Active protection against rotavirus infection of mice following intraperitoneal immunization

VIROLOGY

191,

150-157

(19%‘)

Active Protection against Rotavirus Infection of Mice Following lntraperitoneal Immunization MONICA M. McNEAL,*

JOHN F. SHERIDAN,t

AND

RICHARD L. WARD**’

*Division of Clinical Virology, J. N. Gamble Institute of Medical Research, 2 14 1 Auburn Avenue, Cincinnati, Ohio 452 19; and tDepartment of Oral Biology and Medical Microbiology and Immunology, Colleges of Dentistry and Medicine, Ohio State University, Columbus, Ohio 432 10 Received February 4, 1992; accepted July 22, 1992 Active immunity to rotavirus has been demonstrated following oral inoculation with live virus but little is known about the effects of parenteral immunization. In this study, adult mice were immunized by intraperitoneal (ip) inoculation with live rotaviruses and later orally challenged with murine rotavirus (EDIM) to measure active immunity against infection. Three doses of EDIM (8 agldose) given intraperitoneally (ip) provided full protection against EDIM infection, whether administered with or without Freund’s adjuvant. Only partial protection was found when the quantity of immunogen was reduced to ~2 pgldose. Reduction of the number of doses from three to one (8 pgldose), however, still resulted in protection of all mice. Significant protection was also observed after inoculation with one or three doses (2 pg/dose) of heterologous rotaviruses. Protection provided by the heterologous strains did not correlate with neutralizing antibody to EDIM, which indicated that neutralizing antibody to the challenge virus was not required for protection. uv-lnactivated EDIM also provided significant protection against EDIM, thus demonstrating that viral replication was not required for protection. These results suggest that parenteral immunization may be an effective method to vaccinate Q 1992 Academic Press, Inc. against rotavirus disease.

INTRODUCTION

intraperitoneal adoptive transfer of splenic CD8+ cells from mice immunized with rotavirus can protect neonatal mice from rotavirus disease (Offit and Dudzik, 1990). Furthermore, the same method of adoptive transfer resolved chronic rotavirus shedding in SCID mice (Dharakul et a/., 1990). These findings suggest that active immunity to rotavirus might be developed by parenteral immunization, and that cellular immune mechanisms mediated by T-cells may play a role. A murine model to study the mechanism of active immunity against rotavirus was recently developed in our laboratory (Ward et a/., 1990). This model is based on the finding that adult mice, although resistant to rotavirus-induced diarrhea1 disease, were consistently infected after oral inoculation with a murine rotavirus (EDIM strain) if not previously immunized. It was subsequently found that oral inoculation of mice with infectious EDIM, murine EB, or simian SAl I-FEM rotavirus strains protected against infection when challenged with EDIM while other animal rotavirus strains were not protective (Ward et al., 1990, 1992). We have now utilized this model to determine whether active immunity to rotavirus infection also develops after intraperitoneal immunization and whether the virus strains that were protective when delivered orally are also protective when administered parenterally.

The site of rotavirus infection is usually limited to the mature enterocytes on the tips of intestinal villi (Blacklow and Greenberg, 1991). Therefore, intestinal immune responses are presumed to be required to protect against rotavirus disease. In support of this suggestion, it has been reported that passively acquired intestinal but not circulating neutralizing antibody is associated with protection of animals against rotavirus (Woode et a/., 1975; Snodgrass and Wells, 1976; Offit and Clark, 1985). High titers of passive circulating antibodies have, however, been found to prevent (Snodgrass and Wells, 1978) or moderate (Saif and Smith, 1985) rotavirus disease, possibly due to the transfer of antibody from the circulation to the intestine. Furthermore, children under the age of approximately 3 months are resistant to rotavirus illness which has been attributed to passively acquired transplacental antibody (Tufvesson et a/., 1986; Vial et al., 1988; Bernstein eta/., 1990). These findings suggest that circulating antibody may help provide some protection against rotavirus disease. Several vaccines against rotavirus disease have been tested, all of which were live, attenuated rotaviruses delivered orally, a route intended to mimic natural rotavirus infection. Induction of protective immune responses by parenteral immunization has been largely ignored. It has been repot-ted, however, that

MATERIALS

Rotavirus strains The rotavirus strains used in this study were described in a previous publication (Ward et a/., 1992).

