Cross-Protection among Serotypes of Group A Streptococci

THE JOURNAL OF INFECTIOUS DISEASES VOL. 125 NO.4 APRIL 1972 1972 by the University of Chicago. All rights reserved. ' Cross-Protection among Serotypes of Group A Streptococci Sonia Bergner-Rabinowitz, Itzhak Ofek, and Max D. Moody From the Streptococcal Reference Laboratory, Government Central Laboratories, Ministry of Health, lerusalem, Israel, and the Reagents Evaluation Unit, Center for Disease Control, Health Services and Mental Health Administration, Atlanta, Georgia Cross-protection between two serotypes of Group A streptococci was demonstrated. Type 55 strain 2004 and type 60 strain 365 shared a cross-protective relationship in one direction: type 55 antiserum possessed opsonophagocytic activity against type 60, but type 60 antiserum was inactive against type 55. Agar-gel diffusion tests with both types, their antisera, and reciprocally absorbed sera showed that type 55 possesses two M antigens; one of them is identical to that of type 60 and is responsible for the cross-protection. This cross-protection was found in sera of patients during an outbreak of acute glomerulonephritis caused by type 55. Fifty percent of the patients who developed immunity against the infecting type 55 also developed immunity against type 60 as tested by opsonophagocytic or bactericidal tests. None of the sera tested possessed a significant amount of opsonophagocytic activity against control types of group A streptococci. Evidence for a cross-protective relationship among streptococcal types has been a subject of interest in recent publications [1-3]. Wiley and Bruno [4] summarized the combinations of streptococcal types for which cross-protection has been demonstrated either in mouse-protection or bactericidal tests. These authors stress the importance of crossprotection in immunity acquired from streptococcal infections in humans. The studies discussed in this paper include cross-protection between two serotypes of group A streptococci, M type 55 (strain 2004), and M type 60 (strain 365), demonstrated by agar-gel diffusion, opsonophagocytic, and bactericidal tests on immune rabbit sera and convalescent sera obtained from patients during an outbreak of acute glomerulonephritis caused by M type 55 [5]. Received for publication March 26, 1971, and in revised form August 30, 1971. The authors are indebted to Dr. Kurt Rabinowitz, Head, Hospital Cross-Infection Unit, Ministry of Health, Jerusalem, Israel, who made it possible to use the material from patients at hospitals. Please address requests for reprints to Dr. Sonia Bergner-Rabinowitz, Streptococcal Reference Laboratory, Government Central Laboratories, Ministry of Health, P. O. Box 6115, Jerusalem, Israel. Materials and Methods Strains. The strains used in this work were type 55 strain 2004, isolated from skin lesions of a patient with acute glomerulonephritis [5] and type 60 strain 365, isolated in 1967 from the throat of a patient with acute pharyngitis. These streptococci will subsequently be referred to as type 55 and type 60, respectively. The latter strain reacted with type 4 agglutinating antiserum and was not precipitated by any M sera available [5]. Type 12 strain 1187 was isolated in Israel from the throat of a patient with an acute upper respiratory infection. Type 49 strain 100076 and type 25 strain 100088 were obtained from the Cross-Infection Reference Laboratory, Colindale, London. Double-diffusion microtechnique in agar gel. The agar-gel-coated slides were prepared in our laboratory. One gram of Difco special Noble agar was dissolved in 100 ml of barbitone-acetate buffer, 0.05 ionic strength, ph 8.6 (Oxo Ltd., London, code no. BR 11G) by heating to boiling with constant stirring. Four milliliters of this agar gel was poured onto a slide, 1 X 3 in. After the agar had set, two series of holes were punched, one central and five peripheral, and the bottoms of the holes were coated with the same agar by means of 339

