Voi. 11, 1992
919
I
IIII
II
Variability of Surface-Exposed Antigens of Different Strains of Moraxella catarrhalis I. J 6 n s s o n l , , T. H o l m e 2, A. K r o o k 1, M. Rahrnan 2, M. Thor6n 2
For serological
diagnosis of infection
with
Moraxella (Branhamella) catarrhalis it is important to determine if there is variability of antigenic properties among different strains. Cross-reactions of nine strains were investigated by an immunofluorescence test using sera from immunized rabbits. All titres but one were 1:256 or higher, the highest being 1:4096. Thus a high degree of antigenic similarity was demonstrated among different strains of Moraxella catarrhalis. However, the homologous titres of six sera were 2 to 16 times higher than the titres for other strains indicating strain variations in antigenic properties of some surface components. There was no correlation between lipopolysaccharide type and titre in the immunofluorescence test.
Moraxella catarrhalis has been increasingly recognized as a major pathogen in childhood otitis media and a frequent cause of maxillary sinusitis. It may also cause respiratory disease in patients with chronic bronchitis and in immunoCOmpromised hosts (1). The isolation of Moraxella catarrhalis in clinical samples is, however, not sufficient evidence of a role as pathogen in respiratory disease. It is found in nasopharyngeal samples from healthy children in frequencies up to 45 % and is also found, although at much lower rates, in healthy adults (2, 3). Therefore, serological evidence of infection with Moraxella catarrhaliv is of great importance.
For the detection of an antibody response to Moraxella catarrhalis during infection it is of importance to determine if there is variability of antigens in different strains. In some serological studies whole bacterial cells were used as antigen in enzyme immunoassays. In order to overcome l Department of Infectious Diseases, Huddinge Hospital, Karolinska Institute S-141 86 Huddinge, Sweden. Department of Bacteriology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
errors depending on possible variations in antigens between different strains, a mixture of ten different strains was used (4, 5). In other studies one strain has been considered representative of the species (6) on the basis of the reported similarity in outer membrane proteins of different isolates of Moraxella catarrhalis (7). However, variations in lipopolysaccharide (LPS) as well as outer membrane protein antigens have been demonstrated (8-10). We analysed the variability of antigens exposed on the surface of Moraxella catarrhalis by immunizing rabbits with nine different strains and examining cross-reactivity of the antisera by an immunofluorescence test using whole bacteria as antigen. Materials and Methods, Nine Moraxella catarrhalis strains were used. Four of these were clinical isolates from nasopharyngeal swabs and five were reference strains from the American Type Culture Collection (ATCC 25238) and the Culture Collection of the University of G6teborg, Sweden (CCUG 1497, 3292, 18283, 18284). Strains isolated from nasopharyngeal specimens which were gram-negative diplococci showing a typical colony morphology and a positive oxidase test were provisionally considered as Moraxella catarrhalis. Fermentation of glucose, fructose and maltose and a DNase test was performed to distinguish the isolates from Neisseria species. All isolates were nonfermenters and positive in the DNase test. Antigens for immunization were prepared by culturing strains overnight on horse blood agar plates at 37°C. Bacteria were suspended in saline with 0.5 % (v/v) formalin and incubated for 30 min, washed twice in saline and diluted to OD 0.3 at 600 nm. Nine New Zealand white rabbits were immunized intravenously, each one with a different strain of Moraxella catarrhalis. Two rabbits (immunized with strains CCUG 18283 and 18284) were immunized with 0.5 ml of the bacterial suspension on five occasions over a period of 15 months whereas the other seven rabbits were given 0.5 ml bacterial suspension three times over two months. Rabbits were finally bled 2 to 3 weeks after their last immunization. The antibody response of the rabbits was determined by an enzyme immunoassay (EIA) using whole bacterial cells as antigen. The bacteria were suspended in phosphate buffered saline (PBS) and washed four times. Microtitre plates (M 129 A, Dynatech Laboratories, USA) were coated with 100 ~1 of bacterial suspensions diluted in 0.05 M carbonate buffer, pH 9.6 (OD 0.01 at 600 nm). Plates were incubated overnight at room temperature and then washed four times
920
in saline containing 0.05 % (v/v) Tween 20 (saline-T). Two-fold dilutions of sera were added in 100 ~al amounts to each well. Plates were incubated at 37 °C for 1 h and then rinsed four times in saline-T. Horse radish peroxidase conjugated sheep anti-rabbit immunoglobulin was used in a dilution of 1:4000, 100/al of which was added to each well and incubated for 30 rain at 37 °C followed by washing four times. Finally, 100 ~al of substrate solution (0.1 mg/ml TMB, Merck, Germany) in 0.1 M acetate buffer at pH 6.0 with 0.002 % (v/v) H202 was added to the wells. The reaction was stopped by adding 50 ~ul2 M H2SO4. Absorbance was read at 450 nm in a Titertec Multiscan photometer (Flow Laboratories, UK). Smears for immunofluorescence were prepared by growing strains on horse blood agar plates for 18 h at 37 °C. Bacteria were then transferred to nutrient broth and shaken at 37 °C for 2 h. Bacterial suspensions were centrifuged at 3,000 x g for 5 min and washed twice in PBS. The pellets were diluted in PBS to OD 1.0 at 600 nm. Antigens were coated on eight well multislides washed in ethanol. Ten ~tl of the antigen solution was placed in each well. Smears were allowed to dry at 37 °C and were fixed in acetone for 15 min. They were frozen at -20°C until used. The. slides were thawed at room temperature in a moist chamber, washed twice in PBS for 5 min and dried. Ten/al of serially doubly diluted serum was added to appropriate wells and slides were incubated for 30 min at 37 °C in a chamber, washed twice in PBS for 5 min and let dry. Ten ~1 conjugate (Dakopatts fluorescein conjugated swine anti-rabbit immunoglobulin) was added to the wells, incubated for 20 min at 37 °C in a chamber and again washed twice for 5 min in PBS and dried. Slides were finally mounted under cover glasses with phosphate buffered glycerol (PBS-glycerol 1:10) and kept dark and cool in order not to get bleached. Pre-immune rabbit sera diluted 1:8 were used as negative controls. Smears were read at magnification 1000 times under a Zeiss standard 16 EPIfluorescence microscope. The following criteria for recording the fluorescence were used: 3+ very bright peripheral staining, 2+ bright and clearly visible peripheral staining, 1+ barely visible peripheral staining, 0 no reaction. 2+ and 3+ were considered as positive results. The LPS type was determined by an EIA similar to that described by Vaneechoutte et al. (10), using reference strains ATCC 25238 (type A), F17 (type B), and B3 (type C), kindly supplied by
Eur. J. Clin. Microbiol. Infect. Dis.
