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Rotavirus-Specific Intestinal

Immune

Response in Mice

Assessed

by

Enzyme-Linked

Immunospot

Assay

and

Intestinal Fragment Culture

CHRISTIANA.

KHOURY,'

KURT A. BROWN,1 JOON E.KIM,1 ANDPAUL A. OFFITl 2*

Section of InfectiousDiseasesand TheDivision

of

Gastroenterology

andNutrition, The University

ofPennsylvania School ofMedicine, and The Children'sHospital of

Philadelphia,

1 and

The Wistar InstituteofAnatomy and Biology,2Philadelphia, Pennsylvania 19104

Received1April 1994/Returned for modification13 June1994/Accepted 10August 1994

Primate rotavirus strain RRV and bovine strain WC3 or reassortants made betweenthese animal viruses and human rotaviruses have been administeredtoinfantsascandidate vaccines. WecomparedRRV and WC3 in a murine model of oral infection. We determined the relative capacities of these viruses to induce a

virus-specific humoral immune responseby intestinal lymphocytes as testedby enzyme-linked immunospot

assay,intestinalfragment culture, andenzyme-linked immunosorbent assayofintestinalcontents.We found that inoculation of mice with RRV induced higher frequencies of

virus-specific

immunoglobulin A (IgA)-secreting cells in the lamina propria, greaterquantities ofvirus-specific IgAin intestinal fragmentcultures, and greater quantities of virus-specific IgA in intestinal secretions than did inoculation with WC3 or

inactivated RRV(iRRV). The induction ofanIgA response inserumwaspredictiveofanIgAresponseamong intestinallymphocytes after inoculation with RRV butnotWC3.In

addition,

large

quantities

of

IgG, IgA,

and IgM notspecific for rotaviruswereproduced infragmentculturesfrom mice inoculated with RRV butnotin culturesfrom mice inoculated with WC3oriRRV. Possible mechanisms of RRV-inducedpolyclonalstimulation of intestinal B cellsarediscussed.

Rotaviruses are recognized as the major cause of acute

diarrheal disease in infantsand young children

(51); they

are

responsible for an estimated 830,000 deaths per year

(20).

Rotavirus infections arehighly contagiousandaredifficult to

controlby general

hygienic

measures.

Therefore,

active

immu-nization may be the most successful wayto prevent disease.

Because rotaviruses replicate only in maturevillus

epithelial

cells which line the small intestine

(45), protection against

rotavirus diseaseisprobably mediatedbyanimmuneresponse

active at the intestinal mucosal surface. Development of a

successful vaccine will depend on understanding the most

efficientmeans ofinducing virus-specific effector cells within

gut-associated lymphoid tissue (GALT).

Efforts to develop avaccine have focused on two

animal-originrotavirus strainswhich have attenuated virulence

char-acteristics in humans: primate strain RRV (17) and bovine

strain WC3(8).Inaddition,reassortantvirusesconsistingof all gene segmentsfrom eitherRRV (7, 15, 17, 29, 39, 40, 50) or

WC3

(5,

8, 9, 11,

53)

anda

single

genefromhuman rotavirus

strains have beendevelopedandtested forefficacy. There are

important differences between thesetwoanimal-origin

rotavi-ruses.First, RRV appears to be better adapted to growth in the human intestine than is WC3 (49). RRV is detected in the feces of infants and young children at higher titers and for longer periods than is WC3 (4, 5, 29, 40, 48). In addition, children inoculated with RRV develop fever more frequently than dochildren inoculated with eitherWC3 or placebo (4, 5, 7, 8, 28, 33,48). Second, although studies directly comparing the immune responses to RRV and WC3 are lacking, WC3

*Corresponding author. Mailing address: Section of Infectious

Diseases and Division of Gastroenterology and Nutrition, The Uni-versityofPennsylvaniaSchool of Medicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 590-2020. Fax: (215) 590-3044.

may be less consistent than RRV in inducingavirus-specific

immunoglobulin A (IgA) response in theserumand feces of

infants (9, 28, 29, 40, 50).

As inhumans,primate-originrotavirusesarebetteradapted

togrowthin the murineintestine thanarebovine-originstrains

(3). Therefore, studies of the rotavirus-specific immune

re-sponse inmice may bepredictive of the immune response in humans. In this reportwecomparethe intestinalvirus-specific humoral immune response in mice after oralinoculation with rotavirus strains RRV and WC3 by using enzyme-linked immunospot (ELISPOT) assay, intestinal fragment culture, andenzyme-linked immunosorbent assay (ELISA) of intesti-nalcontents.

MATERIALSAND METHODS

Mice. Conventionally bred,pregnant CD2

(Fl)

mice

(Tac-onicBreeding Laboratories, Germantown, N.Y.)werehoused

in separateisolationunits. Only littersfrom rotavirus-seroneg-ative damswere used inthese experiments.

Cells.Fetal greenmonkey kidneycells(MA-104cells) were grown as previously described (36).

Viruses.Bovine rotavirus WC3 was obtained from H. Fred

Clark, Philadelphia,Pa. Simian rotavirus RRV (MMU 18006)

was obtained from Nathalie Schmidt, Viral and Rickettsial DiseaseLaboratory, Berkeley, Calif. Viral growth, purification, andquantitation by plaque assay were performed as previously

described (36, 37).

RRV wasinactivated by treatment with psoralen plus UV irradiation (19). 4'-Aminomethyl-4,5',8-trimethylpsoralen (HRI Associates Inc., Concord, Calif.) dissolved in 50% ethanol was addedat afinal concentration of 10 ,ug/ml to a tissue

culture-derived, clarified preparation of RRV. The preparation was

placed on ice for 5 min, aliquoted into individual wells of a

24-wellplate (BectonDickinson and Co., Lincoln Park, N.J.),

722

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and exposed to a UV lamp (GBL-100; Geroge W. Gates & Co., New York, N.Y.) at a distance of 7.5 cm for 15 min.

Immunization of animals. CD2 (F1) suckling mice, 7 to 8 days old, were orally inoculated, by proximal esophageal

intubation,with Stoker's medium (mock infection), inactivated

RRV (iRRV) (215 ng per mouse), or tissue culture-derived

WC3 or RRV (each at 107 PFU [approximately 215 ng] per mouse) in a volume of 100 pul.

