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Journal of Virology, September 1999, p. 7565-7573, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Antibody-Dependent and -Independent Protection
following Intranasal Immunization of Mice with Rotavirus
Particles
Monica M.
McNeal,1
Mary N.
Rae,1
Judy A.
Bean,2 and
Richard L.
Ward1,*
Division of Infectious
Diseases1 and Department of
Biostatistics,2 Children's Hospital Medical
Center, Cincinnati, Ohio 45229
Received 12 January 1999/Accepted 25 May 1999
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ABSTRACT |
The ability to elicit protective immune responses after intranasal
immunization with rotavirus particles, either with or without the
attenuated Escherichia coli heat-labile enterotoxin
LT(R192G) as an adjuvant, was examined in the adult mouse model. BALB/c mice were administered one or two inoculations of
psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl)
purified rotavirus particles. Four weeks after immunization, mice were
challenged with the murine rotavirus strain EDIM, and the shedding of
rotavirus antigen was quantified. Rotaviruses used for immunization
included EDIM and heterotypic simian (RRV), bovine (WC3), and human
(89-12) strains. tl EDIM stimulated both systemic and intestinal
rotavirus antibody responses and complete protection with as little as
one 1-µg dose. Inclusion of LT(R192G) (10 µg) significantly
increased rotavirus antibody responses and reduced antigen
concentrations needed for full protection. Both dl EDIM and heterotypic
dl and tl particles stimulated protection, but they did so less than tl
EDIM at comparable concentrations, either with or without LT(R192G). When B-cell-deficient µMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM
and heterotypic particles in BALB/c mice. However, dl EDIM particles
induced similar levels of protection in both mouse strains. The
protection stimulated by tl or dl EDIM particles was not diminished by
CD8 cell depletion prior to immunization in either strain of mice.
These results indicate that tl EDIM induced immunity at least partially
through responses to its outer capsid proteins, presumably by
stimulation of serotype-specific neutralizing antibody. In contrast,
the other particles stimulated protection primarily by an
antibody-independent mechanism. Finally, depletion of CD8 cells had no
effect on protection by either mechanism.
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INTRODUCTION |
Rotaviruses are the primary cause of
severe infantile gastroenteritis and, hence, have been targeted for
vaccine development. Rotavirus vaccines evaluated to date have all been
live viruses that are delivered orally to mimic the protection found
after natural rotavirus infection. These vaccines have provided only partial immunity against subsequent rotavirus disease (2, 3, 5,
18, 33, 34, 36). Because intranasal (i.n.) immunization has been
successful against other mucosal pathogens (19), this route
of immunization should be a promising method to prevent rotavirus
disease which primarily, if not solely, results from infection of the
intestinal mucosa. To test this possibility, we utilized the adult
mouse model developed not only to rapidly evaluate new vaccination
strategies but also to identify immunological effectors of protection
(37).
After oral immunization with live murine rotavirus, BALB/c mice were
found to be completely protected against subsequent murine rotavirus
infection as determined by the absence of viral shedding and by the
lack of significant rises in serum or stool rotavirus antibody
responses (28). Protection correlated with the titers of
serum (24) and stool (10) rotavirus
immunoglobulin A (IgA) and was found to diminish rapidly in genetically
altered mice that were B cell deficient (11, 23). Not only
did antibody appear to be necessary for protection, but even the
resolution of rotavirus infection in immunologically normal mice
correlated with the presence of CD4 cell-dependent antibody production
(26). Thus, rotavirus antibody appeared to play a major role
in immunity after oral inoculation of mice with live virus. CD8 cells
were also found to have a major role in the resolution of rotavirus infection in mice (11, 23) and may have some role in
protection as well, at least during the first weeks after oral,
live-virus immunization (12). Similar to the results found
in mice, CD8 cells appear to be important in the normal resolution of
rotavirus infection in calves, while CD4 cells were crucial for normal
antibody responses (30).
In the study reported here, we examined the protection against
rotavirus infection after i.n. immunization with inactivated triple-layered (tl) and double-layered (dl) (i.e., lacking VP4 and VP7)
rotavirus particles. These particles were of both homologous (murine)
and heterologous (simian, bovine, and human) origin. All particles
examined provided good protection, but the mechanism of this protection
varied depending on the immunogen. Based on the degree of protection in
BALB/c and in B-cell-deficient µMt mice stimulated by the different
murine rotavirus particles, it appeared that tl murine rotaviruses
protected at least partially through an antibody-dependent mechanism,
while the dl particles appeared to protect through an
antibody-independent mechanism.
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MATERIALS AND METHODS |
Mouse strains.
Two strains of mice were used in these
studies. One was pathogen-free BALB/c which were purchased from
Harlan-Sprague-Dawley when 6 weeks of age. No mouse had evidence of
previous rotavirus infection as determined by serum rotavirus antibody
titers. The other strain was genetically engineered and was unable to
produce functional antibody. This strain was produced by Kitamura et
al. (16) by using targeted disruption of a membrane exon of
the gene encoding the µ-chain constant region (µMt mutation) in
mouse embryonic stem cells. The transfected stem cell clone D3 was
injected into blastocysts from C57BL/6 mice, and the derived offspring were backcrossed multiple times to C57BL/6 mice. These mice, containing a µMt mutation on a C57BL/6 background, were purchased as a breeding pair from Jackson Laboratories (Bar Harbor, Maine). Offspring of this
pair were included in this study with the permission of K. Rajewsky.
