineate the transport of dIgA across the mucosal membrane in the upper RT, the nasal anti-HA IgA and IgG Ab concentrations were determined by estimating the nasal mucus volume. The results confirm that S-IgA plays important roles not only in protection against upper RT infection by homologous viruses, which can be substituted by serum IgG, but also cross-protection against upper RT infection by variant viruses, which cannot be substituted by serum IgG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pIgR-transgenic KO mice were generated as previously described (34). Heterozygous mice were generated by mating male chimeric mice and female BALB/c mice, and F1 mice were backcrossed with BALB/c mice eight times (N8). Some experiments were conducted using littermates of the N8 or N9 generation. Additional experiments were conducted by intercrossing pIgR-KO mice with homozygous mice from the N10 generation.
Screening for mice with a disruption at exon 2 of the pIgR
gene in littermates of a cross between heterozygous mice was conducted
by PCR analysis of genomic DNAs extracted from each mouse tail using a
DNeasy Tissue kit (Qiagen, Tokyo, Japan). Sense primer I-1
(5'-GACCCTTTACCAGACTCCTGGTCTT), antisense primer E2–1
(5'-ATTCTCATACAA GAAGCCAACCTGA) of the wild-type genome, and antisense
primer Neo-1 (5'-AAGTATCCATCATGGCTGATGCAAT) of the gene-targeting
region were used in PCR. PCR products were separated by agarose gel
electrophoresis (Fig. 1
A).
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Nasal wash samples were collected from the decapitated heads of
wild-type (pIgR+/+), heterozygous
(pIgR+/-), and homozygous
(pIgR-/-) BALB/c mice as described previously
(15, 34). After SDS-PAGE was conducted, proteins were
blotted onto a polyvinylidene difluoride membrane (Immobilon;
Millipore, Bedford, MA) using the wet blotting system (Bio-Rad,
Hercules, CA). To detect pIgR and SC proteins, the membrane was
incubated with rabbit anti-mouse SC IgG (3 µg/ml)
(34) and then treated with peroxidase-conjugated
anti-rabbit Igs (Amersham, Arlington Heights, IL). Thereafter, the
membrane was soaked in ECL reagents (Pierce, Rockford, IL). The SC
protein was detected as a single band of 100 kDa in the nasal wash
samples from pIgR+/- and
pIgR+/+ mice, but it was not detected in the
nasal wash of pIgR-/- mice (Fig. 1
B). A band of 70 kDa for the nasal wash was regarded as
nonspecific staining, because the band was detected using both rabbit
anti-mouse SC IgG and normal rabbit serum (34).
HA vaccines and influenza viruses
HA vaccines (split-product virus vaccines) were prepared from
influenza viruses A/PR/8/34 (A/PR8; H1N1), A/Yamagata/120/86
(A/Yamagata; H1N1), A/Beijing/262/95 (A/Beijing; H1N1), and
B/Ibaraki/2/85 (B/Ibaraki) according to the method of Davenport et al.
(39) at the Kitasato Institute (Saitama, Japan). The HA
vaccine was produced from the viruses, grown in the allantoic cavity of
10- to 11-day fertile chicken eggs, highly purified, and disintegrated
with ether. The vaccine contained all the proteins from the virus
particle, including HA molecules as the major component (30% of the
total protein). These viruses, with the exception of A/PR8, which have
been used to prepare vaccines in Japan, are immunologically different
from one another (15). The A/Yamagata (H1N1) or A/Beijing
(H1N1) vaccine-immunized mice contained a serum HI Ab that is
cross-reactive to the A/PR8 virus, with titers of 1/128 or <1/128. The
B/Ibaraki vaccine-immunized mice contained no serum HI Ab that is
cross-reactive to A/PR8 virus (titers of <1/128). The A/PR8 virus was
passaged 148 times in the ferret, 596 times in the mouse, and 73 times
in 10-day fertile chicken eggs.