’ To whom requests for reprints should be addressed. 0042-6822192

$5.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

AND METHODS

150

INTRAPERITONEAL

IMMUNIZATION

These included EDIM (murine), EB (murine), RRV (simian), SAl 1-FEM (simian), SAI 1-SEM (simian), and WC3 (bovine). All rotaviruses used to immunize mice were plaque-purified isolates which were grown in MA104 cells and purified by CsCl gradient centrifugation. The EDIM strain used to challenge mice was a viral lysate of the ninth cell culture passage (Ward et a/., 1990). Study design Female BALB/c mice 6 weeks of age were screened for rotavirus IgG by ELISA, then immunized by intraperitoneal (ip) inoculation with defined quantities of purified, live, or uv-inactivated double-shelled virus particles. All mice had undetectable amounts of rotavirus IgG before inoculation. To purify the virus, frozen lysates of rotavirus-infected MA1 04 cells were thawed, virus and cellular debris were pelleted bycentrifugation (100,000 g, 90 min), and the resuspended pellet in Earles balanced salt solution (EBSS) was blended (2 min, 4”) with an equal volume of freon. The aqueous phase was then underlaid with CsCl and further purified as described previously (Ward et a/., 1991). The infectivities of the purified particles [i.e., focus forming units (ffu) per microgram of viral protein], determined as previously described (Bernstein et al., 1989) were as follows: EDIM, 5 X 105; EB, 5 X 106; RRV, 1 X lo*; SAl l-FEM, 3 X 107; SAl l-SEM, 3 X 107; WC3, 7 X 106. Preparations of uninfected cells were purified in the same manner and used to inoculate control mice. If Freund’s adjuvant was used for immunization, complete adjuvant was included in the first inoculation, incomplete adjuvant in the second inoculation, and no adjuvant with the third inoculation. When more than one ip rotavirus inoculation was administered, the inoculations were separated by 2 weeks. Blood specimens were obtained by retro-orbital capillary plexus puncture before the first inoculation and just before mice were challenged by oral inoculation with either 2 X 1O3 or 40 X lo3 ffu of EDIM (ninth cell culture passage). Either dose was sufficient to infect 100% of unimmunized adult mice. These serum specimens were examined for rotavirus antibody. Mice were challenged with EDIM (ninth cell culture passage) at 10 days after the final ip inoculation when given in two or three doses or between 24 and 27 days after ip immunization with a single dose. Stool specimens were collected from every mouse on each day for 7 days after EDIM challenge and examined for infectious rotavirus and rotavirus antigen. A final blood sample was collected at 24 days after mice were challenged with EDIM. uv Inactivation

of purified

rotavirus

Purified rotavirus (0.5 ml) was pipetted directly into the center of a 60-mm tissue culture plate and irra-

151

TO ROTAVIRUS

diated at a distance of 14 cm with a germicidal lamp (Millipore U.V. Sterilizer) while being rotated at 70 rpm. This caused viral infectivity to decrease 1OO-fold within 1 min and >l million-fold within 4 min. To ensure that no live rotavirus was inoculated ip into mice, the purified EDIM used in the study was irradiated for 8 min prior to inoculation. This specimen was then brought to a volume of 2 ml with dialysis buffer (phosphate-buffered saline with 10 mm CaCI,, and 20% glycerol) and titered before inoculation of mice. No infectious virus was found, thus showing uv treatment caused viral titer to decrease > lOO,OOO-fold relative to the untreated control preparation. Detection

of rotavirus

in stool

Shedding of EDIM in stools of mice was initially determined by both production of CPE in tube cultures of MA104 cells (Ward et al., 1990) and by an enzymelinked immunosorbent assay (ELISA) as previously described (Ward et a/., 1986). The two assays gave identical results with 93% of the specimens but rotavirus was detected by only one of the two methods in 17/50 positive specimens (10 by ELISA and 7 by CPE production). Because the ELISA was much less labor intensive and was at least as sensitive as CPE production, it was used for the remainder of the experiments. Detection of rotavirus antigen cavity and in different organs

in the peritoneal

Mice inoculated ip with a single dose of 8 pg of purified, live EDIM (4 x 10” ffu) were examined for the presence of viral antigen in different tissues at 0, 5, 12, and 24 days after inoculation. Two mice were examined on each date. Initially, mice were sacrificed and their peritoneal cavities were thoroughly rinsed with EBSS (5 ml). Spleen, stomach, liver, large intestine, and small intestine were removed and each was homogenized (Omni mixer) with EBSS (5 ml) and centrifuged (2000 g, 10 min). The supernatant fractions of these specimens along with the peritoneal washes were analyzed by ELISA for rotavirus antigen (Ward et a/.,

1986). Determination

of rotavirus

antibody

titers

Serum specimens were tested for both rotavirus IgG (Ward et a/., 1990) and neutralizing antibody (Bernstein et a/., 1989). Radiolabeling

of rotavirus

proteins

To prepare [35S]methionine-labeled rotavirus proteins, monolayers of MA1 04 cells were grown to confluency in minimal essential medium, Dulbecco’s modi-

