INFECTION AND IMMUNITY, Oct. 1979, p. 211-216 0019-9567/79/10-0211/06$02.00/0
Vol. 26, No. 1
Immunogenicity of Soluble Versus Cellular Glycerol Teichoic Acid FRANK W. CHORPENNING,* HAROLD R. COOPER,t JOHN W. OLDFATHER, AND JOHN J. LYNCH, JR. The Ohio State University, Columbus, Ohio 43210
Received for publication 19 July 1979
Guinea pigs which were injected with either whole bacilli or purified soluble glycerol teichoic acid (GTA) usually exhibited a rise in hemolysin titer to GTAcoated erythrocytes. The only exceptions were those animals having high baseline titers of natural anti-GTA antibodies. Rats yielded better responses than guinea pigs and produced significantly higher responses to the soluble antigen than to the cellular GTA. Rats reared on a GTA-free diet were predominantly free of natural antibodies to GTA and furnished a more clear-cut model for assaying immune responses. Using this model, it was shown that adsorption of GTA to homologous erythrocytes before injection resulted in poor responses, suggesting that such spontaneous adsorption does not account for the good responses to soluble antigen. In GTA-deprived rats, positive skin tests were induced only with bacilli, whereas migration inhibitory factor was induced with both bacilli and soluble antigen. Hemolytic plaques in immunized rats were increased over controls with both kinds of immunogen, but the GTA-deprived rats responded better than conventional ones, and hemolytic plaque responses to bacilli were better than those to soluble antigen. This reversal of the serum hemolysin results may be due to delayed suppression by soluble GTA or to antibody cycling. The guinea pig data, combined with results from GTA-deprived rats, suggest that high antibody levels resulted in depressed antibody synthesis, perhaps because antibody cycling was initiated. No evidence was found to explain the superior responses to soluble antigen, but it did not seem related to formation of immune complexes or adsorption to erythrocyte membranes.
GTA, when integral to streptococcal cells, was established by McCarty (22), and responses in rabbits to a crude phenol extract in Freund adjuvant were reported later (18). We have pre-
viously shown that purified GTA is immunogenic in rabbits when coated on human group 0 erythrocytes (10) and that crude acid-precipitated GTA is also immunogenic. Burger (6) showed that purified GTA is immunogenic when complexed with methylated bovine serum albumin or cetyl pyridinium chloride. On the other hand, acid-extracted teichoic acids are not immunogenic in rabbits (19), and it has been suggested that the protein content is important in immunogenicity (29). In a recent paper, Fiedel and Jackson (Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M364, p. 126) reported that phenol-extracted lipoteichoic acid was not immunogenic in rabbits using Freund incomplete adjuvant, although it was immunogenic when precipitated with miethylated bovine serum albumin. To expand these findings, we have examined the immunogenicity of soluble and cellular GTA in guinea pigs and rats.
t Present address: Microbiological Associates, Walkersville, MD 21793.
MATERIALS AND METHODS Animals. Guinea pigs were randomly bred conven-
The teichoic acids are important constituents of gram-positive bacterial cells (1, 2) and exhibit significant biological activity (19). For example, they produce natural delayed hypersensitivity in animals (4, 14) and humans (21), nephritis in rabbits (14), experimental arthritis (26), and hemolytic episodes (8). In addition, immunoglobulin G (IgG) antibodies of teichoic acid (TA) specificity are associated with staphylococcal endocarditis (12) and rheumatic fever (3). Furthermore, glycerol teichoic acid (GTA) suppresses the immune response to sheep erythrocytes
(SRBC) (9, 23, 24). The biological importance of GTA under-
scores the need for information relative to its immunogenicity. Yet, the literature on the subject is not extensive. The immunogenicity of
211
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CHORPENNING ET AL.
tional animals of both sexes fed Purina guinea pig chow (Ralston-Purina Co., St. Louis, Mo.) and were reared in our animal facility. Sprague-Dawley rats were originally obtained from Laboratory Animal Supply (Indianapolis, Ind.) and were bred in our animal facility. Conventional animals were fed laboratory animal chow 5010 (Ralston-Purina Co.), and GTA-deprived rats were fed the Similac-corn oil diet described by us (27). The latter rats were taken as weanlings from randomly mated animals, placed on the diet, and reared as described earlier. Both guinea pigs and rats were injected at 12 weeks of age. TA. The GTA used for both immunization and testing was extracted from Bacillus sp. ATCC 29726 with aqueous phenol and purified as previously described (11, 13). It was essentially free of peptidoglycan and lipid. For immunization with whole bacteria, a heat-killed suspension of Bacillus sp. 29726 (2 x 10"' organisms per injection) in phosphate-buffered saline was employed. Hemolysin tests. Serological assays were performed by passive hemolysis, using the spectrophotometric 50% endpoint method previously described (5). Complement levels and GTA-coated SRBC were carefully standardized (17). SRBC were used because rat erythrocytes fail to lyse in this system. All sera and guinea pig complement pools were absorbed with SRBC before testing. Hemolytic plaque tests. Direct and indirect tests were performed as previously described (5) except that a 1:250 dilution of a monospecific anti-rat IgG (Microbiological Associates, Bethesda, Md.) was employed for indirect tests. This antiserum was made heavychain specific by passing through CNBr Sepharose 4B which had been charged with 25 mg of pure rat IgM per g. The indicator SRBC were coated with purified GTA (11), and plates containing uncoated SRBC were incubated in parallel for comparison. Pooled guinea pig serum (complement) and anti-IgG were absorbed with both SRBC and GTA-bearing Bacillus sp. (ATCC 29726) before use. Cell-mediated responses. Skin tests employed 100 jig of purified GTA injected intradermally on the shaved flank and were observed at 1, 12, 24, and 48 h (4). An area of induration greater than 5 mm in diameter was considered positive. Positive and negative