ELISPOT assay. Lymphocytes from Peyer's patches (PP), mesenteric lymph nodes (MLN), small intestinal lamina pro-pria(LP), and spleens were obtained 21 days after inoculation

aspreviously described (38).

The ELISPOT assay was a modification of published

meth-ods (34, 43). Individual wells of 96-well millititer HA plates

(Millipore Corp., Bedford, Mass.) were coated overnight at

4°Cwith 100 pIofphosphate-buffered saline (PBS) or 200 ng

ofpurified, double-shelledRRV or WC3 suspended in 100 pI

ofPBS. The wells were washed four times with PBS (Wellwash 2;Denley Instruments Inc., Durham, N.C.) and treated for 1 h

at roomtemperature (RT) with 100 ,ul of 1% bovine serum

albumin (BSA) diluted in PBS. Wells were washed four times with PBS, and 100

RI

of asuspension containing 1 x 106, 2 x

105,

or 4 x

104

cells diluted in RPMI 1640 plus 10% fetal bovine serum was added to each well. The plates were incu-batedfor4h at37°C in aCO2incubatorand then washed five times with PBS and five times with PBS-0.05% Tween 20

(PBS-Tween) (Sigma Chemical Co., St. Louis, Mo.).

Rotavi-rus-specific mouse Igs were detected with isotype-specific, horseradish peroxidase-labeled goat anti-mouse IgG (diluted 1:1,000 in 1% BSA-0.05% Tween), goat anti-mouse IgA

(diluted 1:2,000 in BSA-0.05% Tween), or goat anti-mouse

IgM (diluted 1:1,000 in BSA-0.05% Tween) (Southern

Bio-technology Associates, Inc., Birmingham, Ala.). The plates were incubatedovernight at 4°C in a humidified environment. Theywere thenwashed four times with PBS, and 100 p1 of a solution containing 10 ,ug of3-amino-9-ethyl carbazole

(Sig-ma) dissolvedin 1 ml ofN,N-dimethyl formamide(Sigma), 0.1

Msodium acetate (pH 5.0) (filtered through a

0.45-p.m-pore-sizefilter [NalgeCo.,Rochester, N.Y.]), and 1% H202 (Sigma)

was added to each well. After 15min, the wellswere washed

withdistilledwater. The plates were dried for 24 h at RT, and

thewells were examined by light microscopy (MZ800 micro-scope; Swift, SanJose, Calif.) under x8 to x10magnification. Average numbers of spots in duplicate wells coated without

antigen were subtracted from average numbers in duplicate

wells coatedwith antigen.

Intestinal fragment cultures. Intestinal fragment cultures

were prepared 7, 14, and 21 days after inoculation, by a

modificationof themethod described by Logan et al. (27). PP,

MLN, and small intestinal fragments were collected under

sterile conditions and washed three times in calcium- and

magnesium-free Hanks balanced salt solution (Gibco Inc.,

GrandIsland,N.Y.) supplemented with 10 mM

N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic

acid(HEPES)and 50 pug

ofgentamicin (JRH,Lenexa,Kans.)per ml.On average, 10 to

12 PP werecollectedper mouse.EachPPwasincubatedin one wellof a24-well plate(BectonDickinson), suspended in 1 ml ofamediumcontaining Kennet's HYmedium(JRH)

supple-mentedwith 10 mMHEPES, 10%fetalbovineserum(Gibco),

4 mML-glutamine (JRH), 100 U ofpenicillin (JRH) perml,

100

pug

ofstreptomycin (JRH) per ml, 50

pug

of gentamicin

(JRH) per ml, and 0.25

p.g

of amphotericin B (Fungizone;

JRH) per ml (GALT medium). MLN were incubated in Iscove's modifiedDulbecco's medium

(Gibco) supplemented

with 50 ,ug of gentamicin per ml. After removal of the mesentery, connective tissue, and fat, MLN were cut in half

and transferred to two wells of a 24-well plate containing 1 ml of GALT medium per well. After the PP were removed, the small intestine was opened longitudinally and cut into 2-in. (5-cm) segments. Small intestinal fragments were washed twice inHBSS, once in HBSS plus 0.05% EDTA (Sigma), and twice in HBSS. Eight small intestinal fragments (each approximately 0.25 cm long) were each placed in individual wells of a 24-well plate containing 1 ml of GALT medium per well. All tissue specimens were incubated for 7 days at37°C in an atmosphere of 95% 02 to 5% CO2. Supernatant fluids (750 pu) were collected from each well, stored at -20°C, and tested by ELISA for detection of rotavirus-specific antibodies.

Each fragment containing LP cells had a surface area of 1 mm2, whereas the total surface area of LP in the small intestine of suckling mice was calculated to be 1,400mm2. We adjusted the quantities of virus-specific and total antibodies obtained from eight small intestinal fragments to reflect antibody pro-duction by the entire small intestine.

Collection ofintestinal secretions. Mice were fasted for 16 to 18 h prior tocollection of intestinal fluids. A 500-pd portion of a solution containing 1.36 g of Golytely (Braintree Labora-tories Inc., Braintree, Mass.) in 20 ml of distilled water was inoculated orally by proximal esophageal intubation into each mouse four times at 15-minintervals. After 30 min, 150 plIof 1% pilocarpine hydrochloride (Sigma) was injected intraperi-toneally and intestinal contents were collected in a petri dish containing 6 ml of PBS, 0.05 M EDTA (pH 7.4), and 0.1 mg of soybean trypsininhibitor (type I-S; Sigma). Suspensions were vortexed for 30 s and centrifuged at 700xgfor 10minat4°C. Then 60

pI

of 100 mMphenylmethylsulfonyl fluoride (PMSF; Sigma) in PBS was added to each sample. Samples were centrifuged at 27,000xgfor 20minat4°C; 4 ml was collected from each sample, and 40 ,ul of 2% sodium azide (Fisher Scientific Co., Pittsburgh, Pa.) and 40 p1 of 100 mM PMSF were added. The samples were placed on ice for 15 min, and 200

pI

ofFBS and 1.4 ml of glycerol (Sigma) were added. The samples were mixed, divided into 1-ml aliquots, and stored at -700C.

