INFECTION AND IMMUNITY, Feb. 1988, p. 400-404 Vol. 56, No. 2 0019-9567/88/020400-05$02.00/0 Copyright © 1988, American Society for Microbiology Cross-Neutralization of Staphylococcal and Streptococcal Pyrogenic Toxins by Monoclonal and Polyclonal Antibodies GREGORY A. BOHACH,* CAROLYN J. HOVDE,t JEFFREY P. HANDLEY, AND PATRICK M. SCHLIEVERT Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received 3 August 1987/Accepted 2 November 1987 We evaluated cross-reactivity of antibodies against staphylococcal and streptococcal pyrogenic toxins. Monoclonal antibodies against staphylococcal enterotoxin (ET) Cl and streptococcal pyrogenic exotoxin (SPE) A were tested for reactivity with homologous and heterologous pyrogenic toxins in vitro. Ten immunoglobulin Gl anti-ET Cl monoclonal antibodies showed little or no cross-reactivity in an enzyme-linked immunosorbent assay, but many of these could neutralize the mitogenic effect of ET B, SPE A, or both. Two immunoglobulin M anti-ET Cl monoclonal antibodies and eight immunoglobulin M anti-SPE A monoclonal antibodies showed extensive cross-reactivity in the enzyme-linked immunosorbent assay and the mitogenicity neutralization assay. No cross-reactivity was observed with SPE C or toxic shock syndrome toxin 1. Rabbits immunized against ET B, ET Cl, or SPE A were resistant to challenge with the immunizing toxin. In addition, reciprocal immunity was stimulated by the two ETs, and immunity to SPE A provided protection against ET B but not ET C1. These results show that staphylococcal and streptococcal pyrogenic toxins which share sequence homology have common antigenic determinants which may not be detected in Ouchterlony immunodiffusion assays.

Staphylococcus aureus and Streptococcus pyogenes pro- says, can be demonstrated in more sensitive competition and duce a family of pyrogenic toxins (PTs) with similar immu- immunoprecipitation assays (28, 29). nobiological and biochemical properties. These include the The use of neutralization assays has been a sensitive streptococcal pyrogenic exotoxins (SPEs) A through C and method for identification and classification of ETs based on staphylococcal toxic shock syndrome toxin 1 (TSST-1), their antigenic diversity (3, 4, 11). Several biological activi- enterotoxins (ETs) A through E, and pyrogenic exotoxins A ties of the staphylococcal and streptococcal PTs can be and B. All of these toxins are pyrogenic and immunosup- neutralized by homologous antibodies in vitro or through pressive as a result of ability to induce nonspecific T active immunization (3, 4, 7, 10, 11). Likewise, the emetic lymphocyte mitogenicity and enhance host susceptibility to activity of some staphylococcal ETs can be neutralized by lethal endotoxin shock (17, 20, 23, 27, 30). In addition to heterologous anti-ET antibodies (4). However, cross-neu- these shared biological activities, SPEs and ETs also have tralization between staphylococcal and streptococcal PTs by unique properties. SPEs induce cardiac tissue damage (26), heterologous antibodies has not been demonstrated. Brun- and ETs cause vomiting and diarrhea (5, 12). son and Watson (10) showed that immunity to SPEs or ET A Recent reports have provided evidence that the shared does not provide protection against challenge with heterol- immunobiological properties of some PTs may result from ogous toxins. Similar studies have not been done to assess structural similarities. For instance, ET B, ET Cl, and SPE cross-neutralization between SPE A and staphylococcal ET A are small proteins with molecular weights between 25,000 B and ET Cl, toxins with well-documented