McNEAL,

152

SHERIDAN,

fication (D-MEM), containing 10% fetal calf serum, washed twice with D-MEM, and either mock-infected or infected with EDIM (passage 31) at a multiplicity of infection of 5. After a 1 hr adsorption period at 37’, the cells were washed once and D-MEM with actinomycin D (1 pg/ml) was added. After an additional 2 hr at 37”, the medium was changed to methionine-free D-MEM containing 1 rg actinomycin D/ml. Following a 1 hr incubation at 37”, the medium was again replaced with methionine-free D-MEM containing actinomycin D and [36S]methionine (50 &i/ml). Incubation was continued (2 hr, 37”) then the cells were washed twice with EBSS and scraped from the flasks with glass beads. The resuspended cells were washed twice with EBSS then lysed with RIPA buffer (150 mM NaCI, 10 mM Tris, pH 7.2, 1% aprotinin, 1% Triton X-l 00, 0.5% NP-40). After 30 min on ice, the lysed cells were centrifuged (35,000 rpm, SW 50.1 rotor, 30 min) and the supernatants were stored at -20”.

AND WARD TABLE 1 PROTECTIONOF BALB/c MICE AGAINSTEDIM INFECTIONWHEN ORALLY CHALLENGEDAFTERIP IMMUNIZATIONWITH LIVE EDIM GMT of serum rotavirus antibody before EDIM challenge Immunizing virus* Control Control EDIM EDIM

N Adjuvantb

Rotavirus W

Neutralizing antibody

No. shedding EDIM after challenge’ (%)

5 4 4 3

<300 <300 3.6 X lo6 3.0 x 10’

<20 <20 510 2130

5 (100) 4 (100) 0 (Old cl (o)d

+ +

’ Three doses of purified EDIM (8 fig/dose) or MA1 04 cell extract purified in the same manner as virus (control). * Freund’s complete (first dose) and incomplete (second dose). c Oral challenge with 2 X 1O3 ffu of EDIM/mouse. d Significantly (P < 0.05) less than matched control by Fisher’s exact test.

lmmunoprecipitation [36S]Methionine-labeled lysates of mock-infected or EDIM-infected cells were incubated with mouse antisera (diluted 1:15) overnight at 4”. Sepharose CL-4protein A beads (20 PI), preadsorbed with unlabeled EDIM-infected lysate, were added and held for 30 min. The beads were then washed twice with A buffer (0.5 M NaCI, 10 mM Tris, pH 7.2, 0.05% NP-40) B buffer (0.1 M NaCI, 10 mMTris, pH 7.2,0.05% NP-40) and C buffer(10 mMTris, pH 7.2, 0.05% NP-40) (Chen eta/., 1992). SDS-polyacrylamide

gel electrophoresis

lmmunoprecipitated [36S]methionine-labeled proteins were analyzed by the method of Laemmli (1970) using a 10% resolving gel. Specimens were dissolved in sample buffer (30 ~1) and held at 100” for 2 min before loading gels. After electrophoresis (30 mAmps/ gel, 4 hr), the gels were stained with Coomaise brilliant blue to locate viral and molecular weight markers before drying. Gels were then exposed to X-ray film at -70” before developing. RESULTS Protection after ip immunization with EDIM The initial experiment was to determine whether mice could be protected against EDIM infection if immunized ip with three doses of purified, live EDIM and whether inclusion of adjuvant (Freund’s) modified the protective efficacy of the immunogen. Mice immunized with EDIM developed high titers of rotavirus IgG and moderate titers of neutralizing antibody to the immuniz-

ing virus (Table 1). Inclusion of adjuvant caused the geometric mean titer (GMT) to increase four- to eightfold. All mice administered the control preparation, either with or without Freund’s adjuvant, had no detectable rotavirus antibody. When challenged with EDIM, the control animals shed rotavirus beginning at 2 days after inoculation. In contrast, the mice immunized ip with EDIM were consistently protected against EDIM shedding following oral challenge. To ensure that the inability to detect viral shedding was not due merely to steric hindrance by antibody binding, stool specimens collected after oral challenge were examined for the presence of rotavirus by gel electrophoresis of extracted RNA and none was found. Thus, significant protection against EDIM shedding was observed after ip immunization with this virus, even without adjuvant. No adjuvant was included in subsequent experiments. We next determined the dosage of immunogen required to obtain 100% protection against EDIM shedding. Following three ip inoculations with between 0.1 and 8.0 rg of EDIM/inoculation, all mice had high titers of rotavirus IgG (Table 2). These titers increased gradually with increasing dose along with titers of neutralizing antibody. Mice inoculated with the control preparation had no detectable rotavirus antibody. When orally challenged with EDIM, no mice shed rotavirus after immunization with either 2 or 8 pg of purified EDIM but inoculation with either 0.1 or 0.5 pg failed to protect one of four mice in each group. All four control animals shed rotavirus as expected. Thus, only partial protection against shedding was observed when mice were immunized with dosages of < 2 pg of purified EDIM. The effect of the number of doses was next deter-

