Immunological Cross-Reactivity between Streptococcus mutans and ...

Vol. 58, No. 11

INFECTION AND IMMUNITY, Nov. 1990, p. 3545-3552

0019-9567/90/113545-08$02.00/0 Copyright © 1990, American Society for Microbiology

Immunological Cross-Reactivity between Streptococcus and Human Heart Tissue Examined by CrossImmunization Experiments

mutans

HONGYIN WU AND MICHAEL W. RUSSELL*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Received 23 April 1990/Accepted 8 August 1990

Hyperimmunization of rabbits with Streptococcus mutans or other related cariogenic streptococci sometimes induces serum antibodies that react with human heart muscle. To determine whether antigen I/II (AgI/II), a major surface protein antigen present in most human isolates of these organisms, was responsible for inducing cross-reactive antibodies, we tested it for antigenic similarity to heart components, exploiting the ability of immune systems to mount anamnestic responses to antigens previously encountered. Mice immunized with a strain of Streptococcus pyogenes type M6, known to be heart cross-reactive, or with intact S. mutans cells developed antibodies that could be detected on a human heart sarcolemmal preparation. However, mice immunized with AgI/II and boosted with sarcolemma were unable to develop significant antisarcolemmal antibodies attributable to prior sensitization by AgI/ll. Similarly, AgI/II was unable to recall antisarcolemmal responses in mice previously immunized with sarcolemma. Nevertheless, strong immunoglobulin G antibody responses to AgI/Il were detected at the single-cell level in spleens and as circulating antibodies in all mice immunized with AgI/II or AgI/Vl-bearing S. mutans. We conclude that the ability of S. mutans to induce heart-reactive antibodies is not due to antigenic similarity between AgI/II and components of human heart but may be caused by other cross-reactive antigens in the bacterial cells or by nonspecific stimulation of the immune system.

Immunization with Streptococcus mutans cells and purified antigens has been shown to be protective against dental caries in several animal models (21, 25, 35). However, early optimism over the development of a vaccine against dental caries was tempered by a report that sera from rabbits hyperimmunized with mutans streptococci displayed positive immunofluorescence staining on mammalian heart tissue (42). Different patterns of staining were reported by various investigators, and sera from unimmunized rabbits often displayed similar staining reactions, suggesting that several factors might contribute to this phenomenon (for a review, see reference 35a). AgI/Il (antigen I/II [30]), also termed AgB (36), P1 (15), or spaA (18), one of the major antigenic proteins on the surface of mutans streptococci (26), has been implicated in bacterial adherence to tooth surfaces (12, 34), and antibodies against it have been associated with protection against dental caries in monkeys (23, 37). AgIF, which is thought to correspond to AgI/Il, was reported to be responsible for cross-reactivity between the bacteria and human heart (19). However, the evidence for this association has been questioned, and several alternative explanations are possible (35a). In particular, Ayakawa and colleagues have pursued the observation that Streptococcus rattus (formerly S. mutans serotype b), which does not possess AgI/II, may nevertheless induce heart-reactive antibodies and have obtained evidence that membrane rather than cell wall antigens are responsible (3). On the other hand, Swartzwelder and colleagues have concluded that mutans streptococci nonspecifically stimulate clones of autoreactive B cells (41). We have previously found that rabbit anti-mutans streptococcus antisera that display immunofluorescence staining of human *

heart sections contain antibodies detectable with heart antibut that these antibodies are unable to bind AgI/Il (29). However, most previous investigations of this problem, including those discussed above, were performed with rabbits, which are prone to developing nonspecific or polyclonal responses to bacteria, and their interpretation has remained controversial. To investigate antigenic similarity between S. mutans AgI/lI and human heart tissue, we used mice, which preliminary experiments showed were capable of responding to human heart antigens and of developing putative heartreactive antibodies after immunization with S. mutans. In particular, we developed a new approach using cross-immunization, exploiting the capacity of the immune system to mount anamnestic responses to previously encountered antigenic determinants, instead of analyzing antibodies for cross-reactivity against heterologous antigens. If two different antigens share similar epitopes, then immunizing with one should prime the system to make enhanced responses subsequently to the other. In the present context, immunization with AgI/II, which is alleged to be heart cross reactive, should prime the system to make subsequent responses to heart antigens and, conversely, AgI/II should recall antiheart responses in mice primed with heart antigens. These responses were analyzed in terms of serum antibody production and of the development of antibodysecreting cells in the spleen. The advantage of the latter is that, whereas serum antibodies may persist long after immunization, making booster responses to cross-reactive antigens sometimes difficult to discern, splenic antibody-secreting cells usually subside over a few days or weeks but reappear promptly upon restimulation with the same or a cross-reactive antigen. Furthermore, serum antibody responses are polyclonal in nature, whereas individual anti-

gens

Corresponding author. 3545

3546

INFECT. IMMUN.