Immunization
Wild-type (pIgR+/+) and homozygous (pIgR-/-) female and male BALB/c mice, 6–8 wk old, were used in all experiments. Groups of five to six mice for each experiment were anesthetized by i.p. injection of amobarbital sodium (0.25 ml of a 1 µg/ml solution). Then they were immunized by dropping 1 µl of PBS containing the required concentration of a CTB*-combined HA vaccine into each nostril (15). In a single-dose regimen, the mice were immunized with the HA vaccine (5 µg/2 µl) prepared from A/PR8, A/Yamagata, A/Beijing, or B/Ibaraki virus, together with CTB* (1 µg/2 µl CTB from Sigma-Aldrich (St. Louis, MO) supplemented with 0.2% cholera toxin (CT; Sigma-Aldrich)) (16). In a two-dose regimen, the mice immunized primarily with the adjuvant-combined HA vaccine were boosted with the HA vaccine (5 µg/2 µl) alone 4 wk later.
Infection
Two conditions for virus infection were used (14, 40, 41). Under one of these conditions, each mouse was
anesthetized and then infected by i.n. dropping 20 µl of PBS
containing an A/PR8 virus suspension with 104.1
EID50 per mouse. This procedure induced a total
RT infection that caused virus shedding from the nose and lungs and
ultimately death from viral pneumonia 7 days later. The virus
inoculum was 20 x LD50, where
LD50 was determined by the total respiratory
infection method. The nasal and lung wash virus titers on day 3 after
infection were used as indices of protection in the upper and lower RTs
of immunized mice, respectively. Under the second infection condition,
which was mainly used in the present experiments, anesthetized mice
were infected by dropping 1 µl of PBS containing an A/PR8 virus
suspension with 104.1 EID50
into each nostril (a total of 2 µl per mouse). The nose-restricted
volume (2 µl) of the virus suspension induced nose-localized
infection that began in the nasal mucosa and spread to the lungs in
3–7 days but was not lethal. The nasal wash virus titers, the peak of
which was attained 3 days after infection, were used as indices of
protection in the upper RTs of the immunized mice.
Specimens
Mice were anesthetized and then bled from the heart with a
syringe. Serum was separated from blood by centrifugation and used for
Ab titration. After bleeding, the bronchoalveolar wash was collected by
washing the excised trachea and lungs twice with 2 ml of PBS containing
0.1% BSA, as described previously (40). The nasal wash
was collected by washing the nasal cavity of the excised head three
times with the same 1 ml of PBS containing 0.1% BSA. The nasal wash
removed 80% of nasal mucus-associated Abs after three washes and
95% after seven washes, as determined by ELISA for Ab titers of
nasal washes from the immunized mice. The nasal or bronchoalveolar wash
was used for Ab and virus titrations after removing cellular debris by
centrifugation. The physical manipulation of the nose to collect
secretions at the luminal surface caused leakage of blood from the
lamina propria. Blood contamination of the nasal wash was estimated to
be 0.003 ± 0.002% (mean ± SD) by counting the RBCs and
comparing the count with that of RBCs (8 x
109 cells/ml) from 10 specimens.
ELISA
The levels of IgA and IgG Abs against HA molecules purified from
the A/PR8 viruses were determined by ELISA as described previously
(15). Briefly, ELISA was conducted sequentially from the
solid phase (EIA plate; Costar, Cambridge, MA) with a ladder of
reagents consisting of the following: first, HA molecules purified from
the A/PR8 virus according to the procedure of Phelan et al.
(42); second, nasal wash, bronchoalveolar wash or serum;
third, goat anti-mouse IgA Ab (
-chain specific; Amersham) or
goat anti-mouse IgG Ab (
-chain-specific; Amersham) conjugated
with biotin; fourth, streptavidin conjugated with alkaline phosphatase
(Life Technologies, Rockville, MD); and, finally,
p-nitrophenylphosphate. The chromogen produced was measured
for absorbance at 405 nm with a SJeia Autoreader (model er-8000; Sanko
Junyaku, Tokyo, Japan). A 2-fold serial dilution of purified
HA-specific IgA or HA-specific monoclonal IgG (160 ng/ml) was used as a
standard. The Ab concentration of an unknown specimen was determined
from the standard regression curve constructed for each assay with the
programmed SJeia Autoreader. The chromogen produced was measured for
absorbance at 405 nm using an ELISA reader.