ELISA for detection of the titer ofrotavirus-specific anti-bodies and quantities of IgA and IgG.Supernatant fluids from intestinal fragment cultures, supernatant fluids from intestinal contents, and serum from inoculated and uninoculated animals weretested for the presence ofrotavirus-specific antibodies or quantities of total IgA,IgM,and IgG byELISA. For detection of rotavirus-specific antibodies, individual wells of 96-well, flat-bottomed plates were coated with either 100

p1

of PBS or 200 ng of purified WC3 or RRV diluted in 100

pI

ofPBS. The plates were stored overnight at 4°C. The wells were each washed fourtimes with PBS and blocked for 1 h with 200

pI

of 0.25% BSA plus 0.025% Tween diluted in PBS at RT. The wells were thenwashed four times with PBS, 100

p1

ofsample wasadded to each well, and the mixture wasincubated for 1 h at RT. The wells werewashed four times with PBS, and 100

pI

of horseradish peroxidase-conjugated goat anti-mouse IgM, IgA, and IgG (Southern Biotechnology Associates) diluted 1:2,000 in 1% BSA was added to each well and incubated for 1 h at RT. The wells werewashed four times in PBS, and 100

pI

of a 0.04% tetramethylbenzedine peroxidase solution (Ki-erkegaard and Perry, Gaithersburg, Md.) was added; the mixture was incubated for 5 min. A 100-pA portion of 85% o-phosphoric acid (Sigma) was added to each well, and colo-rimetric changes were determined at an optical density(OD) of 450 nm on amicroplate ELISA reader(Microplate reader 2000;Biowhittaker, Walkersville, Md.). Samples were consid-eredpositive if OD values forvirus-coated wells were both 0.1

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unit greater and at least twofold greaterthan OD values for PBS-coated wells.

For detection of the quantities of total IgA and IgG in intestinal fragment cultures, wells were initially coated with

100 [lof PBSor a 1:1,000 dilution of eithergoat anti-mouse IgM, IgG, or IgA (Cappel, Organon Teknika Corp., West Chester, Pa.) diluted in PBS. As described above, 10to12PP

werecollectedpermouseand placed in individual wells, while MLNweredivided intotwoportions andplaced intwowells. The individual PP and MLNOD valueswereadded; theirsums represented the total PP and MLN antibody output per animal. For small intestinal fragments, the sum of the OD values from eightLPsampleswasdivided by 8andmultiplied by 1,400 (onefragment represents approximately 1/1,400thof the surface of the small intestine). Quantities of both virus-specific and total IgG,IgA, and IgMinsupernatantfluidsfrom intestinal fragment cultures and fecal contents were deter-mined by comparison of values with those obtained from a standard curve constructed for each isotype with purified mouseIgM, IgG, andIgA(Sigma).

RESULTS

Frequencies of virus-specific plasma cells assessed by plasma cellELISPOT assay.The specificity andsensitivity of ourplasma cell ELISPOTassaywereassessedby using hybrid-omacells directed againsteither RRV innercapsid protein vp6 (IgGl; provided by Robert Shaw, Department of Veterans Affairs Medical Center, Northport, N.Y.)orinfluenza virus Hi hemagglutinin (IgGl; provided byAndrewCaton,The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.). Similar to previous studies (34), 80 to 100% of putative antibody-secreting cells (ASC)weredetected in wells coated with RRV and incubated with hybridoma cells secreting antibodies

di-rected against RRV. No ASC were detected in wells coated with PBS and incubatedwithhybridomacells directed against RRVorin wells coatedwith RRV but incubated with influenza virus-specific hybridoma cells. Cycloheximide treatment of mononuclear cells reduced the number of rotavirus-specific ASC by more than 90%, indicating that antibody secretion occurred de novo (datanotshown).

We determinedfrequencies of rotavirus-specific plasmacells 3 weeks after oral inoculation of micewith 107 PFU ofeither WC3 or RRV. The frequencies of virus-specific plasma cells bearing IgA in the LP, spleen, PP, and MLN were approxi-mately 50-to80-foldgreaterafterinoculation with RRV than with WC3 (Table 1). There were no detectable rotavirus-specificASC after mock infectionof animals(datanotshown). The total number of IgA-bearing cells in the LPofRRV- or

mock-infected animals was 77,500 and 89,250 per 106 mono-nuclear cells, respectively. Therefore, the increase in the number ofrotavirus-specific ASC in LPwasnotaccompanied by an increase in the total number of IgA-bearing cells. Rotavirus-specific ASC in theLPofmiceinoculatedwith RRV accounted for approximately 14% of all IgA-bearing cells.

Quantities of total and virus-specific Igs produced in intes-tinal fragment culture. The quantities of total and virus-specific antibodies in supernatant fluids from intestinal frag-mentcultures of sucklingmice weredetermined 7, 14, and21

days after oralinoculation with iRRV (215ng permouse), 107

PFU of WC3 (215ngpermouse),or107PFUofRRV(215ng

per mouse).

All of thegroupstested showedanincrease intotalantibody production (IgM, IgA, and IgG) between 7 and 21 days after inoculation (Fig. 1D). Inaddition, greater quantities of total

IgG, IgA, and IgM were produced by lymphocytes obtained

TABLE 1. Frequencies ofrotavirus-specific ASCs in lymphoid tissues of suckling miceafter oral inoculation withrotavirus

strainsWC3or RRVassessedby ELISPOTassay' Immunizing Source of Frequencyof virus-specificASCsb

strain lymphocytes IgA IgG IgM

WC3 Spleen 3 1 1± 0.5 50± 17

MLN 1 0.5 4± 1 3±1

PP 18±11 2±0.5 31±7

LP 158 64 NDC 20±16

RRV Spleen 97 12 5 2 58±32

MLN 85 17 3 1 ND

PP 1,014 82 71 19 6±4

LP 10,668 938 11 4 ND

aGroups of three 7- to 10-day-old CD2

(F1)

suckling mice were orally

inoculated with107PFUof either tissue culture-derivedbovine strainWC3or

primatestrain RRV. At 3weekslater,animals were sacrificed andlymphocyte populationswere obtainedto determine the frequencies ofrotavirus-specific

ASC.

bFrequenciesofvirus-specificASCs are shown per 106 mononuclear cells. Standard errors were calculated onsamples performedinduplicatewells.

cND, notdetected at frequenciesof 1 rotavirus-specificASCper4 x 104

mononuclear cells.

from mice inoculated with RRV (from all tissues and at all

timepoints) than in mice inoculated with iRRVorWC3 (Fig.

