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J Virol, April 1998, p. 3390-3393, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Rotavirus 2/6 Viruslike Particles Administered Intranasally with
Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT),
and LT-R192G Induce Protection from Rotavirus Challenge
Christine M.
O'Neal,1
John D.
Clements,2
Mary K.
Estes,1 and
Margaret
E.
Conner1,*
Division of Molecular Virology, Baylor
College of Medicine, Houston, Texas 77030,1 and
Department of Microbiology and Immunology, Tulane University
Medical Center, New Orleans, Louisiana 701122
Received 1 October 1997/Accepted 16 December 1997
 |
ABSTRACT |
We have shown that rotavirus 2/6 viruslike particles composed of
proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with
cholera toxin (CT) induced protection from rotavirus challenge, as
measured by virus shedding. Since it is unclear if CT will be approved
for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 µg of 2/6-VLPs combined with CT, LT, or
LT-R192G. All three adjuvants induced equivalent geometric mean titers
of rotavirus-specific serum antibody and intestinal immunoglobulin G
(IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the
adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and
LT-R192G were totally protected (100%) from rotavirus challenge,
while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91%
protection from challenge. The availability of a safe, effective
mucosal adjuvant such as LT-R192G will increase the practicality of
administering recombinant vaccines mucosally.
| |
TEXT |
Rotaviruses are the leading
cause of viral gastroenteritis in young children worldwide, leading to
more than 500,000 deaths each year in developing countries
(10). Our laboratory is working on the development of a
subunit vaccine for rotavirus. Viruslike particles (VLPs) are made by
coinfecting insect cells with baculovirus recombinants that express
rotavirus structural proteins; these proteins self-assemble into VLPs
(3). We have previously shown that 10 µg of 2/6-VLPs
composed of VP2 and VP6 (2/6-VLPs) administered intranasally with the
mucosal adjuvant cholera toxin (CT) induced between 70 and 90%
reduction in virus shedding (14). To assess the necessity of
a mucosal adjuvant for protection, 2/6-VLPs were administered
intranasally to mice at doses of 10, 50, and 100 µg without CT. Low
levels of protection (20, 29, and 38%, respectively) from viral
shedding were seen (14), indicating that a mucosal adjuvant
is necessary to achieve high levels of protection from rotavirus
challenge after intranasal administration of 2/6-VLPs. Although CT is a
potent mucosal adjuvant, the holotoxin may not be approved for use in
humans because the toxic dose is low; as little as 5 µg of purified
CT is sufficient to induce significant diarrhea in volunteers
(12). Efforts are under way to dissociate the toxic and
adjuvant properties of CT (16).
Although limited, mild diarrhea in vaccinees may be tolerated when the
perceived benefit of an oral vaccine is very high, there is a need for
alternate mucosal adjuvants that do not cause side effects in the
vaccinee. Escherichia coli heat-labile enterotoxin (LT) is
similar to CT in having adjuvant activity (2, 5), but it may
be less toxic to humans than CT (1). Unlike CT, LT is not
secreted from bacteria or fully biologically active when first isolated
from the cell. LT must undergo cleavage by proteases in the intestine
for it to be toxic. This difference in the need for LT activation
results in differences in the toxic dose of CT versus LT, with LT being
less toxic (1). Although LT is less toxic, there is still a
potential for administration of LT to induce diarrhea, limiting its
benefits as a mucosal adjuvant. A mutant of the E. coli
heat-labile toxin (LT-R192G) has been developed in an effort to
separate the adjuvant and toxic properties of LT (4). The
mutant encodes a glycine instead of an arginine at position 192 of the
protein. This amino acid change eliminates the trypsin-sensitive
cleavage site in the protein, thereby preventing cleavage of the A
subunit, rendering the protein nontoxic at adjuvant-effective doses. In
a randomized placebo-controlled, dose-escalating study in adult
volunteers, 0 of 24 volunteers showed adverse reactions to single oral
doses of 5, 25, or 50 µg of LT-R192G; at 100 µg of LT-R192G, 2 of
12 (16.7%) volunteers developed mild to moderate diarrhea which
resolved after 24 h (15). Maximum antibody responses or
antibody-secreting cells to labile toxin B subunit (LTB) were observed
when administered with 25 µg of LT-R192G. Therefore, LT-R192G retains
its adjuvant activity with greatly reduced toxicity when administered
orally (4, 11, 15).