Experiments were conducted with adult mice (6 to 20 weeks of age). The
µMt mice were found to produce no detectable rotavirus antibody.
Rotaviruses.
The murine EDIM strain of rotavirus used
throughout these studies was originally obtained from M. Collins
(Microbiological Associates, Bethesda, Md.) in 1980. To challenge mice
after immunization, both wild-type (wt) virus from mouse stool and cell
culture-adapted (passage 9) EDIM preparations were used. Because adult
C57BL/6 mice were totally resistant to infection when inoculated with passage 9 of EDIM (i.e., no rotavirus shedding or immune responses were
detected) but were susceptible to infection with wt EDIM based on
development of rotavirus antibody, the wt strain was used to inoculate
µMt mice which have a C57BL/6 background. However, since the passage
9 preparation has been used routinely for all previous studies with
BALB/c mice in our laboratory since the inception of the adult mouse
model for rotavirus (37), it was used to inoculate BALB/c
mice. Even though adult C57BL/6 mice are more susceptible to wt EDIM,
the two virus preparations were nearly equally infective in adult
BALB/c mice; i.e., the doses required to infect 50% of the mice were
240 and 560 focus-forming units (FFU) for the wt and passage 9 preparations, respectively. The large number of mice required to
conduct an infectivity study precluded conducting this analysis in
µMt mice. Thus, the results found with µMt and BALB/c mice may not
be directly comparable because of differences in the EDIM preparations
used for challenge. It was observed, however, that rotavirus shedding
in unimmunized µMt mice after inoculation with wt EDIM was comparable
to that found in unimmunized BALB/c mice administered passage 9 of
EDIM. The wt EDIM preparation used here was obtained from stools of infected neonatal mice and purified as described previously
(23). The final preparation contained 107 FFU
per ml. The passage 9 preparation of EDIM was obtained as previously
reported (37) and contained 2 × 106
FFU/ml.
Immunization of mice was performed with inactivated particles generated
from four different strains of rotavirus: the G3[P16] murine EDIM
strain, the G3[P3] simian RRV strain (provided by Y. Hoshino,
National Institutes of Health, Bethesda, Md., and triply plaque
purified), the G6[P5] WC3 strain (provided by H. F. Clark,
Children's Hospital, Philadelphia, Pa., and triply plaque purified),
and the G1[P8] human 89-12 strain (our laboratory). The EDIM strain
used to generate the particles for immunization was a triply plaque
purified isolate that had been passaged a total of 41 times in MA104
cells and purified by CsCl centrifugation (27). tl
infectious virus (1.36 g/ml) and dl particles lacking VP4 and VP7 (1.38 g/ml) were collected from the same gradient. The dl particles were
further treated with 10 mM EDTA and purified a second time in CsCl to
ensure complete removal of VP4 and VP7 proteins. After purification,
the virus particles were dialyzed against phosphate-buffered saline
containing 20% glycerol and stored frozen at 
70°C. To establish
which proteins were present in the particles, each one was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously
described (27). Furthermore, Western blot analyses of the
antibody generated after administration of these particles to mice by
using methods already described (27) revealed no evidence
for VP4 or VP7 in the dl particles, while the tl particles generated
clear antibody responses to these proteins (results not shown).
Purified tl or dl EDIM particles were inactivated by UV-psoralen
treatment as described by Groene and Shaw (13). In short,
the particles were dispensed into 60-mm petri dishes, and psoralen
(4'-aminomethyltroxsalen HCl; Lee Biomolecular Research, Inc., San
Diego, Calif.) was added at a concentration of 40 µg/ml. The virus
was then placed in ice on a rotating platform at a distance of 10 cm
and irradiated with high-intensity, long-wavelength UV light (365 nm,
40 min). After treatment, the viral titer was determined by a
fluorescent focus assay as previously described (17).
Although the infectivity of tl particles containing 8.4 × 108 FFU/ml was reduced below detectable limits (i.e.,
102 FFU/ml) by 20 min of UV treatment, the irradiation time
was extended to 40 min to ensure that no infectious virus remained.
Evidence that these particles were not infectious was shown in two
ways: no immune responses to nonstructural proteins were found after inoculation of mice with these particles by using Western blot analyses
as previously described (27), and stool rotavirus IgA titers
stimulated following inoculation with these particles were consistently
at least 10-fold less than was found after oral infection with live
EDIM (results not shown).
The other three viruses used for immunization were grown and processed
in the same manner. For this study, these particles
were all considered
to be heterotypic relative to EDIM even though
RRV and EDIM are both G3
strains and were found to share a very
weak, one-way
cross-neutralization response with hyperimmune antisera
(
38).
Properties of the LT adjuvant.
Escherichia coli
heat-labile enterotoxin (LT) has been found to be an effective mucosal
adjuvant with various antigens, including virus-like particles (VLPs)
of rotavirus (31). wt LT is activated by protease cleavage
in the intestine which simultaneously potentiates its toxigenic
properties (6). A mutant LT [i.e., LT(R192G)] has been
developed in which arginine 192 is replaced by glycine which eliminates
the trypsin cleavage site and attenuates the protein (8).