lesions were excised, fixed in alcohol-acetic acid-chloroform (60:10:30 vol/vol/vol), embedded in Epon 812, and stained with pyronine-methyl green. Heavy mononuclear cell infiltrates were observed in positive lesions but were absent from negative ones. Tests for migration inhibitory factor employed 100 jig of purified GTA in the chamber and were performed as previously described (4). Inhibition of migration greater than 20% was interpreted as a positive result. Immunization. Animals were bled before antigen injection for base-line determinations. They were then injected with either whole bacteria, 100 jig of purified GTA, 200 jig of GTA, or phosphate-buffered saline by the intravenous route (see tables). After 3 days, the dosage was repeated by the intraperitoneal route and it was repeated again after 6 days. Additional blood samples were collected on days 5 and 10. RESULTS
Immunization of outbred guinea pigs with purified soluble GTA produced weak responses in 9 out of 11 animals. These nine guinea pigs had very low base-line levels of natural anti-GTA hemolysins. The other two animals had high base-line titers (125 and 137), and injection of soluble antigen produced a continuous drop in hemolysin titer. Animals with low base lines did not show a drop in titer. Four out of five animals injected with whole killed bacilli produced a rise in titer, but again the only one with a high baseline titer exhibited a fall rather than a rise. A variant animal with an abnormally high base line was also seen in the control group. The mean titers and frequencies are shown in Table 1. Mean titers with animals showing high base lines eliminated are shown in parentheses. The guinea pigs receiving a total of 700 jig of GTA yielded a significant rise in mean titer. Animals receiving 400 tig of GTA also exhibited an increased mean titer at 5 days, but the titer fell by day 10. All of the 33 Sprague-Dawley rats (outbred) which were injected with purified soluble GTA
TABLE 1. Immune responses to GTA in guinea pigs Mean hemolysin titer (± SE)"
Frequency of a
Immunogen
Bacilli (2 x 10"' cells)
Base line
5 days
48.0 + 42.8
37.2 ± 28.3
(5.3)
(9.0)
10 days
requn ofta rise in titer
23.1 ± 14.7 4/5 (8.6) Purified GTA (700 jig) 25.7 ± 22.3 20.4 ± 10.1 33.3 ± 21.9 5/6 (3.4) (10.5) (12.4) Purified GTA (400 jig) 26.1 ± 24.7 8.9 ± 2.3 5.7 ± 3.7 4/5 (1.3) (8.4) (2.0) Control 47.6 ± 43.9 21.1 ± 18.9 12.9 ± 12.4 (3.7) (2.2) (0.5) Based on five or six animals per group. Measured by passive hemolysis with purified GTA coated on SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. Ratio of animals showing a rise in titer to total number in group. Mean titers with animals having high base lines eliminated are shown in parentheses.
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IMMUNOGENICITY OF TEICHOIC ACID
exhibited an increase in hemolysin titer, and mean titer increases were significant. Titers were generally higher than seen in immunized guinea pigs, and individual variation in base lines was not as great. Results of a representative experiment are presented in Table 2. Rats injected with bacilli produced much weaker hemolysin responses than did those injected with soluble antigen, in spite of the fact that the GTA dosage was estimated to be at least equivalent to the soluble dose. In our effort to demonstrate differences in responsiveness more clearly, we also carried out immunization experiments in GTA-deprived Sprague-Dawley rats (27), which are usually free of natural anti-GTA hemolysins. These rats were comparable to those described earlier, except that in this larger group we observed that about 10% yielded a very weak hemolysin reaction. Nevertheless, much more clear-cut results could be obtained with this model. One of these experiments is summarized in Table 3. Good titers were obtained in all animals which were injected with soluble GTA and, again, rats injected with whole bacilli yielded lower antibody titers than those given purified soluble antigen. Furthermore, animals injected with 600 Iug of GTA coated on rat erythrocytes also responded
poorly. Responses to whole bacilli and GTAcoated erythrocytes were comparable, and both were significantly lower than those to soluble GTA in four separate experiments. As expected, skin tests for delayed-type hypersensitivity were negative in the rats given soluble antigen but, unexpectedly, tests for migration inhibitory factor were positive. Another interesting observation was that although skin testing (100 Mug of GTA intradermally) 2 days before the final bleeding usually depressed the antibody levels in conventional animals, it appeared to furnish an additional stimulus in GTA-deprived rats (Table 4). The GTA-deprived animals were undergoing a priwhereas the conventional rats had been environmentally primed (27). Hemolytic plaque (PFC) tests with GTAcoated cells were performed on both conventional and GTA-deprived rats at 10 days postimmunization (4 days after the third antigen injection). Whole bacilli produced good responses, and indirect PFC reached a peak at the same time as direct PFC (4 days postimmunization). Comparison of the immunogenicity of bacillary and purified soluble antigen is shown in Table 5. In view of the hemolysin results, responses to the bacilli were unexpectedly good.
mary response,
TABLE 2. Immune responses to GTA in Sprague-Dawley rats Mean hemolysin titer (± SE)a Immunogen Base line 22.8 + 10.2 24.0 ± 4.9 45.0 ± 7.6 15.6 ± 6
213
5 days 62.7 ± 5.3 53.0 ± 13.6 85.0 ± 32.8 11.2 ± 3.4
10 days 40.0 ± 12 126 ± 29
Frequency of a riei ttr
rise in titer
Bacilli (2 x 10' cells) Purified GTA (600 Mg) 326.7 ± 151 Purified GTA (300 Lg) 16.2 ± 3 Control a Based on three to five animals per group. Measured by passive hemolysis with purified GTA SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. b Ratio of animals showing a rise in titer to total number in group.
4/4 3/3 5/5 coated on
TABLE 3. Immune responses of GTA-deprived Sprague-Dawley rats to GTA Mean hemolysin titer' Skin d Frequency MIF Immunogen test of titer rise' 5 days 10 days Base line 42.5 ± 11 0.25 ± 0.25 83.3 ± 39.2 Bacilli (2 x 1010) 4/4 2/4 4/4 0 95.0 ± 13.2 285.0 ± 87 Purified GTA (600 Mg) 4/4 0/5 3/3 230.0 ± 60 73.4 ± 16.9 0.25 ± 0.2 Purified GTA (300jug) 4/4 0/5 4/5 0 57.5 ± 17.5 50 ± 18 GTA on erythrocytes (600 3/3 0/4 2/3 Mg) Control 0 0 0 0.4 0/4 1/4 a Based on four rats per group. Measured by passive hemolysis with purified GTA coated on SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. b Ratio of animals showing a rise in titer to total number in group. 'Ratio of positive animals (induration, >5 mm) at 24 to 48 h to total number tested (different animals than migration inhibitory factor [MIF] and antibody studies). d Ratio of positive animals (inhibition, >20%o) to total number tested.