This article reports studies to determine whether the immunogenicity
and protective efficacy of rotavirus 2/6-VLPs administered intranasally
are retained by coadministration of the nontoxic mutant-labile toxin,
LT-R192G.
Rotavirus VLPs administered mucosally with CT, LT, and LT-R192G are
immunogenic.
The 2/6-VLPs were prepared in insect cells, purified,
and characterized as previously described (3). VLPs were
composed of bovine Rf VP2 and SA11 VP6. Electron microscopic analysis
of the particles showed the 2/6-VLPs were intact and properly formed (data not shown). Coomassie blue staining and Western blot analysis of
the proteins in the VLPs confirmed the presence in the expressed particles of each of the expected structural proteins; no additional proteins were present (data not shown).
Virus antibody-free adult female CD-1 (22 to 24 g) mice were
obtained from Charles River Laboratories (Portage, Mich.). Mice were confirmed to be free of rotavirus antibody by enzyme-linked immunosorbent assay (ELISA) prior to vaccination. We compared the
effects of the different adjuvants on the serologic antibody response,
the intestinal immunoglobulin A (IgA) and intestinal IgG antibody
response, and on protection from rotavirus challenge. Groups
(n = 10) of adult mice (>30 days of age) were
vaccinated intranasally, as previously described (14), with
10 µg of 2/6-VLPs mixed with either 5 µg of CT (Sigma St. Louis,
Mo.), 10 µg of LT (1), or 10 µg of LT-R192G
(4) or with phosphate-buffered saline (PBS) on 0, 14, and 45 days postinoculation. The choice of dose for each adjuvant was the
optimum dose described in the literature for each adjuvant.
Antibody responses were evaluated in serum and fecal samples collected
1 month after the third vaccination. Fecal samples
collected from
individual mice were processed and stored as previously
described
(
8,
14). Measurement of total antirotavirus antibody
in
serum and antirotavirus IgA and IgG in intestinal samples was
performed
as previously described (
14). Antibody titers were
defined
as the reciprocal of the highest dilution giving a net
optical density
(OD) value (OD with SA11 minus OD with 0.5% Blotto)
above 0.1. All
control mice, inoculated with PBS, remained negative
for rotavirus
antibody until challenge.
The choice of adjuvant did not affect the serologic or intestinal IgG
immune response to 2/6-VLPs (Fig.
1A and
B). All groups
of mice
inoculated with 2/6-VLPs had high, but equivalent, rotavirus-specific
serum antibody geometric mean titer (GMT) responses regardless
of the
adjuvant used (range, 51,200 to 819,200) (
P > 0.05, Kruskal-Wallis)
(Fig.
1A). Twenty-eight of the 30 mice inoculated with
2/6-VLPs
had intestinal IgG responses (Fig.
1B) regardless of the
adjuvant
used (range, 10 to 2,560), and the GMTs did not vary
significantly
between the groups (
P > 0.05, Kruskal-Wallis).
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FIG. 1.
Serum and intestinal antibody responses after intranasal
immunization with 2/6-VLPs particles. Total (IgA, IgG, and IgM) serum
(A) and intestinal IgG (B) and IgA (C) antirotavirus antibodies were
measured in mice after three inoculations (72 days postinoculation)
(left panels) with 2/6-VLPs administered intranasally with CT, LT, or
LT-R192G, as indicated along the x axis, and 23 days postchallenge with ECwt (right panels). Antibody
titers were measured for individual mice by ELISA, and results are
plotted as the GMTs of the groups (n = 10 per group).
Error bars represent one standard error of the mean. Significant
differences in GMTs (P < 0.05), measured by
Mann-Whitney U, within a panel are indicated (*, @, and #).
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|
The choice of adjuvant did affect the rotavirus-specific intestinal IgA
responses (Fig.