Only the R192G mutant of LT was used in this study. It was provided by
John Clements and Candice Smith of Tulane University School of
Medicine, New Orleans, La.
Study plan.
After collection of both blood (retroorbital
capillary plexus puncture) and stool specimens (two pellets placed in
0.5 ml of Earle's balanced salt solution [EBSS]) for rotavirus
antibody measurements, groups of six or eight mice were immunized. i.n. immunization was performed under light sedation (i.e., mice were placed
in a closed vessel with metafane until they could no longer stand). The
immunogen was then given by gradual inoculation of the nares of mice
(maximum of 25 µl/nostril). When two doses were administered, the
second dose was given 2 weeks later. At 4 weeks after the final dose,
blood and stool specimens were again collected for antibody
measurements and 1 day later mice were orally (gavage) challenged with
either 2 × 104 FFU of passage 9 EDIM (BALB/c) or
5 × 105 FFU of unpassaged EDIM (µMt). Stools were
then collected for at least 7 days after challenge (two pellets/day
from each mouse placed into 1 ml of EBSS) to be examined for viral
shedding. At 3 weeks after challenge, blood and stool samples were
again collected for antibody determinations.
To determine the effects of CD8 cell depletion prior to immunization,
mice were injected intraperitoneally once per day with
1 mg of ammonium
sulfate-precipitated monoclonal antibody preparation
obtained from the
rat hybridoma cell line 2.43 purchased from
the American Type Culture
Collection. Inoculations were initiated
5 days before immunization and
were performed for four consecutive
days and twice weekly thereafter
until the end of the experiment.
Depletion was monitored by using a
fluorescence-activated cell
sorter (FACS). This analysis was performed
with mesenteric lymph
node tissue collected from mice on the day of the
first immunization
and 21 days after EDIM challenge. Two control mice
and two depleted
mice were included in each analysis. Methods used for
FACS analysis
have been reported previously (
23).
Oral immunization of mice was performed by gavage. Specimens were
collected and mice were challenged by the same procedures
as for
i.n.-immunized
mice.
Detection of rotavirus antigen in stool.
Stool pellets
collected into EBSS on each day following EDIM challenge were stored
frozen (20°C). To test for rotavirus antigen, the specimens were
thawed, homogenized, and analyzed by enzyme-linked immunosorbent assay
(ELISA) as previously described (26) with the following
exception. Instead of basing antigen shedding directly on net optical
density (A490) values, shedding was quantified from a standard curve by using CsCl-purified dl EDIM as the standard. Therefore, the quantities of rotavirus antigen shed were determined in
micrograms of rotavirus protein for each stool specimen. Although rotavirus shedding was often not fully resolved in unimmunized mice
until 9 days after challenge, the amount of rotavirus shed per mouse
within a group was determined only during the first 7 days after
challenge. Since detectable shedding in previously immunized mice was
fully resolved by 7 days after challenge, the degree of protection due
to immunization was somewhat greater than the values calculated. This
protection was determined from the average number of micrograms of
rotavirus antigen excreted for every mouse on each of the 7 days after
challenge. These values were used for statistical comparison between
groups of mice. In addition, the total amount of antigen shed for each
group of mice was used to determine the percentage reduction in
shedding relative to unimmunized control mice.
Determination of rotavirus antibody titers.
Serum rotavirus
IgA and IgG and rotavirus stool IgA were measured by ELISA as
previously described (23). Briefly, EIA plates (Corning
Costar Co., Cambridge, Mass.) were coated overnight at 4°C with
anti-rotavirus rabbit IgG. After being washed with phosphate-buffered saline plus 0.05% Tween 20, EDIM viral lysate and mock-infected cell
lysate were each added to duplicate wells. Serial twofold dilutions of
pooled sera from EDIM infected mice assigned concentrations of 160,000 or 10,000 U of rotavirus IgG or IgA per ml, respectively, were added to
duplicate wells coated with either EDIM-infected or uninfected MA104
cell lysates to generate a standard curve. Serial 10-fold dilutions of
mouse sera to be tested were also added to duplicate wells of each
lysate. This was followed by a sequential addition of biotin-conjugated
goat anti-mouse IgG or IgA (Sigma Chemical Co., St. Louis, Mo.),
peroxidase-conjugated avidin-biotin (Vector Laboratories, Burlingame,
Calif.), and o-phenylenediamine substrate (Sigma). Color
development was stopped after 15 min with 1 M
H2SO4, and the A490 was
measured. Titers of rotavirus IgG or IgA, expressed as units per
milliliter, were determined from the standard curve generated by
subtraction of the average A490 values of the
duplicate cell lysate wells from the average of the wells coated with
EDIM lysate. The same methods were used to measure serum rotavirus IgG1
and IgG2a titers except for two modifications: (1) the biotinylated
goat anti-mouse IgG used to detect bound rotavirus antibody was subtype
specific (i.e., it specifically recognized mouse IgG1 or IgG2a), and
(2) the concentration of rotavirus antibody was determined from a
standard curve that measured rotavirus antibody in nanograms rather
than in units per milliliter. To generate this curve, EIA plates were
initially coated overnight with either goat anti-mouse IgG or normal
goat serum. After the plates were washed, serial twofold dilutions of
purified mouse IgG1 or IgG2a were added to the wells. The remaining steps were identical to those used to measure rotavirus-specific IgG1
or IgG2a.