WU AND RUSSELL

body-secreting cell responses are inherently monoclonal, so that if cross-reacting antibody-secreting cells developed after cross-immunization, they could be immortalized by hybridoma fusion for further analysis of the cross-reactivity. MATERIALS AND METHODS Preparation of bacteria. S. mutans serotype c strains MT8148 (donated by Suzanne M. Michalek) and IB162 (donated by Kenneth W. Knox) and Streptococcus pyogenes type M6 strain D471 (donated by Susan K. Hollingshead) were grown in tryptic soy broth medium (Difco Laboratories, Detroit, Mich.) overnight at 37°C and killed by adding 0.075% (wt/vol) formaldehyde. The killed bacteria were washed in saline and stored at 4°C in sterile saline containing 0.037% (wt/vol) formaldehyde. The concentration of the bacteria was determined turbidimetrically at 660 nm by reference to standard curves constructed from suspensions of bacteria counted under a microscope. Preparation of bacterial antigens. AgI/II was purified chromatographically from culture supernatants of S. mutans IB162 as described previously (30). S. pyogenes D471 was grown in 4 liters of brain heart infusion medium (Difco) overnight at 37°C and washed twice in sterile 0.02 M phosphate buffer (pH 7.0). The bacteria were suspended in 80 ml of 30% (wt/vol) raffinose in phosphate buffer and digested by adding 8 ml of 1-mg/ml mutanolysin (Sigma Chemical Co., St. Louis, Mo.) and incubating the mixture at 37°C for 1 h. The supernatant was dialyzed thoroughly against phosphate-buffered saline (pH 7.4) containing 0.04% (wt/vol) NaN3 to remove raffinose. The concentration of extracted S. pyogenes wall proteins (SPWP) was determined by the Lowry method (24), and the preparation was stored at 4°C. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 15 bands, including a major band corresponding to a molecular mass of approximately 50 kDa, suggestive of an M protein. Preparation of sarcolemma. Human heart ventricular muscle was obtained at autopsy from individuals who were aged under 50 years and who died of conditions unrelated to heart pathology; the heart muscle was supplied in ice-cold sterile saline within 6 h of death (Tissue Procurement Service of the Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham). To avoid the loss of the potential cross-reactive antigens by overrefining, a crude sarcolemma preparation was used. This preparation was obtained by homogenizing 10 g of fresh heart muscle in 50 ml of 0.05 M CaCl2 for 5 min on ice; the homogenate was centrifuged at 14,000 x g for 30 min at 4°C, washed in saline, subjected to autolysis in filtered water, treated with RNase and DNase, and lyophilized (43). Analysis of the product by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed multiple protein bands in the approximate molecular mass range of 40 to 200 kDa. Immunization of mice. BALB/c mice were bred in a pathogen-free colony, and animals of either sex were used at 8 to 15 weeks of age. Intraperitoneal immunizations were performed with AgI/Il (50 jig in 50 ,ul of pyrogen-free saline emulsified with 50 ,ul of Freund incomplete adjuvant [FIA] per animal), sarcolemma (200 pLg in 50 ,u1 of saline-50 ,ul of FIA), S. mutans (1.2 x 109 cells in 1 ml of saline), or S. pyogenes (1.2 x 109 cells in 1 ml of saline), according to the schedule shown in Table 1. Control injections consisted of saline alone, saline-FIA, or 50 ,ug of keyhole limpet hemocyanin (KLH; Calbiochem, San Diego, Calif.)-FIA (Table

TABLE 1. Immunization schedules Expt Group

Primary immunization (n.oof dss (no. doses)

Interval

(wk)

immunization

ntra

Booster

Booster

A

1 2 3 4 5 6 7

AgI/Il + FIA (1) AgI/Il + FIA (1) Saline + FIA (1) Sarcolemma + FIA (1) Sarcolemma + FIA (1) Saline + FIA (1) Saline + FIA (1)

3-4 3-4 3-4 3-4 3-4 3-4 3-4

AgI/Il + FIA Saline + FIA AgI/Il + FIA Sarcolemma + FIA Saline + FIA Sarcolemma + FIA Saline

B

1 2 3 4 5 6

AgI/Il + FIA (1) KLH + FIA (1) AgI/Il + FIA (1) Sarcolemma + FIA (1) KLH + FIA (1) Sarcolemma + FIA (1)

3-4 3-4 3-4 3-4 3-4 3-4

Sarcolemma + FIA Sarcolemma + FIA KLH + FIA AgI/Il + FIA AgI/Il + FIA KLH + FIA

C

1 2 3 4

AgI/Il + FIA (6)a AgI/Il + FIA (6)a

7 7

Sarcolemma + FIA Saline + FIA Sarcolemma + FIA Saline + FIA

1 2 3a 3b 4

Sarcolemma + FIA (1) Sarcolemma + FIA (1) Sarcolemma + FIA (1) Sarcolemma + FIA (1) Sarcolemma + FIA (1)

3-4 3-4

Sarcolemma + FIA S. pyogenes S. mutans MT8148 S. mutans IB162 KLH + FIA

D

None None

3-4 3-4 3-4

a One dose in FIA, followed after 3 weeks by five doses in saline at weekly intervals.

1). Mice were killed 5 days after the booster injection for evaluation of immune responses in terms of splenic antibody-secreting cells and serum antibodies. Previous experiments indicated that these doses and schedules were appropriate for detecting responses to these antigens (32). ELISPOT assay. The enzyme-linked immunospot (ELISPOT) assay for enumerating specific antibody-secreting cells has been described in detail previously (10, 32). Plastic petri dishes (Falcon 1006, 50-mm diameter; Becton Dickinson, Lincoln Park, N.J.) were coated with optimal concentrations of the antigens (AgI/Il, 10 jig/ml; sarcolemma, 50 ,ug/ml; KLH, 50 ,ug/ml; and SPWP, 20 ,ug/ml) or 10% fetal calf serum as a control and blocked with 5% fetal calf serum. Single-cell suspensions of spleens pooled from groups of three mice were prepared in RPMI 1640 medium with 10% fetal calf serum and counted in a hemacytometer with 0.4% trypan blue to assess viability (always >95%). Suspensions of 1 x 105 to 5 x 106 cells were incubated on the dishes for 3 h at 37°C, and the spots of antibodies bound to the antigen coat were revealed by adding peroxidase-conjugated goat anti-mouse immunoglobulin M (IgM) or IgG (Southern Biotechnology Associates, Birmingham, Ala.), followed by a molten agar substrate containing p-phenylenediamine and H202. The spots were counted under a stereomicroscope, and the mean numbers of spot-forming cells (SFC) in duplicate dishes were expressed per 106 spleen cells plated, less the mean numbers of SFC, if any, detected on the fetal calf serum-coated control dishes. Preliminary experiments showed that spleen SFC were inhibited by cycloheximide, indicating the requirement for protein synthesis in the formation of spots in this assay (9). ELISA. For the enzyme-linked immunosorbent assay (ELISA), microtiter plates were coated with antigens as follows: AgI/HI and SPWP, 5 ,ug/ml; sarcolemma, 20 ,ug/ml; and KLH, 10 ,ug/ml (32). Mouse sera were titrated in five