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INFECT. IMMUN.
CHORPENNING ET AL. TABLE 4. Effect of skin testing on the hemolysin response in rats
Skin test'
No skin test' Mean hemolysin titer
Conventional rats 1 2 3 4 Mean (± SE)C
GTA-deprived rats 1 2 3 4 Mean (± SE)
10 days
Base line
10 days
Base line
60 35 40
80 600 300
32 25 30 25
36 45 74 35
45 ± 7.6
326 ± 150.7
28 ± 1.8
47.5 ± 9.1
0 0 0 0
400 200 120 200
0 0 0 0
800 2,000 1,600
230 ± 59.7 of GTA intravenously followed by 100
Itg
600
1,250 ± 330 intraperitoneally on both days 3
a Animals initially received 100 jg and 6. b Skin-tested animals received the same immunization regimen as described above and were injected with an additional 100 ,ug of GTA intradermally on day 8. 'SE, Standard error of the mean.
They were better in GTA-deprived animals. Possible reasons for the low level of PFC response to soluble GTA will be presented in the discussion.
DISCUSSION In view of reports (6, 19, 29) that purified GTA is not immunogenic in rabbits and the wellknown refractoriness of this species to purified polysaccharide antigens (16), we chose guinea pigs and rats as our subject animals. Surprisingly, guinea pigs responded better to soluble purified GTA than to GTA-bearing bacilli. However, they did not respond as well as SpragueDawley rats. Thus, it appears that responsiveness to this purified antigen follows the sequence, rat > guinea pig > rabbit, in which the rabbit may be completely unresponsive. However, optimal dosages of GTA were not determined in this investigation. We attribute the differences between the present study and those of earlier workers essentially to species differences in the subject animals. It is generally believed that cellular antigens usually produce better responses than soluble ones. Therefore, the greater immunogenicity of soluble GTA in rats was unexpected. We have found no similar observation in the literature and have little to offer in explanation. The greater responsiveness to soluble antigen cannot be attributed to formation of antigen-antibody complexes in primed animals because the TAdeprived rats seldom exhibited natural antibody but responded as well or better than conventional rats. Furthermore, data presented here indicate that increased initial levels of antibody
TABLE 5. Splenic PFC response of conventional and GTA-deprived Sprague-Dawley rats to GTA Immunogen
Mean PFC/107 spleen cells' (+ SE) Conventional
GTA deprived
Bacilli' (2 X 10"')
98.8 ± 20.7 172.5 ± 31.0 79.0 ± 95.8 Purified GTA (600 fg) 22.0 ± 11.2 80.3 ± 43.7 Purified GTA (300 ,g) 19.0 ± 8.1 0 1.9 ± 0.6 Unimmunized control 'Tests performed with GTA-coated SRBC. SE, Standard error of the mean; PFC, plaque-forming cells. 'All animals except the controls were skin tested 2 days before sacrifice.
are associated with poor responses (see below). It might be suggested that the unexpectedly high immunogenicity of soluble GTA was due to its demonstrated adsorbability to cell membranes (8, 15, 25). This does not appear to be the case because in four separate experiments the responses to GTA-coated rat cells were comparable to those with whole bacterial cells and both were much lower than those with soluble
antigen. From both guinea pig and rat data, it appears that animals with relatively high initial (base line) hemolysin levels responded poorly to injected antigen, while those with lower base lines responded well. In fact, at 4 days after the last injection the titer was frequently lower than it was initially. Our previous report (5) shows that injection of antigen produces cycling in this species. Antibody production is depressed (presumably) whenever an antibody excess exists (28),
VOL. 26, 1979
which would be the case in animals yielding high-titered base-line sera. Thus, in these rats the antibody level dropped due to immune elimination in the absence of antibody synthesis. In low-titered rats no such depression would occur and antibody synthesis would proceed. In view of the antigen's persistence (5), it is likely that titers in the high-base line animals would have risen again by 16 days after the antigen injection (5). In this connection, it is important to note that 10-day titers in GTA-deprived rats (zero base line) rose considerably higher than in conventional rats (Table 4) when the animals were given a fourth injection of 100 tLg of GTA. Because rats possess fluctuating levels of natural antibodies for GTA (5), determination of responses to GTA in this species is a problem. Such determinations may be based on the frequency of a rise in titer or an increase in the mean titer of this group (Table 2); however, it was believed that more clear-cut results would be obtained in GTA-deprived animals (27). This assumption was partially true because most rats yielded negative base lines. As we reported earlier (4), normal conventional rats may yield positive skin tests and migration inhibitory factor tests to purified GTA. Therefore, these tests for cell-mediated immunity were conducted on GTA-deprived rats because it was believed they would lack such responses before immunization. This assumption was true for skin tests but, unexpectedly, two of the uninjected GTA-deprived control rats yielded positive migration inhibitory factor tests. Nevertheless, the increase in frequency of migration inhibitory factor in rats injected with either soluble GTA or bacilli indicates that cellmediated responses were induced. The separate group of animals which were skin tested may have failed to exhibit cell-mediated immunity because the test dose was excessive, resulting in a shift to humoral responses. Criticism that the emphasis in this study was on IgM responses would be valid except for the fact that we have previously shown (7) that IgM antibodies predominate in this age group and that IgG and IgM plaques peak together (present study). The lower PFC response when the immunogen was soluble GTA may be related to two previously observed phenomena. Miller and Jackson (23) have shown that soluble GTA can suppress SRBC responses, and we have demonstrated that GTA responses are suppressed as well (unpublished data). We have also shown that very small amounts of GTA enhance rather than suppress (20). Thus, it might be expected that the small amount of soluble GTA released
IMMUNOGENICITY OF TEICHOIC ACID
215
by bacilli would not suppress the response, allowing full immunogenic action by the cellbound GTA. With soluble GTA, initial enhancement may account for circulating antibody levels, followed by PFC suppression which is not yet reflected in antibody levels. It is not clear why such responses would be better in GTAdeprived rats, but it should be kept in mind that these animals are relatively unprimed and would respond differently than conventional rats. Even without invoking suppression, antibody cycling could possibly account for the disparity between PFC responses to soluble antigen and bacilli. We showed that injection of bacilli induced synchronized cycling in rats injected simultaneously, with a PFC peak at 4 days and a serum antibody peak at 16 days (5). However, we did not examine the effects of soluble antigen, and it is quite possible that this kind of immunogen would produce cycles of a different interval. ACKNOWLEDGMENTS This study was supported by the Department of Microbiology and the Graduate School, The Ohio State University. We acknowledge the technical assistance of Thomas J. Alexander, Mary Ann Decker, and Dorian L. Bockstiegel.