1D).

Virus-specificIgMwasdetected in the PP and MLN butnot

the LP of mice inoculated with RRV, WC3, and iRRV (Fig.

1A). Quantities ofvirus-specific IgA (and the percentage of

virus-specific IgA comparedwith total IgA) obtained 21 days

after inoculationweresignificantly largerinLP, MLN,and PP of RRV-infected mice than ofWC3- oriRRV-infected mice

(Fig. 1B).Atotal of15%ofIgAproducedinfragmentcultures

ofPP21 dayspostinfectionwith RRVwererotavirusspecific,

comparedwith 1.74 and0% for WC3 andiRRV, respectively.

Quantitiesof total andvirus-specific IgGweresmaller than for

either IgA or IgM; no significant differences invirus-specific IgG secretion were observed among animals inoculated with RRV or WC3 (Fig. 1C). There were nodifferences in

virus-specific or total antibodies secreted by PP taken from the

proximal, medial, or distal segments of the small intestine

(datanotshown).Virus-specificIgM, butnotvirus-specificIgA

andIgG,wasdetected inGALT of mice inoculated with iRRV

(Fig. 1A to C), and neithervirus-specific IgG, IgA, nor IgM

was detected in GALT of mock-infected mice (data not shown).

Quantities of virus-specific antibodiesin feces and serum.

Rotavirus-specific antibodies were detected in the sera of

animals 3 weeks after oral inoculation with RRV, WC3, or

iRRV (Table 2). Circulating rotavirus-specific IgA was

de-tectedonly in mice infected with RRV. High titers of

rotavirus-specific IgGwere detected in mice inoculated with RRV or

WC3 butnotinmice inoculated with iRRV. Rotavirus-specific IgM wasdetected in animals inoculated with RRV, WC3, or iRRV.

Rotavirus-specific and total IgA in intestinal contents were determined in animals 2 months after oral inoculation with RRV, WC3, or iRRV (Table 3). Rotavirus-specific IgA was detected in intestinal contents of animals inoculated with eitherRRV orWC3but not iRRV. The quantity of

rotavirus-specific IgA compared with total IgA was approximately

fivefold greater after inoculation with RRV than with WC3.

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!bASdA

[I

JEImI

IgM

IgA

714 21 7 1421 7 1421 714 21 7 14217 14 21 iRRV WC3 RRV IRRV WC3 RRV

PP MLN

IgG

TABLE 2. Titers ofrotavirus-specific antibodies inserum byELISA after oral inoculationofmice

with RRV, WC3, or iRRVa

Titer ofrotavirus-specific Immunizing Time(days) after antibodies'

strain immunization

IgM IgA IgG

WC3 7 400 <100 <100

14 800 <100 6,400

21 400 <100 12,800

RRV 7 3,200 1,600 1,600

14 1,600 3,200 12,800

21 3,200 12,800 25,600

iRRV 7 3,200 <100 <100

14 1,600 <100 100

21 1,600 <100 <100

7 14 2171 iRRV W

L

2000F

1800 1600 1400 1200 k

days 7 1421 7 14 217 14 21 714 2171421 7 14 21

IRRV WC3 RRV iRRV WC3 RRV

PP MLN

D IgG IgA

+IgM

22

20. 18 16

j14-12

10 E

6

1000 600 400 200 0l

7 14 21 7 1*RR

IRRV V

l

a Serawereobtained fromgroupsoftwoanimals7, 14,and 21days after oral lTOWaAb inoculation with either 107 PFU of RRV (215 ng of virus protein), 107PFUof

*Spe Ab WC3 (215 ng of virus protein),or215ng of iRRV.

bData areshownasthereciprocalofthehighestserumdilutionpositiveby ELISA andrepresenttheaverage resultforseraobtained fromtwoanimals.

fI DISCUSSION

Each of the three assays chosen for these studies addresses

a selected aspect of the intestinal humoral response. The

ELISPOT assay enables one to determine the frequencies of

virus-specific plasma

cells

(of specific isotype)

within a

given

population

of cells.

However,

this assay does not

necessarily

4 21 71421 predict the quantity or isotype of virus-specific antibodies

VC3 RRV located atthe intestinal mucosalsurface, nordoesitallowfor

.P detection of virus-specific neutralizing antibodies. Intestinal

fragment culture preserves the native microenvironment

VI

T"taAb

withinGALT andallows for determination ofthequantities,

SpecAb isotypes, andneutralizing capacitiesof antibodies producedby PP and LP. However, production ofantibodies invitro is not

necessarily predictive of events occurring at the intestinal

mucosal surface. ELISA of intestinal contents allows for a direct determination of the presence ofvirus-specific antibod-ies at the intestinal mucosal surface.Unfortunately, measure-mentof thequantityofvirus-specific secretoryIgA (sIgA) in intestinal fluids may be confoundedbydegradation of sIgAby intestinal proteases, entrapment ofsIgAin mucus, and variable i] dilution ofsIgAbythe osmotic catharsis requiredfor collec-tion. For these reasons, the three assays chosencomplement 14 21 714 21 each other in their capacity to detect the rotavirus-specific

1C3 RRV

humoral immune response in GALT.

Inoculationof mice with RRV inducedsignificantlygreater

frequencies of rotavirus-specific IgA-secreting cells in LP,

MLN, and PP as determined by ELISPOT assay than did ICkAb inoculation with either WC3 oriRRV. Similarly, RRVinduced secretion of greater quantities of rotavirus-specific IgAby PP T and LP in fragment cultures and greater quantities of

rotavi-FIG. 1. Ig responses in intestinal fragment cultures. CD2

(Fl)

sucklingmice(7to8daysold)wereorallyinoculatedwith either 107 PFU of RRV (215 ng per mouse), 107 PFU ofWC3 (215 ng per mouse), or 215 ngof iRRV per mouse. Groups oftwo micewere

sacrificed7, 14, and21daysafterinoculation,and intestinalfragment

cultureswereestablishedforPP,MLN,and LP of the small intestine. Supernatant fluidsfrom theseculturesweretestedbyELISA for the presenceofrotavirus-specificandtotalIgM(A) IgA(B),andIgG (C).

