INFECTION AND IMMUNITY, May 1980, p. 441-4500019-9567/80/05-0441/10$02.00/0

Vol. 28, No. 2

Local and Systemic Antibody Response to OralAdministration of Glucosyltransferase Antigen Complex

DANIEL J. SMITH,* MARTIN A. TAUBMAN, AND JEFFREY L. EBERSOLEForsyth Dental Center, Boston, Massachusetts 02115

The salivary and serum immune responses to orally administered glucosyltrans-ferase antigen complex from Streptococcus mutans strain 6715 were investigatedin hamsters. An enzyme-linked immunosorbent assay was used to measure theantibody quantity and isotype, and a ['4C]glucosyl-labeled sucrose incorporationassay was used to measure functional inhibition of the enzyme. A total of 21 to 27daily doses of antigen administered in hamster oral cavities elicited salivaryimmunoglobulin G and immunoglobulin A antibody responses and functionalinhibitory activity. The salivary response increased throughout the immunizationprocedure, and the amount of salivary antibody was dependent upon the dose ofantigen given. The salivary response to a second oral administration of antigenfor 4 days showed some features of anamnesis. The response after a secondantigen administration was detected sooner than the primary response, andsomewhat higher levels of antibody and inhibitory activity were observed. Serumantibody (immunoglobulin G and immunoglobulin M) and functional inhibitoryresponses were also elicited by oral administration of the soluble enzyme antigen.These responses were lower than responses induced by local injections of antigenin complete Freund adjuvant. The ability to evoke a salivary immune response tothe glucosyltransferase antigen complex may increase the potential of using thisantigen in an effective caries vaccine.

Enteric vaccines have been investigated re-cently for their ability to induce a protectiveresponse against a variety of bacterial infectionsof mucosal surfaces (18, 20, 29). Administrationof antigen by this route has been considered apossible mechanism to stimulate an immuneresponse to protect the teeth, which are bathedby mucosal secretions. Experiments in rodentmodels have shown that dental caries caused byStreptococcus mutans can be reduced signifi-cantly by feeding either whole cells (14, 15) orsoluble antigens (27). Preliminary studies in hu-mans suggest that a salivary immune responsecan be elicited by feeding killed S. mutans cellsin enteric capsules (13). However, little is knownof the characteristics of the salivary and serumimmune responses to oral administration of iso-lated antigens derived from this organism. Theglucosyltransferase (GTF) antigen complex (26)of S. mutans is particularly interesting becausethe products of this constitutively synthesizedenzyme have been implicated in dental plaqueformation by S. mutans (8). In addition, injec-tion or feeding of these antigens has been shownto diminish the extent of disease caused by S.mutans in rodents (25, 27, 30). Since antibodydirected to antigens of the GTF complex may beprotective, an understanding of the kinetics ofantibody appearance in the saliva, the effect of

441

dose of this antigen on the amount of antibodyelicited, and the nature of the secondary re-sponse to orally administered GTF may contrib-ute to the development of a dental caries vac-cine. Therefore, we investigated several of thecharacteristics of the primary and secondarysalivary and serum immune responses after oraladministration of the GTF antigen complex.

MATERIALS AND METHODSAnimals. NIH white, acromelanic hamsters were

bred and raised at the Forsyth Dental Center. LHC/Lak cream hamsters (Charles RiverLakeview) werealso used in this study because of the limited availa-bility of the NIH white hamsters for all experiments.These two hamster strains show similar levels of sali-vary and serum antibodies to GTF after local immu-nization with GTF in adjuvant (unpublished data). Allanimals were individually caged and maintained on apelleted diet (Purina Mouse Chow; Ralston PurinaCo.) unless otherwise indicated. No S. mutans wasobserved on mitis salivarius agar plates after swabbingthe dental surfaces of these two strains of hamsterswhile these animals were housed at the Forsyth DentalCenter.Antigen preparation. GTF for immunization and

antibody analyses was prepared as previously de-scribed (26). Briefly, S. mutans strain 6715 was grownanaerobically in 10% C02-90% N2 for 24 h at 370C in6 to 10 liters of chemically defined medium. A cell-freesupernatant, which was obtained by centrifugation

442 SMITH, TAUBMAN, AND EBERSOLE

(13,000 x g), was brought to pH 6.5 with 1 N NaOH.Water-insoluble polysaccharide was then synthesizedby incubation of the GTF-containing supernatant with10% sucrose for 48 h at 370C. Bacterial growth wasinhibited by the addition of 0.02% sodium azide. Thewater-insoluble polysaccharide which formed was col-lected by centrifugation at 13,000 x g and then washedextensively with cold distilled water and 0.01 M so-dium phosphate (pH 6.8) to which 0.02% sodium azidehad been added. GTF was then eluted from thewashed, water-insoluble polysaccharide by I h of in-cubation at 4VC with an equal volume of 6M guanidinehydrochloride. After elution, guanidine was removedby dialysis, and the enzyme was concentrated by ul-trafiltration. Enzymatic activity was determined bythe Somogyi (28) and Glucostat (Worthington Bio-chemicals Corp.) assays, and protein was measured bythe assay of Lowry et al. (11). Approximately 40% ofthe enzymatic activity which was present in the cul-ture supernatant was recoverable after guanidine elu-tion and dialysis (26). Fructose was the principal sugarreleased (78%) after incubation of the enzyme prepa-ration with sucrose for 2 h at 370C. As previouslydescribed, the guanidine-eluted preparations containGTF, glucan, nonenzyme protein bound to glucan, andlow-molecular-weight material. This type of antigenwas used in all experiments. Antigen for use in theassays for antibody or enzyme-inhibiting activity wasprepared by gel filtration on a column of 8% agarose(Pharmacia Fine Chemicals, Piscataway, N.J.) in 0.01M sodium phosphate, pH 6.8. Column fractions weremonitored for protein spectrophotometrically at 280nm and for enzymatic activity by the chemical assaysdescribed above. GTF activity was eluted in the col-umn void volume. Fractions containing GTF activitywere pooled and concentrated for use as antigen in theassays for antibody and enzyme-inhibiting activity.The gel filtration procedure eliminates the low-molec-ular-weight material. No significant invertase activitywas detected in the column fractions. Glucose repre-

