Vol. 29, No. 8JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1991, p. 1693-17010095-1137/91/081693-09$02.00/0Copyright © 1991, American Society for Microbiology

Murine Intestinal Antibody Response to HeterologousRotavirus Infection

AYUB A. MERCHANT, WILLIAM S. GROENE, EDWARD H. CHENG, AND ROBERT D. SHAW*

Department of Medicine, Northport Veterans Medical Center, Northport, New York 11768, and Department of Medicine,State University ofNew York at Stony Brook, Stony Brook, New York 11790

Received 21 December 1990/Accepted 14 May 1991

Rotavirus is the most important worldwide cause of severe gastroenteritis. Extensive efforts have beendevoted to the design of a vaccine that will prevent disease, but development of a more effective vaccine strategymay require progress in the understanding of the mucosal immune response to replicating viral antigens. In thisarticle, we report the characterization of the intestinal antibody response of a murine model to heterologousinfection with the rhesus rotavirus vaccine strain. We have adapted the enzyme-linked immunospot assay tomeasure this response without the difficulties associated with measurement of antibodies in intestinal contentsor the artifacts associated with culturing of lymphocytes. The predominant response in terms of antibody-secreting cells (ASC) is seen in the small intestine lamina propria, which can be measured within 4 days ofinfection, peaks 3 weeks after infection, and remains near that level for longer than 8 weeks. The magnitudeof the immunoglobulin A (IgA) cell response is approximately 10 times greater than the intestinal IgG cellresponse, and IgM cells are rare. Virus-specific ASC constitute approximately 50% of all ASC in the gut at thepeak of the virus-specific response. This response is considerably greater than responses to nonreplicatingmucosal antigens measured by similar techniques. Enteral infection engenders minimal virus-specific ASCresponse in the spleen. Rhesus rotavirus-specific enzyme-linked immunosorbent assay and neutralization assays

of serum and intestinal contents did not correlate with virus-specific ASC response.

Rotavirus is an important cause of severe gastroenteritis inhumans, as well as in other mammalian and avian species(15, 26). In children under the age of two years, it is thesingle most important cause of severe diarrhea and contrib-utes significantly to infant mortality in less developed coun-tries (29, 56). Although death due to rotavirus gastroenteritisoccurs rarely in developed countries, it is still an extremelycommon cause of morbidity and physician visits (20, 21).Rotavirus can also cause disease in adults, especially theelderly and the immunocompromised (22). The tremendousworldwide impact of this virus has led to efforts to controlthis disease by the development of an effective vaccine.The development of efficient vaccine strategies may be

dependent on an understanding of the host immune responseto natural infections and vaccination. Immunity to rotavirusinfection has been studied with humans and several animalmodels, but the precise nature of the host response toinfection or vaccination is still not clear. Several studieshave suggested the importance of local antibodies at themucosal surface of the small intestine as a major determinantof resistance to rotavirus illness (3, 5, 31, 39, 40, 49, 52, 55).Serum, feces, duodenal aspirate, saliva, and breast milkantibody titers have all been measured by using variousimmunoassays to define the antibody response. Unfortu-nately, most assays may not provide reliable measures of theintestinal response because of a variety of artifacts, includ-ing antibody degradation, mucus entrapment, dilution insecretions, or simply because of the inherent compartmen-talization of the systemic and local antibody responses.Furthermore, the passive acquisition of antibodies from themother in utero and later from breast milk can interfere withthe quantitation of immunoglobulin production as measuredin titers of serum or intestinal contents.

* Corresponding author.

Studies on specific antibody responses at the mucosallevel have been limited by technical difficulties of immuno-histochemical staining and plaque assays as well as inherentdifficulties of quantitation in these assays. A modification ofthe enzyme-linked immunospot (ELISPOT) technique hasallowed us to accurately enumerate functionally active anti-body-secreting cells (ASC) within intestinal tissues anddefine the local antibody production in terms of specificlocation, kinetics, antigen specificity, and isotype. Use ofthis assay directly on freshly isolated lymphocytes from thegut-associated lymphoid tissues without significant in vitromanipulations allows for the most accurate estimation oftheir in vivo activity.Though various animal models have been developed to

study the pathogenesis of and immune response to rotavirusinfection, none represents a perfect system. The short ges-tation period, modest price, ease of isolation of animals,commercial availability of immunological reagents, and thewell-characterized immune system make the suckling mousea convenient small-animal model which has been widelyused (16, 23, 35-38, 46). Moreover, the infection in sucklingmice closely parallels that seen in human infants in patho-genesis, pathological lesions, and symptomatic but self-limiting diarrheal illness. For these reasons, we chose thismodel for the study of intestinal immune response.The vaccine strategy most widely tested to date utilizes

animal viruses that are naturally attenuated in humans (27).Rhesus rotavirus (RRV), a simian strain, has been effectiveat stimulating only type-specific immunity in various fieldtrials (8, 17-19, 44, 45, 67). This rotavirus strain has previ-ously been shown to replicate and cause disease in micewhen administered at sufficient doses (43). Because of theextensive prior studies on murine and human infection andthe substantial virulence of this rotavirus strain in a heterol-ogous mouse model, we chose this well-characterized strainas the infecting agent.