Total andvirus-specific IgM, IgA,and IgGresponseswerecombined (D).Ab, antibody.

I

I

.2

U

days

B

10

8

6

I

I

I

U

2

days

C 12

10

6

I

I

I I

I

if

.U 4

days 714 21 7 14217 14 21 714 21 7 1421 7 14 21 IRRV WC3 RRV IRRV WC3 RRV

PP MLN

2

01'

F-h -,

r-

L.1i 1.

e-t

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rus-specific IgAinintestinalcontentsthan didWC3oriRRV.

Oral inoculation of mice with WC3 induced lowfrequenciesof virus-specific IgA-secreting cellsbyELISPOTassay; this find-ing correlated with the small quantities of IgA secreted by GALT infragmentcultures and the low levels ofvirus-specific IgA in intestinalcontents. The induction ofavigorous, virus-specific IgA response in GALT by RRV probably occurs

because RRV is better adapted to growth at the murine intestinal mucosal surface than is WC3(3).Theimportance of RRVreplicationin induction ofavirus-specific IgAresponse

in GALTis supported bythefinding that noninfectious RRV doesnotinducevirus-specific IgA. Ourfindings aresimilarto those of VanCott et al. (46), who found that the capacity of porcine coronaviruses to inducevirus-specific IgA-producing ASC in GALT after oral inoculation was related to the virulence of the immunizingstrain. Virus replicationin intes-tinal epithelial cells (which expressclass II antigens [6]) may

cause antigen to be presented in a manner more likely to induce secretion of cytokines associated with production of

IgA (i.e., interleukin-5 IL-5 and IL-6 [1, 2]) than does virus which does notreplicate. Alternatively, induction of a virus-specific IgAresponse in GALT maybe related simplytothe amountofantigen presentedtothe intestinal mucosal surface. The quantityof RRV(butnotWC3) presentedtothe intesti-nal mucosal surface is amplified by replication in intestinal

epithelial cells (3).Therefore, it maybepossible to inoculate mice with aquantity ofWC3 oriRRV sufficient to induce a vigorous, virus-specific IgAresponse.

We found thatapproximately 14%of allIgA-bearingcellsin the LP ofsucklingmice inoculated with RRVwere rotavirus

specific.Thesefindingsaresimilartothosepreviously reported for suckling mice orally inoculated with RRV or murine rotavirus (16, 34). It is known thatat theageof 10daysvery

fewIgA plasma cells are present in the LP ofsuckling mice.

However, by2 weeksofage,there isadramaticincrease in the number of IgA plasma cells (47). The large percentage of

rotavirus-specific plasmacellsmaybe due to the virusbeingthe firstforeign antigenthatthe mucosal immune systemsees,and hence theresponseisvigorous.

The presence ofrotavirus-specific IgA in serumparalleled the presence of a rotavirus-specific IgA response in GALT detected byELISPOT assay, intestinalfragment culture, and ELISA of intestinal contents. However, a WC3-specific IgA responsedetectedbyintestinalfragmentculture andbyELISA of intestinal contents was not detected in serum. Serum antibodiesmaynotparalleltheimmuneresponseoccurringat the intestinalsurface.OurfindingswithRRV-inoculated mice

are similar to those of a number of studies with mice and humans in which a correlation was demonstrated between antigen-specific ASC in GALT or antigen-specific antibodies

in intestinal contents and the presence of antigen-specific

antibodies in the circulation (12, 14, 18, 22, 41, 52). These findings are consistent with the observation that antigen-specificASCareinduced in PP andtraffic via the circulationto the laminapropria (13).

The titersofvirus-specific IgG inducedinthe circulationand the quantities of virus-specific IgG produced by GALT in fragment culture were similar after inoculation of mice with

RRV or WC3. However, the frequencies of virus-specific IgG-secreting plasma cellsin GALT byELISPOTassayafter

RRV or WC3 inoculation were low. These findings suggest

that intestinal fragment culture is a more sensitive assay for

detection ofvirus-specific, IgG-secreting cells in GALT than theELISPOTassayis(andthatthevirus-specificIgGresponse

detected inserumispredictive ofavirus-specificIgGresponse

inGALT).Insupportof thishypothesis, virus-specificIgGwas

not detected in either serum or fragment cultures of mice orally inoculated with iRRV;similarly,virus-specific IgM was detected in both serum and intestinal fragment cultures. In addition, Ward et al. (52) found a correlation between the presenceofrotavirus-specific IgG inserumandjejunal fluid in adults orally inoculated with a human rotavirus strain. Alter-natively,virus-specific IgG may be made atsites distant from the intestine after oral inoculation with RRVorWC3. Anti-gen-presenting cells in PP may transport and presentantigen toTcellsatsites other than the intestine(e.g., thespleen). For example, Liu and MacPherson (25, 26) found that dendritic cells in PP and LP can acquire antigen after oral administra-tion and present antigen to primed splenic lymphocytes. In addition, infectious virus or viral antigens may enter the circulation, travel to thespleen, and stimulate a virus-specific IgG response. Rotavirus antigen has been detected in the PP and MLN of mice(16), and infectious virus has been detected in the MLN of cows (32), afterhomologous host infection.

Production of large quantities of IgG, IgA, and IgM not specific for rotavirus was observed in intestinal fragment cultures from mice orally inoculated with RRV but not in fragment cultures from mice inoculated with WC3 or iRRV. Increased secretion of antibodieswas notassociated with an increase in the frequencies of IgA-bearing cells in GALT in mice inoculated with RRV as compared with uninoculated animals. Similarly, bystander stimulation of GALT

antibody-secreting cells occurs in mice orally inoculated with reovirus

type 1 (24a).There areseveralpossible hypothesestoexplain these observations. First, infection with RRV may induce synthesis ofcytokinesassociated withthe Th2phenotype(e.g., interleukin-4 [IL-4], IL-5, and IL-6) in LP. It has been shown that IL-5 and IL-6 are important in the induction of IgA

synthesisfrom B cellsalreadycommittedtoIgAsynthesis (1,2)

and that the LP of adult mice is rich in Th2 cells(31).Weare

presentlydeterminingthecapacityof RRVorWC3 toinduce

Thl or Th2 cells in GALT after oral inoculation of mice.