1C). All mice inoculated intranasally
with 2/6-VLPs had
detectable rotavirus-specific intestinal IgA
regardless of the adjuvant
used. However, mice inoculated with
2/6-VLPs mixed with LT had
extremely high IgA titers (range, 320
to 2,560) that were significantly
higher than those of mice inoculated
with 2/6-VLPs mixed with CT
(range, 80 to 640) (
P = 0.029, Mann-Whitney
U). Mice
inoculated with 2/6-VLPs with LT-R192G had an IgA GMT
that was
intermediate between those of groups receiving CT or
LT (range, 80 to
1,280) but was not significantly different from
that of either group
(
P > 0.05, Kruskal-Wallis).
2/6-VLPs administered intranasally with LT and LT-R192G induced
significantly higher protection against rotavirus challenge than
CT.
One month following the third vaccination, mice were
challenged with 10 50% shedding doses (SD50) of wild-type
murine virus (ECwt), obtained from Harry Greenberg
(Stanford University Medical School, Palo Alto, Calif.) (7),
following oral administration of 40 µl of 5% bicarbonate buffer.
Protection from infection was determined (Fig.
2). Fecal samples from individual mice
were collected daily for 11 days, starting on the day of challenge.
Fecal samples were processed and stored as previously described
(14). The level of viral antigen in fecal samples was
measured by ELISA as previously described (14). Percent
reduction in shedding was calculated for each animal, and the mean
percent reduction was calculated for each vaccine group.
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FIG. 2.
Protection from rotavirus challenge in mice receiving
three intranasal inoculations of 2/6-VLPs. 2/6-VLPs were administered
intranasally with CT, LT, or LT-R192G, as indicated along the
x axis. Mice were challenged at 73 days postinoculation
with 10 SD50 of ECwt rotavirus. Individual
stool samples were collected daily and quantitated for levels of
rotavirus antigen by ELISA. Virus shedding curves were plotted, and the
area under the curve was calculated for each animal along with the mean
for the group. Results are plotted as percent reduction in antigen
shedding for each individual animal ( ) (n = 10 per
group) as well as the mean for each group ( ), calculated by comparing
the mean area under the curve of each vaccine group to the mean area
under the curve of the control group (1  mean area of vaccine
group/mean area of control group). The number of mice with overlapping
levels of protection is indicated on the graph (n = 6).
Significant differences in protection, measured by the Mann
Whitney U, are indicated (# and @).
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All mice inoculated intranasally with 2/6-VLPs mixed with LT or
LT-R192G were totally protected from challenge; there was
no
detectable virus shedding. The reduction in virus shedding
in mice
inoculated with 2/6-VLPs mixed with CT ranged from 66%
to 100%, with
mean protection for the group being 91%; this was
significantly lower
protection than that achieved using LT or
LT-R192G
(
P = 0.013, Mann Whitney U).
Following challenge with murine rotavirus, all mice previously mock
inoculated with PBS developed serum antibody and intestinal
IgA but no
intestinal IgG (Fig.
1). Mice inoculated with PBS and
then challenged
with EC
wt murine rotavirus had equivalent levels
of serum
antibody and intestinal IgA but lower levels of intestinal
IgG compared
to mice inoculated intranasally with 2/6-VLPs with
any adjuvant prior
to challenge. After challenge, significant
elevations in serum antibody
and intestinal IgA GMTs were only
observed in mice inoculated with PBS
(
P = 0.04, Wilcoxon signed
ranks test). Significant
decreases in intestinal IgG following
challenge were only observed in
mice inoculated with LT-R192G
(
P = 0.018, Wilcoxon
signed ranks test). Following challenge,
mice inoculated with PBS had
significantly lower serum antibody
and intestinal IgG GMTs than mice
inoculated with VLPs with any
adjuvant (
P = 0.001 and
P = 0.001, respectively, Kruskal-Wallis)
but higher
intestinal IgA titers (
P = 0.007, Kruskal-Wallis).