INTRAPERITONEAL

IMMUNIZATION

153

TO ROTAVIRUS

TABLE 2

TABLE 4

EFFECTOF QUANTIW OF PURIFIEDEDIM USED FOR IP INOCULATIONON PROTECTIONAGAINSTSHEDDINGOF EDIM FOLLOWINGORAL CHALLENGE

PROTECTIONAGAINSTEDIM INFECTIONAFTERA SINGLE IP IMMUNIZATION WITH HETEROLOG~USROTAVIRUSES’

Quantity of immunizing virus” (pg/dose) 0 0.1 0.5 2.0 8.0

GMT of serum rotavirus antibody before EDIM challenge

N 4 4 4 4 4

Rotavirus bG <3.0 2.6 4.1 5.2 2.0

Neutralizing antibody

Outcome post-EDIM challenge No. shedding EDIM after challengeb (%)

x 102 X 106c

<20 126’

4 (100) 1 (25)

x lo6 X lo6 x loe=

104 281d

1 (25) 0w

364

0 (0)’

o Three doses of virus administered without adjuvant, separated by 2-week intervals. b Challenge dose: 2 X 1O3ffu/mouse. c Significantly (P K 0.001) greater than group given next lower dose (Student’s t test). d Significantly (P = 0.02) greater than group given next lower dose (Student’s t test). * Significantly (P c 0.05) less than group that received no immunizing virus (Fisher’s exact test).

mined. Mice immunized with between one and three doses of EDIM (8 pg/dose) developed correspondingly higher titers of rotavirus IgG (Table 3). However, all groups were significantly protected against EDIM infection relative to the unimmunized control group. Thus, a single dose of purified, live EDIM administered ip was found to protect mice against EDIM infection when subsequently challenged.

TABLE 3 EFFECTOF NUMBER OF DOSES OF EDIM ADMINISTEREDip ON PROTECTIONAGAINSTEDIM INFECTION

GMT of serum rotavirus antibody before challenge

No. of doses of immunizing virus”

N

0 1 2 3

4 4 4 4

Rotavirus I@ c3.0 5.5 4.9 2.0

x x x x

lo* lo’= 106= 10EC

Neutralizing antibody

No. shedding EDIM after challengeb (%)

<20 74=

4 (100) 0 Kod

126

0 (o)d

364*

0 (Old

a Inoculation ip with 8 fig of purified virus per dose. b Challenge dose: 2 X 1O3Wmouse. c Significantly (P < 0.001) greater than group given next lower number of doses (Student’s t test). d Significantly (P < 0.05) less than group receiving no immunizing virus (Fisher’s exact test). ’ Significantly (P < 0.01) greater than group given next lower number of doses (Student’s t test).

Immunizing virus

N

Control EDIM EB RRV

8 8 8 8

SAl I-FEM

8

SAll-SEM WC3

8 8

GMT of rotavirus IgG before EDIM challenge
3.8X104 4.9 x lo4 3.3 x lo4 5.4 x lo4 4.8 X lo4 4.1X104

Shed virus 8 5 5 7 7 7 8

Mean days of viral shedding

Mean quantity of virus shedb

4.38 o.aac

7.76 0.58”

1.50”

1.25’ 1.05=

2.25c 2.75d 2.3ad 2.8ad

2.37’ 1.08’ 2.08’

* Groups of 6-week-old mice were inoculated ip with 2 ag of purified live rotavirus and monitored for serum antibody responses at 24 days postinoculation. On the following day, they were orally challenged with 4 X lo4 ffu of EDIM (passage 9) per mouse. Stools collected daily for 7 days post-challenge were tested for viral antigen by ELISA. b Quantity defined by total A4gOvalues above background for every mouse on the 7 days after challenge determined by ELISA. c P < 0.001 relative to control mice by Student’s t test. d P < 0.01 relative to control mice by Student’s r test.