Second, RRV replication inintestinal epithelial cells, aswith

transmissiblegastroenteritisvirus,maycausedisruptionof the mucosal surface (24); penetration of commensal bacterial,

fungal,orenvironmental antigens; andstimulation of

antigen-specific antibodyresponses. Third, replicationof RRV within

matureintestinal epithelial cells may break the oral tolerance

todietaryormicrobial antigens,asinthecasewith

inflamma-torybowel disease (21, 54)which results in the production of autoantibodies and antibodiesagainst environmental antigens.

Finally, RRV, like human immunodeficiency virus (44) and

Epstein-Barr virus (23, 42), causes a bystander B-cell

poly-clonal stimulation.

The immunologic response which clearly correlates with

protection against rotavirusdisease has not been determined.

Several studies found that the presence of rotavirus-specific

TABLE 3. Quantities of rotavirus-specific IgA and total IgA inintestinalcontents of animals inoculated

withWC3, RRV, oriRRV0

Strain Amt of total sIgA Amt of virus-specific Virus-specificIgA/ (ng/ml) sIgA(ng/ml) total IgA(%)

iRRV 19,000 <4 <0.02

WC3 23,000 41 0.18

RRV 15,000 148 1.02

aIntestinalcontentswereobtained 2 monthsafteroralinoculation of suckling mice witheither107PFU of RRV(215ng of virusprotein),107PFU ofWC3 (215ng ofvirus protein), or 215 ng of iRRV. Intestinal contents were pooled

fromtwomice in each group.

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IgA in serum or feces correlated with protection against challenge (12, 14, 18, 30, 52). We found that induction of rotavirus-specificIgAresponses amongGALT lymphocytes in

mice by RRVwasbetter than thatinduced by WC3. Similarly, WC3 may be less consistent than RRV in inducing

virus-specific IgAresponsesin theserumand fecesofinfants (9,28, 35). However, the capacity of RRVto protectagainstrotavirus disease in vaccine trials is not clearly different from that of WC3 (9, 10, 40). Either rotavirus-specific sIgA alone is not predictive of protection against diseaseorRRV andWC3do

not differ in their capacity to induce a virus-specific IgA

response in infants. A studycomparing the relative capacities of RRV and WC3 to induce avirus-specific IgAresponse in infantsremains tobe performed.

ACKNOWLEDGMENTS

This workwassupported inpartbygrants R01 AI26251 and K04 AI00889toP.A.O.from theNational Institutes of Health.

We thank Charlotte Moser, Edward S. Bell, Xanthipi Travias, Yin-Shan Lai, and Jeong Yoon for theirtechnicalsupport, aswell as

John J. Cebra and David Kramer for theirhelpful discussions. REFERENCES

1. Beagley, K. W.,J. H.Eldridge,H. Kiyono, M. P. Everson, W. J. Koopman, T. Honjo, and J. R McGhee. 1988. Recombinant murine IL-5 induced high rate IgA synthesis in cycling IgA-positivePeyer's patch B cells. J. Immunol. 141:2035-2042. 2. Beagley, K. W., J. H. Eldridge, F. Lee,H.Kiyono,M. P. Everson,

W.J. Koopman, T.Honjo,and J. R McGhee. 1989.Interleukins andIgAsynthesis: human and murine IL-6 induce highrateIgA secretioninIgA-committed B cells. J. Exp. Med. 169:2133-2139. 3. Bell, L M., H. F. Clark, E. A. O'Brien,M. J. Kornstein, S. A. Plotkin, and P. A. Ollit. 1987. Gastroenteritis caused by human rotaviruses(serotype 3) inasucklingmousemodel. Proc. Soc. Exp.

Biol. Med.184:127-132.

4. Bernstein,D.I., M.A.Kacica, M. M. McNeal,G. M.Schif;and R L Ward. 1989. Localandsystemic antibodyresponsetorotavirus WC3 vaccineinadult volunteers.Antiviral Res.12:293-300.

5. Bernstein, D. I.,V. E.Smith, D. S. Sander, K. A. Pax, G. M. Schif, andR L.Ward. 1990. Evaluation of WC3 rotavirusvaccine and correlates ofprotection in healthy infants. J. Clin. Infect. Dis. 162:1055-1062.

6. Bland, P. W., and L. G.Warren. 1986. Antigen presentation by epithelial cells of the rat small intestine. 1. Kinetics, antigen specificityandblocking byanti-Iaantisera. Immunology 58:1-9. 7. Ceyhan, M.,G.Kanra, G. Seecmeer,K.Midthum,B. L.Davidson,

E.T.Zito,andT.Vesikari.1993. Take ofrhesus-human reassort-ment tetravalent rotavirus vaccine in breast-fed infants. Acta Paediatr.82:223-227.

8. Clark,H.F.,F.E.Borian,L. M.Bell,K.Modesto,V.Gouvea,and S. A. Plotkin. 1988. Protection effect of WC3 vaccine against

severerotavirusdiarrheain infantsduringapredominantly

sero-group1rotavirusseason.J. Infect. Dis. 158:570-587.

9. Clark,H.F.,F.E.Borian,and S. A. Plotkin. 1990. Immuneprotection

ofinfantsagainstrotavirusgastroenteritis byaserotype 1

reassort-mentofbovine rotavirusWC3. J. Infect. Dis. 161:1099-1104. 10. Clark,H.F.,and P. A. Offit.Rotavirus vaccines. In S.A.Plotkin

and E. A. Mortimer, Jr. (ed.), Vaccines, 2nd ed.,in press. The

W. B.Saunders Co.,Philadelphia.

11. Clark,H.F.,P. A.Ofilt,K.T.Dolan,A.Tezza,K.Gogalin,E. M.

Twist,and S. A. Plotkin. 19*. Responseof adult human volun-teers tooraladministration of bovine andbovine/human reassort-mentrotavirus. Vaccine 4:25-31.