VOL. 58, 1990

S. MUTANS AND HEART CROSS-REACTIVITY

TABLE 2. Antibody-secreting cells specific for AgI/IH and sarcolemma in spleens of mice immunized with AgI/IH or sarcolemma Antibodies toa:

Immunization

AgI/Il

Group First

Second

AgI/Il Agl/Il AgI/IH FIA FIA AgIl/II Sarcolemma Sarcolemma Sarcolemma FIA FIA Sarcolemma FIA Saline a Mean SFC per 106 spleen cells (pooled

Al A2 A3 A4 A5 A6 A7

Sarcolemma

IgM

IgG

IgM

IgG

0.3 0 0.6 0 0 0 0

215 24 2.9 0.2 0 0 0

0 0 0 0 0 0

0 0 0 18 0 0 ND

ND"

from three mice) in duplicate

dishes. b

ND, Not determined.

wells by serial twofold dilution in phosphate-buffered saline containing 0.05% Tween 20, starting at a dilution appropriate for the expected antibody level, and incubated overnight. Bound antibodies were developed with appropriately diluted peroxidase-conjugated goat anti-mouse IgM or IgG for 4 h, and the color developed with 2,2'-azino-bis(3-ethylbenzthiazoline sulfonic acid)-H202 substrate was measured at 414 nm on a Vmax microplate reader (Molecular Devices Corp., Menlo Park, Calif.). Antibody concentrations were calibrated against mouse reference serum standards assayed simultaneously, by means of a computer program based on four-parameter logistic or logit-log algorithms (31). Statistics. Serum antibody data obtained from individual mice are presented as group means ± 1 standard'deviation and were analyzed statistically by Student's t test or analysis of variance. Splenic antibody-secreting cell data were obtained from pooled spleens and are presented as the means of duplicate determinations, which were in close agreement. RESULTS Response to immunization with AgI/II or sarcolemma. To assess primary and secondary antibody responses to AgI/II or to sarcolemma and to determine whether homologous immunization with either antigen could induce cross-reactivity to the other, we gave mice one or two doses of AgI/Il or sarcolemma or sham immunized them (Table 1, experiment A). Splenic antibody responses were assayed 5 days after the second immunization (Table 2). In mice primed and boosted with AgI/II, a strong response to AgI/HI dominated by IgG antibody-secreting cells was observed. In mice given one

3547

dose of AgI/Il 5 days previously (Table 2, group A3), only a weak response was detected, but mice primed with AgI/Il 1 month previously (Table 2, group A2) showed moderate numbers of IgG antibody-secreting cells. No cells secreting antibodies to sarcolemma were detected in the spleens of these mice. Only mice primed and boosted with sarcolemma developed splenic antibody-secreting cells specific for sarcolemma (Table 2, group A4). The results of the serum antibody assays were generally in agreement with those of the ELISPOT assay of splenic antibody-forming cells (Table 3). High levels of IgG antibodies were found in mice immunized with AgI/II, especially after a secondary immunization (group Al). Mice primed and boosted with AgI/II (group Al) showed somewhat higher levels of IgM and IgG antibodies to sarcolemma than did the control group (A7; P < 0.05, Student's t test), indicating the possible presence of heart-cross-reactive antibodies. Mice immunized with sarcolemma 1 month prior to testing (group A5) displayed serum IgG antibodies to sarcolemma but not to AgI/II. Control mice sham immunized with FIA and saline appeared to have low levels of antibodies to both AgI/II and sarcolemma. Repetition of this experiment again showed slightly higher levels of IgM (but not IgG) antibodies to sarcolemma in mice primed and boosted with AgI/II than in control mice sham immunized with FIA and saline. Cross-immunization experiments. The validity of the crossimmunization approach was tested with a known heartcross-reactive organism, S. pyogenes type M6 (5, 11). Mice were primed by immunization with four doses of S. pyogenes cells (1.2 x 109 cells), rested for 3 weeks, and then boosted with either S. pyogenes cells or sarcolemma in FIA. Serum antibody and splenic antibody-forming cell responses were determined against SPWP and sarcolemma (Fig. 1). Responses to homologous cell wall protein and sarcolemma, especially of the IgM isotype, could be induced by immunization with S. pyogenes and recalled by S. pyogenes or sarcolemma. Mice primed by repeated injections of S. mutans cells showed a greater serum IgM antibody response to sarcolemma after boosting with sarcolemma (6.55 + 2.90 jxg/ml) than after boosting with FIA (1.78 + 1.09 ,ug/ml), indicating that cross-immunization can detect putative heart cross-reactivity induced by S. mutans cells. Response to cross-immunization with AgI/II and sarcolemma. To test whether the antisarcolemmal antibodies found in mice immunized with AgI/II (Table 3, experiment A) were due to antigenic cross-reactivity, we primed mice with AgI/II or with another strongly immunogenic, unrelated protein antigen, KLH, and boosted them with sarcolemma. Conversely, other mice were primed with sarcolemma and