LITERATURE CITED 1. Archibald, A. R., and J. Baddiley. 1966. The teichoic acids. Adv. Carbohydr. Chem. 21:323-375. 2. Archibald, A. R., J. Baddiley, and N. L. Blumson. 1968. The teichoic acids. Adv. Enzymol. 30:223-253. 3. Beachey, E. H., L. Ofek, and A. L. Bisno. 1973. Studies of antibodies to non-type-specific antigens associated with streptococcal M protein in the sera of patients with rheumatic fever. J. Immunol. 111:1361-1366. 4. Bolton, R. W., and F. W. Chorpenning. 1974. Naturally occurring cellular and humoral immunity to teichoic acid in rats. Immunology 27:517-524. 5. Bolton, R. W., H. Rozmiarek, and F. W. Chorpenning. 1977. Cyclic antibody formation to polyglycerophosphate in normal and injected rats. J. Immunol. 118:1154-1158. 6. M. M. Burger. 1966. Teichoic acids: antigenic determinants, chain separation, and their location in the cell wall. Proc. Natl. Acad. Sci. U.S.A. 56:910-917. 7. Chorpenning, F. W., R. W. Bolton, G. T. Frederick, and H. Rozmiarek. 1979. Development of cellular and humoral responses to teichoic acid. Dev. Comp. Immunol., in press. 8. Chorpenning, F. W., and M. C. Dodd. 1965. Polyagglutinable erythrocytes associated with bacteriogenic transfusion reactions. Vox Sang. 10:460-471. 9. Chorpenning, F. W., J. J. Lynch, Jr., H. R. Cooper, and J. W. Oldfather. 1979. Modulation of the immune response to sheep erythrocytes by lipid-free glycerol teichoic acid. Infect. Immun. 26:262-269. 10. Chorpenning, F. W., and H. B. Stamper. 1973. Spontaneous adsorption of teichoic acid to erythrocytes. Immunochemistry 10:15-20. 11. Cooper, H. R., F. W. Chorpenning, and S. Rosen. 1978. Lipid-free glycerol teichoic acids with potent membrane-binding activity. Infect. Immun. 19:462-469. 12. Crowder, J. G., and A. White. 1972. Teichoic acid antibodies in staphylococcal and nonstaphylococcal endocarditis. Ann. Intern. Med. 77:87-90.
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13. Decker, G. P., F. W. Chorpenning, and G. T. Frederick. 1972. Naturally occurring antibodies to bacillary teichoic acids. J. Immunol. 108:214-222. 14. Frederick, G. T., R. A. Holmes, and F. W. Chorpenning. 1972. Naturally occurring cell-mediated immunity to purified glycerol-teichoic acid antigen in guinea pigs. J. Immunol. 109:1399-1401. 15. Jackson, R. W., and M. Moskowitz. 1966. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205-2209. 16. Kabat, E. A., and M. M. Mayer. 1964. In E. A. Kabat (ed.), Experimental immunochemistry, 2nd ed., p. 7-8. Charles C Thomas, Publisher, Springfield, Ill. 17. Kent, J. F., and E. H. Fife. 1963. Precise standardization of reagents for complement fixation. Am. J. Trop. Med. 12:103-116. 18. Knox, K. W., M. J. Hewett, and A. J. Wicken. 1970. Studies on the group F antigen of lactobacilli: detection of antibodies by hemagglutination. J. Gen. Microbiol. 60:303-313. 19. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 20. Lynch, J. J., Jr., and F. W. Chorpenning. 1978. Effects of dosage modulaton of sheep red cell responses by teichoic acid. Fed. Proc. 37:1489. 21. Martin, R. R., H. Daugherty, and A. White. 1966. Staphylococcal antibodies and hypersensitivity to tei-
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22.
23. 24. 25.
26.
27. 28.
29.
choic acids in man, p. 91-96. Antimicrob. Agents Chemother. 1965. McCarty, M. 1959. The occurrence of polyglycerophosphate as an antigenic component of various gram-positive bacterial species. J. Exp. Med. 109:361-378. Miller, G. A., and R. W. Jackson. 1973. The effects of Streptococcus pyogenes teichoic acid on the immune response of mice. J. Immunol. 110:148-156. Miller, G. A., J. Urban, and R. W. Jackson. 1976. Effects of streptococcal lipoteichoic acid in host responses in mice. Infect. Immun. 13:1408-1417. Moskowitz, M. 1966. Separation and properties of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2200-2204. Ne'eman, N., and I. Ginsburg. 1972. Cell sensitizing antigen of group A streptococci. II. Immunological and immunopathological properties. Isr. J. Med. Sci. 8: 1807-1816. Rozmiarek, H., R. W. Bolton, and F. W. Chorpenning. 1977. Environmental origin of natural antibodies to teichoic acid. Infect. Immun. 16:505-509. Weigle, W. 0. 1975. Cyclical production of antibody as a regulatory mechanism in the immune response. Adv. Immunol. 21:87-111. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigen. Science 187: 1161-1167.