sented more than 97% of the labeled carbohydrate inthe ethanol-insoluble polysaccharide synthesized fromsucrose by GTF eluted from water-insoluble polysac-charide of S. mutans 6715 when [U-'4C]sucrose and[fructose-1-3H(N)]sucrose incorporation assays wereused (26). All guanidine-eluted GTF preparationsformed both water-insoluble and water-soluble poly-saccharide when incubated with 0.125 M sucrose for 4h at 370C.Antigen administration protocol. Figure 1

shows the immunization protocols for all of the exper-iments. In the first experiment (experiment A), 23-day-old LHC/Lak cream hamsters were placed intothe following two groups: (i) animals sham immunizedorally with 0.2 ml of 0.01 M sodium phosphate buffer(n = 14) and (ii) animals immunized orally with 0.2 mlofGTF in phosphate buffer (n = 14). Antigen or bufferwas administered daily for 27 consecutive days intothe oral cavity with an automatic 0.2-ml pipette. Eachdose of antigen contained 0.9 IU of enzyme activity in400 jig of protein. A total of 24.3 IU of enzyme activityin 10.8 mg of protein was administered during the 27-day immunization period. Hamsters in this experimentwere bled and salivated at 10, 16, 22, 28, and 70 daysafter oral immunization was begun. Hamsters ingroups i and ii were infected 30 days after the initialoral administration with approximately 10' colony-forming units of a cariogenic streptomycin-resistantstrain of S. mutans 6715. The purpose of this infectionwas a subsequent study on the effect of oral immuni-zation on S. mutans infections. At this time all animalswere placed on cariogenic diet 2000 (10). Infectionswere confirmed in all animals of groups i and ii byswabbing and plating on mitis salivarius agar contain-ing 200 mg of streptomycin sulfate per ml.

In the second experiment (experiment B), 24-day-old LHC/Lak cream hamsters (n = 28) were placedinto two equal groups as in experiment A. Antigen orbuffer was administered orally daily for 26 consecutivedays. Each dose of antigen contained 0.25 IU in 250

Immunization ProtocolDose

Exp. A (0.90 U, 400 pg) _

Exp. B (O.254 2504

Exp. C (0.204 200 uo)

Exp. D (0.144 200pWg2 * * * <

Exp. E (0.23U, 47lg) t + t t 0

0 1 2 3 4 5 6 7 8 9 10 '

Weeks

a * = blood. saliva+ = injection29 = oral administration

(no LeTspneb,, *V 0 9 0 0 ;To

0

I Lonh -s.I3 4 5 6 7 8

Months

FIG. 1. Immunization protocols for oral administration or injection ofGTF antigen complex in hamsters.Orally administered antigen or buffer was given via a 0.2-ml pipette once a day for the times indicated.Injected antigen was given subcutaneously in the salivary gland vicinity in complete Freund adjuvant. Theenzymatic activity (in international units [IUJ) and protein content (in micrograms) of each single dose ofantigen are shown. The times (weeks or months) after initiation of immunization are shown on the abscissa.Hamsters in experiment A were also infected with S. mutans 6715 between weeks 4 and 10.

jr

INFECT. IMMUN.

VOL. 28, 1980

[Lg of protein. A total of 6.5 mg of protein was admin-istered to each hamster during the immunization reg-imen. Hamsters were bled and salivated as previouslydescribed (30) at 1 day after the final daily dose ofantigen.

In a third experiment (experiment C), 24-day-oldwhite hamsters (n = 28) were separated into two equalgroups as in experiment A. Antigen or buffer wasadministered orally daily for 21 consecutive days. Eachdose contained 0.2 IU in 200 jg of protein. A total of4.2 IU of enzyme activity in 4.2 mg of protein wasadministered to each hamster during the immuniza-tion regimen. Hamsters were bled and salivated at 1day after the final daily dose of antigen.

In a fourth experiment (experiment D), 24-day-oldwhite hamsters were divided into the following twogroups: (i) animals sham immunized orally with 0.2 mlof phosphate buffer (n = 6) and (ii) animals orallyimmunized with 0.2 ml of GTF in buffer (n = 11).Antigen or buffer was administered orally for 21 con-secutive days. Each dose of antigen contained 0.14 IUof enzyme activity in 200 pg of protein. A total of 4.2mg of protein was administered during the 21-dayperiod. All hamsters were bled and salivated at 7, 14,22, 28, 35, and 42 days after the start of the immuni-zation protocol. Those animals showing an immuneresponse (n = 3) to this minimal antigen dose duringthis time were periodically bled and salivated duringan additional 2.5 months. These hamsters were thenorally immunized again with 0.2 ml of the same GTFpreparation daily for 4 days. Salivas and sera werethen periodically collected during an additional 4months. At the end of the 4 months, hamsters (n = 7)which had not shown a response to either the primaryor secondary antigenic challenge were injected with0.1 IU of GTF enzyme activity in 75 pg of protein incomplete Freund adjuvant once in the salivary glandvicinity. Five untreated hamsters of approximately thesame age were also injected once. Hamsters were bledand salivated at 7 days after the injection.

In a fifth experiment (experiment E), 29-day-oldLHC/Lak cream hamsters were injected with 0.1 mlcontaining 0.23 IU of enzyme activity in 47 pg ofprotein in 0.1 ml of complete Freund adjuvant. Antigenwas injected into the vicinity of the parotid and sub-mandibular glands once weekly for 4 weeks. Hamsterswere bled and salivated at 6 and 47 days after the finalinjection.