For determination of stool rotavirus IgA, two stool pellets were
collected into 0.5 ml of EBSS, homogenized, and centrifuged
(1,500 ×
g, 5 min). Stool rotavirus IgA was then
measured by the
method described
above.
Neutralizing antibody to EDIM was determined by using an antigen
reduction ELISA assay as described previously (
17).
Statistical methods.
All analyses were performed by using
SAS version 6.12 (SAS Institute, Inc., Cary, N.C.). Differences in the
mean quantities of rotavirus antigen shed per mouse were determined by
analysis of variance since there were more than two groups being
compared. The variances in the different groups were not equal.
Therefore, the analyses were performed on the log transformations. The
same procedure, analysis of variance, was used to determine whether there were differences in geometric mean titers of rotavirus antibody between groups of mice. Since there were numerous comparisons, an
adjustment to the level of significance was made, and only when the
P value is <0.01 are the differences reported.
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RESULTS |
Dose-dependent protection stimulated by homologous tl
rotaviruses.
The initial experiment was to determine the level of
protection against murine rotaviruses that could be stimulated by i.n. inoculation with the homologous strain of tl, inactivated rotavirus particles. Groups of BALB/c mice were administered one or two doses of
psoralen/UV-inactivated rotavirus strain EDIM ranging from 10 to 0.1 µg/dose and challenged with live EDIM 4 weeks after the last
immunization. Mice were monitored for antibody responses both after
immunization and challenge and for shedding of rotavirus after challenge.
Immune responses after immunization were significantly associated with
both the quantity of the immunogen and the number of
doses as
determined by serum rotavirus IgG and IgA and stool rotavirus
IgA
titers (Table
1). Only the highest
concentration of immunogen
administered in a two-dose regimen
stimulated detectable stool
rotavirus IgG or serum neutralizing
antibody to EDIM. The quantity
and number of doses were also
significantly associated with protection
against rotavirus shedding and
immune responses to rotavirus after
EDIM challenge. None of the mice
immunized with one or two doses
of 10 µg of EDIM or two doses of 1 µg shed detectable rotavirus
antigen after challenge (Fig.
1), and none experienced a significant
(i.e.,
4-fold) boost in serum rotavirus IgG or IgA or stool rotavirus
IgA titer. In contrast, one dose of 1 µg stimulated a partial
but
significant (
P = 0.002) reduction in rotavirus
shedding, and
three of the eight mice in this group did not shed
detectable
rotavirus antigen or seroconvert. Finally, immunization with
either
one or two doses of 0.1 µg of rotavirus provided no
significant
protection.
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TABLE 1.
Geometric mean titers (GMTs) of serum and stool rotavirus
antibody after i.n. immunization with different doses of
tl EDIMa
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FIG. 1.
Dose-dependent protection after i.n. immunization with
tl EDIM. Groups of mice were immunized and challenged as described in
the legend to Table 1. Stools collected for the 7 days following
challenge were quantified for rotavirus shedding by an ELISA. Shedding
was compared to that found in mock-immunized control mice. *,
P < 0.001 versus mock-immunized mice; **,
P = 0.002 versus mock-immunized mice.
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Protection stimulated by tl and dl homotypic and heterotypic
rotavirus particles.
It was next determined whether protection in
this model was dependent on the presence of homotypic VP4 and VP7
proteins, the outer capsid proteins of rotavirus responsible for
stimulating neutralizing antibody. For this determination, either EDIM
particles lacking these proteins were used for immunization (i.e., dl
particles) or mice were immunized with heterotypic tl or dl rotavirus particles.
After two doses of 10 µg each of psoralen/UV-treated tl or dl EDIM or
heterotypic rotavirus particles, the only rotavirus
antibody response
against dl particles that was significantly
different from the response
elicited by the same concentration
of tl particles was stool IgA after
WC3 inoculation (Table
2).
As expected,
serum neutralizing antibody to EDIM was undetectable
following
immunization with any particles except tl EDIM (results
not shown). The
level of protection stimulated by all dl particles
and tl heterotypic
particles was consistently less than that stimulated
by tl EDIM
particles as determined by antigen shedding after EDIM
challenge and,
in most cases, this difference was significant
(
P 
0.01; Fig.
2). In contrast to
results found after immunization
with tl EDIM particles, none of the
other particles (tl or dl)
stimulated complete protection. Furthermore,
there were no consistent
differences between the protection stimulated
by the two types
of heterotypic particles. These results indicate that
heterotypic
tl and dl particles and homotypic dl EDIM particles
consistently
stimulate partial protection in this model, while
homotypic tl
EDIM particles induce complete protection.
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TABLE 2.
Geometric mean titers (GMTs) of rotavirus antibodies
stimulated by i.n. immunization with tl or dl particles of homotypic
and heterotypic rotavirusesa
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FIG. 2.
Protection against EDIM shedding after i.n. immunization
with homotypic and heterotypic dl and tl rotavirus particles. Mice
immunized as stated in the legend of Table 1 were challenged with
4 × 104 infectious EDIM, and stool samples collected
for the 7 days after challenge were quantified for rotavirus antigen.