TABLE 3. Serum antibodies to AgI/Il and sarcolemma in mice immunized with AgI/Il or sarcolemma Immunization

Group

First

Antibodies toa: Second

IgM 6.00 ± 1.50 Al AgI/Il AgI/Il 1.40 ± 0.62 FIA A2 AgI/Il 3.06 ± 1.36 A3 FIA AgI/Il 1.24 ± 0.54 Sarcolemma A4 Sarcolemma 0.49 ± 0.05 FIA Sarcolemma A5 0.51 ± 0.15 Sarcolemma FIA A6 1.03 ± 0.24 Saline FIA A7 a Mean concentration (micrograms per milliliter) ± standard deviation (n = 3).

AgI/Il

Sarcolemma

1,060 ± 406 233 1.85 0.25 0.05 0.04 0.22

± 154 ± 1.90

± ± ± ±

IgG

IgM

IgG

0.28 0.05 0.01 0.04

1.93 1.70 1.36 2.43 1.17 1.29 1.04

± 0.06 ± 0.59

± ± ± ± ±

0.47 1.22 0.17 0.24 0.29

0.76 0.52 0.16 10.6 8.19

± 0.07 ± 0.18

± 0.11 ± 3.1 ± 3.10

0 0.27 ± 0.08

INFECT. IMMUN.

WU AND RUSSELL

3548

A

20

A

60

a

0

a

a

0

0 VI

0

IgG

40

coa

10-

* IgM

50

30

a.

0

co

06

20

(0

U.

IL.

n)

10.

Bi B2 B3 B4 B5 B6 Group

S. pyo. Sarc. Booster Immunization

B

1000

100.

* IgM

a

IgM anti-SPWP IgG anti-SPWP IgM anti-sarc. IgG anti-sarc.

0gG

. 0 (0

0

a.

.E 10-

cL (0

0

IL

a-

co

11 Bi B2 B3 B4 B5 B6 Group

C

.1

S. pyo. Sarc. Booster Immunization FIG. 1. Splenic SFC (A) and serum antibodies (B) in mice immunized with S. pyogenes and boosted with either S. pyogenes (S. pyo.) or sarcolemma (Sarc.). Mice were killed 5 days after booster immunizations, and the ELISPOT assay or the ELISA, respectively, was carried out on SPWP or sarcolemma antigens for detecting IgM and IgG responses.

boosted with AgI/II or KLH, while others were primed with Agl/II and boosted with KLH or vice versa (Table 1, experiment B). Priming with AgI/lI elicited homologous responses, as expected, and appeared to promote slightly greater IgM, but not IgG, antisarcolemmal antibody responses upon boosting with sarcolemma, as compared with priming with KLH (Fig. 2, groups Bi and B2; P < 0.05). Furthermore, priming with AgI/Il and boosting with sarcolemma induced higher levels of IgM antisarcolemmal antibodies than did boosting with KLH (Fig. 2, groups Bi and B3; P < 0.05). Only small numbers of IgM antisarcolemmal antibody-secreting cells were found in the spleens of these mice (Fig. 2). Mice primed with sarcolemma and boosted with AgI/lI did not show greater recall responses (serum antibody or splenic SFC) to sarcolemma than did mice boosted with KLH (Fig. 2, groups B4 and B6) or than did mice primed with KLH and boosted with AgI/II (Fig. 2, group B5). Mice boosted with AgI/II after priming with sarcolemma or KLH developed antibodies to AgI/Il, as expected (Fig. 2, groups B4 and B5). In other cross-immunization experiments, mice primed with AgI/II and boosted with sarcolemma developed serum IgM antisarcolemmal responses that were not significantly greater than those of mice primed with AgI/Il and boosted with FIA (P > 0.05).

100001

* IgM

1000

-E

100 1

v10

.1

BI B2 B3 B4 B5 B6

Group

D 10000 1000-

* IgM

bIgG

.1

Bi B2 B3 B4 B5 B6

Group FIG. 2. Splenic SFC (A and B) and serum antibodies (C and D) in mice cross-immunized with AgI/Il and sarcolemma. Mice were killed 5 days after booster immunizations, and the ELISPOT assay or the ELISA, respectively, was carried out on AgI/Il (A and C) or sarcolemma (B and D) antigens for detecting IgM and IgG responses. For immunization groups, see Table 1, experiment B.