Vol. 26, No. 1
Immunogenicity of Soluble Versus Cellular Glycerol Teichoic Acid FRANK W. CHORPENNING,* HAROLD R. COOPER,t JOHN W. OLDFATHER, AND JOHN J. LYNCH, JR. The Ohio State University, Columbus, Ohio 43210
Received for publication 19 July 1979
Guinea pigs which were injected with either whole bacilli or purified soluble glycerol teichoic acid (GTA) usually exhibited a rise in hemolysin titer to GTAcoated erythrocytes. The only exceptions were those animals having high baseline titers of natural anti-GTA antibodies. Rats yielded better responses than guinea pigs and produced significantly higher responses to the soluble antigen than to the cellular GTA. Rats reared on a GTA-free diet were predominantly free of natural antibodies to GTA and furnished a more clear-cut model for assaying immune responses. Using this model, it was shown that adsorption of GTA to homologous erythrocytes before injection resulted in poor responses, suggesting that such spontaneous adsorption does not account for the good responses to soluble antigen. In GTA-deprived rats, positive skin tests were induced only with bacilli, whereas migration inhibitory factor was induced with both bacilli and soluble antigen. Hemolytic plaques in immunized rats were increased over controls with both kinds of immunogen, but the GTA-deprived rats responded better than conventional ones, and hemolytic plaque responses to bacilli were better than those to soluble antigen. This reversal of the serum hemolysin results may be due to delayed suppression by soluble GTA or to antibody cycling. The guinea pig data, combined with results from GTA-deprived rats, suggest that high antibody levels resulted in depressed antibody synthesis, perhaps because antibody cycling was initiated. No evidence was found to explain the superior responses to soluble antigen, but it did not seem related to formation of immune complexes or adsorption to erythrocyte membranes.
GTA, when integral to streptococcal cells, was established by McCarty (22), and responses in rabbits to a crude phenol extract in Freund adjuvant were reported later (18). We have pre-
viously shown that purified GTA is immunogenic in rabbits when coated on human group 0 erythrocytes (10) and that crude acid-precipitated GTA is also immunogenic. Burger (6) showed that purified GTA is immunogenic when complexed with methylated bovine serum albumin or cetyl pyridinium chloride. On the other hand, acid-extracted teichoic acids are not immunogenic in rabbits (19), and it has been suggested that the protein content is important in immunogenicity (29). In a recent paper, Fiedel and Jackson (Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M364, p. 126) reported that phenol-extracted lipoteichoic acid was not immunogenic in rabbits using Freund incomplete adjuvant, although it was immunogenic when precipitated with miethylated bovine serum albumin. To expand these findings, we have examined the immunogenicity of soluble and cellular GTA in guinea pigs and rats.
t Present address: Microbiological Associates, Walkersville, MD 21793.
MATERIALS AND METHODS Animals. Guinea pigs were randomly bred conven-
The teichoic acids are important constituents of gram-positive bacterial cells (1, 2) and exhibit significant biological activity (19). For example, they produce natural delayed hypersensitivity in animals (4, 14) and humans (21), nephritis in rabbits (14), experimental arthritis (26), and hemolytic episodes (8). In addition, immunoglobulin G (IgG) antibodies of teichoic acid (TA) specificity are associated with staphylococcal endocarditis (12) and rheumatic fever (3). Furthermore, glycerol teichoic acid (GTA) suppresses the immune response to sheep erythrocytes
(SRBC) (9, 23, 24). The biological importance of GTA under-
scores the need for information relative to its immunogenicity. Yet, the literature on the subject is not extensive. The immunogenicity of
211
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INFECT. IMMUN.
CHORPENNING ET AL.
tional animals of both sexes fed Purina guinea pig chow (Ralston-Purina Co., St. Louis, Mo.) and were reared in our animal facility. Sprague-Dawley rats were originally obtained from Laboratory Animal Supply (Indianapolis, Ind.) and were bred in our animal facility. Conventional animals were fed laboratory animal chow 5010 (Ralston-Purina Co.), and GTA-deprived rats were fed the Similac-corn oil diet described by us (27). The latter rats were taken as weanlings from randomly mated animals, placed on the diet, and reared as described earlier. Both guinea pigs and rats were injected at 12 weeks of age. TA. The GTA used for both immunization and testing was extracted from Bacillus sp. ATCC 29726 with aqueous phenol and purified as previously described (11, 13). It was essentially free of peptidoglycan and lipid. For immunization with whole bacteria, a heat-killed suspension of Bacillus sp. 29726 (2 x 10"' organisms per injection) in phosphate-buffered saline was employed. Hemolysin tests. Serological assays were performed by passive hemolysis, using the spectrophotometric 50% endpoint method previously described (5). Complement levels and GTA-coated SRBC were carefully standardized (17). SRBC were used because rat erythrocytes fail to lyse in this system. All sera and guinea pig complement pools were absorbed with SRBC before testing. Hemolytic plaque tests. Direct and indirect tests were performed as previously described (5) except that a 1:250 dilution of a monospecific anti-rat IgG (Microbiological Associates, Bethesda, Md.) was employed for indirect tests. This antiserum was made heavychain specific by passing through CNBr Sepharose 4B which had been charged with 25 mg of pure rat IgM per g. The indicator SRBC were coated with purified GTA (11), and plates containing uncoated SRBC were incubated in parallel for comparison. Pooled guinea pig serum (complement) and anti-IgG were absorbed with both SRBC and GTA-bearing Bacillus sp. (ATCC 29726) before use. Cell-mediated responses. Skin tests employed 100 jig of purified GTA injected intradermally on the shaved flank and were observed at 1, 12, 24, and 48 h (4). An area of induration greater than 5 mm in diameter was considered positive. Positive and negative