12. Coulson, B. S., K.Grimwood, P.J. Masendyycz, J. S.Lund, N.

Mermelstein, RF.Bishop,andG.Barnes. 1990.Comparaisonof

rotavirusimmunoglobulinAcoproconversionwith other indices of

rotavirus infection in a longitudinal study inchildhood. J. Clin.

Microbiol. 28:1367-1374.

13. Craig,S.W.,andJ. J.Cebra.1971.Peyer's patches.An enriched

sourceofprecursorsforIgA-producing immunocytesinthe rabbit.

J.Exp. Med.134:188-200.

14. Czerkinsky,C. C., S. J.Prince, S. M. Michalek, S. Jackson, M. W. Russel, Z.Moldoveneau, J. R. McGhee, and J. Mestecky. 1987. IgA antibody-producing cells in peripheral blood after antigen ingestion: evidence for a common mucosal immune system in humans. Proc.Natl. Acad. Sci. USA 84:2449-2453.

15. Dagan, R.,I.Kassis, B. Sarov, K. Midthun, B. L. Davidson, T. Vesikari, andI. Sarov. 1992. Safety and immunogenicity of oral tetravalent human-rhesus reassortment rotavirus vaccine in neo-nates. Pediatr. Infect. Dis. J. 11:991-996.

16. Dharakul, T., M.Riepenhof-Talty,B. Albini, and P. L.Ogra.1988. Distribution of rotavirus antigen in intestinal lymphoid tissues: potential role indevelopment of the mucosal immune response to rotavirus. Clin. Exp. Immunol. 74:14-19.

17. Flores, J., G. Daoud, N. Daoud, M. Puig, M. Martinez, I. Perez-Schael, R. Shaw, H. B. Greenberg, and A. Z. Kapikian. 1988. Reactogenicity and antigenicity of rhesus rotavirus vaccine (MMU-18006) in newborninfants in Venezuela. Pediatr. Infect. Dis. J.7:776-780.

18. Grimwood, K., J. C. S. Lund, B. S. Coulson, I. L. Hudson, R. F. Bishop, and G. L. Barnes. 1988. Comparison of serum and mucosal antibody responses following severe acute rotavirus gas-troenteritis in children. J. Pediatr. Gastroenterol. Nutr. 4:60-66. 19. Groene, W. S., and R. D. Shaw. 1992. Psoralen preparation of

antigenically intact noninfectious rotavirus particles. J. Virol. Methods 38:93-102.

20. Ho, M., R. Glass, P. Pinsky, and L. Anderson. 1988. Rotaviruses as acause of diarrheal morbidity and mortality in the U.S. J. Infect. Dis. 158:1112-1116.

21. Ibbotson,J. P., J. R.Lowes, H. Chahal, J. S. H. Gaston, P. Life, D. S.Kumararatne, H. Sharif, J. A. Williams, and R N. Allan. 1992. Mucosal cell-mediated immunity to mycobacterial, enter-obacterial and other microbial antigens in inflammatory bowel disease. Clin. Exp. Immunol.87:224-230.

22. Kantele, A., H. Arvilommi, and I. Jokinen. 1986. Specific immu-noglobulin-secreting human blood cells after peroral vaccination againstsalmonella typhi. J. Infect. Dis. 153:1126-1131.

23. Kataaha, P. K., S. M. Mortazavi-Milani, G. Russell, and E. J. Holborow. 1985.Anti-intermediate filament antibodies, antikera-tin antibody, and antiperinuclear factor in rheumatoid arthritis and infectious mononucleosis. Ann. Rheum. Dis. 44:446-449. 24. Keljo, D. J., D. G. Butler, and J. R Hamilton. 1985. Altered

jejunalpermeabilitytomacromolecules during viral enteritis in the piglet. Gastroenterology 88:998-1004.

24a.Kramer, D. Personal communication.

25. Liu, L. M., and G. G. MacPherson. 1991. Lymph-borne (veiled) dendritic cells can acquire and present intestinally administered antigens. Immunology 73:281-286.

26. Liu, L. M., and G. G.MacPherson. 1993. Antigen acquisition by dendritic cells:intestinaldendriticcells acquire antigen admines-tered orally and can prime naive T cells in vivo. J. Exp. Med. 177:1299-1307.

27. Logan, A. C., K. N. Chow, A. George, P. D. Weinstein, and J. J. Cebra. 1991. Use of Peyer's patch and lymph node fragment cultures tocomparelocal immune responses to Morganella

mor-ganii. Infect.Immun.59:1024-1031.

28. Losonsky, G. A., M. B. Rennels, Y. Lim, G. Krall, A. Z. Kapikian, M. M.Levine. 1988.Systemic and mucosalimmune responses to rhesus rotavirus vaccine MMU 18006. Pediatr. Infect. Dis. J. 7:388-393.

29. Madore, H. P., C.Christy,M. Pichechero,C. Long, P. Pincus, D. Vosefski, A. Z. Kapikian, R Dolin, and the Elmwood Panorama andWestfall PediatricGroups. 1992.Field trial ofrhesusrotavirus orhuman-rhesus rotavirusreassortmentvaccine ofVP7 serotype3 or 1 specificityininfants. J. Infect. Dis. 166:235-243.

30. Matson, D. O., M. L. O'Ryan, I. Herrera,L. K. Pickering,and M.K. Estes. 1993. Fecal antibodyresponsestosymptomaticand asymptomatic rotavirusinfections. J. Infect. Dis.167:577-583. 31. McGhee, J. R, J. Mestecky,M.T.Dertzbaugh,J.H.Eldridge,M.

Hirasawa, and H. Kiyono. 1992. The mucosal immune system: fromfundamentalconceptstovaccinedevelopment.Vaccine 10: 75-88.