Protection after ip immunization with heterologous rotaviruses Although oral immunization with most heterologous rotavirus strains was not found to provide active immunity against EDIM infection (Ward et a/., 1992) it was possible that ip immunization with these same strains may consistently protect. Therefore, the abilities of heterologous rotavirus strains to protect against EDIM infection was determined. A single ip dose (2 pg) of every rotavirus strain analyzed provided significant protection against EDIM as determined by both days of virus shedding and quantity of virus shed (Table 4). Although protection against EDIM shedding was not absolute following a single ip immunization with 2 pg of purified virus, even in mice immunized with EDIM, the breadth of strains that provided protection was much greater than found after oral immunization (Table 5). It was previously reported that protection following oral immunization did not appear to be dependent on neutralizing antibody to the challenge virus (Ward et a/., 1992). It was, therefore, of interest to determine whether this was also found following ip immunization by heterologous rotavirus strains. To increase the probability for these strains to produce a neutralizing antibody response to EDIM after ip inoculation, mice were inoculated with three doses (2 pg/dose) of each virus

McNEAL,

154

SHERIDAN,

TABLE 5 PROTECTIONAGAINSTEDIM INFECTIONFOLLOWINGIMMUNIZATIONWITH DIFFERENT ROTAVIRUSSTRAINS BY ORAL VERSUS ip INOCULATIONWITH LIVE VIRUSES Protection

Immunizing virus Control EDIM EB RRV SAl 1-FEM SAl 1-SEM WC3

against EDIM

Oral inoculation

b inoculation

-

-

+ + -

+ + + + + +

+ -

separated by 2-week intervals. Although this dosing regimen consistently stimulated neutralizing antibody to the immunizing strains, only mice inoculated with EDIM made detectable neutralizing antibody to EDIM (Table 6). However, all rotavirus strains tested provided significant protection against EDIM infection. Thus, protection following ip immunization was not found to be dependent on neutralizing antibody. Is rotavirus replication needed for protection following ip immunization? Rotavirus replication is normally confined to epithelial cells on the tips of the intestinal villi. It is possible, however, that protection against intestinal infection

AND WARD

with EDIM observed after ip inoculation with live rotaviruses depended on replication of the immunizing virus at or near the site of inoculation. To analyze this possibility, we compared the protective response against EDIM infection induced by a single ip inoculation with 8 gg of either live or UV-inactivated purified EDIM. At 26 days after immunization (the day before mice were challenged with EDIM), the GMT of serum rotavirus IgG was nearly identical for these two groups of mice (Table 7). This suggested that if viral replication occurred following ip immunization with live EDIM, it was not extensive. When the mice were subsequently orally challenged with EDIM, those immunized with inactivated virus were less well protected than mice given live virus (Table 7). Both groups of mice, however, shed significantly less viral antigen and for fewer days than control mice. Thus, ip inoculation with uv-inactivated EDIM at least partially protected against subsequent intestinal EDIM infection. The greater protection provided by live versus uv-inactivated virus could have been due to viral replication. To examine this possibility, mice were inoculated ip with 8 rg of live EDIM, then at different times postinoculation, the peritoneal cavity as well as different visceral organs were examined for the presence of rotavirus antigen. In addition, mice were examined for evidence of intestinal shedding of viral antigen between Days 1 through 7 following ip inoculation. Rotavirus antigen was detected in the washed peritoneal cavity immediately after inoculation (Day O), but was not observed in either of two mice sacrificed on Days 5, 12, or

TABLE 6 PROTECTIONAGAINST EDIM INFECTIONAFTER THREE ip INOCULATIONSWITH HETEROLOGOUSROTAVIRUSES IN THE ABSENCEOF DETECTABLENEUTRALIZINGANTIBODYTO EDIMB GMT of serum rotavirus antibody before EDIM challenge Outcome Neutralizing Immunizing virus

N

Control EDIM EB RRV SAl 1-FEM WC3

4 4 8 4 4 4

Roatvirus IgG <3.0 5.2 8.0 7.8 8.6 1.2

x x X x x x

lo2 lo6 lo6 lo6 lo6 lo6

Homologous

103 4820 3080 1570

post-EDIM

challenge

antibody EDIM

Shed virus

120 281 <20 <20 t20 t20

4 OC 4 OC 2 1

Mean quantity of virus shed* 5.87 0* 0.40d 0* 0.23’ 0.65’

B Groups of 6-week-old mice were inoculated ip three times at 2-week intervals (2 pg/dose) and monitored for serum antibody reponse 10 days after the third inoculation. One day later, the mice were orally challenged with EDIM (3 x 1O3ffu/mouse). stools were collected daily for 7 days and these were tested for viral antigen by ELISA. * See legend of Table 4. c P < 0.05 relative to control mice by Fisher’s exact test. d P < 0.001 relative to control mice by Student’s t test. * P i 0.01 relative to control mice by Student’s t test.