structural simi- and 29,000 and share significant amino acid and nucleotide larities and sequence homology. In this report, the cross- sequence homology (8a, 14, 25, 31). In contrast, TSST-1, reactivity of monoclonal antibodies (MAbs) and polyclonal also a small protein of molecular weight 22,049, does not antibodies against several PTs was evaluated in vitro and in share homology with these three proteins (6). Other PTs vivo. have not been characterized sufficiently to permit compari- son. MATERIALS AND METHODS Despite the structural and immunobiological similarities, Toxin purification. S. aureus MNDon (8) was used to most PTs have classically been regarded as being antigeni- prepare ET Cl. TSST-1 was isolated from S. aureus MN8 cally distinct (5, 11). Except for cross-reactivity between ET (22). SPE A and SPE C were prepared from cultures of S. A and ET E (4) and between ET Cl, ET C2, and ET C3 (1, pyogenes strains NY5 (2) and T18P (21), respectively. ET B 19), serological assays do not indicate the presence of shared was purified from S. aureus MNHO, a clinical isolate from a antigenic determinants. In our laboratory and others (3), patient with nonmenstrual toxic shock syndrome. Each hyperimmune antisera against purified PTs do not cross- strain was grown in pyrogen-free dialyzable beef heart react in vitro with heterologous toxins in immunodiffusion medium, and toxins were purified from ethanol precipitates assays. Different results were obtained by Hynes et al. (13), by preparative thin-layer isoelectric focusing (24). Toxin who reported cross-reactivity between ET B, ET Cl, and identity was confirmed by Ouchterlony immunodiffusion SPE A. Others have shown that ET B and ET Cl possess with hyperimmune antisera. Purity was assessed by sodium shared antigenic determinants which, despite their apparent dodecyl sulfate-polyacrylamide gel electrophoresis followed lack of serological cross-reactivity in immunodiffusion as- by Coomassie blue staining of gels. Each PT preparation contained a homogeneous protein band with a size identical * Corresponding author. to the established molecular weight for the toxin. t Present address: Department of Microbiology and Molecular MAb production. Female BALB/c WAT mice (8 weeks Genetics, Harvard Medical School, Boston, MA 02115. old) were immunized twice weekly with ET Cl for 6 weeks 400 VOL. 56, 1988 CROSS-NEUTRALIZATION OF PYROGENIC TOXINS 401

TABLE 1. Characterization of anti-ET Cl MAbs: neutralization of PT'-induced mitogenicity ELISA titer' against: 103 cpm (P) when MAb were preincubated withc: MAb Isotype ET C1 ET B SPE A ET C1d ET Bd SPE A' 3F6A10 IgM 8,000 16,000 2,000 83.4 (<0.02) 84.3 (-) 158.1 (<0.009) 3B8F8 IgM 256,000 2,000 256,000 3.3 ( 0.1). Results are means of these test wells. d Murine splenocyte indicators. Rabbit splenocyte indicators. f Controls were prepared from ascites induced by X63-Ag8.653 cells. or with SPE A for 6 to 24 weeks. Initial doses of ET Cl or of ascites used. X63-Ag8.653 control ascites was treated SPE A (25 ,ug) were prepared in Freund complete adjuvant identically. (day 1) or Freund incomplete adjuvant (days 5, 12, and 19). Each PT (0.1 or 0.01 ,ug in RPMI 1640 medium) was mixed Adjuvant was omitted from subsequent doses. Toxin immu- with 5 ,ul of MAb solution in a final volume of 50 ,u1. The nizations were administered by the intraperitoneal route. mixture was incubated for 2 h (37°C) to promote antigen- The mice were sacrificed 2 days postimmunization. Their antibody binding. Indicator splenocytes were added in 200 ,u1 splenocytes were fused with X63-Ag8.653 murine myeloma