32. Mebus, C. 1976.Reovirus-likecalfenteritis.Dig.Dis.21:592-598. 33. Mei-shang, H., R L.Flyod, R. I.Glass,M.A.Pallansch,B.Jones,

on August 17, 2020 by guest

http://cvi.asm.org/

(7)

B. Hamby, P. Woods, M. E. Penaranda, A. Z.Kapikian, G. Bohan, W.D. Wilcox, andR.Blumberg. 1989. Simultaneous administra-tion of rhesus rotavirus vaccine and oral poliovirus vaccine: immunogenicity and reactgenicity. Pediatr. Infect. Dis. J. 8:692-696.

34. Merchant, A. A., W. S. Groene, E. H. Cheng,and R.D.Shaw. 1991. Murine intestinal antibody response to heterologous rotavirus infection. J. Clin.Microbiol. 29:1693-1701.

35. Midthum, K L. Pang, J. Flores, and A. Z. Kapikian. 1989. Comparison ofimmunoglobulin A(IgA), IgG, and IgM enzyme-linked immunosorbent assays, plaque reduction neutralization assay, and complement fixation in detecting seroresponses to rotavirus vaccine candidates. J. Clin.Microbiol. 172:2799-2804. 36. Oflit,P.A.,H. F.Clark, and S.A. Plotkin.1983.Response of mice

torotavirus of bovineorprimate originas assessedby radioim-munoassay,radioimmunoprecipitation, and plaque reduction neu-tralization. Infect.Immun.42:293-300.

37. Offit, P. A.,H. F. Clark,W. G. Stroop, E. M.Twist, and S. A. Plotkin.1983.Thecultivation of humanrotavirus, strain 'Wa',to high titerincell culture andcharacterization of the viral structural polypeptides.J.Virol. Methods 7:29-40.

38. Offit,P.A.,S. L.Cunningham,and KI. Dudzik. 1991.Memory anddistributionofvirus-specific cytotoxicTlymphocytes (CTLs) and CTL precursors after rotavirus infection. J. Virol. 65:1318-1324.

39. Perez-Schael, I., M. Blanco, M.Vilar, D. Garcia, L.White, R. Gonzalez,A. Z.Kapikian,andJ.Flores.1990. Clinicalstudiesof a quadrivalent rotavirus vaccine in Venezuelan infants. J. Clin. Microbiol. 28:553-558.

40. Perez-Schael,I.,D.Garcia,M.Gonzalez,N.Daoud,M.Perez,W. Cunto,A. Z.Kapikian,andJ.Flores.1990. Prospective study of diarrheal diseasesinVenezuelan childrentoevaluatetheefficacy ofrhesus rotavirusvaccine.J.Med. Virol. 30:219-229.

41. Quiding, M., I. Nordstrom, A. Kilander, G. Andersson, L. A. Hanson, J.Holmgren,and C.Czerkinsky.1991.Intestinal immune response in humans: oral cholera vaccination induces strong intestinal antibody responses and interferon-y production and evokes localimmunological memory.J.Clin.Invest. 88:143-148. 42. Robinson, J.,and K.C. Stevens.1984.Production of

autoantibod-iestocellularantigensby humanBcells transformedby Epstein-Barrvirus.Clin. Immunol. Immunopathol. 33:339-350.

43. Shaw, R,A.Merchant,W. Groene,and E. Cheng. 1993. Persis-tence ofintestinal antibody response to heterologous rotavirus infection in a murine model beyond 1 year. J. Clin. Microbiol. 31:188-191.

44. Shirai, A., M. Cosentino, S. F. Leitman-Klinman, and D. M. Klinman. 1992. Human immunodeficiency virus infection induces both polyclonal andvirus-specificBcell activation. J.Clin. Invest. 89:561-566.

45. Starkey,W.G., J.Collins,T.S. Wallis, G. J.Clarke,A.J.Spencer, S.J. Haddon, M. P. Osbourne, D. C. A. Candy, and J. Stephen. 1986. Kinetics, tissue specificity, and pathological changes in murine rotavirus infection of mice. J. Gen. Virol. 67:2625-2634. 46. VanCott, J. L.,T. A. Brim,R A.Simkins, and L.J. Saif. 1993.

Isotype-specific antibody-secreting cells to transmissible gastroen-teritis virus and porcine respiratory coronavirus in gut- and bronchus-associated lymphoid tissues of suckling pigs. J. Immunol. 150:3990-4000.

47. Van der HeUden, P. J., A. T. J. Bianchi, W. Stock, and B. A. Bokhout. 1988. Background(spontaneous) immunoglobulin pro-duction in the murine small intestine as a function of age. Immunology 65:243-350.

48. Vesikari, T., A. Z. Kapikian, A. Delem, and G. Zissis. 1986. A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) oral rotavirus vaccines in young children. J. Infect. Dis. 153: 832-839.

49. Vesikari, T., T. Ruuska, A. Delem, and F. Andre. 1986. Oral rotavirusvaccination in breast- and bottle-fed infants aged6 to 12 months.Acta.Paediatr. Scand. 75:573-578.

50. Vesikari, T.,T.Ruuska,K. Y.Green, J. Flores,and A. Z.Kapikian. 1992. Protectiveefficacy against serotype 1 rotavirus diarrhea by live oral rhesus-human reassortment rotavirus vaccines with hu-manrotavirusVP7serotype1or2specificity. Pediatr. Infect. Dis. J.11:535-542.

51. Walsh, J., andK. Warren. 1979.Selective primary health care: an interim strategy for disease control in developing countries. N. Engl.J.Med. 301:967-974.

52. Ward, R L., D. J. Bernstein, R Shukla, E. C. Young, J. R Sherwood,M. M.McNeal,M.C.Walker, and G.L.Schiff.1989. Effects of antibody to rotavirus on protection of adultschallenged with a human rotavirus. J. Infect. Dis. 159:79-88.

53. Ward,R.L.,D.RKnowlton,H.B.Greenberg, G.M.Schiff,and D.Bernstein. 1990.Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavi-rus.J.Virol. 64:2687-2691.

54. Wu,K. C., Y. R. Mahida, J. D. Priddle, and D. P. Jewell. 1990. Effect of human intestinalmacrophages on immunoglobulin pro-duction by human intestinal mononuclear cells isolated from patients with inflammatory bowel disease. Clin. Exp. Immunol. 79: 35-40.

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http://cvi.asm.org/

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