INTRAPERITONEAL

IMMUNIZATION

TABLE 7 ANTIBODY

lnoculum

RESPONSES AND PROTECTION FOLLOWING ip INOCULATION WITH LIVE VERSUS UV-INACTIVATED EDIM’

N

Control

8

Live EDIM

8

uvinactivated EDIM

8

GMT of serum rotavirus IgG following immunization (range of titers)

Outcome post-EDIM challenge

Shed virus

Mean days of virus shedding

Mean quantity of virus shedb

<300 (<300) 28,550 (14,500-48,000)

8

3.38

1.03

oc

Od

Od

26,650 (2,450-46,000)

5

1 .25e

0.19’

a Groups of 6-week-old mice were inoculated ip with 8 pg of purified live or uv-inactivated EDIM per mouse and monitored for serum antibody responses at 26 days postinoculation. On the following day, they were orally challenged with 4 X 1O4ffu of EDIM per mouse. Stools collected daily for 7 days post-challenge were evaluated for viral antigen by ELISA. b See legend of Table 4. c P < 0.0001 relative to control mice by Fisher’s exact test. d P < 0.0001 relative to control mice by Student’s f test. e P < 0.001 relative to control mice by Student’s f test. ‘P < 0.02 relative to control mice by Student’s t test.

TO ROTAVIRUS

155

Antisera from mice immunized ip with uv-inactivated virus precipitated only structural rotavirus proteins while antisera from mice inoculated either orally or ip with live EDIM precipitated nonstructural as well as structural rotavirus proteins (Fig. 1). Therefore, even though extensive viral replication had not been found following ip inoculation with live EDIM, at least some viral protein synthesis must have occurred. This could account for the better protection provided by live versus uv-inactivated virus.

DISCUSSION Development of human rotavirus vaccines has focused primarily on the use of live, attenuated viruses which have been delivered orally. The rationale is that these viruses will replicate in the intestine and stimulate local immunity at the site normally associated with rotavirus infection and disease. Rotavirus vaccines that have been tested, however, have been found to provide only partial protection against rotavirus dis-

1

2

3

4

5

6

7

8

9

10

97kdr VP

24. Furthermore, no evidence of rotavirus antigen was found in homogenized tissue from spleen, liver, stomach, small intestine, or large intestine of these mice at any of these times. As a positive control, we examined liver specimens of SCID (severe combined immunodeficiency) mice with rotavirus-induced hepatitis for the presence of viral antigen. These mice had been infected ip 3.5 months earlier with live SAl 1 and rotavirus antigen was readily detected in their homogenized liver tissue. Finally, 0 of 12 mice inoculated ip with EDIM shed detectable quantities of rotavirus antigen as determined by ELISA. These results indicated that the EDIM virus did not undergo extensive replication following ip inoculation. Evidence of viral replication following ip inoculation was further analyzed by examination of serum antibody responses. In order to produce antibody to nonstructural rotavirus proteins in inoculated mice, at least some viral protein synthesis must occur. Therefore, we compared the ability of antisera obtained from mice 26 days after ip inoculation with either live or uv-inactivated EDIM to immunoprecipitate EDIM proteins. Preand postimmune antisera obtained from mice orally immunized with live EDIM were included as negative and positive controls, respectively.

66kdb

45kdb

31 kdb

21 kd,

FIG. 1. Polyacrylamide gel electrophoresis of [35S]methioninelabeled proteins immunoprecipitated from EDIM-infected or mockinfected MA104 cells with mouse antisera. Labeled proteins from infected (lanes l-5) or mock-infected (lanes 6-l 0) cells were precipitated with Sepharose CL-4-protein A beads following overnight incubation with convalescent serum from mice inoculated orally with live EDIM (lanes 1 and 6) intraperitoneally with uv-inactivated EDIM (lanes 2 and 7) or intraperitoneally with live EDIM (lanes 3 and 8). Labeled proteins precipitated after incubation with preimmune serum (lanes 4 and 9) or no serum (lanes 5 and 10) were included as controls. Positions of the molecular weight marker (kDa) and structural proteins from purified EDIM (VP) are designated. Assignment of nonstructural proteins (NS) is based on their molecular weights as described by Estes and Cohen (1989).