of RPMI 1640 medium supplemented with 2% fetal bovine cells (16) by using 50o (vol/vol) polyethylene glycol PEG serum, 2 mM L-glutamine, penicillin (100 U/ml), and strep- 500 (M. A. Bioproducts, Walkersville, Md.). The fused cells tomycin (100 ,ug/ml), and the residual mitogenic activity of were grown in the presence of hypoxanthine (1 x 10-1 mM), the toxins was measured. aminopterin (4 x 10-4 mM), and thymidine (3.2 x 10-2 mM) Rabbit protection assays. For use in protection assays to select for lymphocyte-myeloma hybrids. American Dutch Belted rabbits (1.3 to 2.3 kg) were immu- Culture supernatants were screened for antibody by an nized biweekly with ET B, ET Cl, or SPE A as described enzyme-linked immunosorbent assay (ELISA) as described previously (21). Immunizations were continued until serum previously (7). Cells in. positive wells were cloned by limit antibodies against the immunizing toxin were detected by dilution and injected into pristane-sensitized mice by stan- Ouchterlony immunodiffusion. Seven days after the final dard techniques (9). Control ascites fluid was prepared by booster, the rabbits were challenged with the immunizing or injection of mice with unfused X63-Ag8.653 cells. heterologous PT. Neutralization of the toxin in vivo was MAb characterization. To measure the relative titer of evaluated by measuring the susceptibility of the animals to each MAb, serial twofold dilutions (1:500 to 1:1,024,000) of lethal endotoxin shock (16). Briefly, immune rabbits were the ascitic fluids were tested by ELISA for reactivity with given an initial intravenous injection of PT (5 ,ug/kg). En- the homologous and other PTs. The titer was arbitrarily hanced susceptibility of the animals to lethal endotoxin designated as the reciprocal of the highest dilution that gave shock was indicated by death of the animals after receiving an A490 of greater than or equal to 0.1. Absorbance measure- a second injection consisting of 1/50 of a 50% lethal dose of ments were made with a Minireader II (Dynatech Laborato- endotoxin (10 p.g/kg). Endotoxin was prepared from Salmo- ries, Inc., Alexandria, Va.). MAb typing was also performed nella typhimurium by the method of Westphal and Jann (32) by ELISA with light chain- and subclass-specific antibody and was administered 4 h after the initial dose of PT. conjugates (Southern Biotechnology Associates, Inc., Bir- Statistics. Mitogenicity neutralization data were analyzed mingham, Ala.). by the Student t test of unpaired, normally distributed data. Mitogenicity neutralization assays. Mitogenicity of PTs was Data from rabbit protection studies were analyzed by the assessed using murine (BALB/cWAT) or rabbit (Atmerican Fisher exact probability test. Dutch-Belted) splenocytes as indicators of proliferation. Incorporation of [3H]thymidine (Amersham Corp., Arling- RESULTS ton Heights, Ill.) into splenocyte cellular DNA was used as an index of proliferation in a 4-day assay (18). MAb characterization. Twelve anti-ET Cl MAbs and eight For use in neutralization assays, MAbs were partially anti-SPE A MAbs were tested for immunological reactivity purified from ascitic fluids by precipitation with (NH4)2SO4 with homologous and heterologous PTs by ELISA. The (33%, wt/vol). The antibodies were dissolved and dialyzed baseline titer of control ascites induced by unfused X63- exhaustively against 0.1 M Na2CO3-0.5 M NaCl. The anti- Ag8.653 cells was consistently 500 or less against all PTs. body solutions were adjusted to contain the original volume Table 1 shows the results of antibody typing and titers of 402 BOHACH ET AL. INFECT. IMMUN.