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The present study defines the kinetics, isotype specificity,and the anatomy of the murine intestinal antibody responsesto a single oral inoculum of the heterologous RRV strain. Wealso studied the antibody titers in the serum and intestinalcontents in an attempt to seek correlation of ASC numberswith these previously described parameters.

MATERIALS AND METHODS

Animals and immunization. Pregnant CD2F1 hybrid mice(pathogen free) were obtained from Taconic Farms (Ger-mantown, N.Y.) and housed in microisolator cages contain-ing sterile bedding, food, and water throughout the experi-ment. Sera obtained from mice upon arrival were tested byfocus neutralization assay to ensure no prior exposure torotavirus. Litters were infected with RRV (ATCC VR 954) atthe age of 9 to 12 days. The virus was grown in MA104 cellsin serum-free M199 culture medium as previously described,released by freezing and thawing, and stored at -90°C (25).The dose of virus administered by gastric intubation was 107PFU administered in 100 [lI of tissue culture supernatant.The virus was not trypsin activated in vitro prior to admin-istration. Pups were restricted from suckling for 45 minbefore and after immunization. This dose of virus resulted indiarrhea in more than 95% of pups, usually between 2 to 4days after infection. Two littermates were sacrificed atdefined time intervals, and the spleen, mesenteric lymphnodes (MLN), small intestine, intestinal contents, and serumfrom each were obtained. Experiments included pups ofboth sexes whenever possible to minimize the significance ofsex-specific influences on the immune response, if any.Naive mice housed in adjacent cages showed no evidence ofdiarrhea or a specific immune response in parallel experi-ments, indicating the absence of cage-to-cage transmissionof RRV. Sentinel mice were tested by serum immunofluo-rescence assay for the murine rotaviruses (which causeepizootic diarrhea of infant mice) by Anmed Biosafe Inc.(Rockville, Md.) and were reported to test negative.

Intestinal fluid collection. The contents of the small intes-tine were collected by a method previously described, withminor modification (14). Briefly, after the small intestineswere removed, the outside of each was rinsed twice withcold Hanks balanced salt solution, and about 5 ml of coldHanks balanced salt solution was then rinsed through theinside of each intestine and mixed 1:1 with protease inhibitorsolution (50 mM EDTA, soybean trypsin inhibitor [0.1mg/ml], pH 7.4). After being vortexed for 1 min the tube wascentrifuged at 850 x g at 4°C for 10 min, and 100 plI ofphenymethylsulfonyl fluoride (100 mM in 95% ethanol) wasadded to the supernatant in a clean tube. The tube was thencentrifuged at 1,300 x g at 4°C for 25 min. One hundredmicroliters of phenylmethylsulfonyl fluoride, 0.01% sodiumazide, and 300 pl of fetal bovine serum (FBS) were added tothe supernatant in a clean tube. The contents were aliquotedand frozen at -90°C.Lymphocyte isolation procedure. Small intestinal contents

were removed as described above, and the intestine wascollated over a narrow spatula. Macroscopically visiblePeyer's patches (PP) were dissected. Lamina propria (LP)lymphocyte isolation was accomplished by a modification ofthe procedure first described by Davies and Parrott andmodified by Van der Heijden et al. by using EDTA andcollagenase, which allows recovery of functionally activeintestinal lymphoid cells (12, 63). The cell suspension wassubjected to a discontinuous Percoll gradient centrifugationto isolate viable mononuclear cells, which were enumerated

by trypan blue exclusion. Spleen, PP, and MLN tissues wereminced and forced through steel mesh; clumps were allowedto settle, and the supernatant was decanted. Splenic eryth-rocytes were lysed with 0.83% ammonium chloride. Mono-nuclear cells from these tissues could be reliably countedwithout Percoll gradient centrifugation.ELISPOT. We utilized a modification of the ELISPOT