All particles provided significant (P < 0.001)
protection from shedding relative to mock-immunized control mice.
Significant (P 0.01) increases in shedding between
groups relative to mice immunized with tl EDIM, are indicated by an
asterisk.
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Effect of E. coli LT(R192G) on immune responses and
protection after i.n. immunization with rotavirus particles.
Mucosal adjuvants such as cholera toxin and E. coli
heat-labile toxin (LT) have been found to stimulate increased immune
responses after mucosal immunization with other antigens
(9). To determine the effects of such adjuvants, mice were
immunized i.n. with homotypic and heterotypic, inactivated, tl and dl
particles in the presence of 10 µg of genetically attenuated E. coli LT [i.e., LT(R192G)], and both rotavirus antibody responses
and protection were compared to that induced without LT(R192G). In this
experiment, the concentration of tl EDIM used for immunization was
reduced to 0.25 µg/dose in order to detect potential effects of
LT(R192G) in protection.
Inclusion of attenuated LT(R192G) during immunization normally resulted
in at least small and often significant increases
in serum and stool
rotavirus antibody titers (Table
3). In
every
case, the protection associated with inclusion of LT(R192G) was
substantial but not always significant because of differences
in
shedding between individual mice (Table
4). This effect was
most evident after tl
EDIM was administered at low concentrations.
Immune responses and protection induced by oral immunization with
inactivated rotavirus particles.
Although only small volumes of
immunogen were administered in these studies and mice were anesthetized
during immunization, it was possible that some of the responses were
due to intestinal exposure after swallowing a portion of the inoculum.
To determine the possible effects of intestinal exposure, the
immunogens were administered by oral gavage either with or without
LT(R192G). Rotavirus antibody responses stimulated by oral immunization
with rotavirus particles were almost always significantly less
(P 0.004) than that found after i.n. immunization,
including stool IgA (Table 5). Even so,
oral immunization, even without LT(R192G), resulted in significant
(P < 0.001) reductions in shedding and inclusion of
LT(R192G) caused further significant (P < 0.001) decreases in shedding with all three antigens tested (Table
6). In five of six cases, the level of
protection was less than after i.n. immunization under the same
conditions, and in three of these cases the difference was significant
(P < 0.001). These results indicate that at least some
of the protection observed after i.n. immunization was not due to
intestinal exposure but do not rule out the possibility that swallowing
a portion of the i.n. inoculum contributed to protection.
Differences in protection stimulated by tl EDIM versus other
rotavirus particles is accentuated at reduced doses.
Based on the
level of protection stimulated by two i.n. immunizations with 10 µg
of rotavirus particles, tl EDIM was superior to the other particles.
Because protection associated with tl EDIM immunization was dose
dependent (Fig. 1), it was of interest to determine whether protection
stimulated by other rotavirus particles also decreased when the dose
was reduced 10-fold, possibly increasing the difference in the levels
of protection stimulated by tl EDIM and other rotavirus particles in
this model system. This possibility was tested by using dl EDIM and tl
RRV as immunogens, both in the absence and in the presence of LT(R192G).
The rotavirus antibody responses stimulated with 1 µg of dl EDIM per
dose were comparable to those induced with 10 µg/dose;
however, those
induced by the lower concentration of tl RRV were
consistently less
(2.1- to 12.5-fold) after immunization with
the lower quantity of
antigen (results not shown). Protection
was consistently less with the
lower dose and was particularly
evident in mice immunized without
LT(R192G) (Table
7). Therefore,
differences in the level of protection between tl EDIM and other
rotavirus particles was even more evident after two doses when
the
dosage was reduced to 1 µg. At this dose, tl EDIM still provided
100% protection, even without adjuvant (Fig.
1).
Ratios of rotavirus IgG1/IgG2a stimulated by different rotavirus
particles.
The degrees of protection found after i.n. immunization
with rotavirus particles in the experiments already described showed distinct differences between that stimulated by homotypic tl EDIM particles versus those induced by heterotypic tl rotavirus particles and all dl particles. This suggested that the mechanisms of protection associated with the two groups of particles may be different. Likewise,
inclusion of LT(R192G) as adjuvant with all particles dramatically
increased their protective effects, a result possibly also due to
mechanistic differences.
The processes by which protection is induced after immunization or
natural infection rely on the production of immunomodulating
cytokines.
The induced cytokines direct the development of immune
responses along
specific pathways (
29). Key differences in pathways
often
concern the relative dominance of T-helper-cell subclasses,
T
H1 and T
H2. One outcome of T
H1
induction is the eventual production
of IgG2a, while T
H2
responses are associated with IgG1 production.
The ratios of IgG1/IgG2
have, therefore, been used in numerous
studies to determine whether
immunity is primarily associated
with T
H1 or
T
H2 responses. Therefore, IgG1/IgG2a ratios were measured
after immunization with different rotavirus particles, either
with or
without LT(R192G), as surrogate markers of the relative
induction of
T
H1 and T
H2
responses.