The approximate amount of anti-A/PR8 IgM Abs was also determined
indirectly by ELISA using the purified HA-specific monoclonal IgG as
the standard, after purification of IgM in nasal wash and serum samples
by passing them through an affinity column conjugated with goat
anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove,
PA), because a reliable standard for the IgM assay was not obtained.
Briefly, ELISA was conducted sequentially from the solid phase with a
ladder of reagents consisting of the following: first, purified HA
molecules; second, nasal wash or serum IgM; third, goat anti-mouse
-chain-specific Ab conjugated with biotin (BioSource International,
Camarillo, CA), which could detect >95% of IgM in mice
(43); fourth, streptavidin conjugated with alkaline
phosphatase (Life Technologies); and, finally,
p-nitrophenylphosphate.
The level of albumin in the nasal wash, bronchoalveolar wash, or serum was also determined by ELISA using purified mouse albumin (Biogenesis, Poole, U.K.) as the standard (35). Briefly, ELISA plates were coated with goat anti-mouse albumin IgG (Bethyl Laboratories, Montgomery, TX) and then incubated with nasal wash, bronchoalveolar wash, or serum specimens after blocking the coated plate with 1% BSA in PBS. The bound albumin was detected by the sequential addition of goat anti-mouse albumin Ab conjugated with alkaline phosphatase (Bethyl Laboratories) and p-nitrophenylphosphate.
Virus titrations
Serial 10-fold dilutions of the nasal or bronchoalveolar wash were prepared, and 0.2-ml aliquots of each dilution were added to Madin-Darby canine kidney cells in a six-well plate. After 1 h of adsorption, each well in the plate was overlaid with 2 ml of agar medium according to Tobita et al. (44, 45). After 2 days of incubation in a CO2 incubator, the plaques were counted. The virus titer was expressed in PFU per milliliter. The virus titer of each experimental group was represented by the mean ± SD of the virus titers per milliliter of specimens from five mice in each group.
Estimation of mucus volume in the nasal cavity of mice
The nasal wash concentrations of anti-A/PR8 HA IgA and IgG
Abs per milliliter of mucus volume, which covers the surface of the
nasal cavity of BALB/c mice (10-wk-old female), were estimated as
follows. First, the surface area of the nasal cavity, which is covered
with squamous, columnar, and olfactory epithelia, was estimated.
Briefly, serial tissue sections (each 3-µm thick) from the upper jaw,
which were fixed with 4% formaldehyde in PBS and decalcified with EDTA
before embedding in paraffin, were prepared and stained with H&E
(46). The circumference of the nasal cavity was measured
based on photographs of each of the 104 tissue sections that were taken
at 110-µm intervals of the serial sections from the nostrils to the
posterior nasopharynx pore. The total surface area of the
nasal cavity was estimated by integration using the trapezoidal
rule for the surface area of 104 section blocks calculated based on
both the circumference of the nasal cavity and the thickness of the
tissue blocks (113 µm). The circumference was calculated using
software PIXS 2000 Ver 2.0 (Inotech, Hiroshima, Japan). Next, the mucus
volume was estimated by assuming that the thickness of the mucus layer,
including both the mucus blanket (4- to 8-µm thickness) and the
periciliary space (cilia, 5–7 µm long), was 0.01 mm (47, 48). Table I shows that the
surface area of the nasal cavity is 300 mm2
and the mucus volume is 3 mm3 when the
thickness of the mucus layer is 0.01 mm.