Saliva and serum. Salivas and sera were collectedand treated before the antibody assay, as previouslydescribed (30). In addition, salivas to be used in theinhibition of ['4C]glucose incorporation assays weredialyzed, first against phosphate-buffered saline con-taining 0.001 M ethylenediaminetetraacetate and thenagainst phosphate buffer. All samples were stored at-20'C until antibody analysis.Assay for inhibition of GTF activity. The pro-

cedure for determining inhibition of GTF activity byserum and saliva is a modification (24) of the methodof Evans and Genco (7). This assay reflects the totalamount of glucan synthesized by GTF. GTF activitywas determined by ['4C]glucose incorporation fromglucosyl-labeled sucrose into ethanol-insoluble poly-saccharide. Additional modifications were incorpo-rated into the assay to facilitate pipetting, increase

ORAL ADMINISTRATION OF GTF 443

reproducibility, and decrease variability resulting fromlow protein concentrations (manuscript in prepara-tion). In our assay for the measurement of inhibitingactivity in serum, 5 A1 of serum was preincubated with0.80 mIU of GTF (guanidine eluted from S. mutans6715) in 100 pA of 0.02 M phosphate buffer (pH 6.8) for1 h at 37°C in a shaking water bath. A total of 330 pgof primer dextran T10 (Pharmacia Fine Chemicals)and 0.018 pg of ['4C]glucose-labeled sucrose (specificactivity, 275 mCi/mmol; approximately 13,000 cpm;New England Nuclear Corp.) in 0.2 ml of 0.01 Msodium phosphate (pH 6.8) were then added. Thismixture was incubated at 37°C for 2 h, and the reactionwas stopped by precipitation with ethanol (final con-centration, 75% vol/vol). After centrifugation of theprecipitate at 13,000 x g, the pellet was dissolved indistilled water and reprecipitated with ethanol. Thecentrifuged pellet was dissolved in 0.2 ml of distilledwater and mixed with 4 ml of Ready-Solv Solution VI(Beckman Instruments Inc.) and counted in a liquidscintillation spectrometer (model LS-100C; Beckman).Under these conditions approximately 1,000 cpm of['4C]glucose is incorporated into ethanol-insoluble pol-ysaccharide in the presence of buffer. Salivary inhibit-ing activity was assayed by incubating 20p1 of dialyzed,unconcentrated saliva with 20 ,ul of enzyme (0.38 mIUof activity) and 10 pl of 1% bovine serum albumin (23)(both in 0.01 M sodium phosphate, pH 6.8) for 1 h at37°C in a shaking water bath. A total of 330 pg ofprimer dextran and 0.018 pLg of ['4C]glucosyl-labeledsucrose were then added in 50 tl of sodium phosphatebuffer. Mixtures were incubated for 5 h, after whichpolysaccharide was precipitated, washed, and countedas described above for the serum assay.The inhibiting ability of an immune serum or saliva

sample was determined as follows. The counts perminute incorporated into precipitated polysaccharideby the enzyme in the presence of immune fluid wasfirst expressed as a percentage of the counts per min-ute incorporated by the enzyme in the presence of theappropriate control fluid. This value subtracted from100 gave the percent inhibition. This measure of theinhibitory activity of immune fluids was found todeviate from linearity at high antibody concentrations(unpublished data). Thus, to determine accurately theinhibitory capacity of a given serum or saliva over arange of antibody concentrations, standard curveswere prepared, in which the percent inhibitions ofserum and saliva standards were measured at severaltwofold dilutions. The percent inhibition versus thereciprocal dilution (loglo) of the standards showed alinear relationship, with a correlation coefficient of>0.999; 50% inhibition in the saliva and serum assayswas assigned a value of 100 inhibition units so that theinhibitory activities in the two fluids could be com-pared. The inhibitory capacities of experimental serumand saliva samples were determined from these stan-dard curves by using the following equations: for se-rum, percent inhibition = 48.7(logio inhibition units)- 45.9; and for saliva, percent inhibition = 38.9(logioinhibition units) - 27.9. The values from these equa-tions were multiplied by the appropriate dilution fac-tor.

Experimental sera were tested at 10- to 200-folddilutions, depending on the level of inhibition found in

444 SMITH, TAUBMAN, AND EBERSOLE

these sera. At these dilutions enzyme in the presenceof normal serum incorporated 0 to 10% more radioac-tivity into polysaccharide than did enzyme in thepresence of buffer. Experimental salivas were testedundiluted. Enzyme in the presence of undiluted, di-alyzed normal saliva incorporated approximately 35%fewer counts per minute into polysaccharide than didenzyme in the presence of buffer. Therefore, little orno stimulation of enzyme activity was seen in theseassays.

Antisera. Antisera specific for hamster gammachains were obtained by injecting rabbits with hamsterimmunoglobulin G (IgG) (the 0.005 M sodium phos-phate, pH 8.0, eluate after diethylaminoethyl cellulose[DE52; Whatman] chromatography of hamster se-rum). Light chain activity was removed with an im-munosorbent prepared by linkage of IgA-rich hamsterintestinal perfusates (50% ammonium sulfate precipi-tates [1, 9]) to cyanogen bromide-activated Sepharose4B. Rabbit antisera directed to hamster alpha or muchains and prepared as described previously (1, 2)were provided by John Bienenstock, McMaster Uni-versity, London, Ontario, Canada.Antibody analysis. Antibody in saliva or serum

was determined by a modification of the indirect en-zyme-linked immunosorbent assay (ELISA) (5,6). TheGTF antigen used for this assay was the same as thatused for oral immunization in the respective experi-ments. Antigen in 0.1 M sodium carbonate (pH 9.6)was added to wells of polystyrene microtiter plates(Linbro Scientific) and incubated for 3 h at 370C. Aftersubsequent incubation overnight at 40C, plates werewashed and then incubated with appropriate dilutionsof hamster saliva or serum. The amount of boundantibody was determined by reaction with monospe-cific rabbit anti-hamster antisera directed againsthamster gamma, alpha, or mu heavy chains. After 2 hof incubation and washing, plates were reacted withgoat anti-rabbit IgG (Miles Laboratories) conjugatedto alkaline phosphatase (type VII; Sigma ChemicalCo., St. Louis, Mo.). After overnight incubation atroom temperature, the substrate (p-nitrophenylphos-phate; Sigma 104) was added, and the degree of reac-tion was determined spectrophotometrically at 400nm. To determine relative differences in the concen-trations of salivary IgG and IgA antibodies or serumIgM and IgG antibodies, the salivas or sera giving thehighest absorbance at 400 nm (A4.0) were seriallydiluted and retested in the ELISA assay by using theappropriate monospecific antisera. The resulting A4wvalues were plotted against the dilution logoe) toobtain standard curves for each immunoglobulin. Lin-ear relationships were generally obtained with A400and the reciprocal dilution logol) between opticaldensities of 0.250 and 1.200 nm. The following A4w0values from the most active fluids were designated as100 ELISA units (EU): anti-y chain (saliva), 0.475;anti-y chain (serum), 1.054; anti-a chain (saliva), 1.117;and anti-Ii chain (serum), 0.650. The relative amountsof immunoglobulin bound of the remaining sera orsalivas were then compared with these standard curvesby using the following equations: for anti-y chain (sa-liva), A4oo = 0.423(logloEU) - 0.489; for anti-y chain(serum), A4 = 0.749(logoEU) - 0.443; for anti-a chain(saliva), A4 = 1.04(logloEU) - 0.963; and for anti-ctchain (serum), A400 = 0.525(logloEU) - 0.400.