The majority of human pathogens enter the body through interactions
with mucosal surfaces. Therefore, the development of vaccines
that
induce effective mucosal immune responses is vital. We have
recently
shown that nonreplicating rotaviruslike particles administered
orally
or intranasally can protect mice against a live rotavirus
challenge
(
14). Significantly higher levels of protection were
achieved when the mucosal adjuvant CT was used in conjunction
with the
VLPs, showing the necessity of an effective mucosal adjuvant.
CT can
cause diarrhea in humans at very low doses and therefore
cannot be
considered for use as an adjuvant in humans. Other,
potentially safer
adjuvants have been developed; these include
LT, mutants of CT,
and mutants of LT. In this study, we compared
the adjuvanticities of
CT, LT, and mutant LT-R192G when administered
intranasally with
rotavirus 2/6- VLPs. Although
91% protective
efficacy was
achieved with 2/6-VLPs in all adjuvants, 2/6-VLPs
administered with LT
or the nontoxic mutant LT-R192G induced higher
levels of protection
against rotavirus challenge than did CT.
However, the increased
protective efficacy of VLPs with LT or
LT-R192G may be due to the
higher dose of LT or LT-R192G (10 µg)
compared to that of CT (5 µg) or to the induction of different
immune responses by the
different adjuvants (
16). Of note is
the high level of
protective efficacy achieved with the mutant
LT-R192G, which was at
least equivalent to the protection obtained
with native CT and LT,
indicating that LT-R192G is an effective
mucosal adjuvant.
A number of mutants of LT and CT have been developed principally for
use as homologous vaccines to prevent
E. coli-induced
diarrhea (
4,
6,
9,
13,
16). Some of these mutant
toxins have
been examined to determine if they retain their ability
to function as
mucosal adjuvants. The two most widely studied
are LT(S63K)
(
6) and LT-R192G (
4). LT(S63K) is an
active-site
mutant and has been shown to lack in vitro
ADP-ribosyltransferase
and enterotoxicity and is completely devoid of
activity in the
mouse Y-1 adrenal tumor cell assay (a surrogate
indicator for
induction of cyclic AMP). LT-R192G is a protease site
mutant and
has been shown to lack in vitro ADP-ribosyltransferase and
enterotoxicity,
but LT-R192G retains a basal level of activity in
the mouse Y-1
adrenal tumor cell assay. Importantly for the development
of mucosal
vaccines, LT-R192G retains the adjuvanticity of the
native molecule.
Both intranasal immunization of rotavirus VLPs and
oral immunization
of inactivated influenza virus with LT-R192G
induced protection
against live-virus challenge (
11).
Nonreplicating VLPs administered mucosally can induce protection
against rotavirus challenge. Nonreplicating vaccines are
safer than
replicating vaccines but are generally less immunogenic;
therefore
safe, mucosal adjuvants are needed. Mutants of CT and
LT have been
developed and shown to have adjuvant activity. Our
results show that
equivalent or better responses, either antibody
or protection from
rotavirus challenge, can be achieved with a
nontoxic mutant of LT
(LT-R192G). Mutants of LT and CT need to
be tested further in
animals and humans for safety and adjuvanticity
but offer promising
alternatives.
| |
ACKNOWLEDGMENTS |
We thank Sue Crawford and Andrea Bertolotti-Ciarlet for production
of the VLPs and Juan Alvarado for assistance in processing of fecal
samples.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Rm. 936E,
Division of Molecular Virology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030. Phone: (713) 798-3590. Fax: (713) 798-3586. E-mail: mconner{at}bcm.tmc.edu.
| |
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J Virol, April 1998, p. 3390-3393, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Choi, A. H.-C., Basu, M., McNeal, M. M., Clements, J. D., Ward, R. L.
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Ryan, E. T., Crean, T. I., John, M., Butterton, J. R., Clements, J. D., Calderwood, S. B.
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Cardenas-Freytag, L., Cheng, E., Mayeux, P., Domer, J. E., Clements, J. D.
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Ciarlet, M., Crawford, S. E., Barone, C., Bertolotti-Ciarlet, A., Ramig, R. F., Estes, M. K., Conner, M. E.
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