VOL. 58, 1990

However, mice primed with sarcolemma and boosted with AgI/Il developed higher levels of serum IgM antibodies to sarcolemma (3.72 + 0.22 jig/ml) than did mice primed with sarcolemma and boosted with FIA (2.01 + 0.51 ,ug/ml) (P < 0.05). The above-described results suggested that a weak IgM antisarcolemmal response could be induced by immunization with AgI/Il but also that an unrelated antigen, KLH, could sometimes induce similar effects. Therefore, we increased the immunological stimulus by repeated immunization with AgI/Il, in the expectation that weak responses to minor epitopes that cross-react with sarcolemma would thereby be enhanced (Table 1, experiment C). In mice hyperimmunized with AgI/Il and boosted with sarcolemma (Table 1, group Cl), no antisarcolemmal antibody-forming cells were detected (data not shown) and serum antibodies to sarcolemma remained at or below the levels found in similarly hyperimmunized mice that were sham boosted with FIA (Fig. 3, group C2) or in control, unimmunized mice that were given booster injections (Fig. 3, groups C3 and C4). All mice hyperimmunized with AgI/II developed strong IgGdominated antibody responses to AgI/lI (Fig. 3). Recall of responses in mice immunized with sarcolemma. The results described above indicated that the soluble proteins AgI/Il and KLH could not effectively recall responses in mice primed with sarcolemma (Fig. 2), whereas boosting with sarcolemma elicited homologous IgM and IgG responses (Tables 2 and 3). To investigate whether streptococcal cells could recall responses to sarcolemma, we primed mice with sarcolemma and boosted them with sarcolemma, KLH, or cells of S. mutans or S. pyogenes (Table 1, experiment D). Two strains of S. mutans, MT8148 and IB162 (which releases most of its surface AgI/II into the culture medium), were used. Boosting with S. mutans MT8148 or S. pyogenes resulted in IgM antibody responses to AgI/Il and to S. pyogenes cell wall protein, respectively. However, S. mutans IB162, which lacks cell wall AgI/II, failed to elicit antibodies to AgI/I1 (Fig. 4). Levels of IgM antisarcolemmal antibody-secreting spleen cells and antibodies were highest in mice boosted with sarcolemma and were lower, although similar, in mice boosted with any of the streptococcal strains (Fig. 4). However, boosting with KLH also resulted in detectable IgM antisarcolemmal antibody responses. No significant IgG responses were generated in these animals. In a further experiment, mice were immunized four times with sarcolemma and boosted after 3 weeks with sarcolemma or other antigens, and serum IgM and IgG antibodies to sarcolemma were determined (Table 4). Although the trend of these data suggested that S. pyogenes or S. mutans was as effective as sarcolemma and more effective than AgI/II or KLH in recalling antisarcolemmal antibodies, multiple-comparison analysis of variance revealed no significant differences among the groups. DISCUSSION The original observation of van de Rijn et al. (42) that sera from rabbits hyperimmunized with cells of mutans streptococci sometimes show reactivity with human heart antigens has been reproduced by several other groups, including ours (14, 15, 29, 39), but the mechanisms responsible have been difficult to establish. Bleiweis et al. have concentrated on their early discovery that a cross-reactive antigen was present in the cell membrane of mutans streptococci (42) and have subsequently identified several membrane components that may be similar to those found in group A streptococci

S. MUTANS AND HEART CROSS-REACTIVITY

3549

A

50

* IgM 0

40

0

30

a

40

40

U-

cnLCL C1

C2 Group

C3

C4

B

10000] * IgM

m IgG

1000

100 10 1

C1

C2 C3 Groiup

C4

C

1000

* IgM 100 E

10

Ii .1

C1

C2

C4 C3 Group FIG. 3. Splenic SFC (A) and serum antibodies (B and C) in mice repeatedly immunized with AgI/Il. Mice were killed 5 days after booster immunizations, and the ELISPOT assay or the ELISA, respectively, was carried out on AgI/Il (A and B) or sarcolemma (C) antigens for detecting IgM and IgG responses. For immunization groups, see Table 1, experiment C. No splenic SFC responses were detected for sarcolemma in any group (data not shown).

(1, 3, 13), which are acknowledged to induce heart-crossreactive antibodies in acute rheumatic fever (6, 16). Lee et al. have demonstrated that a clone of S. mutans in which the AgI/II gene has been specifically inactivated (22) retains the capacity of the parent strain to elicit heart-reactive antibodies (S. F. Lee, P. J. Crowley, and A. S. Bleiweis, J. Dent. Res. 68:963, 1989). In contrast, Stinson et al. (40) and Swartzwelder et al. (41) have pursued the frequent finding that preimmune rabbit sera often show similar immunofluo-

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WU AND RUSSELL

TABLE 4. Serum antibodies to sarcolemma in mice repeatedly immunized with sarcolemma and boosted with various antigens

A * IgM

10-

Immunization

Antisarcolemmal antibodies':

Group U

1 2 3 4 5 6

0

10 I 0 IUL

Primary

Booster

Sarcolemma Sarcolemma Sarcolemma Sarcolemma Sarcolemma Sarcolemma

Sarcolemma S. pyogenes S. mutans

AgI/Il KLH FIA

IgM

6.84 6.20 6.92 4.05 3.54 2.77

" Mean concentration (micrograms per milliliter)

± ± ± ± ± ±

IgG

3.31 4.95 0.46 1.59 1.02 0.20

1.22 1.31 1.35 0.54 1.28 0.70

± ± ± ± ± ±

0.28 0.83 0.51 0.38 0.33 0.74

± standard deviation (n =

3).