technique to measure the number of total and RRV-specificASC of each isotype (immunoglobulin G [IgG], IgA, andIgM) present in spleen, LP, PP, and MLN tissues (11).Immobilon PVDF (Millipore Corp., Bedford, Mass.), awhite hydrophobic polyvinylidene difluoride membrane, wasused as the protein-binding matrix. The membrane wasmounted in a 16- or 28-lane miniblotter (Immunetics, Cam-bridge, Mass.). Lanes on the template-mounted membranewere coated with the desired capture antigens, which in-cluded RRV diluted in 0.01 M Tris, 0.01 M NaCl, and 2 mMCaCI2, pH 7.5 (TNC buffer), or rabbit anti-mouse IgG, goatanti-mouse IgM, or goat anti-mouse IgA diluted in phos-phate-buffered saline (PBS). The optimum concentrationsfor the capture antigen substances were previously deter-mined in titration experiments with appropriate hybridomacells as positive and negative controls. Rotavirus used ascapture antigen was purified on cesium chloride densitygradients as previously described (51). This template wasincubated at 37°C for 2 h on a rocking platform. Lanes werewashed with PBS to remove unbound capture antigen, andthe membrane was blocked with PBS containing 5% FBS for1 h at 37°C.Lymphocyte cell suspensions in the appropriate dilutions

in RPMI 1640 containing 2% FBS were made so that acountable number of spots resulted in each lane after a fixedvolume of the cell suspension was incubated on the captureantigen for 2 h at 37°C (the optimal incubation time and cellconcentrations were determined in preliminary experi-ments). Once the incubation was complete, the cells werewashed from the membrane with PBS. Detection of boundmouse immunoglobulin was accomplished with isotype-spe-cific biotinylated anti-mouse immunoglobulin (Kirkegaard &Perry Laboratories, Inc., Gaithersburg, Md.) and avidin-horseradish peroxidase conjugate (Vector Labs, Burlin-game, Calif.) by employing the precipitating substrate 3-ami-no-9-ethylcarbazole (Sigma Chemical Co., St. Louis, Mo.),which resulted in red-brown spots. Figure 1 is a repre-sentative photograph of the ELISPOT assay. The spots wereenumerated under x 2 to x 6 magnification with an OlympusSZ-PT zoom stereo microscope with a halogen fiberopticlight source (by the same individual). Within experiments,multiple lanes of a particular type were run, and the meanwas used to determine the total number of spots per 106viable mononuclear cells. Very high numbers of spots perlane (>300) were difficult to count because of the coales-cence of spots, and the results were taken from wellsincubated with fewer cells.ELISA. IgG and IgA isotype-specific antibodies to rotavi-

rus were measured in the serum and intestinal contents byusing an indirect double-sandwich enzyme-linked immuno-sorbent assay (ELISA). IgM ELISA was not performed. Aguinea pig anti-RRV hyperimmune serum was developed,and IgG was affinity purified from this serum by the Mem-brane Affinity Separation System (Nygene Corp., Yonkers,N.Y.). This IgG was used as capture antibody on Immulon IImicrotiter plates (Dynatech, Alexandria, Va.) to enhance thebinding of cesium chloride gradient-purified rotavirus. Opti-mum dilution for the capture antibody and rotavirus wasdetermined by checkerboard titration in preliminary studies

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INTESTINAL ANTIBODY RESPONSE TO ROTAVIRUS INFECTION 1695

a b c d e f g h

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FIG. 1. Photograph of the membrane used for ELISPOT, show-ing sample spots. Lanes a and b were produced by positive controlhybridomas against complete RRV particles and IgG capture. Lanesc, d, and e were produced by experimental lymphocytes againstimmunoglobulin capture, RRV capture, and no capture, respec-tively. Lanes f and g contain LP lymphocytes against RRV capturewith >300 spots per lane. Lanes h and i contain LP lymphocytes ata higher dilution, resulting in discrete countable spots.

to ensure adequate rotavirus binding and minimal nonspe-cific interactions. The plates were blocked with 2% FBS, andtwofold dilutions of serum or intestinal content samples inPBS with 1% FBS were added and incubated for 2 h.Subsequently, the bound anti-rotavirus antibodies were de-tected by using biotinylated isotype-specific goat anti-mouseimmunoglobulin and avidin-horseradish peroxidase conju-gate (as above); o-phenylenediamine dihydrochloride wasused as the soluble substrate (Sigma Chemical Co.) in a0.4-mg/ml solution in citrate buffer, pH 5.3, and activatedwith 0.2 ,ul of 30% hydrogen peroxide per ml. The reactionwas stopped after 10 min by the addition of 3 N sulfuric acid,and the A490 of the reactants was read on an EL309 auto-mated microplate reader (Biotek, Winooski, Vt.). Parallelwells that were not coated with RRV were used as controlsto quantitate nonspecific binding; control values were sub-tracted from those for the RRV-coated wells. All sampleswere run in triplicate, and titers are reported as the recipro-cal of the highest dilution giving a mean absorbance readingof >0.100 optical density units.