RESULTSImmune responses to oral administration

of GTF-containing antigens. The develop-ment of salivary and serum immune responseswas followed in hamsters given 27 daily doses ofGTF from S. mutans 6715 perorally (experimentA). The salivary IgA antibody responses (Fig.2A) in the hamsters given GTF were signifi-cantly higher than the responses of the controlgroup (data not shown) at 16 days after immu-nization was begun and throughout the durationof the experimental period. The salivary IgA

601

501w

> 40

-C

Z 2C

oJ

IgG 50 IgA

40-

30-

20-

10I^ mchb i 0.|1!fed inject fed

Days After Start of Immunization

A

7Oc

500 50

SIgG. 10M

5 300- ~~~~~~~30-

~200- 20-

100- 10-

0 122 28 70 70 27 68 10 1 22 21 70 70c 27 68fed inject fed u*ct

Das After Start d lanzation

FIG. 2. Relative amounts of antibody in salivasand sera of hamsters given 0.9 IU of GTF antigencomplex perorally once a day for 27 days. Antibodyactivity is expressed as EU, which were obtained byusing separate equations for each isotope (see text).Thus, identical EU for different isotopes do not nec-essarily represent equivalent amounts of antibody.(A) Open bars, Salivary IgG antibody from orallyimmunized hamsters (5 to 11 animals); cross-hatchedbars, salivary IgG antibody from injected hamsters(3 animals); solid bars, salivary IgA antibody (5 to 11animals). Brackets enclose two standard errors. Thecolumns marked 70c indicate antibody levels ofsham-immunized control hamsters. Salivas were tested at1:10 dilutions. (B) Open bars, Serum IgG antibodyfrom orally immunized hamsters (5 to 11 animals);broken bars, serum IgM antibody from orally immu-nized hamsters (5 to 11 animals); cross-hatched bars,serum responses of injected hamsters (4 animals).Brackets enclose two standard errors. The columnsmarked 70c indicate antibody levels of sham-immu-nized hamsters. Sera were tested at 1:400 dilutions.

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antibody responses continued to increasethroughout the immunization period. At 6 weeksafter completion of the immunization protocol,the salivary IgG and IgA antibody responseswere still detectable. Both the sham-immunizedcontrol group and the GTF-immunized groupwere infected with cariogenic S. mutans 6715 onday 30. However, salivary antibody activity inthe infected control group was not significantlydifferent than activity in saliva from nonin-fected, nonimmunized hamsters at either day 30or 70 (less than 1 EU for either IgG or IgA). Thesalivary IgG responses of animals given fourweekly local injections of GTF emulsified incomplete Freund adjuvant (experiment E) werenearly eightfold lower (Fig. 2A) than the re-sponses of perorally immunized hamsters im-mediately upon completion of the respectiveimmunization protocols. The salivary IgG anti-body levels decreased twofold by day 68 in in-jected animals. A comparison (oral versus injec-tion) ofthe salivary IgA responses was preventedby insufficient amounts of material.The serum responses to the oral administra-

tion of GTF antigens are shown in Fig. 2B. Alow but significant antibody response was de-tected in the IgM and IgG isotypes of sera ob-tained from immunized hamsters 10 days afterGTF antigen feeding was begun. The peak IgMantibody response occurred by day 16. IgG an-tibody continued to increase until day 22. Theserum IgG response in the orally immunizedhamsters then gradually declined during the 6-week infection period but remained elevated. Noserum IgA antibody was detected at any sam-pling period. Serum IgG antibody responses inthe control infected group that had receivedbuffer orally and in the uninfected group (datanot shown) did not differ significantly during theexperiment. The sera of injected hamsters con-tained 5.7 times as much IgG antibody and 14.6times as much IgM antibody as the sera of orallyimmunized hamsters immediately after the com-pletion ofthe respective immunization protocols.Antibody of the IgG isotype showed a modestincrease in the sera of injected hamsters on day68, whereas the IgM antibody fell to 39% of theday 27 levels.

Salivary and serum responses of orally im-munized and injected hamsters were also ana-

lyzed for their ability to inhibit GTF-mediatedglucose incorporation into total polysaccharide(Table 1). Although insufficient volumes ofham-ster saliva were available to complete the assayon days 10 and 16, significant inhibitory activitywas observed in the salivas of orally immunizedhamsters by day 22. Inhibitory activity was high-est in the day 28 sample and decreased 1.8-foldby day 70, which corresponds to the decrease inspecific antibody determined by the ELISA (Fig.

ORAL ADMINISTRATION OF GTF 445

TABLE 1. Comparison ofGTF inhibitory activitiesin salivas and sera ofhamsters administered GTForally or by injection in the salivary gland vicinity

in Freund complete adjuvant

Salivary inhibitory ac- Serum inhibitory activ-tilvity itylovcDays after tiv(ihityion units xstart of (inhibition units) 1o-3)bGTF ad-

ministra- Salivary Salivarytion Oral gland vi- Oral gland vi-

cinity cinity10 2.9 ± 0.6' NDd16 15.5 ± 1.2 ND22 22.9 ± 1.8 ND 18.5 ± 4.9 ND28 (27)' 47.7 ± 3.7 14.0 ± 4.2 15.6 ± 5.1 43.2 ± 2.670 (68) 26.2 ± 4.4 7.8 ± 6.9 6.0 ± 0.7 49.5 ± 2.270 (sham) 1.7 ± 1.7 <1

a For oral administration each dose was 0.90 IU of enzymeactivity in 400 pg of protein (27 doses), and for salivary glandvicinity injection each dose was 0.23 IU of enzyme activity in47 pg of protein (4 doses).

bValues for saliva and serum were independently derived(see text). However, the 100-inhibition unit level for salivasand sera represents 50% inhibition of enzyme activity. Thevalues shown were adjusted for the differences in dilutionrequired to achieve 50% inhibition.