DI

D4

A lgM

10 _

D3a D3b Group

D2

O

Ag 1/Il

SPUP

Ag I/1I

2-

0

4I

n

D3a D3b Group

D2

KLH

[ D4

C 1001

* IgM

0 IgG 10

T

0

I

.1

Dl

D2

D3a D3b Group

D4

D 100

U IgM SPWP

Ag l/ll

0 IgG

10

|/I| Ag

KLH

rescence reactions to human heart and have concluded that immunization with mutans streptococci results either in nonspecific stimulation of preexisting clones of heart-reactive B lymphocytes or in muscle fiber injury which exposes hidden determinants and provokes autoantibodies. Despite the fact that S. rattus (formerly S. mutans serotype b), which was known not to express AgI/Il (33, 36), could nevertheless induce heart-reactive antibodies in rabbits, Hughes et al. reported that AgIF (AgI/Il) was responsible for cross-reactivity with human heart tissue (19). However, these findings are amenable to several alternative explanations (reviewed in reference 35a). Polyclonal antisera to AgI/II, as well as a large number of monoclonal antibodies, do not react with human heart (2, 4, 38; M. W. Russell, unpublished observations) and, furthermore, heart-reactive antibodies in the sera of rabbits immunized with S. mutans or Streptococcus sobrinus cells also do not recognize AgI/Il (29). The present experiments subject our previous contention that AgI/Il does not display antigenic similarity to human heart tissue to more rigorous testing by means of a new approach based on anamnestic responses to previously encountered antigens. Responses were analyzed at the single-spleen-cell level and in terms of serum antibodies in mice immunized with AgI/II and sarcolemma. In addition to the IgM and IgG isotype data presented, we also determined IgA antibody responses, but the results did not add to or alter the conclusions. The results from experiment A showed that homologous primary and secondary immunizations with either AgI/II or sarcolemma induced antibody responses against the immunizing antigen. Furthermore, secondary responses to AgI/I1 were substantially greater than were primary responses, and antibody-secreting cells specific for sarcolemma were detected only after secondary immunization, although low levels of serum antibodies to sarcolemma persisted after primary immunization 1 month previously. In mice cross-immunized first with AgI/II and then with sarcolemma (experiment B), a weak response to sarcolemma occurred, but this was not enhanced by repeated immunization with AgI/II (experiment C, groups 1 and 2). Furthermore, immunization with KLH was nearly as effective as that with AgI/Il in priming a subsequent response to sarco-

1

.1 D2

D3b D3a Group

D4

FIG. 4. Splenic SFC (A and B) and serum antibodies (C and D) in mice immunized with sarcolemma and boosted with sarcolemma, S. pyogenes, S. mutans, or KLH. Mice were killed 5 days after booster immunizations, and the ELISPOT assay or the ELISA, respectively, was carried out on sarcolemma antigens (A and C) or antigens appropriate to the booster immunization (B and D) for detecting IgM and IgG responses. For immunization groups, see Table 1, experiment D.

VOL. 58, 1990

lemma and, conversely, boosting with KLH was as effective as that with AgI/II in recalling a response to primary immunization with sarcolemma (experiment B). In contrast, sham boosting with FIA, even after repeated immunization with AgI/II, did not elicit significant numbers of antisarcolemmal antibody-secreting cells. We are therefore left with the conclusion that potent immunogens such as AgI/Il and KLH have the capacity to promote weak, nonspecific responses to unrelated antigens, possibly by polyclonal or bystander stimulation of antibody-secreting cells. In contrast, immunization with S. pyogenes cells did induce IgM responses to sarcolemma, and these were also recalled by boosting with sarcolemma. Thus, our assays for serum antibodies and antibody-secreting cells were capable of detecting heart-cross-reactive antibodies induced by an organism known to have this property. Finally, boosting with S. pyogenes appeared to recall antisarcolemmal antibodysecreting cells in mice primed with sarcolemma to a slightly greater extent than did boosting with S. mutans or with KLH but not as much as did boosting with sarcolemma (experiment D). Antisarcolemmal antibodies in mice primed once with sarcolemma and boosted with S. pyogenes, S. mutans, or KLH were similar, although small differences in antisarcolemmal antibodies, which were parallel in IgM and IgG isotypes, were apparent when mice were repeatedly immunized with sarcolemma and boosted with these antigens. In this instance, sarcolemma, S. pyogenes, or S. mutans recalled similar levels of antisarcolemmal antibodies, higher than those recalled by AgI/Il, KLH, or FIA. Furthermore, the ability of different strains of S. mutans to recall responses to sarcolemma did not vary according to their content of AgI/II and their consequent ability to stimulate anti-AgL/II antibodies (experiment D, groups 3a and 3b). Although we showed that our mouse model was capable of responding to human heart antigen and of developing heartcross-reactive antibodies upon immunization of mice with S. pyogenes, it is possible that other inbred strains of mice would react differently because of genetic factors, which are known to be involved in human susceptibility to acute rheumatic fever (16). Nevertheless, our conclusions from this model agree with those obtained by us and others with outbred rabbits (29, 41); furthermore, our analysis of samples of human sera having high levels of anti-AgI/II antibodies, due to subacute bacterial endocarditis associated with S. mutans, indicated no cross-reactivity with sarcolemma (H. Wu and M. W. Russell, FASEB J. 4:A1025, 1990). These results, together with those described previously, demonstrate that AgI/II does not express determinants that induce antibodies which cross-react with epitopes on human sarcolemma preparations. Although such preparations contained multiple components and could detect cross-reactive antibodies induced by S. pyogenes (43) as well as by intact S. mutans, antibodies to antigens not present in these preparations would have been missed. AgI/Il is a major surface protein antigen on most mutans streptococci, except for S. rattus (26), but it has no homology with the M proteins, which are implicated in the heart cross-reactivity displayed by some types of S. pyogenes (5, 6, 11, 16). The sequence of AgI/II has been independently determined by two groups (20, 27), and it does not show the characteristics of the sequences of the M proteins, nor does it contain a sequence similar to Gln-Lys-Ser-Lys-Gln, which has been found to be involved in the heart cross-reactivity of the M5 protein (7). Nevertheless, these experiments illustrate the potent immunogenicity of AgI/II described previously (32). Whether this property can promote nonspecific stimulation of unrelated