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Differences in virus titers of groups between wild-type and pIgR-KO mice were compared by ANOVA. Comparisons between experimental groups were performed by the Student t test. Values of p < 0.05 were considered significant unless otherwise indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were designed to confirm that the cross-protection
against variant influenza virus infection in the upper RT is dependent
primarily on S-IgA Abs and is much stronger than that in the lower RT
(14, 15). BALB/c mice were immunized i.n. with different
CTB*-combined, inactivated vaccines prepared from A/PR8 (H1N1),
A/Yamagata (H1N1), A/Beijing (H1N1), and B/Ibaraki viruses. Four weeks
after immunization, the mice were challenged by the total RT infection
method with an A/PR8 virus suspension (20 µl). Three days later,
nasal wash, lung wash, and serum specimens were obtained for
anti-A/PR8 HA IgA and IgG titration by ELISA and for
virus titration as an index of protection. Fig. 2 shows that i.n. immunization with the
A/PR8 vaccine and its variant (A/Yamagata or A/Beijing) vaccine
conferred complete protection and partial cross-protection,
respectively, against A/PR8 virus infection in the upper RT. The
protection was accompanied by the induction of A/PR8 HA-reactive IgA
with low IgG levels in the nasal wash, while the cross-protection was
accompanied by induction of lower levels of A/PR8 HA cross-reactive IgA
in the nasal wash. In contrast, the immunization conferred complete
protection against homologous virus infection but negligible
cross-protection against variant virus infection in the lower RT. The
protection was accompanied by the induction of high levels of A/PR8
HA-reactive IgG in the lung wash. Thus, cross-protection against
variant influenza virus infection, which depends primarily on
anti-A/PR8 HA-reactive IgA, was detected in the upper RT. To
clarify the roles of S-IgA Abs in cross-protection against variant
virus infection in the upper RT, the nonlethal upper RT infection
method using an A/PR8 virus suspension (2 µl) was used in subsequent
experiments.
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The roles of S-IgA in providing cross-protection against variant
virus infection in the upper RT were examined in pIgR-KO mice immunized
i.n. with different CTB*-combined, inactivated A/PR8 (H1N1), A/Yamagata
(H1N1), A/Beijing (H1N1), and B/Ibaraki virus vaccines. Four weeks
after immunization, the mice were challenged by the upper RT infection
with the PR8 virus. Three days later, nasal wash and serum specimens
were obtained. First, anti-A/PR8 HA Ab and albumin levels in the
nasal wash and serum were compared between pIgR-KO mice immunized with
CTB*-combined A/PR8 vaccine and wild-type mice immunized under the same
conditions. Table II shows that the
blockade of transepithelial transport of dIgA in pIgR-KO mice caused a
marked increase in the serum IgA concentration, leaving serum IgG and
IgM levels almost unaltered. Contrary to the accumulation of serum IgA,
the blockade of transepithelial transport of dIgA caused a decrease in
the IgA level in the nasal wash, leaving the nasal wash IgG level
almost unaltered. In both wild-type and pIgR-KO mice, serum IgM was a
minor Ab component at less than one-tenth of serum IgG.
Therefore, in the subsequent experiments, A/PR8 HA-reactive IgA and IgG
were assayed as Ab components involved in protective immunity. Table II
also shows that there was no significant difference between wild-type
and pIgR-KO mice in the albumin level in the nasal wash, which was an
index of the leakage of blood from the lamina propria by physical
manipulation of the nose to collect secretions, or the disruption of
the pIgR gene. The nasal wash albumin level was 0.01–0.02%
of the serum, which corresponded to the blood contamination estimated
by counting the RBCs in the nasal wash (see Materials and
Methods). Thus, the leakage of blood in the nose was not enhanced
by the disruption of the pIgR gene in pIgR-KO mice. In
addition, the leakage of blood did not affect the concentrations of IgA
and IgG.
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). In the wild type-mice, the
immunization with the A/PR8 vaccine conferred complete protection
against the A/PR8 virus challenge and was accompanied by a predominant
A/PR8 HA-reactive IgA Ab titer in the nasal wash. The immunization with
A/Yamagata or A/Beijing vaccine conferred or tended to confer
partial cross-protection against the A/PR8 virus challenge, when the
specific protection afforded by the respective influenza A vaccines was
statistically compared with the nonspecific protection afforded by the
B/Ibaraki vaccine (40, 49). The cross-protection
was accompanied by relatively low A/PR8 HA-reactive IgA titers in the
nasal wash.