Immunohistochemical focus neutralization assay. Serumand intestinal contents were assayed for RRV neutralizationby an immunohistochemical focus neutralization assay aspreviously described (50). The neutralization titer was thatdilution of the sample that resulted in >50% reduction ofRRV antigen-positive MA104 cells.

Statistical analysis. Cells were harvested from spleen, PP,MLN, and small intestine LP tissues from animals 4 to 63days after infection with RRV. These cells were indepen-dently assayed for secretion of total IgG, IgM, and IgAantibodies as well as specific antibodies for gradient-purifieddouble-shelled rotavirus particles. The data from the 33experiments were analyzed by grouping these experimentsover five time intervals of 10, 20, 30, 45, and 60 dayspostinfection. Comparisons among means of groups weredone by analysis of variance and multiple comparisons bythe Scheffe F test by using StatView II (Abacus Concepts,Berkeley, Calif.) on a Macintosh computer. An additionalnine experiments were done with naive mice 14 to 45 daysafter mock infection with virus-free tissue culture superna-tants.

TABLE 1. Total ASC isolated from indicated tissues between 20and 60 days after RRV infectiona

Tissue IgM ASC IgG ASC IgA ASC

Spleen 2,498 + 232 2,310 + 169 199 ± 9Peyer's patches 541 + 69 2,575 + 297 4,927 + 611Mesenteric nodes 91 + 10 394 + 43 339 ± 51Lamina propria 109 + 30 19,440 ± 3,907 142,061 + 10,241

' Results are expressed as the number of ASC per 106 mononuclear cells ±SEM (n = 29).

RESULTS

Specificity of ELISPOT. The groups of naive or of mock-infected animals showed <33 ASC per 106 specific ASC ofany one isotype in all tissues, except IgG and IgM in spleen,in which up to 133 ASC per 106 specific ASC were seen insome experiments. This phenomenon, previously described,is believed to be secondary to the secretion of cross-reactiveantibodies against a variety of antigens, which is mostmarked in the spleens of 1- to 5-week-old mice during theacquisition and maturation of the B-cell repertoire (28).Hybridoma cells secreting rotavirus-specific antibodiesagainst VP6, VP7, and VP4 (255/60, 4F8, and 7A12, respec-tively) were included as positive and negative controls ineach experiment and consistently showed equivalent num-bers of spots in the immunoglobulin and rotavirus capturelanes. Lanes coated with irrelevant antigens such as choleratoxin or no capture also showed <33 ASC per 106 specificASC.Cycloheximide treatment of the hybridoma cells prior to

and during the incubation decreased the number of spots,indicating that the assay accurately defined active antibodysecretion and did not detect membrane-bound fragments(10). At a concentration of 100 p.g/ml, monoclonal antibody4F8 hybridomas showed a 76% reduction in spot formation,and monoclonal antibody 7A12 hybridomas showed an 83%reduction.

Sensitivity of ELISPOT. The percentage of hybridomacells mentioned above secreting IgG was 64 to 72%, varyingwith the condition of the cell culture at the time of the assay.The efficiency of RRV capture varied from 74 to 100% of theIgG-secreting cells. An IgM-secreting hybridoma, M14, wasalso tested and gave comparable numbers and quality ofspots, suggesting a comparable sensitivity of this assay todetect these immunoglobulin isotypes. In addition, we reportequivalent numbers of IgG and IgM secretors in the spleen.This is consistent with previously described data and indi-cates an equal sensitivity for the two isotypes (64). As IgAhybridomas against RRV are not generally available, wewere not able to test the sensitivity of IgA ASC detectionwith the hybridoma cells. However, the large number ofIgA-secreting cells in the LP attest to the sensitivity fordetection of that isotype and correlate with the numbersfound by other investigators (see Discussion).

Total or background ASC. The numbers of ASC of eachisotype in the four tissues studied are shown in Table 1. Thistable includes all time points beyond day 10 postinfection, asthere was no significant difference in their numbers over theduration of these experiments. The LP showed the greatestnumber of active ASC, predominantly of the IgA isotype.The total number of IgA-secreting cells of over 100,000 per106 mononuclear cells was also reported by Van der Heijdenet al. in 20-week-old mice (64). The number of ASC 10 dayspostinfection was significantly smaller than at all later times

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FIG. 2. Ten-day-old mice were enterally administered tissue culture supernatant from uninfected or rotavirus-infected MA104 cellcultures. (a) Lymphocytes from small intestinal LP tissues were harvested at 14 to 40 days after mock infection and assayed for the numberof all IgG-, IgM-, and IgA-secreting cells, regardless of antigen specificity. (b) The above process was applied to mice infected with RRV, andlymphocytes were harvested at 4 to 63 days after infection (data beyond day 40 not shown). Symbols: O, IgG-secreting cells; M, IgM-secretingcells; + , IgA-secreting cells.