lesions were excised, fixed in alcohol-acetic acid-chloroform (60:10:30 vol/vol/vol), embedded in Epon 812, and stained with pyronine-methyl green. Heavy mononuclear cell infiltrates were observed in positive lesions but were absent from negative ones. Tests for migration inhibitory factor employed 100 jig of purified GTA in the chamber and were performed as previously described (4). Inhibition of migration greater than 20% was interpreted as a positive result. Immunization. Animals were bled before antigen injection for base-line determinations. They were then injected with either whole bacteria, 100 jig of purified GTA, 200 jig of GTA, or phosphate-buffered saline by the intravenous route (see tables). After 3 days, the dosage was repeated by the intraperitoneal route and it was repeated again after 6 days. Additional blood samples were collected on days 5 and 10. RESULTS
Immunization of outbred guinea pigs with purified soluble GTA produced weak responses in 9 out of 11 animals. These nine guinea pigs had very low base-line levels of natural anti-GTA hemolysins. The other two animals had high base-line titers (125 and 137), and injection of soluble antigen produced a continuous drop in hemolysin titer. Animals with low base lines did not show a drop in titer. Four out of five animals injected with whole killed bacilli produced a rise in titer, but again the only one with a high baseline titer exhibited a fall rather than a rise. A variant animal with an abnormally high base line was also seen in the control group. The mean titers and frequencies are shown in Table 1. Mean titers with animals showing high base lines eliminated are shown in parentheses. The guinea pigs receiving a total of 700 jig of GTA yielded a significant rise in mean titer. Animals receiving 400 tig of GTA also exhibited an increased mean titer at 5 days, but the titer fell by day 10. All of the 33 Sprague-Dawley rats (outbred) which were injected with purified soluble GTA
TABLE 1. Immune responses to GTA in guinea pigs Mean hemolysin titer (± SE)"
Frequency of a
Immunogen
Bacilli (2 x 10"' cells)
Base line
5 days
48.0 + 42.8
37.2 ± 28.3
(5.3)
(9.0)
10 days
requn ofta rise in titer
23.1 ± 14.7 4/5 (8.6) Purified GTA (700 jig) 25.7 ± 22.3 20.4 ± 10.1 33.3 ± 21.9 5/6 (3.4) (10.5) (12.4) Purified GTA (400 jig) 26.1 ± 24.7 8.9 ± 2.3 5.7 ± 3.7 4/5 (1.3) (8.4) (2.0) Control 47.6 ± 43.9 21.1 ± 18.9 12.9 ± 12.4 (3.7) (2.2) (0.5) Based on five or six animals per group. Measured by passive hemolysis with purified GTA coated on SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. Ratio of animals showing a rise in titer to total number in group. Mean titers with animals having high base lines eliminated are shown in parentheses.
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IMMUNOGENICITY OF TEICHOIC ACID
exhibited an increase in hemolysin titer, and mean titer increases were significant. Titers were generally higher than seen in immunized guinea pigs, and individual variation in base lines was not as great. Results of a representative experiment are presented in Table 2. Rats injected with bacilli produced much weaker hemolysin responses than did those injected with soluble antigen, in spite of the fact that the GTA dosage was estimated to be at least equivalent to the soluble dose. In our effort to demonstrate differences in responsiveness more clearly, we also carried out immunization experiments in GTA-deprived Sprague-Dawley rats (27), which are usually free of natural anti-GTA hemolysins. These rats were comparable to those described earlier, except that in this larger group we observed that about 10% yielded a very weak hemolysin reaction. Nevertheless, much more clear-cut results could be obtained with this model. One of these experiments is summarized in Table 3. Good titers were obtained in all animals which were injected with soluble GTA and, again, rats injected with whole bacilli yielded lower antibody titers than those given purified soluble antigen. Furthermore, animals injected with 600 Iug of GTA coated on rat erythrocytes also responded
poorly. Responses to whole bacilli and GTAcoated erythrocytes were comparable, and both were significantly lower than those to soluble GTA in four separate experiments. As expected, skin tests for delayed-type hypersensitivity were negative in the rats given soluble antigen but, unexpectedly, tests for migration inhibitory factor were positive. Another interesting observation was that although skin testing (100 Mug of GTA intradermally) 2 days before the final bleeding usually depressed the antibody levels in conventional animals, it appeared to furnish an additional stimulus in GTA-deprived rats (Table 4). The GTA-deprived animals were undergoing a priwhereas the conventional rats had been environmentally primed (27). Hemolytic plaque (PFC) tests with GTAcoated cells were performed on both conventional and GTA-deprived rats at 10 days postimmunization (4 days after the third antigen injection). Whole bacilli produced good responses, and indirect PFC reached a peak at the same time as direct PFC (4 days postimmunization). Comparison of the immunogenicity of bacillary and purified soluble antigen is shown in Table 5. In view of the hemolysin results, responses to the bacilli were unexpectedly good.
mary response,
TABLE 2. Immune responses to GTA in Sprague-Dawley rats Mean hemolysin titer (± SE)a Immunogen Base line 22.8 + 10.2 24.0 ± 4.9 45.0 ± 7.6 15.6 ± 6
213
5 days 62.7 ± 5.3 53.0 ± 13.6 85.0 ± 32.8 11.2 ± 3.4
10 days 40.0 ± 12 126 ± 29
Frequency of a riei ttr
rise in titer
Bacilli (2 x 10' cells) Purified GTA (600 Mg) 326.7 ± 151 Purified GTA (300 Lg) 16.2 ± 3 Control a Based on three to five animals per group. Measured by passive hemolysis with purified GTA SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. b Ratio of animals showing a rise in titer to total number in group.
4/4 3/3 5/5 coated on
TABLE 3. Immune responses of GTA-deprived Sprague-Dawley rats to GTA Mean hemolysin titer' Skin d Frequency MIF Immunogen test of titer rise' 5 days 10 days Base line 42.5 ± 11 0.25 ± 0.25 83.3 ± 39.2 Bacilli (2 x 1010) 4/4 2/4 4/4 0 95.0 ± 13.2 285.0 ± 87 Purified GTA (600 Mg) 4/4 0/5 3/3 230.0 ± 60 73.4 ± 16.9 0.25 ± 0.2 Purified GTA (300jug) 4/4 0/5 4/5 0 57.5 ± 17.5 50 ± 18 GTA on erythrocytes (600 3/3 0/4 2/3 Mg) Control 0 0 0 0.4 0/4 1/4 a Based on four rats per group. Measured by passive hemolysis with purified GTA coated on SRBC (all sera absorbed with SRBC before testing). SE, Standard error of the mean. b Ratio of animals showing a rise in titer to total number in group. 'Ratio of positive animals (induration, >5 mm) at 24 to 48 h to total number tested (different animals than migration inhibitory factor [MIF] and antibody studies). d Ratio of positive animals (inhibition, >20%o) to total number tested.