' Mean ± standard error.dND, Not determined.' Numbers in parentheses refer to days after start of injec-

tion schedule.

2A). Also similar to the antibody data (Fig. 2A),the salivas of injected hamsters contained ap-proximately 30% of the inhibitory activity ob-served in the salivas of orally immunized ham-sters at either time period. Analyses of the serafrom orally immunized hamsters for inhibitoryactivity supported the ELISA data. A serumresponse was detected on day 10 (Table 1). Theincrease and decrease in serum inhibition cor-responded to the changes observed for antibodyof the IgG isotype (Fig. 2B), as did the differencebetween serum inhibitory activities after oralversus subcutaneous antigen administration.Thus, oral administration of soluble GTF-con-taining antigens over a 4-week period was suffi-cient to give rise to salivary IgG and IgA anti-bodies, as well as to serum IgM and IgG anti-bodies, which were detectable by assays whichmeasured either antibody or functional inhibi-tion of enzyme activity.Effect of dose on immune response to

orally administered antigen. Table 2 showsthe effect of antigen dose on the induction ofsalivary and serum immune responses by oraladministration of GTF antigen. Three differentdoses of enzyme antigen were tested for abilityto elicit a salivary and/or serum immune re-sponse measured by both ELISA and functionalinhibition. The highest dose of orally adminis-tered antigen (experiment A) induced salivaryIgG and IgA antibody responses in all animals(14 of 14). The salivas of these hamsters also

446 SMITH, TAUBMAN, AND EBERSOLE

contained relatively high levels of inhibitory ac-tivity. When the antigen dosage was lowered toa total of 6.5 IU of enzyme (experiment B), animmune response could be detected in only 9 of11 hamsters. Furthermore, the levels of salivaryantibody or inhibitory activity in the hamsterswhich did respond to the 6.5 IU dose were sig-nificantly lower than the levels of response tothe 24.3 IU dose. Less than one-half the ham-sters (5 of 11) given the lowest antigen dosage(4.2 IU of enzyme) (experiment C) had demon-strable salivary inhibitory activity. However, themean levels of antibody and inhibitory activitiesin hamsters responding to the lowest dose werenot significantly different than the mean positiveresponses to the 6.5 IU dose.Some of these dose-response relationships

were also observed with the serum responses(Table 2). Increasing the amount of total enzymeantigen administered from 6.5 to 24.3 IU resultedin a 10-fold increase in serum IgG antibody anda 7-fold increase in serum inhibitory activity. Nosignificant difference in the amount of antibodyof the IgM isotype occurred over the range ofantigen doses tested in those hamsters showinga response. However, few animals given the low-est amount of antigen (4.2 IU) showed a signifi-cant serum antibody or serum inhibitory re-sponse (3 of 11 animals). Thus, both serum andsalivary immune responses were apparently in-fluenced by the dosage of orally administeredGTF antigen.

It was also of interest to determine whether

the animals (6 of 11) which did not respond tooral administration of the lowest antigen dose(4.2 IU, 21 days) (Table 2) had been renderedtolerant to this antigen. These animals did notshow a serum or salivary inhibitory responseeither after the initial 21-day oral administrationof antigen or after a subsequent 4-day adminis-tration of the same antigen concentration (Table3). The ability of these hamsters to respond toan injection of the same antigen 4 months afterthe second oral administration was comparedwith the response in injected hamsters that hadnot been exposed to antigen. The serum andsalivary inhibitory responses of the previouslyorally immunized hamsters were in fact slightlyhigher than the responses of the previously un-treated controls (Table 3). This may indicatethat, rather than making the hamsters tolerant,some priming had in fact occurred in the ham-sters given GTF orally, despite the inability todetect an immune response previously.Anamnestic responses to orally admin-

istered soluble GTF antigens. Salivary andserum responses to a second oral exposure of thesoluble GTF antigen preparation were investi-gated in weaning white hamsters given 0.14 IUof GTF activity in 200 jig of protein daily for 21days (experiment D). Under this regimen 3 of 11hamsters showed significant salivary and serumresponses by both ELISA and functional inhi-bition techniques. The antibody responses of thethree responding hamsters were followed for 4months (Fig. 3A and B) after completion of oral

TABLE 2. Effect of dose on immune responses to orally administered antigen in salivas and sera ofhamsters

Total amt Total amt Days of EUh No. show-Fluid" of protein of enzyme oral admin- unito nginhibi-PH~~~~~~~~~~~~~~~~~~~~unait tionteOenyeotalam(mg) (IU) istration IgA IgG IgM no.

Salivas 4.2 4.2 21 10.0 ± 1.0c 2.0 ± 0.5 10,1 ± 1.3c 5/116.5 6.5 26 13.9 ± 2.1 4.8 ± 2.0 9.5 ± 1.3 9/11

10.8 24.3 27 28.1 ± 6.8 42.6 ± 12.9 46.5 ± 4.1 14/14Sera 4.2 4.2 21 1.3 ± 0.1 7.2 ± 1.6 0.601x 3± 3/11

6.5 6.5 26 5.2 ± 1.9 6.6 ± 1.1 2.1 x 103 8/11

10.8 24.3 27 54.8 ± 13.4 5.1 ± 0.8 15.6 X 10:' 14/14111 1 1 ~~~~~~~~~~~~5.1x 103

a Hamsters were bled and salivated 1 to 2 days after the last day or oral administration of the GTF antigencomplex.

b EU values were derived independently, and the 100-EU level was based on different optical densities forsalivas and sera (see text). The EU values are intended to reflect relative differences in antibody activitieswithin the respective fluids. The EU values for salivas were determined from 1:10 dilutions, and the EU valuesfor sera were determined from 1:400 dilutions.

c Mean ± standard error of hamsters which showed a positive response. A response was considered positiveif it was more than 2 standard deviations above the responses of buffer-immunized hamsters. Salivary responsesfor buffer-immunized animals were 1.5 ± 0.6 EU for IgA, 1.0 ± 0.1 EU for IgG, and 0.4 ± 0.3 inhibition units forfunctional inhibition. Serum responses for buffer-immunized animals were 0.5 ± 0.2 EU for IgM, 1.1 ± 0.1 forIgG, and <100 inhibition units for functional inhibition.