Although differences in the IgG1/IgG2a ratios were found following
immunization with the different particles, no consistent
pattern was
evident. In three different experiments, the ratio
found after
immunization with two doses of tl EDIM was consistently
0.5, and this
ratio varied little with either dose number or quantity
(Table
8). Likewise, i.n. immunization with dl
EDIM stimulated
an almost identical IgG1/IgG2a ratio. Heterotypic tl
particles
stimulated various IgG1/IgG2a ratios ranging from 0.2 to 2.4,
but neither the ratio nor actual quantity of either IgG subclass
could
be associated with increased protection. Inclusion of LT(R192G)
during
immunization caused increases in the geometric mean titers
in both IgG
subclasses in every case but no consistent change
in IgG1/IgG2a ratios.
For example, LT(R192G) caused the ratio
of IgG subclasses to decrease
from 2.4 to 2.0 for tl RRV and increase
from 0.2 to 0.9 for tl 89-12. Therefore, possible differences
in the mechanism of protection induced
by different particles
or due to the inclusion of LT(R192G) could not
be detected by
changes in IgG1/IgG2a ratios.
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TABLE 8.
Ratios of serum rotavirus IgG1/IgG2a stimulated by
different rotavirus particles either with or
without LT(R192G)a
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Increased protection stimulated by tl EDIM was associated with
antibody, while most protection induced by dl EDIM appeared to be
independent of antibody.
Mechanistic studies on rotavirus immunity
conducted to date have all suggested that rotavirus antibody plays at
least some role. For example, it was found that the normal, lifelong
protection against rotavirus infection in mice induced by a single oral
immunization with live murine rotavirus (28) was not
obtained in B-cell-deficient JHD or µMt mice (references
11 and 23 and unpublished
observations). To determine the importance of antibody after i.n.
immunization with inactivated rotavirus particles, shedding of
rotavirus antigen was compared after EDIM challenge of immunologically
normal BALB/c and B-cell-deficient µMt mice.
The total protection from shedding observed after immunization with tl
EDIM, either with or without LT(R192G), in BALB/c mice
was lost in
µMt mice and simulated that found with dl EDIM and
heterotypic tl and
dl particles in BALB/c mice (Table
9; see
also Table
4). In contrast, the degree of protection induced
by dl EDIM
particles was similar in BALB/c and µMt mice, i.e.,
protection after
immunization in the presence of LT(R192G) was
nearly identical in the
two mice strains while protection was
slightly but significantly
(
P < 0.001) less in µMt mice immunized
without
adjuvant. Thus, the added protection due to the presence
of VP4 and VP7
proteins in EDIM particles after i.n. immunization
of BALB/c mice was
lost in the absence of antibody. However, protection
stimulated by EDIM
particles that lacked these proteins occurred
in the absence of
antibody.
CD8 cell depletion did not alter the level of protection following
i.n. immunization.
The final experiments in this study were to
determine the importance of CD8 T cells in protection after i.n.
immunization with tl EDIM particles. BALB/c mice depleted of CD8 cells
with anti-CD8 cell antibody prior to immunization and kept depleted throughout the remainder of the experiment maintained complete protection against rotavirus shedding after EDIM challenge (Table 10). Similarly, µMt mice depleted of
CD8 cells were protected to the same extent against rotavirus shedding
as were the nondepleted mice. This experiment was repeated using either
tl or dl EDIM particles as immunogens and incorporated LT(R192G) as
adjuvant during i.n. immunization of both BALB/c and µMt mice. As
expected, tl particles stimulated 100% protection against EDIM
shedding in BALB/c mice and nearly complete protection (i.e., >99%
reduction in EDIM shedding) in µMt mice (results not shown). Also, as
expected, dl particles with LT(R192G) stimulated >99% protection in
both strains of mice as well. Depletion of CD8 cells beginning 5 days before challenge caused no loss in protection (results not shown). Therefore, the absence of CD8 cells in either BALB/c or
B-cell-deficient µMt mice did not alter the degree of protection
afforded by i.n. immunization with tl or dl EDIM.
| |
DISCUSSION |
Rotaviruses are ubiquitous pathogens that infect the mature
enterocytes on the tips of the intestinal villi, subsequently leading
to gastrointestinal disease, particularly severe diarrhea. Protection
in both humans and animals is believed to be due to the presence of
intestinal immunological effectors and, in some instances, protection
has been correlated with intestinal antibody (7, 10, 21).
Therefore, immunization schemes that have the greatest probability of
stimulating intestinal immune responses are the most likely to
stimulate protection against rotavirus. Based on evidence for a common
mucosal immune system (22, 39), i.n. immunization with
rotavirus antigens may be a feasible method for protecting against
rotavirus disease.
To evaluate the possible protective effects of i.n. immunization
against rotavirus, we have used the adult mouse model which we
developed for studies on active immunity (37). Mice have been found to be protected against reinfection with rotavirus for at
least 14 months after oral immunization with live murine rotavirus
(28). Therefore, we chose to immunize i.n. with inactivated rotavirus in order to avoid confounding effects of potential intestinal replication. Furthermore, we chose to anesthetize the mice during immunization to limit the quantity of antigen that was swallowed. Finally, we have evaluated the potential stimulating effects of a
mucosal adjuvant [E. coli LT(R192G)] on both immune
responses and protection. A similar study was reported by O'Neal et
al. following immunization with VLPs of rotavirus (32).