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INFECT. IMMUN.
CHORPENNING ET AL. TABLE 4. Effect of skin testing on the hemolysin response in rats
Skin test'
No skin test' Mean hemolysin titer
Conventional rats 1 2 3 4 Mean (± SE)C
GTA-deprived rats 1 2 3 4 Mean (± SE)
10 days
Base line
10 days
Base line
60 35 40
80 600 300
32 25 30 25
36 45 74 35
45 ± 7.6
326 ± 150.7
28 ± 1.8
47.5 ± 9.1
0 0 0 0
400 200 120 200
0 0 0 0
800 2,000 1,600
230 ± 59.7 of GTA intravenously followed by 100
Itg
600
1,250 ± 330 intraperitoneally on both days 3
a Animals initially received 100 jg and 6. b Skin-tested animals received the same immunization regimen as described above and were injected with an additional 100 ,ug of GTA intradermally on day 8. 'SE, Standard error of the mean.
They were better in GTA-deprived animals. Possible reasons for the low level of PFC response to soluble GTA will be presented in the discussion.
DISCUSSION In view of reports (6, 19, 29) that purified GTA is not immunogenic in rabbits and the wellknown refractoriness of this species to purified polysaccharide antigens (16), we chose guinea pigs and rats as our subject animals. Surprisingly, guinea pigs responded better to soluble purified GTA than to GTA-bearing bacilli. However, they did not respond as well as SpragueDawley rats. Thus, it appears that responsiveness to this purified antigen follows the sequence, rat > guinea pig > rabbit, in which the rabbit may be completely unresponsive. However, optimal dosages of GTA were not determined in this investigation. We attribute the differences between the present study and those of earlier workers essentially to species differences in the subject animals. It is generally believed that cellular antigens usually produce better responses than soluble ones. Therefore, the greater immunogenicity of soluble GTA in rats was unexpected. We have found no similar observation in the literature and have little to offer in explanation. The greater responsiveness to soluble antigen cannot be attributed to formation of antigen-antibody complexes in primed animals because the TAdeprived rats seldom exhibited natural antibody but responded as well or better than conventional rats. Furthermore, data presented here indicate that increased initial levels of antibody
TABLE 5. Splenic PFC response of conventional and GTA-deprived Sprague-Dawley rats to GTA Immunogen
Mean PFC/107 spleen cells' (+ SE) Conventional
GTA deprived
Bacilli' (2 X 10"')
98.8 ± 20.7 172.5 ± 31.0 79.0 ± 95.8 Purified GTA (600 fg) 22.0 ± 11.2 80.3 ± 43.7 Purified GTA (300 ,g) 19.0 ± 8.1 0 1.9 ± 0.6 Unimmunized control 'Tests performed with GTA-coated SRBC. SE, Standard error of the mean; PFC, plaque-forming cells. 'All animals except the controls were skin tested 2 days before sacrifice.
are associated with poor responses (see below). It might be suggested that the unexpectedly high immunogenicity of soluble GTA was due to its demonstrated adsorbability to cell membranes (8, 15, 25). This does not appear to be the case because in four separate experiments the responses to GTA-coated rat cells were comparable to those with whole bacterial cells and both were much lower than those with soluble
antigen. From both guinea pig and rat data, it appears that animals with relatively high initial (base line) hemolysin levels responded poorly to injected antigen, while those with lower base lines responded well. In fact, at 4 days after the last injection the titer was frequently lower than it was initially. Our previous report (5) shows that injection of antigen produces cycling in this species. Antibody production is depressed (presumably) whenever an antibody excess exists (28),
VOL. 26, 1979
which would be the case in animals yielding high-titered base-line sera. Thus, in these rats the antibody level dropped due to immune elimination in the absence of antibody synthesis. In low-titered rats no such depression would occur and antibody synthesis would proceed. In view of the antigen's persistence (5), it is likely that titers in the high-base line animals would have risen again by 16 days after the antigen injection (5). In this connection, it is important to note that 10-day titers in GTA-deprived rats (zero base line) rose considerably higher than in conventional rats (Table 4) when the animals were given a fourth injection of 100 tLg of GTA. Because rats possess fluctuating levels of natural antibodies for GTA (5), determination of responses to GTA in this species is a problem. Such determinations may be based on the frequency of a rise in titer or an increase in the mean titer of this group (Table 2); however, it was believed that more clear-cut results would be obtained in GTA-deprived animals (27). This assumption was partially true because most rats yielded negative base lines. As we reported earlier (4), normal conventional rats may yield positive skin tests and migration inhibitory factor tests to purified GTA. Therefore, these tests for cell-mediated immunity were conducted on GTA-deprived rats because it was believed they would lack such responses before immunization. This assumption was true for skin tests but, unexpectedly, two of the uninjected GTA-deprived control rats yielded positive migration inhibitory factor tests. Nevertheless, the increase in frequency of migration inhibitory factor in rats injected with either soluble GTA or bacilli indicates that cellmediated responses were induced. The separate group of animals which were skin tested may have failed to exhibit cell-mediated immunity because the test dose was excessive, resulting in a shift to humoral responses. Criticism that the emphasis in this study was on IgM responses would be valid except for the fact that we have previously shown (7) that IgM antibodies predominate in this age group and that IgG and IgM plaques peak together (present study). The lower PFC response when the immunogen was soluble GTA may be related to two previously observed phenomena. Miller and Jackson (23) have shown that soluble GTA can suppress SRBC responses, and we have demonstrated that GTA responses are suppressed as well (unpublished data). We have also shown that very small amounts of GTA enhance rather than suppress (20). Thus, it might be expected that the small amount of soluble GTA released
IMMUNOGENICITY OF TEICHOIC ACID
215
by bacilli would not suppress the response, allowing full immunogenic action by the cellbound GTA. With soluble GTA, initial enhancement may account for circulating antibody levels, followed by PFC suppression which is not yet reflected in antibody levels. It is not clear why such responses would be better in GTAdeprived rats, but it should be kept in mind that these animals are relatively unprimed and would respond differently than conventional rats. Even without invoking suppression, antibody cycling could possibly account for the disparity between PFC responses to soluble antigen and bacilli. We showed that injection of bacilli induced synchronized cycling in rats injected simultaneously, with a PFC peak at 4 days and a serum antibody peak at 16 days (5). However, we did not examine the effects of soluble antigen, and it is quite possible that this kind of immunogen would produce cycles of a different interval. ACKNOWLEDGMENTS This study was supported by the Department of Microbiology and the Graduate School, The Ohio State University. We acknowledge the technical assistance of Thomas J. Alexander, Mary Ann Decker, and Dorian L. Bockstiegel.