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). The incomplete protection was accompanied by a decrease
in nasal wash anti-A/PR8 HA IgA titer (approximately one-fourth of
that for the wild-type mice) and by a marked increase in serum
anti-A/PR8 HA IgA titer (20 times higher than that in the
wild-type mice). The immunization with the A/Yamagata vaccine reduced
cross-protection against the A/PR8 virus challenge to a greater degree
than that observed in wild-type mice, and this difference in the degree
of protection between wild-type and pIgR-KO mice was statistically
significant (t test). The immunization with the
A/Beijing vaccine also tended to reduce partial cross-protection to a
greater degree than that observed in the wild-type mice (statistically
not significant in t test). The difference in the degree of
protection and cross-protection between the wild-type and pIgR-KO mice
was statistically significant when three groups immunized with
H1N1-subtype vaccines were compared between the wild-type and pIgR-KO
mice by analysis of variance (p < 0.006). The
reduction of the ability to provide cross-protection in pIgR-KO mice
immunized with A/Yamagata and A/Beijing vaccines was accompanied by the
reduction of nasal wash anti-A/PR8 HA IgA titers, when compared
with those in the wild-type mice. For the B/Ibaraki vaccine group,
there was no difference in the influenza-types nonspecific protection
between the wild-type and pIgR-KO mice. These results indicate that the
degree of protection or cross-protection provided by immunization with
A/PR8 or other variant virus vaccines within the same subtype (H1N1)
was reduced in pIgR-KO mice, when compared with that in the wild-type
mice, in parallel with the reduction of nasal wash A/PR8 HA-reactive
IgA Ab titers in the pIgR-KO mice. In addition, the presence of a
non-negligible level of A/PR8 HA-reactive IgA Abs in the nasal wash of
pIgR-KO mice suggests that they are involved in compensation for the
reduction in the degree of protection or cross-protection. Characterization of A/PR8 HA-reactive IgA and IgG Abs in upper RT of the vaccinated pIgR-KO mice
To investigate in detail the transport of dIgA across the mucosal
membrane of the upper RT, the nasal wash A/PR8 HA-reactive IgA or IgG
titer, shown in Fig. 3, was further represented by the Ab concentration
in the nasal mucus and compared with the concentration of the serum Ab.
The IgA and IgG concentrations in the nasal mucus were determined as
follows. The mucus volume was estimated to be 3
mm3 by multiplying the surface area of the nasal
cavity (299.8 mm2) by the thickness of the mucus
blanket including the periciliary space (0.01 mm) (Table I). The nasal
wash specimens that were collected from the immunized mice by washing
three times were estimated to contain 80% of all the
mucus-associated Abs. Thus, the anti-A/PR8 HA IgA and IgG
concentrations in the nasal mucus (micrograms per milliliter of mucus
volume) were calculated by multiplying each nasal wash-ELISA Ab titer
(micrograms per milliliter) in Fig. 3 by 333 (1000/3
mm3) and 1.25 (100/80%), respectively.
Table III shows the characteristics of
nasal and serum A/PR8 HA-reactive Abs in the wild-type and pIgR-KO mice
immunized i.n. with the A/PR8 vaccine. In the wild-type mice, the
concentration of anti-A/PR8 HA IgA in the nasal mucus was 30
times higher than that of IgG. The nasal mucus IgA concentration was
40 times higher than the serum IgA concentration, which suggests
that IgA was secreted actively by pIgR-mediated transcytosis
across the mucosal membrane. In contrast, the IgG was the major
component in the serum and serum IgA concentration was approximately
one-fifth of the serum IgG concentration. The nasal mucus IgG
concentration was approximately one-fifth of the serum IgG
concentration.
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20 times higher than the serum IgA concentration in the wild-type
mice. The accumulation of serum IgA resulted in the appearance of a
small quantity of nasal mucus IgA via pathways other than pIgR-mediated
transcytosis. The nasal mucus IgA concentration of was 10 times
higher than the nasal mucus IgG concentration and approximately
one-fourth of the nasal IgA concentration of the wild-type mice. This
low but non-negligible level of nasal mucus IgA seemed to be sufficient
to provide partial protection against A/PR8 virus infection, because
the reduction in the degree of protection, accompanied by the reduction
in the nasal IgA level, was only partial (Fig. 3). These results,
together with the data that the IgG levels in the nasal mucus and serum
of the pIgR-KO mice were almost the same as those of the wild-type
mice, confirm that the A/PR8 HA-reactive IgA in the nasal mucus is
essential for providing complete protection against homologous virus
infection.