156

McNEAL,

SHERIDAN,

ease in children. Therefore, development of candidates that consistently prevent rotavirus disease in vaccinees are clearly needed. This report suggests an alternative route of immunization. Mice inoculated ip with live EDIM virus were protected against EDIM infection, even when administered as a single dose in the absence of adjuvant. Furthermore, protection against EDIM infection was provided by ip inoculation with heterologous strains of rotavirus. In a previous study (Ward et a/., 1992) we reported that mice could be protected against EDIM infection following oral inoculation by some but not all rotavirus strains. For example, inoculation of neonatal mice with RRV, WC3, or SAl 1-SEM caused illness and development of high rotavirus IgG titers in the infected mice, but did not protect against EDIM challenge. In the present study, these same viruses produced significant protection when administered ip. This suggested that the mechanisms of protection following immunization by the different routes were not identical and that ip immunization may provide broader protection. It is also possible, however, that the mechanisms were the same but higher levels of the protective immune response were more consistently induced following ip versus oral immunization. Previous studies by other investigators have indicated that neutralizing antibody delivered to the intestine can passively protect against rotavirus disease in animals (Woode et al., 1975; Snodgrass and Wells, 1976; Offit and Clark, 1985; Offit et a/., 1986a,b; Hoshino et a/., 1988; Matsui et a/., 1989). We found no evidence, however, to suggest that the mechanism of active protection following either oral or ip immunization in our mouse model was due to neutralizing antibody to the challenge virus. In both cases, immunity to EDIM infection following inoculation with heterologous rotaviruses was observed in the absence of detectable (i.e., titers of 220) serum neutralizing antibody to EDIM. This finding does not rule out the possibility that very low titers of neutralizing antibody to EDIM were developed following immunization with these viruses and that these low titers were sufficient to protect against EDIM infection. Infectious rather than inactivated rotavirus was used as the immunogen in this study. This was done in order to ensure that the structural proteins of the virus were in their native configuration. Thus, we attempted to maximize the possibility that protection would be developed following ip inoculation. Zissis et al. (1983) reported that intramuscular (im) immunization of piglets with live serotype 6 bovine rotavirus provided partial protection against viral replication when challenged with heterotypic human rotaviruses. However, when the piglets were immunized with formalin-inactivated

AND WARD

bovine rotavirus, a loss of protection was observed, possibly due to protein denaturation. It was also possible, however, that loss of protection in that study was due to the inability of the virus to replicate. Rotavirus replication is generally restricted to the mature enterocytes on the villi of the small intestine, but this is not always the case (Grunow et al., 1985; Uhnoo et a/., 1990; Offit et a/., 1991; Zheng et al., 1991). Therefore, it was possible that some rotavirus replication may have occurred following either ip or im inoculation. To determine whether protection against EDIM infection in mice following ip immunization required viral replication, a comparison of outcomes was made following inoculation with live versus uv-inactivated EDIM. A single inoculation with either viral preparation stimulated large and nearly equal serum rotavirus IgG responses which suggested that little viral replication occurred following inoculation with live EDIM. More importantly, uv-inactivated EDIM provided significant protection against EDIM infection although it was not as effective as live EDIM. This demonstrated that inactivated rotavirus could provide protection following ip inoculation. Because live EDIM provided better protection when delivered ip than inactivated virus, however, it was possible that the difference was due to viral replication. Although no evidence of viral replication was found by examination of visceral organs and fecal specimens for rotavirus antigen following ip inoculation with live EDIM, these mice had antibody responses to nonstructural proteins which did not occur when administered inactivated virus. Therefore, some viral protein synthesis did occur following ip inoculation with live EDIM which may have resulted in better protection against EDIM infection. Viral protein synthesis may be needed to stimulate cytotoxic lymphocytes to rotavirus-infected cells (Braciale et al., 1987) which have been reported to be protective against rotavirus illness (Offit and Dudzik, 1990) and resolve shedding (Dharakul et a/., 1991) in mice following adoptive transfer. These results show that ip immunization with rotavirus can, in the absence of viral replication or neutralizing antibody to the challenge virus, provide significant protection of mice against a subsequent rotavirus infection. It will now be important to determine whether protection can be obtained after immunization by other parenteral routes, can be produced after parenteral immunization with viral subunits such as proteins or peptides, and is effective against multiple strains of rotavirus. ACKNOWLEDGMENT We thank D. I. Bernstein for his insightful comments and suggestions concerning the planning and writing of this study.