TABLE 2. Characterization of anti-SPE A MAbs: neutralization of PT-induced mitogenicitya ELISA titer against: 103 cpm (P) when MAb were preincubated with: MAb Isotype SPE A ET B ET C1 SPE A ET B ET C1 EllB IgM 32,000 32,000 32,000 26.1 (<<0.001) 52.6 (<0.05) 88.1 (-) 2C1C7 IgM 8,000 500 500 46.2 (<0.001) 64.7 (<0.02) 59.4 (<0.008) 8E9E3 IgM 8,000 8,000 8,000 24.8 (<-0.001) 30.7 (<0.008) 27.8 (<0.001) 11-1-E5 IgM 16,000 500 500 42.5 (<0.001) 78.9 (-) 42.8 (<0.003) 11-15-D8 IgM 8,000 4,000 8,000 72.2 (<0.02) 72.1 (-) 67.0 (<0.02) 11-29-C3 IgM 128,000 128,000 128,000 99.4 (-) 89.1 (-) 65.0 (<0.008) 11-17-B7 IgM 8,000 8,000 8,000 NDb ND ND 11-28-A2 IgM 8,000 500 500 ND ND ND Control 500 <500 500 110.6 (-) 85.8 (-) 96.4 (-) a See footnotes a through f of Table 1. b ND, Not determined. anti-ET Cl ascitic fluid against ET Cl, ET B, and SPE A. from homologous exotoxin plus endotoxin challenge. None Except for two immunoglobulin M (IgM) MAbs, the anti-ET of the animals had detectable serum antibodies against any Cl antibodies were of the IgGl subclass (all had kappa light PT other than the one used for immunization. chains). All 12 MAbs reacted strongly with ET Cl. The two Unimmunized rabbits succumbed after receiving a stan- IgM antibodies cross-reacted with ET B and SPE A. Several dard dose of PT followed by a second injection of endotoxin IgGl anti-ET Cl MAbs reacted weakly with ET B (titers, (Table 3). The same dose of endotoxin given alone did not 1,000). No reactivity with SPE C or TSST-1 was observed result in death (data not shown). This demonstrated the (data not shown). ability of the toxins to enhance host susceptibility to lethal All eight anti-SPE A MAbs were of the IgM class (Table endotoxin shock. In contrast, immune rabbits did not display 2). Except for 11-15-D8, all anti-SPE A MAbs had kappa this enhanced susceptibility when challenged with the ho- light chains. Cross-reactivity with heterologous toxins was mologous toxin. In addition, reciprocal immunity was stim- more evident than observed with the anti-ET Cl MAbs. ulated by the two ETs. Likewise, immunity to SPE A Several anti-SPE A MAbs had the same titers or differed by provided protection against ET B but not ET Cl. Immunity a twofold dilution against SPE A, ET B, and ET Cl. to either ET did not protect the animals against challenge Mitogenicity neutralization by MAbs. Several MAbs were with SPE A. tested for their ability to neutralize homologous and heter- ologous PTs. Toxins were preincubated with partially puri- DISCUSSION fied MAbs, and the residual mitogenic capacity of the PTs was assessed in standard assays with splenocyte indicators. This study was undertaken to evaluate cross-reactive Nonspecific mitogenicity of staphylococcal toxins was de- antigenic epitopes on staphylococcal and streptococcal PTs. termined with either murine or rabbit splenocytes. However, It was prompted by demonstration that the genes encoding since murine lymphocytes are nonresponsive to SPEs (2), ET B, ET Cl, and SPE A and their translation products the streptococcal toxins were tested only with rabbit sple- share significant sequence homology. Thus, it seemed pos- nocytes. sible that antibodies against some PTs could neutralize Each anti-ET Cl MAb tested at least partially (43 to 99%) heterologous PTs in the absence of demonstrable serological neutralized the mitogenicity of ET Cl (Table 1). Several cross-reactions. Our experimental approach in addressing MAbs also neutralized Ent B (33 to 86%), SPE A (14 to this question included (i) an assessment of the reactivity of 34%), or both; neutralization of ET B was generally more anti-PT MAbs with heterologous PTs in an ELISA, (ii) a effective than for SPE A. However, the two IgM MAbs determination of the ability of these MAbs to neutralize (3F6A10 and 3B8F8) partially neutralized SPE A but had no biological activity of heterologous toxins in vitro, and (iii) an effect on ET B-induced mitogenesis. None of the anti-ET Cl MAbs neutralized TSST-1 (data not shown). TABLE 3. Protection of immune rabbits against homologous The effect of six anti-SPE A MAbs on mitogenicity in- or heterologous PTsa duced by SPE A and heterologous PTs is shown in Table 2. Five MAbs significantly neutralized SPE A (35 to 78%). All No. of dead animals/total no. of animals six MAbs could also neutralize either ET