The characteristics of the nasal and serum anti-A/Yamagata IgA and
IgG, which were cross-reactive to A/PR8 HA molecules, were also
examined in the A/Yamagata vaccine-immunized wild-type and pIgR-KO mice
(Table III). The levels of A/PR8 HA-reactive IgA and IgG in the
A/Yamagata vaccine-immunized wild-type and pIgR-KO mice corresponded to
the reduced levels of those in the A/PR8 vaccine-immunized wild-type
and pIgR-KO mice, respectively. Thus, the degree of cross-protection
seemed to be dependent on the nasal IgA concentration. These results
suggest that the nasal A/PR8 HA-reactive IgA in the A/Yamagata
vaccine-immunized mice plays an essential role in providing
cross-protection against A/PR8 virus infection.
Ab responses and protection against upper respiratory infection by A/PR8 viruses in pIgR-KO mice vaccinated in a two-dose regimen
To further confirm the superior role of S-IgA to IgG in the upper
RT in providing protection against influenza, A/PR8 HA-reactive
Ab responses and protection against A/PR8 virus infection were compared
between the wild-type and pIgR-KO mice immunized i.n. with various
vaccines (5 µg) together with CTB* (1 µg) and boosted with each
vaccine alone 4 wk later (5 µg). Two weeks after the second
immunization, the immunized mice were challenged by upper RT infection
with the A/PR8 virus, and 3 days later nasal wash and serum specimens
were collected for virus and Ab titration. Fig. 4 shows A/PR8 HA-reactive IgA and IgG
responses in both the nasal wash and serum, and nasal wash virus titers
of the immunized wild-type and pIgR-KO mice. The nasal and serum A/PR8
HA-reactive Ab titers in wild-type and pIgR-KO mice immunized in a
two-dose regimen were 
10 times those in the wild-type and pIgR-KO
mice immunized in a single-dose regimen (Fig. 3). In the wild-type
mice, the immunization with the A/PR8 virus vaccine in a two-dose
regimen conferred complete protection against the A/PR8 virus challenge
and was accompanied by the highest nasal wash anti-A/PR8 HA IgA
titer. The immunization with the A/Yamagata and A/Beijing vaccines
conferred complete cross-protection against the A/PR8 virus challenge
and was accompanied by a relatively high level of nasal anti-A/PR8
HA IgA, which was >20 times higher than that in mice immunized in a
single-dose regimen. Thus, the degree of protection or cross-protection
correlated with the level of anti-A/PR8 HA IgA.
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), was reproduced in a two-dose
regimen. This further confirms that A/PR8 HA-reactive IgA in the nasal
mucus is essential for providing protection or cross-protection against
homologous or heterologous virus infection in the upper RT. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B) and
that a marked increase in anti-A/PR8 HA IgA Ab level and unaltered
levels of the IgM and IgG Abs were observed in the serum with reduced
but not negligible IgA levels in the nasal wash (Tables II and III).
These data demonstrate that the transepithelial transport of dIgA by
pIgR is severely blocked in the epithelial cells of the respiratory and
digestive tracts of the pIgR-KO mice. In addition, the data showing
that the IgM was not a major serum Ab component with an unaltered level
between the wild-type and pIgR-KO mice (Table II) suggest that the role
of pIgM in providing protection in the upper RT is minimal. Table II
also shows that the level of albumin was not altered in the nasal wash
of pIgR-KO mice (Table II). In this regard, Johansen et al.
(35) showed that pIgR-KO mice had elevated levels of
albumin in the saliva and feces, suggesting that deterioration of their
epithelial barrier function in the absence of S-IgA (and S-IgM) results
in leakage of serum proteins. The discrepancy between our results and
theirs remains to be clarified, although it may be due to the
difference in the strains of pIgR-KO mice used.