INFECT. IMMUN.

ORAL ADMINISTRATION OF GTF 447

TABLE 3. Response to a single injection of GTF in untreated hamsters and in hamsters unresponsive toorally administered GTFa

Inhibition units (xlo ') at:

Group Fluid 7XDay afdemin - 7 Days after 4-day 7 Days after singleday oral admiis oral boost with injection withtration of PBS or PBS or GTF GTFGTFA

Untreated/injected (n = 5) Sera 0 NDW 6.9 ± 2.9dSalivas 0.4 ± 0.4d 0 2.6 ± 1.2

Oral (nonresponder)/injected Sera 0 0.2 ± 0.05d 10.3 ± 2.9(n = 6) Salivas 0.3 ± 0.1 0.2 ± 0.1 6.1 ± 1.0

a The single injection of GTF (0.1 IU of GTF enzyme activity in 75 jig of protein) was incorporated intocomplete Freund adjuvant (injection into salivary gland region). The hamsters unresponsive to orally admin-istered GTF received a total of 4.2 mg of GTF (0.14 IU of GTF enzyme activity in 200 I.g of protein per dose).

b Approximately 4 months elapsed between antigen exposures. PBS, Phosphate-buffered saline.'ND, Not determined.d Mean ± standard error.

GTF administration. During the first 2.5 monthsthe mean salivary IgA and IgG antibody levelsremained at or near the maximum primary re-sponse level, which occurred within 1 month ofthe start of antigen administration (Fig. 3A). Onthe other hand, the serum IgG antibody re-sponse (ELISA) (Fig. 3B) fell during the firstmonth and was at pre-immunization levels 4months after completion of the primary immu-nization regimen. A second course of antigenadministration (0.14 IU/dose; duration, 4 days)was initiated at this time in the animals thatresponded to the primary series of antigen ad-ministrations. The peak salivary antibody re-sponse in either the IgA or IgG isotype occurredbetween 8 and 13 days after the second immu-nization protocol was begun. This was in con-trast to the 21 to 28 days before a peak responsewas attained after primary immunization in thisexperiment or in the first experiment (Fig. 2A).The peak salivary IgG response after boostingshowed a threefold increase compared with thepeak primary response, whereas the peak sali-vary IgA response increased by approximately1.5-fold after boosting. However, the salivaryantibody of each isotype fell to levels within therange of the primary response during the 2months after the second antigenic challenge.The inhibitory response in the salivas of boostedhamsters (Fig. 4) was similar to the salivaryantibody response in general (Fig. 3A). Peaksalivary inhibitory activity increased 1.7-foldand remained in the range of the peak primaryresponse more than 4 months after the secondadministration of antigen.The serum antibody response elicited by the

second oral administration of the GTF antigenalso demonstrated aspects of anamnesis (Fig.3B). The peak IgG antibody response was notachieved until approximately 1 month after oralboosting. This response was threefold greater

than the primary IgG response. The secondaryIgG antibody response declined within 2 months.The increase and duration ofthe serum responsewere also reflected in the pattern of inhibitoryactivity observed in the sera of the three animalsto which the second antigen regimen was admin-istered (Fig. 4).

DISCUSSIONEnteric administration of soluble antigens, in-

cluding bovine serum albumin (3, 22), dinitro-phenylated bovine gamma globulin (16, 17), andcholera toxoid (19, 21, 29), has resulted in theappearance of antibody in secretions such asintestinal and respiratory fluid, saliva, and milk.In the present study, 21 to 27 daily doses ofGTFantigen complex administered into hamster oralcavities were sufficient to elicit an immune re-sponse in the saliva. Salivary antibody could bedetected from 6 weeks (Fig. 2) to 4 months (Fig.3 and 4) after primary oral challenge with GTFantigens. In addition, peroral antigen adminis-tration evoked a higher salivary response thandid subcutaneous injection of antigen at thetimes tested (Fig. 2A).Both IgA and IgG isotypes have been reported

to occur in hamster intestinal washings and milk(1, 9). IgA has also been regularly identified inhamster saliva (1, 9). On the other hand, al-though Haakenstad and Coe (9) identified theIgG2 subclass in saliva, Bienenstock (1) was onlyable to detect IgA in this secretion. Salivaryantibody directed against antigens contained inthe GTF preparation was detected in both theIgA and IgG isotypes (Fig. 2A). Although thesite of IgG synthesis was not studied directly, itis unlikely that the salivary anti-GTF antibodyof the IgG isotype observed in the present studywas a serum transudate. Salivary IgG antibodycould be detected in hamsters in the absence ofa detectable serum IgG antibody response, for

VOL. 28, 1980

448 SMITH, TAUBMAN, AND EBERSOLE

2 3

Months

BSerum

10 20

M aI.te~~~~~~~~~~~~~~~~to~~~~~~ ".

0 1 2 3 4 5

Months

FIG. 3. Duration and anamnesis of salivary andserum antibodies in hamsters responding to oraladministration of 0.14 IU of GTF antigen complexfor 21 days (primary immunization [1)]) and, 4months later, for 4 days (secondary immunization[20]). Antibody activity is expressed as EU, whichwere obtained by using separate equations for eachisotype (see text). Thus, identical EU for differentisotopes do not necessarily represent equivalentamounts of antibody. The abcissa shows the numberof months after completion of the primary immuni-zation regimen. (A) Salivary IgA (Q) and IgG (0)responses of two to three animals. Brackets enclosetwo standard errors. The slashed bars indicate thedurations ofprimary and secondary oral administra-tions of GTF antigen. Salivas were tested at 1:10dilutions. (B) Serum IgG (0) responses oftwo to threeanimals. Brackets and bars have the same meaningas in (A). Sera were tested at 1:400 dilutions.

example, after the use of low antigen doses (Ta-ble 2) or during the late stages of the primaryresponse in some hamsters (Fig. 3A). Second,the peak serum IgG antibody response occurred21 days after enteric antigen administration wasbegun (Fig. 2B), whereas the salivary IgG re-sponse was still increasing by day 28 (unpub-lished data). The local nature of the salivary IgAantibody response is emphasized by the absenceof a detectable serum IgA response to any oraldose of antigen employed.