340 Bergner-Rabinowitz, Ojek, and Moody a capillary tube. The. holes were 0.15 mm in diameter and the center-to-center distance between central and peripheral wells was 7 mm. The reactants were put into the holes with a calibrated capillary tube. The antiserum was placed in the center and the antigens at the periphery. The glass slides were then kept at room temperature in a humid atmosphere for 24 hr (adequate time for the occurrence of complete reactions). Staining procedure. The precipitin lines formed were stained with amidoblack. The slides were soaked in 0.9% NaCI for two days; the saline was changed twice daily, with occasional agitation to remove all the soluble material. The washed agar gel was then covered with filter paper in which small holes were punched over the sites of the holes to prevent adhesion of the filter paper during the drying process, which was carried out at 37 C, for 24 hr. The filter paper was then removed, and the dried agar-gel slide was carefully washed under tap water to remove the adherent paper fibers and was submerged in amidoblack stain for 10 min [6]. The preparation was next washed with tap water, and the excess dye was removed with two changes of a solution of 5% acetic acid in 50% methanol for half an hour, and the slide was then dried. Preparation of antigen. Hydrochloric-acid extracts were prepared according to the technique of Swift et al. [7]. Antisera and typing. The streptococci were examined for T antigen by the slide-agglutination method of Griffith [8]; capillary-precipitin tests were performed by the technique of Swift et al. [7]. Typing antisera used for M and T antigens were those prepared in the Streptococcus Reference Center, Jerusalem [9], where M antisera for strains 2004 and 365 (hereafter referred to as M55 and M60 antiserum, respectively) were also produced. The M55 antiserum was absorbed with streptococci of types 6 and 25, while the M60 antiserum was absorbed with streptococci of types 6 and 30. Cross-absorption of M antisera for the types 55 and 60 was done. The cross-absorbed antisera were tested by agar-gel immunodiffusion and for their reciprocal opsonophagocytic activity. Bactericidal test. The test was performed and the results were interpreted as described previously [10]. Patients' sera. Sera were collected from 45 glomerulonephritic patients during an outbreak of infections due to type 55 streptococci [5]. They were kept frozen at -20 C until used in the opsonophagocytic or bactericidal test. Microtest tor type-specific antibody. The methods employed have been described in detail in a separate study [11] in which evidence of the overall reproducibility of the test, and its advantages in comparison to the bactericidal test, were presented. Results Immunodiffusion analysis of the relation between types 55 and 60. Double-diffusion tests demonstrated precipitin bands that corresponded with the homologous and cross-reactive opsonophagocytic properties of the antisera in each case. The cross-precipitin reaction between types 55 and 60 was demonstrated on agar gel (figure 1) by four tests. In the first test, central well A contained M60 antiserum, and the peripheral wells the homologous and heterologous M extracts. The homologous reaction of type 60 consisted of one precipitin line which formed a line of identity with the cross-reacting type 55 line. In the second test, the central well B contained M55 antiserum. The homologous reaction consisted of two lines of precipitate appearing close to each other. One of the bands joined the single band produced by type 60 in a reaction of identity. The third test showed the cross-reaction that occurred when the M55 extract was applied against M55 and M60 antisera. Reciprocal absorption removed from both antisera the antibodies responsible for the crossreactions. The fourth test showed this for the M55 antiserum. The central well C contained the absorbed M55 antiserum as did well B, which was, in addition, absorbed with type 60 cells. This absorption removed the type 60 band, leaving only one band. Identical results were obtained when M55 antiserum was absorbed with type 60 cells only. (Thus, the type 60 cells proved to be the best-absorbing cells for removal of all crossreactions from M55 antiserum.) The M60 antiserum absorbed with type 55 cells lost its homologous line (the one common with type 55). The type-specific precipitin lines produced by both types 55 and 60 were not produced when the M extracts were prepared from cells that had been grown in culture with trypsin at a final concentration of 0.02%.