S. MUTANS AND HEART CROSS-REACTIVITY

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clones of B cells destined to produce autoreactive antibodies, as indicated by some results with AgI/II (and also with another potent large protein antigen, KLH), needs further investigation. This explanation of heart cross-reactivity is favored by Stinson et al. (40). It is interesting that monoclonal and polyclonal anti-S. pyogenes antibodies that crossreact with heart muscle myosin also react with membrane components of S. mutans and S. pyogenes (1, 3, 13). It is thus possible that a factor, perhaps associated with certain types of M proteins, promotes the induction of crossreactive antibodies by streptococci and that, once formed, these antibodies can react with a broader range of antigens. In this regard, it is notable that autoantibodies often display a broad specificity for a variety of seemingly unrelated

antigens (8, 17, 28). AgI/II has been found to induce protective immunity against dental caries in monkey models when given by parenteral injection, resulting in a predominant serum IgG antibody response (23). However, any vaccine proposed for use against dental caries must be shown free of significant adverse effects beyond the induction of heart-reactive antibodies. Furthermore, most individuals are colonized by mutans streptococci during their lifetimes and have detectable serum antibodies to AgI/II and other antigens. If these are potentially harmful, it is desirable to identify them to and assess their pathological significance. Our results, together with those of others, indicate that AgI/Il is not responsible for the induction of heart-cross-reactive antibodies, despite earlier allegations, although other components of mutans streptococci may have this property. ACKNOWLEDGMENTS We thank Pamela White for technical assistance in growing bacteria and purifying antigens, Lucia Kulhavy and Dorothy Hooks for animal care, Cecil Czerkinsky for valuable suggestions relating to this investigation, Katherine A. Kirk for help with analysis of variance, Suzanne M. Michalek and Susan Jackson for critical assessment of the manuscript, and Maria Bethune for secretarial assistance during the course of the study. This work was supported by Public Health Service grants DE06746, DE08182, and DE08228 from the National Institutes of Health. LITERATURE CITED 1. Ayakawa, G. Y., A. S. Bleiweis, P. J. Crowley, an,) M. W. Cunningham. 1988. Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin. J. Immunol. 140:253-257. 2. Ayakawa, G. Y., L. W. Boushell, P. J. Crowley, G. W. Erdos, W. P. McArthur, and A. S. Bleiweis. 1987. Isolation and characterization of monoclonal antibodies specific for antigen P1, a major surface protein of mutans streptococci. Infect. Immun. 55:2759-2767. 3. Ayakawa, G. Y., J. L. Siegel, P. J. Crowley, and A. S. Bleiweis. 1985. Immunochemistry of the Streptococcus mutans BHT cell membrane: detection of determinants cross-reactive with human heart tissue. Infect. Immun. 48:280-286. 4. Bergmeier, L. A., and T. Lehner. 1983. Lack of antibodies to human heart tissue in sera of rhesus monkeys immunized with Streptococcus mutans antigens and comparative study with rabbit antisera. Infect. Immun. 40:1075-1082. 5. Bessen, D., K. F. Jones, and V. A. Fischetti. 1989. Evidence for two distinct classes of streptococcal M protein and their relationship to rheumatic fever. J. Exp. Med. 169:269-283. 6. Cunningham, M. W., K. K. Krisher, R. A. Swerlick, L. A. Barnett, and P. F. Guderian. 1988. Molecular mimicry: streptococci and myosin, p. 413-423. In H. Kohler and P. T. LoVerde (ed.), Vaccines: new concepts and developments. Longman Scientific and Technical, Harlow, England.

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7. Cunningham, M. W., J. M. McCormack, P. G. Fenderson, M.-K. Ho, E. H. Beachey, and J. B. Dale. 1989. Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SER-LYS-GLN in M protein. J. Immunol. 143:2677-2683. 8. Cunningham, M. W., and R. A. Swerlick. 1986. Polyspecificity of antistreptococcal murine monoclonal antibodies and their implications in autoimmunity. J. Exp. Med. 164:998-1012. 9. Czerkinsky, C. 1986. Antibody secreting cells, p. 23-44. In H. U. Bergmeyer, J. Bergmeyer, and M. Grassl (ed.), Methods of enzymatic analysis, vol. 3. VCH Verlagsgesellschaft mbH, Weinheim, Federal Republic of Germany. 10. Czerkinsky, C., L.-A. Nilsson, H. Nygren, 0. Ouchterlony, and A. Tarkowski. 1983. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65:109-121. 11. Dale, J. B., and E. H. Beachey. 1985. Multiple heart-crossreactive epitopes of streptococcal M proteins. J. Exp. Med. 161:113-122. 12. Douglas, C. W. I., and R. R. B. Russell. 1984. Effect of specific antisera upon Streptococcus mutans adherence to saliva-coated hydroxyapatite. FEMS Microbiol. Lett. 25:211-214. 13. Doyle, G., D. Everhart, C. Mallett, G. Ayakawa, and A. S. Bleiweis. 1986. Demonstration of shared antigenic determinants between Streptococcus mutans BHT cell membrane, human heart tissue and myosin using monoclonal antibodies to S. mutans. J. Gen. Microbiol. 132:2885-2892. 14. Ferretti, J. J., C. Shea, and M. W. Humphrey. 1980. Crossreactivity of Streptococcus mutans antigens and human heart tissue. Infect. Immun. 30:69-73. 15. Forester, H., N. Hunter, and K. W. Knox. 1983. Characteristics of a high molecular weight extracellular protein of Streptococcus mutans. J. Gen. Microbiol. 129:2779-2788. 16. Froude, J., A. Gibofsky, D. R. Buskirk, A. Khanna, and J. B. Zabriskie. 1989. Cross-reactivity between streptococcus and human tissue: a model of molecular mimicry and autoimmunity. Curr. Top. Microbiol. Immunol. 145:5-26. 17. Guilbert, B., G. Dighiero, and S. Avrameas. 1982. Naturally occurring antibodies against nine common antigens in human sera. I. Detection, isolation, and characterization. J. Immunol. 128:2779-2787. 18. Holt, R. G., Y. Abiko, S. Saito, M. Smorawinska, J. B. Hansen, and R. Curtiss. 1982. Streptococcuis mutans genes that code for extracellular proteins in Escherichia coli K-12. Infect. Immun. 38:147-156. 19. Hughes, M., S. M. Machardy, A. J. Sheppard, and N. C. Woods. 1980. Evidence for an immunological relationship between Streptococcus mutans and human cardiac tissue. Infect. Immun. 27:576-588. 20. Kelly, C., P. Evans, L. Bergmeier, S. F. Lee, A. Progulske-Fox, A. C. Harris, A. Aitken, A. S. Bleiweis, and T. Lehner. 1989. Sequence analysis of the cloned streptococcal cell surface antigen I/Il. FEBS Lett. 258:127-132. 21. Krasse, B., C.-G. Emilson, and L. Gahnberg. 1987. An anticaries vaccine: report on the status of research. Caries Res. 21:255-