O-. IgA Administration of soluble antigens by the oral,0-0 IgG intragastric, or intraduodenal route has been

reported to result in the formation of serumantibody in a variety of animal model systems(17, 19, 22, 29), although the oral route does notseem to favor a serum response to particulatestreptococcal antigens (13, 16). In the presentstudy oral administration of soluble GTF anti-

'Ad gens for 26 or 27 days resulted in a demonstrableserum response, which was predominantly oftheIgG isotype (Fig. 2B and Table 2). However, the

.. serum response to prolonged feeding with the5 6 7 highest dose of GTF (a total of 24.3 IU in 27

days) was 3-fold (IgG) to 15-fold (IgM) less (Fig.2B) than that observed after subcutaneous in-jection of GTF antigen. Also, the serum IgG

lgG antibody response could be selectively elimi-nated by lowering the antigen dosage (Table 2).The measurement ofsalivary and serum activ-

ities by both ELISA and functional inhibitiontechniques allowed the amount of antibody ac-tivity to be compared with the ability of therespective fluids to inhibit enzyme activity. Ingeneral, the data obtained in the two assayscorrelated quite closely, despite the possibilitythat antibody specificities in addition to anti-GTF may be detected by the ELISA. For ex-

6 7 ample, comparison of the serum IgG antibody(EU) (Fig. 2B) with the inhibition units from

1100-

s 900

700

s 500b

f 300

0 100Cl

U.

*-Serum

I10VIIoI 6I

40130 o-o Saliva20

0 1 2 3 4 5 6 7 8Months

FIG. 4. Duration and anamnesis of salivary (0)and serum (0) responses to oral administration of0.14 IU ofGTF antigen complex for 21 days (primaryimmunization [10 ]) and, four months later, for 4 days(secondary immunization [201). Responses weremeasured by the functional inhibition assay and areexpressed as inhibitory units (IU). Brackets enclosetwo standard errors. The slashed bars indicate du-rations of primary and secondary immunizations.The abscissa shows the number ofmonths after com-pletion of the primary immunization regimen.

uw 20

' 150

>s 10

ac 5

0L

60

D 50w

* 400

>, 30

c 20

10

n -- - .

INFECT. IMMUN.

ORAL ADMINISTRATION OF GTF 449

hamsters to which 0.9 IU of GTF was orallyadministered (Table 1) gave a correlation coef-ficient of 0.98 (P < 0.01). Likewise, a similarcomparison of the salivary IgA antibody (Fig.2A) with inhibition in the same samples (Table1) in this experiment showed a correlation coef-ficient of 0.98 (P < 0.01). Although a significantcorrelation was not observed with serum- IgMantibody, this relationship was probably maskedby the high serum IgG antibody concentration.Although the ELISA technique is 8- to 30-foldmore sensitive than the functional inhibitionassay (unpublished data), these results suggestthat the functional inhibition assay can give areliable estimate of the relative amount of anti-GTF antibody activity in serum or saliva.The primary response in the intestine to orally

administered soluble antigen has been reportedto be rather short lived in mice (29) and ham-sters (3). These findings are presumably relatedin part to the relatively short half-life (4.8 days)of intestinal plasma cells (12). Pierce (19) hasreported that primary immune responses in ratintestines to enteric immunization can be mark-edly increased by altering the structural char-acteristics of the antigen. In the present experi-ments the salivary immune response to pro-longed oral antigen administration appeared tobe influenced by dose. However, the salivaryantibody response or inhibitory response inhamsters that received a low dose (4.2 IU in 21days) of antigen (Fig. 3A and 4) decreased rela-tively slowly during the subsequent 2 to 3months, whereas the salivary response in ham-sters given the highest dose of antigen (Fig. 2Band Table 1) dropped severalfold in 6 weeks. Inthe latter experiment hamsters were infectedwith S. mutans 6715 (the organism from whichthe antigen had been obtained) after the primaryimmunization regimen. This dramatic decreasein salivary response may be explained by theadsorption of salivary antibody by organismscolonizing the teeth.

Until recently, the secretary immune systemhad seldom been reported to exhibit aspects ofimmunological memory (31). However, the je-junal immune response increased 19- to 43-foldwhen rats that were locally sensitized to choleraantigens were boosted intraduodenally withcholera toxoid (19). Furthermore, the ability toboost this response lasted for up to 8 months(19). In the present study hamsters orally primedwith 0.14 IU of enzyme antigen per day for 21days showed additional aspects of a local sec-ondary response after oral boosting for 4 days(Fig. 3A and 4). The secondary antibody re-sponse in both salivary isotypes was more rapidand attained somewhat higher levels of salivaryIgG and IgA antibodies. Also, the levels of func-

tional inhibitory activity observed 3.5 monthsafter boosting were similar to levels of inhibitoryactivity seen at the peak of the primary response(Fig. 4). Thus, the form of the antigen, theamount of the antigen, or the mode of entericadministration may influence the degree towhich memory is exhibited by the secretary IgAsystem after local stimulation.The ability to evoke a salivary immune re-

sponse with GTF administered by the oral routeis significant in that immune responses to thisantigen complex can be protective against thepathogenic effects of cariogenic streptococci (25,27, 30). Our studies have shown that groups ofimmune hamsters fed any of the three antigendoses of GTF reported in Table 2 had fewerdental caries than similarly infected controlgroups (27). The results of the present studysuggest, therefore, that potentially protectivelevels of antibody can be expected to occur formany months, provided that occasional boostingdoses of antigen are applied enterically. Furtherinvestigations of enhanced immunogenicity andincreased concentration at the critical site(s) fortriggering a secretary immune response will as-sure more effective use of GTF in oral vaccines.

ACKNOWLEDGMENwIThis research was performed persuant to Public Health

Service grant DE-04733 from the National Institute of DentalResearch and was also supported by Public Health ServiceResearch Career Development awards DE-00024 (to D.J.S.)and DE-00075 (to J.L.E.) from the National Institute of DentalResearch.We thank W. King for helpful suggestions and expert

technical assistance, C. Raymond for secretarial assistance,and J. Bienenstock for the generous gifts of rabbit anti-ham-ster a and u chain sera.

LITERATURE CITED1. Bienenstock, J. 1970. Immunoglobulins of the hamster.