Cross-Protective Streptococcal Serotypes 341 Figure 1. Immunodiffusion showing the cross-precipitin reaction between types 55 and 60. Wells were charged as follows. Streptococcal antisera wells: (A) M60 antiserum absorbed with cells of types 6 and 30; (B) M55 antiserum absorbed with cells of types 6 and 25; (C) same as B, except additionally absorbed with cells of type 60. Acid extract of strains: (1) type 60; (2) type 55; (3) type 25; (4) type 12; (5) type 49. Table 1. Reactions in agar-gel diffusion and opsonophagocytosis of strains 2004 and 365 with homologous and heterologous antiserum. Strain T M 2004 8/25lImp 19155 55 365 4 60 No. lines in agar-gel diffusion* 2004 365 2 1 1 1 Opsonophagocytic relationship of types 55 and 60. Results of opsonophagocytic tests with the antisera are compared with results of precipitin tests in table 1. Antisera 55 and 60 promoted phagocytosis of each of the corresponding homologous strains. The relationship of the two antisera in reciprocal opsonophagocytic tests showed a cross-protection in one direction only. The M55 antiserum had an opsonophagocytic effect on strain 60, whereas M60 antiserum did not have an opso- Opsonophagocytic index 2004 365 >100 <40 >100 >100 * Strain 2004 antiserum absorbed twice with type 6 cells and 365 antiserum absorbed once with type 6 and once with type 30 cells. nophagocytic effect on strain 55, although they shared a common antigen. These results are similar to those obtained by Wiley et al. [3] concerning type 14-51; we deduced accordingly that type 60 has a single M protein shared by type 55, and the latter has an additional M antigen not present in type 60. Therefore, although the antibodies in M60 antiserum react with the corresponding M antigen (M60) on type 55, the additional M antigen of type 55 for which no antibodies were present in M60 antiserum enables type 55 to resist phagocytosis. The reactions of cross-absorbed antisera (table 2) in immunodiffusion and opsonophagocytosis tests were in accordance with the scheme suggested for the antigenic composition of types 55 and 60. Absorption of M55 antiserum with type 60 cells removed one of the two precipitating lines produced by M55 antiserum against its homologous strain and eliminated the opsonophagocytic effect on type 60 but not on type 55. Absorption of M60 antiserum with type 55 cells removed the precipi-

342 Bergner-Rabinowitz, Ojek, and Moody Table 2. Agar-gel diffusion and opsonophagocytosis of special absorbed antisera against homologous and heterologous strains of Group A streptococci. 2004 Antiserum absorbed with cells 365 Antiserum absorbed with cells Reaction Strain 0* 365t 2004t 0* 2004t 365t No. of lines in 2004 2 1 0 1 0 0 agar-gel diffusion 365 0 0 1 0 0 Opsonophagocytic index 2004 ~120 80-119 <40 <40 <40 <40 365 ~120 <40 <40 ~120 <40 <40 * The serum used was absorbed as described in table 1. t Additional absorption to that described in table 1. tating line produced against types 60 and 55, as well as the opsonophagocytic activity on type 60. Opsonophagocytic activity was examined in 31 sera from convalescent patients with glomerulonephritis associated with type 55, Group A streptococci [5]. The sera were collected three months after onset of the disease. All sera were tested for antibodies to types 55 and 60, and some of them were also tested for antibodies to types 12,25, and 49. Forty-two percent of the sera did not contain opsonophagocytic antibodies against any of the types tested. The results of the opsonic activity of the remaining 58% (18 antisera) against the dif- Table 3. Opsonophagocytic tests showing cross-protection between strains 2004 and 365 in human sera. Opsonophagocytic index Cross- Patient no. 2004 365 protection 76* 99 14 -t 6468 111 2 6471 170 8 7788 154 32 5307* 85 116 7396 98 54 ± 4290 116 48 ± 5701 79 20 5946* 91 140 6885* 90 80 6792 150 13 7666* 89 80 4251 116 86 657* 96 140 7262* 97 80 8625* 140 96 7155* 72 17 7509 89 78 * No opsonophagocytic activity against types 12, 25, and 49. t(-) =None, (±) = borderline, and () = positive. ferent antigens are outlined in table 3. Of the 18 sera, nine contained antibodies to types 55 and 60, and two showed a borderline cross-protecting reaction against type 60. None of 10 sera tested contained a significant amount of antibody to types 12, 25, and 49. This cross-protection was confirmed by the bactericidal test on sera of 14 patients, which was collected three to four months after onset of the disease. Three sera were negative against both types 55 and 60. Of the remaining 11 sera, five contained antibodies to types 55 and 60, and two showed a borderline cross-protecting reaction against type 60 (table 4). Table 4. Bactericidal and opsonophagocytic tests showing cross-protection between strains 2004 and 365 in sera from humans and rabbits. Strain 2004 Strain 365 Bacteri- Opsonopha- Bacteri- Opsonopha- No. of cidal gocytic cidal gocytic patients index* indext index* indext 292 ± ± 383 ± ± 477 577 360 ± 301 304 305 351 407 197 Rabbit antiserum M55 M60 *(-)=<5; (±) = 5-24; () = 25-99; () = ~ 100. t (-) = < 40; (±) = 40-79; () = 80-119; () = 120.