The characteristics of nasal IgA in the immunized mice were first
analyzed in this study by estimating the IgA and IgG Ab concentrations
in the mucus that covered the surface of the nasal cavity (Tables I and III). The estimation of mucus volume seemed to be very useful, because
it enabled us to understand directly the relationship of Ab
concentration in serum and mucus and to illustrate the transport of Abs
from serum to nasal secretion. The concentration of anti-A/PR8 HA
IgA in the nasal mucus of the wild-type mice immunized with
CTB*-combined A/PR8 vaccine was 30 times higher than that of IgG,
whereas the concentration of the nasal IgA in the immunized pIgR-KO
mice was 10 times higher than that of IgG. This result confirms that
IgA is a major Ab component in the upper RT. Also, as the nasal IgA
concentration in the wild-type mice was 40 times higher than the
serum IgA concentration, it was evident that IgA is secreted actively
across the mucosal membrane. In contrast, as the nasal IgA
concentration in the pIgR-KO mice was lower than the serum IgA
concentration (about one-half), it was evident that the transepithelial
transport of dIgA was blocked in pIgR-KO mice. These results reinforce
the idea that nasal anti-A/PR8 HA IgA plays a major role in
providing protection or cross-protection against influenza virus
infection in the upper RT.
This study showed that in the pIgR-KO mice immunized i.n. with A/PR8
and variant H1N1 virus vaccines the degree of protection or
cross-protection against A/PR8 virus infection in the upper RT was
reduced, compared with that in the wild-type mice, in parallel with a
decrease in nasal anti-A/PR8 HA IgA titer (Figs. 3 and 4). The
results indicate that S-IgA is associated with the mechanisms of
protection or cross-protection against influenza virus infection in the
upper RT. In contrast, Mbawuike et al. (33) demonstrated
that IgA is not required for the protection in IgA-KO mice immunized
i.n. with a CTB*-combined vaccine and challenged with a lethal dose of
homologous virus. However, in their experiments, the role of IgA in the
cross-protection against heterologous virus infection in the upper RT,
which cannot be conferred by serum IgG, remained to be examined. Thus,
the present results imply that the IgA-KO mice are less protected than
the wild-type mice against a variant influenza virus infection in the
upper RT. We cannot exclude the possibility that other protective
mechanisms that are not the primary means of protection in the upper RT
of wild-type mice, such as elevated levels of IgM and IgG, may become
active in congenitally S-IgA-deficient mice. In this regard, Renegar et
al. (50, 51) have demonstrated that genetically normal
immune mice, given chemically defined total parenteral nutrition by the
i.v. route, results in loss of nasal anti-influenza immunity with a
significant drop in influenza-specific S-IgA titer in the upper RT
compared with chow-fed mice, suggesting that the impairment of mucosal
immunity in genetically normal mice cannot be substituted by serum IgG
for the role of S-IgA in the protective immunity against influenza
virus infection.
This study also showed that in the pIgR-KO mice the blockade of
transepithelial transport of dIgA by pIgR resulted in the accumulation
of IgA in the serum and the reduction of nasal mucus IgA titer (Table III). However, the level of nasal IgA was not negligible,and was 10
times higher than that of nasal mucus IgG Abs. This result suggests
that pathways other than pIgR-mediated transcytosis, such as
asialoglycoprotein receptor-mediated endocytosis (2, 52),
CD89 (FcR)-mediated binding (53), or
intercellular diffusion from the mucosal tissue (serum) to the mucosal
surface are involved in the IgA transport. With respect to IgG Abs,
Wagner et al. (54) have shown that anti-HA IgG in the
nasal wash is a transudate from serum according to the concentration
gradient of the IgG. The non-negligible nasal wash IgA level in the
pIgR-KO mice, transudates from the accumulated serum IgA, probably
provided compensation for the reduction of the degree of protection or
cross-protection against influenza virus infection in the upper RT
(Figs. 3 and 4) (2).