II. Characterization of the yA and other immunoglob-ulins in serum and secretions. J. Immunol. 104:1228-1235.

2. Bienenstock, J., and K. J. Block. 1970. Immunoglobu-lins of the hamster. I. Antibody activity in four immu-noglobulin classes. J. Immunol. 104:1220-1227.

3. Dolezel, J., and J. Bienenstock. 1971. yA and non-yAimmune response after oral and parenteral immuniza-tion of the hamster. Cell. Immunol. 2:458-461.

4. Ebersole, J. L., and J. A. Molinari. 1978. The inductionof salivary antibodies by topical sensitization with par-ticulate and soluble bacterial immunogens. Immunology34:969-979.

5. Ebersole, J. L., M. A. Taubman, and D. J. Smith.1979. Thyinic control of secretary antibody responses.J. Immunol. 123:19-24.

6. Engvall, E., and P. Perlmann. 1972. Enzyme-linkedimmunosorbent assay, ELISA. III. Quantitation of spe-cific antibodies by enzyme-labeled anti-immunoglobulinin antigen-coated tubes. J. Immunol. 109:129-135.

7. Evans, R. T., and R. J. Genco. 1973. Inhibition ofglucosyltransferase activity by antisera to known sero-types of Streptococcus mutans. Infect. Immun. 7:237-241.

8. Gibbons, R. J., and J. van Houte. 1975. Bacterial

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450 SMITH, TAUBMAN, AND EBERSOLE

adherence in oral microbial ecology. Annu. Rev. Micro-biol. 29:19844.

9. Haakenstad, A. O., and J. E. Coe. 1971. The immuneresponse in the hamster. IV. Studies on IgA. J. Immu-nol. 106:1026-1034.

10. Keyes, P. H., and H. V. Jordan. 1964. Periodontallesions in the syrian hamster. III. Findings related to aninfectious and transmissible component. Arch. OralBiol. 9:377-400.

11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

12. Mattioli, C. A., and T. B. Tomasi. 1973. The life span ofIgA plasma cells from the mouse intestine. J. Exp. Med.138:452-460.

13. Mestecky, J., J. R. McGhee, R. R. Arnold, S. M.Michalek, S. J. Prince, and J. L Babb. 1978. Selec-tive induction ofan immune response in human externalsecretions by ingestion of bacterial antigen. J. Clin.Invest. 61:731-737.

14. Michalek, S. M., J. R. McGhee, and J. L. Babb. 1978.Effective immunity to dental caries: dose-dependentstudies of secretary immunity by oral administration ofStreptococcus mutans to rats. Infect. Immun. 19:217-224.

15. Michalek, S. M., J. R. McGhee, J. M. Mestecky, R. R.Arnold, and L. Bozzo. 1976. Ingestion of Streptococ-cus mutans induces secretary immunoglobulin A andcaries immunity. Science 192:1238-1240.

16. Montgomery, P. C., J. Cohn, and E. T. Lally. 1974.The induction and characterization of secretary IgAantibodies. Adv. Exp. Biol. Med. 45:453-462.

17. Montgomery, P. C., K. M. Connelly, J. Cohn, and C.A. Skandera. 1978. Remote-site stimulation of secre-tory IgA antibodies following bronchial and gastricstimulation. Adv. Exp. Biol. Med. 107:113-122.

18. Nichols, R. L., E. S. Murray, and P. E. Nisson. 1978.Use of enteric vaccines in protection against chlamydialinfections of the genital tract and the eye of guinea pigs.J. Infect. Dis. 138:742-746.

19. Pierce, N. F. 1978. The role of antigen form and functionin the primary and secondary intestinal immune re-sponses to cholera toxin and toxoid in rats. J. Exp. Med.148:195-206.

20. Pierce, N. F., W. C. Cray, Jr., and B. K. Sircar. 1978.

Induction of a mucosal antitoxin response and its rolein immunity to experimental canine cholera. Infect.Immun. 21:185-193.

21. Pierce, N. F., and J. L. Gowans. 1975. Cellular kineticsof the intestinal immune response to cholera toxoid inrats. J. Exp. Med. 142:1550-1563.

22. Rothberg, R. M., S. C. Kraft, and R. S. Farr. 1967.Similarities between rabbit antibodies produced follow-ing ingestion of bovine serum albumin and followingparenteral immunization. J. Immunol. 98:386-395.

23. Russell, M. W., S. J. Challacombe, and T. Lehner.1976. Serum glucosyltransferase-inhibiting antibodiesand dental caries in rhesus monkeys immunized againstStreptococcus mutans. Immunology 30:619-627.

24. Smith, D. J., and M. A. Taubman. 1977. Antigenicrelatedness of glucosyltransferase enzymes from Strep-tococcus mutans. Infect. Immun. 15:91-103.

25. Smith, D. J., M. A. Taubman, and J. L. Ebersole.1978. Effects of local immunization with glucosyltrans-ferase fractions from Streptococcus mutans on dentalcaries in hamsters caused by homologous and heterol-ogous serotypes of Streptococcus mutans. Infect. Im-mun. 21:843-851.

26. Smith, D. J., M. A. Taubman, and J. L. Ebersole.1979. Preparation of glucosyltransferase from Strepto-coccus mutans by elution from water-insoluble polysac-charide with a dissociating solvent. Infect. Immun. 23:446-452.

27. Smith, D. J., M. A. Taubman, and J. L. Ebersole.1979. Effect of oral administration of glucosyltransfer-ase antigens on experimental dental caries. Infect. Im-mun. 26:82-89.

28. Somogyi, M. 1945. A new reagent for the determinationof sugars. J. Biol. Chem. 160:61-73.

29. Svennerholm, A.-M., S. Lange, and J. Holmgren.1978. Correlation between intestinal synthesis of spe-cific immunoglobulin A and protection against experi-mental cholera in mice. Infect. Immun. 21:1-6.

30. Taubman, M. A., and D. J. Smith. 1977. Effects of localimmunization with glucosyltransferase fractions fromStreptococcus mutans on dental caries in rats and ham-sters. J. Immunol. 118:710-720.

31. Tomasi, T. B. 1976. The immune system of secretions.Prentice-Hall, Inc., Englewood Cliffs, N.J.

INFECT. IMMUN.