Cross-Protective Streptococcal Serotypes 343 Discussion This investigation revealed cross-protection between two serotypes of Group A streptococci, types 55 and 60. An excellent opportunity to study this cross-protection in humans was provided by an outbreak of acute glomerulonephritis caused by type 55 [5]. The results presented in this paper (tables 3 and 4) show that 50% of the patients who developed immunity against the infecting type 55 exhibited immunity against the other crossreacting type, strain 365, type 4 (by agglutination), which was identified by the Bacteriology Section of the Center for Disease Control in Atlanta, Georgia, as M type 60 (table 5). At least 13 types show similar combinations of cross-protection in experimental streptococcal infection [4]. The importance of the role that these cross-protective reactions play in human streptococcal immunity is now of great interest [4] and has not been clearly demonstrated in previous publications [12]. Fox et ai. [2] did bactericidal tests which showed cross-protection between types 3 and 12 in two of nine convalescent sera from patients who had infection due to type 3. In their study, the cross-protection observed in the two patients was not conclusive, since the earliest sera in these pairs were not studied by the bactericidal test, and antibodies to type 12 resulting from carriage of type 12 were not excluded. This is supported by unpublished data obtained in our laboratory five years ago on type-specific antibodies found in the sera of children from a single classroom. The bactericidal tests on these sera showed that antibodies to different types may develop in the same subject, due to asymptomatic or Table 5. Precipitin reactions demonstrated at the Center for Disease Control. Precipitin reactions withstrains M antisera 365* 2033900SSt 1-6,8,9,11-15,17-19,22, Negative Negative 23,26,27,29,30-32,36-47,49,50,53-57 M6o Strong Strong 2033900SS Moderate Moderate *T type 4 strain from Israel. t Isolated in Trinidad by Dr. Elizabeth V. Potter, Northwestern University Medical School, Chicago (M60). :t: Prepared from Dillon's strain 2797S. Table 6. Bactericidal antibodies of sera from children with types 3, 12, or 14 streptococcal infections of the upper-respiratory tract and their class contacts from whom the streptococci were not isolated. Bactericidal index* Patient Type 3 Type 12 Type 14 With type 12 1 2 3 With type 3 4 With types 4 and 12 5 6 * (-) =< 5; () = ;;:: 25. symptomatic residence in the throat of the corresponding streptococcal types. Table 6 gives the results of bactericidal tests on 14 such children who had type 3, 12, or 14. Type-specific antibodies against types 3 and 12 were found in two sera, whereas antibodies against types 12 and 14 were found in one serum. The different serotypes were isolated from two of these patients. Since the three serotypes, 3, 12, and 14, circulated in the classroom for months, the appearance of two or more different antibodies in this group of children was not entirely surprising. The present study excludes infection with type 60, as no type 60 streptococci were isolated from any of the patients with glomerulonephritis during or before the outbreak, and none of the earliest sera of the patients studied contained antibodies against type 60. Moreover, whereas Fox et al. [2] were unable to show cross-protection between types 3 and 12 in immune rabbit sera, our tests clearly demonstrated the cross-protection between M types 55 and 60 by opsonophagocytic and bactericidal tests (table 4). From the results obtained after extensive experimental comparison in a previous study [11] and in the present study (table 4), it would appear that the opsonophagocytic test compares favorably to the bactericidal test in both human and immune rabbit sera. No isolation 7, 8, and 9 10 11, 12, 13, and 14

344 Bergner-Rabinowitz, Ojek, and Moody References 1. Wiley, G. G., Bruno, P. N. Cross-reactions among group A streptococci. I. Precipitin and bactericidal cross-reactions among types 33, 41, 43, 52 and Ross. J. Exp. Med. 128:959-968, 1968. 2. Fox, E. N., Wittner, M. K. Antigenicity of the M proteins of group A hemolytic streptococci. IV. Cross-reactivity between serotypes. J. Immunol. 100:39-45, 1968. 3. Wiley, G. G., Wilson, A. T. The occurrence of two M antigens in certain group A streptococci related to type 14. J. Exp. Med. 113:451-465, 1961. 4. Wiley, G. G., Bruno, P. N. Cross-reactions among group A streptococci. II. Further analysis of antigens related to type-specificity and protection. J. Immunol. 103: 149-154, 1969. 5. Lasch, E. E., Frankel, V., Vardy, P. A., Bergner Rabinowitz, S., Ofek, I., Rabinowitz, K. Epidemic glomerulonephritis in Israel. J. Infect. Dis. 124 : 141-147, 1971. 6. Crowle, A. J. Immunodiffusion. Academic Press, New York, 1961. 333 p. 7. Swift, H. F., Wilson, A. T., Lancefield, R. C. Typing group A hemolytic streptococci by M precipitin reactions in capillary pipettes. J. Exp. Med. 78: 127-133, 1943. 8. Griffith, F. The serological classification of Streptococcus pyogenes. J. Hyg, (Lond.) 34:542-584, 1934. 9. Bergner-Rabinowitz, S., Sklut, 0., Haimowici, E., Davies, A. M. Streptococcal types in Israel hospitals. A four-year study. Israel J. Med, Sci. 2:428 435, 1966. 10. Bergner-Rabinowitz, S., Beck, A., Ofek, I., Davies, A. M. Identification of type specific streptococcal antibodies by in vitro phagocytosis. Israel J. Med. Sci. 5:285-292, 1969. 11. Bergner-Rabinowitz, S., Ofek, I., Davies, A. M., Rabinowitz, K. Type-specific streptococcal antibodies in pyodermal nephritis. J. Infect. Dis. 124: 488-493, 1971. 12. Wiley, G. G., Bruno, P. N. Cross-reactions among group A streptococci. III. The M and R antigens of type 43 and serologically related streptococci. J. Immunol. 105:1124-1130, 1970.