B (25 to 64%), ET Immunizing toxin when challenged with: Cl (31 to 71%), or both. One MAb, 11-29-C3, neutralized ET ET B ET C1 SPE A Cl but did not significantly alter the mitogenic capacity of ET B 0/4b"14b 4/4 SPE A or ET B. ET Cl 2/4C 014b 4/4 Rabbit protection assay. To assess cross-neutralization of SPE A 114b 4/4 0/3d PTs by polyclonal antibodies in vivo, rabbits were immu- None 8/8 4/4 4/4 nized against one PT and then challenged with the same or a different PT. The level of protection provided by immunity a Immune rabbits were challenged with PT followed by 1/50 of a 50% lethal dose of endotoxin. Survival indicates immunity to enhancement of lethal was assessed by determining their susceptibility to lethal endotoxin shock by PT. endotoxin shock after the PT challenge. Generally two to b p < 0.01 when immunized animals were compared with nonimmune four biweekly immunizations were sufficient to induce anti- animals challenged with designated exotoxin. levels c p < 0.09 when immunized animals were compared with nonimmune body detectable by Ouchterlony immunodiffusion; animals challenged with ET B. previous experience indicated that this level of antibody is d p < 0.03 when immunized animals were compared with nonimmune higher than that necessary to provide complete protection animals challenged with SPE A. VOL. 56, 1988 CROSS-NEUTRALIZATION OF PYROGENIC TOXINS 403 evaluation of protection against heterologous PTs provided the titer of cross-reactive antibodies required for protection by immunity in vivo. in vivo is below that required for detection in precipitin We initially found that anti-ET Cl and anti-SPE A MAbs reactions. both cross-reacted with heterologous PTs in ELISA and ET B and ET Cl stimulated reciprocal immunity to each neutralization assays. However, the degree of cross-reac- other but not to SPE A in vivo at the doses used. Therefore, tivity in the ELISA appeared to be dependent upon immu- as expected, cross-reactive epitopes on the two ETs are noglobulin class. Our findings suggest that SPE A is ineffi- more similar to each other than to those on SPE A. Com- cient at inducing immunoglobulin class switching in mice and puter alignments of amino acid sequences of these three its associated affinity maturation of murine antibodies. In proteins shows that ET Cl and ET B share 69% homology, spite of extended immunization, all of the anti-SPE A MAbs whereas ET Cl and SPE A share 49% homology (8a). SPE A obtained for this study were IgM. The ELISA titers of these is probably more closely related to ET B than to ET Cl. IgM MAbs showed a high level of cross-reactivity against Rabbits immunized with SPE A were protected against ET B ET B, ET Cl, or both. In contrast, most of the anti-ET Cl but not against ET Cl. This is consistent with the finding that MAbs were IgGl. These reacted strongly with ET Cl but SPE A shares more sequence homology with ET B (52%) weakly with heterologous PTs. The IgM anti-ET Cl MAbs than ET Cl (49%) (8a). Had dose-response assays been displayed cross-reactivity similar to that of the anti-SPE A done, it is possible that additional cross-neutralization might MAbs in ELISAs. have been observed in immunized animals. Both the anti-ET Cl and anti-SPE A MAbs could neutral- Despite sharing many biological activities with SPE A, ET ize mitogenicity induced by homologous and heterologous B, and ET Cl, other PTs may not share close structural and PTs. Although immunoglobulin concentrations were not immunological similarities. For instance, the amino acid determined for the MAb preparations, IgM MAbs generally sequence of TSST-1 does not share significant homology appeared to have less specificity and thus lower affinity for with SPE A, ET Cl, or ET B (6). None of the anti-ET Cl PTs than IgGl MAbs; these were more cross-reactive but MAbs tested in this study cross-reacted with TSST-1 or SPE only partially neutralized the homologous toxins. In con- C in ELISAs or neutralized TSST-1-induced mitogenicity. trast, the IgGl anti-ET Cl MAbs were less effective at Therefore, shared epitopes may not be found on all PTs. neutralizing heterologous PTs but caused nearly complete Additional characterization of other PTs is required to neutralization of ET Cl. determine their relatedness to those used in this study. ET B and ET Cl are believed to possess several