(see Fig. 3), probably reflecting the immaturity of the im-mune system at an age of 3 weeks or less as previouslydescribed (1).

Figure 2 shows a comparison of the ASC (IgG, IgM, andIgA) in the small intestine LP tissue from mice that were

enterally infected with rotavirus with similar cells frommock-infected animals. The marked rise of IgA ASC be-tween 15 and 25 days of age (approximately 5 to 15 dayspostinfection) is approximately 7 days earlier than in theuninfected control animals. The data for uninfected is con-sistent with previous studies on murine intestinal IgA ontog-eny (see Discussion) (1, 62). The relatively early develop-ment of a plateau may have been enhanced by the RRVinfection, although that conclusion cannot be drawn fromthese data alone.

RRV-specific ASC. Figure 3a through d shows the mean (±

standard error of the mean [SEMI) of total and rotavirus-specific ASC of each isotype, expressed as the number ofASC found per one million viable mononuclear cells derivedfrom each of the four tissues studied at the specified timegroups. The data from all time groups except day 10 postin-fection are pooled in Table 2, which shows the mean numberof ASC (± SEM) of each isotype in the four tissues studied.

In our experiments, the RRV-specific IgA responses in LPwere evident by 4 days postinfection, rose to peak around 30days postinfection, and persisted at high levels for up to 63days postinfection. As seen in Fig. 3a, at the height of theRRV-specific response, it accounted for more than 50% of allIgA-secreting cells. This confirms that a single dose of thisheterologous virus is highly immunogenic in the mucosalimmune system of mice.A rise in RRV-specific IgA activity was also seen in the

PP, which correlated with the response in LP but was muchsmaller in magnitude (Fig. 3b). It is possible that thisrepresented contamination of PP with LP cells, which was

inevitable in spite of careful dissection. As expected, thespleen showed a predominance of IgM- and IgG-secreting

cells, with little IgA activity. To be noted is the extremelysmall number of rotavirus-specific ASC of any isotype foundin the spleen, a statistically significant difference from thesmall intestinal response. In the four experiments done at 4,7, 8, and 11 days postinfection, significantly increased IgMand IgG responses was seen in the MLN (Fig. 3c). Theseearly IgG and IgM responses were not seen in any othertissue and disappeared by 14 days postinfection.ELISA. Serum and intestinal contents obtained from naive

controls and the mice sacrificed on day 4 postinfectionshowed IgA titers of <1,000 and <50 and IgG titers of <500and <25, respectively. A fourfold or higher rise up to 64,000and 3,200 for IgA and up to 32,000 and 1,600 for IgG,respectively, was noted in animals sacrificed after day 14,and the mean titers for the different groups are shown in Fig.4. The kinetics of the response are similar to those of theASC by the ELISPOT technique. However, the peak serum

IgG titer occurred at a later time, possibly reflecting thecontinued accumulation of IgG in the serum due to the longhalf-life of this immunoglobulin class.When individual animals were compared, a fairly good

correlation between serum and intestinal IgA titers wasobserved (r = 0.82). The correlation between the smallintestinal and serum IgA titers may be related to the rela-tively efficient clearance of serum IgA by the hepatocytesinto bile via a receptor-mediated pathway present in miceand some other rodents but not in humans (4, 57). However,neither correlated with the number of ASC in the LP (r =0.40 for RRV-specific serum IgA versus LP IgA ASC and0.53 for intestinal contents), indicating other possible varia-bles that influence antibody titer in these fluids.