Using these methods, we found that two i.n. immunizations with tl
murine rotavirus strain EDIM, with as little as 1 µg of inactivated
virus per dose, provided complete protection against rotavirus shedding
after an oral challenge with a 50% infectious dose (ID50)
of EDIM of ca. 100. Furthermore, when lower doses of immunogen were
administered and only partial protection against shedding was obtained,
the level of shedding was greatly reduced by the inclusion of LT(R192G)
during immunization. Immunization with inactivated EDIM particles that
lacked their outer capsid proteins (i.e., dl particles) as well as
heterotypic tl and dl rotavirus particles of nonmurine origin also
stimulated protection against EDIM shedding. However, the level of
protection was reduced relative to tl EDIM at comparable doses. As
found with tl EDIM, protection was also increased with these particles
by the inclusion of LT(R192G) during immunization.
The mechanism by which LT(R192G) increases protection is unclear.
Suggestions include, among others, enhanced mucosal permeability (20), selective induction of TH2-mediated
antibody responses (35), induction of antigen-specific
T-cell responses (15, 40), and increased antigen
presentation (4). In our studies, we observed that both
serum and intestinal (i.e., stool) antibody responses stimulated by
i.n. immunization were enhanced by the inclusion of LT(R192G), an
observation also reported by O'Neal et al. (31, 32) after
immunization with VLPs with either LT, LT(R192G), or cholera toxin.
However, higher titers of any specific rotavirus antibody measured
could not be correlated with protection of individual mice. These
findings suggested the possibility that rotavirus antibodies may only
be markers of protection and not the actual effectors.
The immune responses and protection generated by immunization are, to a
large extent, determined by the T-helper-cell subset that dominates the
response (29). This is reflected in the titers of IgG1 and
IgG2 subtypes stimulated by TH2 and TH1
responses, respectively. The increased rotavirus IgG responses and
protection observed after i.n. immunization with LT(R192G),
however, was not reflected in a consistent preferential increase in IgG
subtype. Therefore, selective increases in either a TH1 or
TH2 response could also not be associated with increased
immunity due to LT(R192G). It should be noted that levels of
TH1 and TH2 in serum as determined by the
surrogate markers used here may not reflect the levels of these helper
cells in the intestinal mucosa where rotavirus replication occurs.
The greater protection stimulated by i.n. immunization with inactivated
tl EDIM relative to either dl EDIM or tl or dl heterotypic rotaviruses
following a subsequent EDIM challenge indicated that immune responses
to homotypic VP4 and VP7 proteins resulted in increased protection.
This was not unexpected and was likely due to stimulation of
serotype-specific neutralizing antibody. If this is the case, the titer
of this antibody required for protection may be quite low because even
serum neutralizing antibody to EDIM was detected only after
immunization with two doses of the highest quantity of tl EDIM
particles (i.e., 10 µg). Immunization with lesser doses or a 10-fold
reduction in antigen still resulted in complete protection but no
detectable serum neutralizing antibody. Although not examined in this
study, intestinal neutralizing antibody concentrations were expected to
be even lower based on the relative concentrations of serum and stool
rotavirus IgA and IgG observed after i.n. immunization. Previous
studies using oral immunization of mice with live rotaviruses revealed
associations between intestinal rotavirus IgA and protection
(10) but no association with neutralizing antibody
(38), possibly due to its instability, low concentrations, or the insensitivity of the assay.
Protection stimulated by rotavirus particles other than tl EDIM in this
study appeared to be due to a mechanism other than neutralizing
antibody. dl particles and heterotypic tl particles should not
stimulate neutralizing antibody to EDIM, and none was found.
Furthermore, the levels of protection stimulated by these particles
were comparable and somewhat less than that induced by tl EDIM
immunization. Since total rotavirus IgA and IgG titers stimulated by
these particles were comparable to those induced by tl EDIM following
immunization with the same concentrations of antigen, differences in
protection were not due to generally poorer immune responses. Finally,
these differences also appeared to not be due to variations in
TH1 vs. TH2 responses since IgG1/IgG2a ratios
after immunization were similar for all the particles tested.
To examine the importance of rotavirus antibody in general and
neutralizing antibody to EDIM in particular following i.n. immunization
with different rotavirus particles, protection was measured in
B-cell-deficient µMt mice. Shedding of rotavirus antigen in these
mice after i.n. immunization with dl EDIM with LT(R192G) was comparable
to that found in immunologically normal BALB/c mice. However, in the
absence of LT(R192G), dl EDIM stimulated somewhat better protection
(P < 0.001) in BALB/c than in µMt mice. This
suggests the possibility that antibody may play some role in protection
after inoculation with these particles but inclusion of LT(R192G)
during immunization masked this role. Antibody appeared to play a more
important role in protection following immunization with tl EDIM
particles because the additional protection stimulated in BALB/c mice
due to the presence of homotypic VP4 and VP7 proteins was lost in µMt
mice. This result not only supports the suggestion that the additional
protection in BALB/c mice was due to serotype-specific neutralizing
antibody but indicates that protection by dl EDIM and, by extension, dl
and tl heterotypic rotaviruses occurs in the absence of antibody.
Several studies with the adult mouse model have shown that CD8 cells,
the major effectors of CTL activity, are involved in the resolution of
rotavirus shedding in BALB/c mice (11, 12, 23). It has also
been suggested that these cells may play a role in the subsequent
protection against rotavirus reinfection in this model (12).