antigenic determinants distributed throughout the molecules (28). The ACKNOWLEDGMENTS results obtained with MAb studies show that some cross- reactive antigenic determinants on SPE A, ET B, and ET Cl This work was supported by Public Health Service grant HL36611 from the National Institutes of Health and by the Kimberly Clark are located at or near a site on the protein required for Corporation. G.A.B. was supported by a fellowship from the mitogenicity, a biological property shared by all PTs. All of American Heart Association, Minnesota Affiliate. these PTs show extensive amino acid homology throughout Renee Kayon and Steve Broberg are gratefully acknowledged for the proteins, with major stretches of homology located near typing the manuscript. their C-terminal ends (8a, 25, 31). It is likely that one or more of these homologous regions is recognized by cross-reactive MAbs and is also involved in mitogenicity and possibly other LITERATURE CITED shared biological properties of PTs. Unlike the C termini 1. Avena, R. M., and M. S. Bergdoll. 1967. Purification and some there are long stretches of amino acids in the N termini that physicochemical properties of enterotoxin C, Staphylococcus are shared between ET Cl and ET B but not SPE A. Unique aureus strain 361. Biochemistry 6:1474-1480. biological properties such as cardiotoxicity of SPE A and 2. Barsumian, E. L., P. M. Schlievert, and D. W. Watson. 1978. ET B and ET Cl are more to be Nonspecific and specific immunological mitogenicity by group emetic activity of likely A streptococcal pyrogenic exotoxins. Infect. Immun. 22:681- determined by this portion of the proteins, where the staphy- 688. lococcal and streptococcal PTs have diverged. Alterna- 3. Bergdoll, M. S., C. R. Borja, and R. M. Avena. 1965. Identifi- tively, the structures which direct these biological functions cation of a new enterotoxin as enterotoxin C. J. Bacteriol. may be derived from the folded PT, which creates three-di- 90:1481-1485. mensional amino acid arrangements. If this is the case short 4. Bergdoli, M. S., C. R. Borja, R. N. Robbins, and K. F. Weiss. stretches of amino acid homology from regions far apart on 1971. Identification of enterotoxin E. Infect. Immun. 4:593-595. the linear sequence may fold to form homologous structures. 5. Bergdoli, M. S., R. N. Robbins, K. Weiss, C. R. Borja, I. Y. SPE A, ET B, and ET Cl stimulated immunity in vivo that Huang, and F. S. Chu. 1973. The staphylococcal enterotoxins: with similarities. Contrib. Microbiol. Immunol. 1:390-396. in some cases protected against challenge heterologous 6. Blomster-Hautamaa, D. A., B. N. Kreiswirth, J. S. Kornblum, toxins. This cross-neutralization of PTs was observed in the R. P. Novick, and P. M. Schlievert. 1986. The nucleotide and absence of detectable cross-reactive antibody by Ouchter- partial amino acid sequence of toxic shock syndrome toxin-1. J. lony immunodiffusion. Others have shown that reactivity of Biol. Chem. 261:15783-15786. ET B and ET Cl with reciprocal antibodies can be demon- 7. Blomster-Hautamaa, D. A., R. P. Novick, and P. M. Schlievert. strated by competition assays and also by immunoprecipita- 1986. Localization of biological functions of toxic shock syn- tion of antigen-antibody complexes with protein A-con- drome toxin-1 by use of monoclonal antibodies and cyanogen taining strains of S. aureus (28, 29). Also, cross-reactivity bromide-generated toxin fragments. J. Immunol. 137:3572- among these three toxins has been demonstrated by Western 3576. 8. Bohach, G. A., and P. M. Schlievert. 1987. Expression of blot analysis (7, 13). Thus, it is possible that cross-reactive staphylococcal enterotoxin Cl in Escherichia coli. Infect. Im- antibodies cannot effectively participate in precipitin reac- mun. 55:428-432. tions with heterologous PTs. This might be expected if the 8a.Bohach, G. A., and P. M. Schlievert. 1987. Nucleotide sequence shared stretches of homology on heterologous toxins are of the staphylococcal enterotoxin. Cl gene and relatedness to exposed differently due to altered configurations in other other pyrogenic toxins. Molec. Gen. Genet. 209:15-20. parts of the toxin molecules. An alternative possibility is that 9. Bohach, G. A., and I. S. Snyder. 1985. Chemical and immuno- 404 BOHACH ET AL. INFECT. IMMUN.

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