Neutralization. Serum and intestinal content samples were

collected at the time of lymphocyte harvest and assayed forRRV neutralization titers by immunohistochemical focusreduction assay. The results for samples from 12 experi-ments from 10 to 60 days postinfection are shown in Fig. 5.While there is a suggestion of peaking titers 20 to 30 days

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INTESTINAL ANTIBODY RESPONSE TO ROTAVIRUS INFECTION 1697

20 30 45 60 10 20 30 45Days Post-Infection Days Post-Infection

FIG. 3. Time course of the antibody response to murine enteral infection with RRV in four tissues as measured by the ELISPOT assay.Mice were orally infected at age 10 days, and cells from the indicated tissues were removed and assayed at 10 to 60 days postinfection. Resultswere expressed as the total number of ASC per million mononuclear cells ± SEM; n = 4 to 8 experiments at each time group; each experimentutilized two mice. Total IgG, IgM, and IgA are indicated by lines, while RRV-specific ASC are indicated by columns. Significant differencesby analysis of variance and Scheffe F multiple comparisons (95% confidence) are marked * for total IgG, IgM, or IgA and with a * forRRV-specific IgG, IgM, or IgA. (a) Small intestine LP IgG, no significant difference (NS); IgM, NS; IgA, P = 0.0025, day 10 versus days 30,45, and 60. RRV-specific IgG, NS; IgM, NS; IgA, P = 0.0015, day 10 versus days 30 and 45. (b) PP IgG, P = 0.0364, day 10 versus day 20;IgM, NS; IgA, NS. RRV-specific IgG, NS; IgM, NS; IgA, P = 0.0054, day 10 versus day 45. (c) MLN IgG, NS; IgM, P = 0.0001, day 10versus all other groups; IgA, NS. RRV-specific IgG, P = 0.0004, day 10 versus all; IgM, P = 0.0021, day 10 versus all; IgA, NS. (d) Spleen,all NS. Symbols: *, RRV-specific IgG; E, RRV-specific 1gM; El, RRV-specific IgA; 0, total IgG; A, total IgM; *, total IgA.

postinfection, no statistical trend can be identified. Neutral-izing antibody titers in nonimmunized animals and mothersprior to their litters being immunized were always <200 inserum and <20 in the intestinal contents. A representativegroup of dams tested at the same time as the immunized micewere sacrificed and showed significant neutralizing titersapproaching those seen in the immunized litter (data notshown). Since the mice immunized were of suckling age, themothers were housed in the same cage and were thereforeexposed to the virus shed in the feces of immunized litter.The possibility of passive transfer of neutralizing antibodyfrom mothers, in the serum by immunoglobulin uptakebefore intestinal closure and in the intestinal lumen ofsuckling mice, makes the interpretation of these data difficultand emphasizes the advantage of ELISPOT assay.

DISCUSSION

Rotaviruses are a common cause of severe gastroenteritisand produce disease by replicating in the terminally differen-tiated epithelium of the small intestine. Several studies haveindicated the importance of local mucosal immunity in theprotection against this disease. Passive transfer of gammaglobulins, neutralizing monoclonal antibodies, or antibodiesin milk or colostrum have been shown to be protective whenadministered together with infectious doses of viruses (3, 5,31, 39, 40, 49, 52, 55). Antibodies in serum seem not to protectagainst infection in most studies (34, 54, 70). However, somestudies have suggested that high titers of passive circulatingantibodies may moderate the severity of rotavirus infectionsvia possible resecretion into the intestine (48).

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TABLE 2. RRV-specific ASC isolated from indicated tissuesbetween 20 and 60 days after RRV infection"

Tissue IgM ASC IgG ASC IgA ASC

Spleen 39 7 58 13 54 18Peyer's patches 370 ± 121 774 ± 158 2,600 ± 321Mesenteric nodes 7 + 2 29 ± 9 38 ± 11Lamina propria 69 ± 30 8,483 ± 2,849 68,183 ± 8,530

' Results are expressed as the number of ASC per 106 mononuclear cells +SEM (n = 29).

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Rotavirus-specific plasma cells in the small intestine start-ing at 10 days postinfection have previously been demon-strated by Dharakul et al. by using immunohistochemicalmethods in a murine model of homologous rotavirus infec-tion (13). Ninety-five percent of these specific cells were ofthe IgA isotype. We have come remarkably close to con-firming this number by using a completely different tech-nique and a heterologous rather than a homologous murinemodel. On the basis of data from Table 2, we have shownthat 89% of small intestine LP RRV-specific ASC weresecreting IgA. This finding may not be surprising, however,as IgA-secreting cells of all specificities constitute 88% ofASC in this tissue (Table 1).Another important point of agreement between the

present study and the data reported by Dharakul et al. is thatapproximately half of the cells bearing stainable immuno-globulins were virus specific in their study. This correlateswell to our finding of almost 50% of ASC in the LP beingvirus specific at 20 to 60 days postinfection.A similar predominance of local IgA response has also

been demonstrated with other intestinal antigens such as thecholera toxoid, and a correlation with protection has beendemonstrated (30, 58). The local secretion of IgA is thereforenow considered to be the important effector mechanism ofmucosal immunity. It has been estimated that more than 40mg/kg of body weight of IgA is secreted into the gut lumendaily, which is more than the total production of IgG, the

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FIG. 4. ELISA titers in serum and intestinal contents at indi-cated time intervals postinfection (p.i.). Analysis of variance andScheffe F multiple comparisons showed significant differences (95%)between the day 10 group and groups marked with a *. Symbols: 0,

intestinal content IgG; 0, serum IgG; *, intestinal content IgA; A,

serum IgA. OD450, optical density at 450 nm.