Therefore, it was of interest to determine whether CD8 cells were
required for protection following i.n. immunization with inactivated
rotaviruses. These studies were initially performed with tl EDIM
without adjuvant as the i.n. immunogen which should elicit protection
by at least two separate mechanisms based on the results already
discussed. No loss of protection was found in either BALB/c or µMt
mice after CD8 cell depletion, even when depletion was initiated before
immunization and maintained throughout the course of the experiment.
Furthermore, the level of CD8 cell depletion was 98% in both strains
of mice, supporting the conclusion that these cells were not needed for protection. The same conclusions were obtained during a subsequent study in which dl or tl EDIM particles with LT(R192G) were administered i.n. to either BALB/c or µMt mice. Lack of involvement of CD8 cells
after i.n. immunization with inactivated rotavirus is in agreement with
the proposed mechanism by which CD8 cell stimulation occurs, i.e.,
internal processing of antigen followed by MHC-1-associated presentation of the processed peptide to the cognate CD8 cells (14).
If B cells and CD8 cells are not required for protection following i.n.
immunization with rotavirus particles, this leaves only one established
immunological effector of memory-associated protection, i.e., CD4
cells. These cells are known to both produce and stimulate the
production of cytokines that block rotavirus replication
(1). Studies that use anti-CD4 cell depletion designed to
directly determine the involvement of CD4 cells in protection after
i.n. immunization are in progress. Previous results obtained with this
method have shown that CD4 cells are needed for the complete normal
resolution of rotavirus shedding in BALB/c mice (26). We now
anticipate that they will be shown to be a major effector of protection
after i.n. immunization. It should be noted that protection did not
diminish when mice were challenged at 3 months rather than 1 month
following i.n. immunization with either tl or dl EDIM particles plus
LT(R192G) (results not shown). Therefore, if CD4 cells are responsible
for protection, they must be retained for extended periods as memory
cells at an inducible site in sufficient concentrations to provide the
rapid and dramatic reduction in rotavirus shedding observed in this study.
The finding that protection following i.n. immunization with
inactivated dl EDIM was not dependent on antibody contrasts with results found after oral immunization with live rotavirus.
Immunologically normal mice are fully protected against reinfection for
at least 14 months after oral inoculation with live murine rotavirus
(28), whereas B-cell-deficient mice are susceptible to
reinfection within weeks following this method of immunization
(11, 23). Although protection could not be associated with
the presence of serotype-specific neutralizing antibody following oral
immunization (38), it may play some role as suggested from
the results found after i.n. immunization with tl homotypic rotavirus
in this study. Also of interest was the finding that oral immunization
with live heterotypic rotaviruses generally provided little protection
against subsequent murine rotavirus infection (38), while
i.n. immunization with inactivated dl or tl heterotypic rotaviruses in
the presence of LT(R192G) provided excellent protection equivalent to
that induced by dl EDIM. Finally, it should be noted that inactivated
tl and dl EDIM administered intramuscularly with the adjuvant (QS-21) both stimulated excellent protection against EDIM infection but that
the tl particles were more effective (25). Taken together, these results indicate that inclusion of homotypic VP4 and VP7 during
immunization by any of the routes examined produced increased protection, possibly due to neutralizing antibody production. However,
particles lacking these homotypic proteins also stimulated excellent
protection when administered with an effective adjuvant. Furthermore,
protection induced by particles lacking homotypic VP4 and VP7 proteins
may not depend on antibody, as shown in this study for i.n. immunization.
It should be noted that oral immunization with inactivated dl or tl
EDIM and tl RRV also stimulated rotavirus antibody responses and
substantial protection in this study, particularly when LT(R192G) was
included during immunization. Interestingly, this protection was much
greater than that found after oral immunization with larger doses of
VLPs in the presence of cholera toxin even though the ID50
used to challenge mice in that study was 10-fold less than in our study
(32). Reasons for these different levels of protection
following oral immunization are unclear, especially since protection
levels stimulated by i.n. immunization with inactivated dl particles
and VLPs were very similar (reference 32 and this study). Although neither serum nor stool rotavirus antibody responses were as great after oral immunization with inactivated particles as
that found after i.n. immunization with the same dosages of antigen and
although protection was significantly reduced as well, oral
immunization with inactivated rotavirus could have been partially responsible for the protection induced by i.n. immunization in this
study. The finding that oral immunization with inactivated rotavirus
results in protection also suggests the possibility that sequential
i.n. and oral immunization will be an even more effective method of
stimulating protection against this mucosal pathogen than either route
individually. This remains to be determined.
| |
ACKNOWLEDGMENTS |
This work was funded in part by NIH NIAID contract NO1 AI-45242
to Children's Hospital Medical Center.
The LT(R192G) was generously provided by John Clements and Candice
Smith of Tulane University School of Medicine, New Orleans, La.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Children's
Hospital Medical Center, Division of Infectious Diseases, 3333 Burnet
Ave., CH-1, Cincinnati, OH 45229-3039. Phone: (513) 636-7628. Fax:
(513) 636-7682. E-mail: wardd0{at}chmcc.org.
| |
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Journal of Virology, September 1999, p. 7565-7573, Vol. 73, No. 9
0022-538X/99/$04.00+0
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Abstract
Introduction