276. 22. Lee, S. F., A. Progulske-Fox, G. W. Erdos, D. A. Piacentini, G. Y. Ayakawa, P. J. Crowley, and A. S. Bleiweis. 1989. Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II). Infect. Immun. 57:3306-3313. 23. Lehner, T., M. W. Russell, J. Caldwell, and R. Smith. 1981. Immunization with purified protein antigens from Streptococcus mutans against dental caries in rhesus monkeys. Infect. Immun. 34:407-415. 24. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

INFECT. IMMUN. 25. Michalek, S. M., and N. K. Childers. 1990. Development and outlook for a caries vaccine. Crit. Rev. Oral Biol. Med. 1:37-54. 26. Moro, I., and M. W. Russell. 1983. Ultrastructural localization of protein antigens I/II and III of Streptococcus mutans. Infect. Immun. 41:410-413. 27. Okahashi, N., C. Sasakawa, M. Yoshikawa, S. Hamada, and T. Koga. 1989. Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries. Mol. Microbiol. 3:673-678. 28. Prabhakar, B. S., J. Saegusa, T. Onodera, and A. L. Notkins. 1984. Lymphocytes capable of making monoclonal autoantibodies that react with multiple organs are a common feature of the normal B cell repertoire. J. Immunol. 133:2815-2817. 29. Russell, M. W. 1987. Analysis of heart-reactive antibodies induced in rabbits by immunization with Streptococcus mutans. J. Oral Pathol. 16:234-240. 30. Russell, M. W., L. A. Bergmeier, E. D. Zanders, and T. Lehner. 1980. Protein antigens of Streptococcus mutans: purification and properties of a double antigen and its protease-resistant component. Infect. Immun. 28:486-493. 31. Russell, M. W., T. A. Brown, J. Radl, J. J. Haaijman, and J. Mestecky. 1986. Assay of human IgA subclass antibodies in serum and secretions by means of monoclonal antibodies. J. Immunol. Methods 87:87-93. 32. Russell, M. W., C. Czerkinsky, and Z. Moldoveanu. 1986. Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans. Infect. Immun. 53:317-323. 33. Russell, M. W., and T. Lehner. 1978. Characterization of antigens extracted from cells and culture fluids of Streptococcus mutans serotype c. Arch. Oral Biol. 23:7-15. 34. Russell, M. W., and B. Mansson-Rahemtulla. 1989. Interaction between surface protein antigens of Streptococcus mutans and human salivary components. Oral Microbiol. Immunol. 4:106111. 35. Russell, M. W., and J. Mestecky. 1986. Potential for immune intervention against dental caries. J. Biol. Buccale 14:159-175. 35a.Russell, M. W., and H. Wu. 1990. Steptococcus mutans and the problem of heart cross-reactivity. Crit. Rev. Oral Biol. Med. 1: 191-205. 36. Russell, R. R. B. 1979. Wall-associated protein antigens of Streptococcus mutans. J. Gen. Microbiol. 114:109-115. 37. Russell, R. R. B., D. Beighton, and B. Cohen. 1982. Immunization of monkeys (Macaca fascicularis) with antigens purified from Streptococcus muitans. Br. Dent. J. 152:81-84. 38. Smith, R., T. Lehner, and P. C. L. Beverley. 1984. Characterization of monoclonal antibodies to Streptococcus mutans antigenic determinants I/Il, I, II, and III and their serotype specificities. Infect. Immun. 46:168-175. 39. Stinson, M. W., R. J. Nisengard, M. E. Neiders, and B. Albini. 1983. Serology and tissue lesions in rabbits immunized with Streptococcus mutans. J. Immunol. 131:3021-3027. 40. Stinson, M. W., F. J. Swartzwelder, R. J. Nisengard, and B. Albini. 1988. Antigens shared by Streptococcus mutans and cardiac muscle-a reevaluation, p. 424-434. In H. Kohler and P. T. LoVerde (ed.), Vaccines: new concepts and developments. Longman Scientific and Technical, Harlow, England. 41. Swartzwelder, F. J., P. K. Barua, B. Albini, and M. W. Stinson. 1988. Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans. J. Immunol. 140:954-961. 42. van de Rijn, I., A. S. Bleiweis, and J. B. Zabriskie. 1976. Antigens in Streptococcus mutans cross reactive with human heart muscle. J. Dent. Res. 55(Special Issue C):C59-C64. 43. van de Rijn, I., J. B. Zabriskie, and M. McCarty. 1977. Group A streptococcal antigens cross-reactive with myocardium. Purification of heart-reactive antibody and isolation and characterization of the streptococcal antigen. J. Exp. Med. 146:579-599.