FIG. 5. Serum (0) and intestinal contents (0) were collected atthe time cells were harvested for the ELISPOT assay, and immu-nochemical focus reduction neutralization assays were performedon MA104 cell monolayers. Titers indicated are the greatest dilu-tions of fluid that resulted in .50% reduction of virus-infected cells.All naive mice tested showed titers of <200 and <20 for serum andintestinal contents, respectively (data not shown). This was thelowest dilution tested.

dominant immunoglobulin in serum (9). The generation ofthese large amounts of secretory antibodies is dependent onthe large number of IgA-producing cells in the LP, estimatedto account for 70 to 80% of all immunoglobulin-producingcells in humans as well as mice (61, 62). The ELISPOTtechnique developed for quantitating intestinal ASC in aprecise and efficient manner now offers the means to studymore effectively the response to rotavirus in this importantorgan of immunity. The detection of rotavirus-specific ASCin the LP as early as 4 days postinfection (compared with 10days by immunohistochemical techniques) indicates the im-proved sensitivity of the ELISPOT in detection of smallerspecific responses.The overall sensitivity of this system for the identification

of ASC in the small intestinal LP can be compared withresults in published articles that reported the backgroundproduction of immunoglobulin in the mouse small intestineas a function of age (62). A rapid rise was seen in smallintestinal IgA ASC beginning at 3 weeks of age and reachinga plateau of about 107 IgA ASC by 6 weeks of age. Mice inthis study designated 20 days postinfection are approxi-mately 4 to 5 weeks of age, and other groups range up toabout 10 weeks. In our study, IgA ASC were not signifi-cantly different in any of the age groups beyond 20 dayspostinfection, with the average IgA ASC in LP being about150,000 per 106 mononuclear cells. For comparison with theresults of Van der Heijden et al., who reported 107 ASC from108 LP mononuclear cells, our rate of 150,000 per 106mononuclear cells is about 1.5 x 107 per animal, which isvirtually the same result. The fact that we observed total IgAlevels at mature levels in mice by the time they were 3 weeksof age suggests that rotavirus infection hastened normalontogeny. A single experiment on naive mice 14 days of ageshowed smaller number of IgA ASC in the LP comparedwith that in infected mice, but a larger number of uninfectedcontrols at ages less than 3 to 4 weeks would be required todirectly address this problem.The incidence of surface and internal T- and B-cell mark-

ers in the lymphocyte populations of spleen and the differentgut-associated lymphoid tissues of mice have been describedand reviewed previously (6, 24, 33, 41, 42, 59, 60). Thoughthe ratio of T to B cells varies somewhat depending on thetechnique used, the reported differences between thesetissues do not account for the marked differences we report

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INTESTINAL ANTIBODY RESPONSE TO ROTAVIRUS INFECTION 1699

in the number of ASC. The use of the total number of viablemononuclear cells as the denominator is a convention usedby most authors studying the immune response in terms ofASC. This approach circumvents the discrepancies involvedin identifying T and B cells by the use of different tests andattempting to distinguish immunoglobulin-containing plasmacells and membrane immunoglobulin-bearing B cells whichmay or may not be secreting antibodies. Moreover, since thetotal number of mononuclear cells in spleen and LP is knownto be in the order of 108 per mouse and in the order of 107 inthe PP and MLN, the relative contribution of each organ tothe total body response can be appreciated (64).Although the location and isotype of the intestinal anti-

body response to rotavirus reported herein is similar to theresponses to other intestinal antigens that have been previ-ously described, the overall magnitude of the response torotavirus is very strong compared with those to otherantigens that have been studied with similar techniques. Forinstance, ASC specific for cholera toxin were quantitated inthe LP by ELISPOT using petri dishes as the solid phase,and only 23 ASC per 106 mononuclear cells were found to bespecific after a single oral dose of 10 pLg of cholera toxin,which could be enhanced to 700 ASC per 106 mononuclearcells after four boosters (32). This was calculated as 7.8% ofthe total active ASC in this version of the ELISPOT assay,whereas the overall RRV-specific response in the LP in ourstudy was 48%. Keyhole limpet hemocyanin, which alonedoes not stimulate a response when orally administered tomice, can be rendered antigenic if administered with choleratoxin. When the ELISPOT assay was used to quantitate thisresponse, peak numbers of small intestinal LP lymphocytesof between 1,000 to 10,000 ASC per 106 mononuclear cellswere noted, but no information regarding the total ASCpresent was available (69).The response to another orally administered (but nonrep-