LITERATURE CITED 1. Archibald, A. R., and J. Baddiley. 1966. The teichoic acids. Adv. Carbohydr. Chem. 21:323-375. 2. Archibald, A. R., J. Baddiley, and N. L. Blumson. 1968. The teichoic acids. Adv. Enzymol. 30:223-253. 3. Beachey, E. H., L. Ofek, and A. L. Bisno. 1973. Studies of antibodies to non-type-specific antigens associated with streptococcal M protein in the sera of patients with rheumatic fever. J. Immunol. 111:1361-1366. 4. Bolton, R. W., and F. W. Chorpenning. 1974. Naturally occurring cellular and humoral immunity to teichoic acid in rats. Immunology 27:517-524. 5. Bolton, R. W., H. Rozmiarek, and F. W. Chorpenning. 1977. Cyclic antibody formation to polyglycerophosphate in normal and injected rats. J. Immunol. 118:1154-1158. 6. M. M. Burger. 1966. Teichoic acids: antigenic determinants, chain separation, and their location in the cell wall. Proc. Natl. Acad. Sci. U.S.A. 56:910-917. 7. Chorpenning, F. W., R. W. Bolton, G. T. Frederick, and H. Rozmiarek. 1979. Development of cellular and humoral responses to teichoic acid. Dev. Comp. Immunol., in press. 8. Chorpenning, F. W., and M. C. Dodd. 1965. Polyagglutinable erythrocytes associated with bacteriogenic transfusion reactions. Vox Sang. 10:460-471. 9. Chorpenning, F. W., J. J. Lynch, Jr., H. R. Cooper, and J. W. Oldfather. 1979. Modulation of the immune response to sheep erythrocytes by lipid-free glycerol teichoic acid. Infect. Immun. 26:262-269. 10. Chorpenning, F. W., and H. B. Stamper. 1973. Spontaneous adsorption of teichoic acid to erythrocytes. Immunochemistry 10:15-20. 11. Cooper, H. R., F. W. Chorpenning, and S. Rosen. 1978. Lipid-free glycerol teichoic acids with potent membrane-binding activity. Infect. Immun. 19:462-469. 12. Crowder, J. G., and A. White. 1972. Teichoic acid antibodies in staphylococcal and nonstaphylococcal endocarditis. Ann. Intern. Med. 77:87-90.
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13. Decker, G. P., F. W. Chorpenning, and G. T. Frederick. 1972. Naturally occurring antibodies to bacillary teichoic acids. J. Immunol. 108:214-222. 14. Frederick, G. T., R. A. Holmes, and F. W. Chorpenning. 1972. Naturally occurring cell-mediated immunity to purified glycerol-teichoic acid antigen in guinea pigs. J. Immunol. 109:1399-1401. 15. Jackson, R. W., and M. Moskowitz. 1966. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205-2209. 16. Kabat, E. A., and M. M. Mayer. 1964. In E. A. Kabat (ed.), Experimental immunochemistry, 2nd ed., p. 7-8. Charles C Thomas, Publisher, Springfield, Ill. 17. Kent, J. F., and E. H. Fife. 1963. Precise standardization of reagents for complement fixation. Am. J. Trop. Med. 12:103-116. 18. Knox, K. W., M. J. Hewett, and A. J. Wicken. 1970. Studies on the group F antigen of lactobacilli: detection of antibodies by hemagglutination. J. Gen. Microbiol. 60:303-313. 19. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 20. Lynch, J. J., Jr., and F. W. Chorpenning. 1978. Effects of dosage modulaton of sheep red cell responses by teichoic acid. Fed. Proc. 37:1489. 21. Martin, R. R., H. Daugherty, and A. White. 1966. Staphylococcal antibodies and hypersensitivity to tei-
INFECT. IMMUN.
22.
23. 24. 25.
26.
27. 28.
29.
choic acids in man, p. 91-96. Antimicrob. Agents Chemother. 1965. McCarty, M. 1959. The occurrence of polyglycerophosphate as an antigenic component of various gram-positive bacterial species. J. Exp. Med. 109:361-378. Miller, G. A., and R. W. Jackson. 1973. The effects of Streptococcus pyogenes teichoic acid on the immune response of mice. J. Immunol. 110:148-156. Miller, G. A., J. Urban, and R. W. Jackson. 1976. Effects of streptococcal lipoteichoic acid in host responses in mice. Infect. Immun. 13:1408-1417. Moskowitz, M. 1966. Separation and properties of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2200-2204. Ne'eman, N., and I. Ginsburg. 1972. Cell sensitizing antigen of group A streptococci. II. Immunological and immunopathological properties. Isr. J. Med. Sci. 8: 1807-1816. Rozmiarek, H., R. W. Bolton, and F. W. Chorpenning. 1977. Environmental origin of natural antibodies to teichoic acid. Infect. Immun. 16:505-509. Weigle, W. 0. 1975. Cyclical production of antibody as a regulatory mechanism in the immune response. Adv. Immunol. 21:87-111. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigen. Science 187: 1161-1167.