INTRAPERITONEAL

IMMUNIZATION

REFERENCES BERNSTEIN,D. I., KACICA, M. A., MCNEAL, M. M.. SCHIFF, G. M., and WARD, R. L. (1989). Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers. Antiviral Res. 12, 293-300. BERNSTEIN.D. I., SMITH, V. E., SANDER,D. S., PA& K. A., SCHIFF, G. M., and WARD, R. L. (1990). Evaluation of WC3 rotavirus vaccine and correlates of protection in healthy infants. /. lnfecf. Dis. 162, 1055-l 062. BLACKLOW,N. R., and GREENBERG,H. B. (1991). Viral gastroenteritis. N. Engl. J. Med. 325, 252-264. BRACIALE, T. J., MORRISON, L. A., SWEETSER.M. T., SAMBROOK, J., GETHING, M. J., and BRACIALE, V. L. (1987). Antigen presentation pathways to class I and class II MHC-restricted lymphocytes. /mmunol. Rev. 98, 95-96. CHEN, D., ESTES,M. K., and RAMIG, R. F. (1992). Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope. J. Viral. 66, 432-439. DHARAKUL,T., Ro-rr, L.. and GREENBERG,H. B. (1990). Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes. J. Viral. 64,4375-4382. ESTES, M. K., and COHEN, J. (1989). Rotavirus gene structure and function. Microbial. Rev. 53, 41 O-449. GRUNOW,J. E., DUNSTON,S. F., and WANER, J. L. (1985). Human rotavirus-like particles in a hepatic abscess. J. Pediatr. 106, 73-76. HOSHINO, Y., SAIF, L. J., SERENO, M. M., CHANOCK, R. M., and KAPIKIAN, A. Z. (1988). Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Viral. 62, 744-748. LAEMMLI, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. MATSUI, S. M., OFFIT, P. A., Vo, P. T.. MACKOW, E. R., BENFIELD.D.A., SHAW, R. D., PADILLA-NORIEGA,L., and GREENBERG,H. B. (1989). Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to the heterotypic neutralization domain of VP7 and the VP8 fragment of VP4. J. Clin. Microbial. 27, 780-782. OFFIT, P. A., and CLARK, H. F. (1985). Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies. 1. Viral. 54, 58-64. OFFIT, P. A., CLARK, H. F., BLAVAT, G., and GREENBERG,H. B. (1986a). Reassortant rotaviruses containing structural proteins VP3 and VP7 from different parents induce antibodies protective against each parental serotype. 1. Viral. 60, 491-496. OFFIT, P. A., SHAW, R. D., and GREENBERG,H. B. (1986b). Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins of VP3 and VP7.J. Viral. 58, 700-703.

TO

ROTAVIRUS

157

OFFIT. P. A., and DUDZIK, K. I. (1990). Rotavirus-specific cytotoxic T-lymphocytes passively protect against gastroenteritis in suckling mice. /. viral. 64, 6325-6328. OFFIT, P. A., CUNNINGHAM, S. L., and DUDZIK, K. I. (1991). Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection. J. Viral. 65, 13181324. SAIF, L., and SMITH, K. (1985). Enteric viral infection of calves and passive immunity. J. Dairy Sci. 68, 206-208. SNODGRASS,D. R., and WELLS, P. W. (1976). Rotavirus infection in lambs: studies on passive protection. Arch. Viral. 52, 201-205. SNODGRASS,D. R., and WELLS, P. W. (1978). Passive immunity in rotaviral infections. J. Am. Vet. Med. Assoc. 173, 565-568. TUFVESSON,B.. POLBERGER,S., SVANBERG,L., and SVEGER,T. (1986). A prospective study of rotavirus infections in neonatal and maternity wards. Acta. Paediatr. Stand. 75, 2 1 l-2 15. UHNOO, I,, RIEPENHOFF-TALTY, M., DHARAKUL,T., CHEGAS, P., FISHER, J. E., GREENBERG,H. B., and OGRA, P. L. (1990). Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus. 1. Viral. 64, 36 l-368. VIAL, P. A., KATLOFF, K. L., and LOSONSKY,G. A. (1988). Molecular epidemiology of rotavirus infection in a room for convalescing newborns. 1. Infect. Dis. 157, 668-673. WARD, R. L., BERNSTEIN,D. I., KNOWLTON,D. R., SHERWOOD,J. R., YOUNG, E. C., CUSACK, T. M., RUBINO, J. R., and SCHIFF, G. M. (1991). Prevention of surface-to-human transmission of rotavirus by treatment with disinfectant spray. J. C/in. Microbial. 29, 19911996. WARD, R. L., BERNSTEIN,D. I., YOUNG, E. C., SHERWOOD,J. R., KNOWLTON, D. R.. and SCHIFF, G. M. (1986). Human rotavirus studies in volunteers: determination of infectious dose and serological response to infection. J. infect. Dis. 154, 87 l-880. WARD, R. L., MCNEAL, M. M., and SHERIDAN,J. F. (1990). Development of an adult mouse model for studies on protection against rotavirus. J. Viral. 64, 5070-5075. WARD, R. L., MCNEAL, M. M., and SHERIDAN,J. F. (1992). Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody. Virology 188, 57-66. WOODE, G. N., JONES,J., and BRIDGER.J. C. (1975). Levels of colostral antibodies against neonatal calf diarrhoea virus. Vet. Rec. 97, 148-149. ZHENG, B. J., CHANG, R. X., MA, G. Z., XIE. J. M., LIU, Q., LIANG, X. R., and NG, M. H. (1991). Rotavirus infection of the oropharynx and respiratory tract in young children. J. Med. Viral. 34, 29-37. ZISSIS, G., LAMBERT,J. P., MARBEHANT, P., MARISSENS,D., LOBMANN, M., CHARLIER,P., DELEM. A., and ZYGRAICH,N. (1983). Protection studies in colostrum-deprived piglets of a bovine rotavirus vaccine candidate using human rotavirus strains for challenge. J. lnfecr. Dis. 148, 1061-1068.