licating) viral antigen, the formalin-inactivated influenzavirus, was studied by using the ELISPOT technique (7). LPresponse was not studied, but the virus-specific ASC in PPcells were quantitated. The peak response was among PP-derived IgA-secreting cells and was 27 ± 1.6 ASC per 106mononuclear cells for virus alone and 326 + 1.5 ASC per 106mononuclear cells when virus was administered with choleratoxin and after PP cells had been cultured in vitro for 2 daysin the presence of viral antigen. These levels are markedlylower than we report for the same tissue and isotype (RRV-specific IgA ASC in PP = 2,600 ± 321) without eitheradjuvant or in vitro culture with antigen. These comparisonsdramatically demonstrate the power of replicating rotavirusas a mucosal antigen. Though the other studies have usedpetri dishes as the solid phase (which, in our experience,may result in an underestimate of the total number of spots),the exponential differences in the small intestinal LP re-sponses could not be attributed to such technical differences,especially considering that the responses in the other tissuesare comparable.The responses to rotavirus in the MLN and the spleen are

more similar to that described for other antigens. When theresponse to the parasite Trichinella spiralis was quantitatedin rat MLN, ASC were found to peak at 751 ± 298 IgA ASCper 106 mononuclear cells, with a peak at 12 days postinfec-tion (65), but IgG and IgM responses were not studied. Thecomparable RRV-specific IgA response in MLN at 10 dayspostinfection was 91 ± 37 ASC per 106 mononuclear cells,though IgG andIgM responses were dominant at these earlytime points (601 ± 300). Splenic lymphocytes have generallybeen studied forIgM or IgG after parenteral immunization

with agents such as Streptococcus pneumoniae polysaccha-ride or Streptococcus mutans, leading to 150 to 500 ASC per106 mononuclear cells (47, 66).The circulating and intestinal virus-specific immunoglob-

ulins after a homologous infection in seronegative mice havebeen described by Sheridan et al. (53). They demonstratedan early IgM response in serum, followed by the appearanceof IgA and IgG in serum and a predominant IgA response inintestinal washings starting at day 7 and continuing to risethrough their period of study (21 days). In our study, specificantibody titers for IgA and IgG in serum and IgG in fecescontinued to rise to 20 days postinfection and persisted athigh levels, with some downward trend at 60 days postin-fection (IgM titers were not studied). The peak serum IgGoccurred at a later time, possibly reflecting the continuedaccumulation of IgG in the serum due to the longer half-lifeof this immunoglobulin class. This delayed peak of serumIgG has previously been described in studies of humanvolunteers (2).Mice are susceptible to rotavirus illness only between 1 to

13 days of age and are therefore generally considered unsuit-able for use in studying the determinants of active diseaseprotection. But Ward et al. have recently described the useof homologous murine rotaviruses (which cause epizooticdiarrhea in infant mice) in adult mice with an end point ofinfection (viral shedding, but not diarrhea) which the authorsproposed as a model for studies on rotavirus disease protec-tion (68). Adaptation of precise measures of antibody re-sponses, such as the ELISPOT assay, may prove useful inattempts to determine the antibody correlates of diseaseprotection.The effects of virus dose and virus strain in this murine

model of rotavirus infection have also been well studied.RRV has been shown to replicate in the intestinal epitheliumand cause severe disease. However, only at very low dosesof virus does replication occur to levels above the inoculateddose (43). In this study, a relatively large dose of rotaviruswas administered, approximately 1 log unit more than thedose needed to cause disease in 50% of the pups (35), andinfection therefore resulted in grossly evident diarrhea inmore than 95% of the pups. This large dose results in thepresence of considerable amounts of both replicating andnonreplicating antigen available in the intestine for presen-tation to the intestinal immune system. The relative contri-bution of each and relative importance of epithelial or theM-cell pathway taken by the replicating and nonreplicatingantigen remain to be elucidated.

This model allows for the study of the local antibody-secreting cells and can be modified to study not only thevirus-specific response but also protein- or epitope-specificresponses. Recent developments in biotechnology have en-hanced the enthusiasm for efforts at immunoprophylaxis byvaccination with synthetic viral peptides, bacterial and viralvectors, and reassortant strains. The effectiveness of theseagents depends on the determination and optimization oftheir immunogenic properties. We believe that this model forstudying local immune response will help realize the poten-tial of these approaches.

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