In this study, i.n. immunizations with the A/PR8 (H1N1) vaccine and its
variant (A/Yamagata or A/Beijing) vaccines conferred complete
protection and a partial cross-protection against the A/PR8 virus
infection in the upper RT of wild-type mice, respectively, and was
accompanied by the induction of predominant S-IgA reactive to A/PR8 HA
in the nasal wash (Fig. 2 and 3). In addition, in mice immunized in a
two-dose regimen, the immunization with the variant vaccines conferred
complete cross-protection against the A/PR8 virus infection, in
parallel with the induction of the higher cross-reactive S-IgA Ab
responses (Fig. 4). Moreover, we previously showed that almost complete
cross-protection by variant virus vaccines within the H3N2 subtype and
the challenge virus (H3N2) was induced with the induction of the
cross-reactive S-IgA in the upper RT (14). Recently,
Tumpey et al. (49) demonstrated that complete
cross-protection by the H3N2 vaccine and the H5N1 (or H1N1) virus was
induced with the subtype cross-reactive IgA and IgG in the RT of mice
immunized three times at weekly intervals with adjuvant-combined H3N2
vaccine and challenged with lethal H5N1 (or nonlethal H1N1) virus.
These results suggest that the degree of cross-protection depends on
the concentration of cross-reactive IgA or IgG Abs, which increases
with the increase in the dose of the vaccine, the frequency of
vaccination, and the antigenic similarity between virus strain used for
nasal vaccination and challenge virus strain. Under the immunization
conditions described above, the nasal IgA was detected by ELISA but
failed to neutralize the A/PR8 virus in vitro. The data regarding NT
activities were not included in the results, because the protection or
cross-protection in the upper RT did not correlate with NT activities
of nasal IgA. This finding, as also reported by other investigators
(49, 55), suggests that anti-HA IgA Abs act through
other in vivo mechanisms to neutralize viruses and/or to enhance
clearance of virus-infected cells.
A slight but significant reduction in nasal virus titers was observed
in wild-type and pIgR-KO mice immunized with the B/Ibaraki vaccine and
challenged with an immunologically unrelated influenza A (A/PR8) virus
(
Figs. 2–4). We and others (40, 49) have already shown
that a partial nonspecific reduction of virus titers was observed in
mice immunized i.n. with CTB*-, or Escherichia coli
heat-labile toxin-combined B-type virus Ags and challenged with A/PR8
or H5N1 virus. Moreover, we have demonstrated that CTB* can confer the
ability to reduce the virus replication nonspecifically, when given
i.n. into mice, suggesting that the effector cells involved in innate
immunity, such as NK or 
T cells, could be stimulated by i.n.
immunization with CT or Escherichia coli heat-labile toxin
derivatives as an adjuvant for nasal influenza vaccine
(56, 57, 58, 59, 60, 61). The restimulation of NK or T cells could
result in partial reduction of virus replication observed after viral
challenge. It has also been observed that the number of NK and T
cells increases in the lung tissue of mice following infection with
influenza virus (59) and that T cells proliferate
nonspecifically in response to virus-infected cells (60, 61).
The development of a mucosal vaccine that stimulates cross-protection
against variant viruses, including viruses with pandemic potential,
would be strategically important for improving the efficacy of
currently inactivated vaccines that induce high levels of serum
antiviral IgG Abs to provide protection against homologous viral
infection. The i.n. immunization procedure using adjuvant-combined
inactivated vaccines was useful, because mucosal IgA Abs that are
capable of providing cross-protection against a variant virus infection
in the upper RT could be induced by this procedure (Figs. 3 and 4)
(14, 62, 63). Field trials will be required to confirm the
effectiveness of the adjuvant-combined vaccine in preventing or in
attenuating illness without negative side effects, and also to develop
an effective nasal influenza vaccine in the near future.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: S-IgA, secretory IgA; p, polymeric; KO, knockout; A/PR8, A/PR/8/34; A/Yamagata, A/Yamagata/120/86; A/Beijing, A/Beijing/262/95; B/Ibaraki, B/Ibaraki/2/85; RT, respiratory tract; HA, hemagglutinin; CT, cholera toxin; CTB, CT B subunit; CTB*, CTB containing a trace amount of CT; i.n., intranasal(ly); dIgA, dimeric IgA; HI, hemagglutination inhibition; NT, virus neutralization; SC, secretory component.
Received for publication October 2, 2001. Accepted for publication January 11, 2002.
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, The significant
difference from the B/Ibaraki vaccine-immunized mice
(p < 0.05) in mean values among the groups within
the nasal wash virus titer or within the lung wash virus titer. The
values shown in parentheses are the numbers of mice that failed to
clear nasal viruses/total mice.