M. Vaneechoutte. The strains to be tested were grown overnight on blood agar plates at 37 °C. The bacteria were suspended in saline and washed three times. Finally, they were suspended in PBS and the protein content was estimated using the BioRad protein assay (BioRad Laboratories, USA). For absorption of sera, a bacterial suspension at a density corresponding to OD 0.7 at 595 nm in the BioRad assay was used. The suspension (500 ~al)was mixed with 500 lal of serum diluted 1:250 an d kept for 4 h at 37 °C. It was then centrifuged at 5,000 x g and the supernatant collected. Each antiserum was absorbed separately with the homologous strain and the reference strains. For typing, fiat bottom microtitre plates (Immunolon II, Dynatech Laboratories) were coated with LPS (extracted by the phenol/water procedure) from the strains to be tested. Coating was done in carbonate buffer at pH 9.6 and the plates kept overnight at room temperature. After washing in PBS, each LPS was incubated with homologous unabsorbed serum (positive control), homologous absorbed serum (negative control) and antisera absorbed with the reference strains for types A, B and C in paired wells and incubated for 2 h at 37 °C. The serum dilutions used were 1:500 to 1:50,000. Horse radish peroxidase conjugated sheep antirabbit immunoglobulin was then added to each well at a dilution of 1:1,000 and incubated for i h at 37 °C. After washing four times 100 tal of substrate solution (TMB, Merck) in 0.1 M acetate buffer (pH 6.0) with 0.002 % (v/v) H202 was added. The reaction was stopped after 10 min by adding 50 lal 2 M H2SO4. Absorbance was read at 450 nm in a Titertec Multiscan photometer (Flow Laboratories, UK). The absorbance was plotted on a logarithmic scale and the degree of inhibition calculated (10). Results and Discussion. The sera from the immunized rabbits were tested in an EIA using whole bacterial cells as antigen. Despite differences in number of booster doses and time for the immunization, all sera showed the same high antibody titres of 1:25,000 for their homologous strain. Sera from three rabbits obtained before immunization and sera from non-immunized rabbits did not show any antibody activity against strains of Moraxella catarrhatis in the EIA. Immunofluorescence tests for surface antigens were read without difficulty in differentiating between positive and negative smears. The antibody titres, represented as the reciprocal value of the highest serum dilution giving a positive reading in the immunofluorescence tests of the nine sera for the
VoI. 11, 1992
921
Table 1: Cross-reactionswith Moraxella catarrhal&surface-exposedantigensas detected by immunofluorescence.Figures represent the reciprocalvalueof the highestserumdilutiongivinga positivereading.Homologoustitres are underlined.The LPS type was determinedby inhibitionEIA (10).
Antigen
ATCC25238 CCUG1497 CCUG18283 CCUG18284 RS272 CCUG3292 RS 13 RS10 RS26 --
LPS type A A A A A B B C C
Serum no. 1
2
3
4
5
6
7
8
9
2048 512 1024 1024 512 1024 1024 512 256
256 4096 256 256 256 256 128 256 256
1024 512 2048 1024 1024 512 1024 1024 1024
1024 512 1024 2048 512 512 512 512 512
512 512 1024 256 1024 512 512 512 1024
1024 1024 1024 512 1024 1024 512 1024 512
1024 512 512 2048 1024 1024 4096 512 512
512 512 1024 512 2048 512 1024 4096 1024
1024 512 1024 2048 4096 1024 1024 2048 204~
,,,,
nine strains, are shown in Table 1. The heterologous reactions ranged, with one exception, from 256 to 4096. When the sera were tested against their homologous antigens (underlined figures in Table 1) the titres were 1024 in two, 2048 in four and 4096 in three sera. The titres of the CCUG 1497 antiserum against other strains were low but the homologous titre was high indicating that this strain might differ from the other strains in antigenic composition.
Moraxella catarrhalis has been divided into tl~ree serotypes on 'the basis of LPS antigens (10). The LPS serotypes of our strains are listed in Table 1. The serotype was determined in order to find out if there was a correlation between LPS antigen type and titre in the immunofluorescence test. This was tested by comparing the median titres of reactions between strains and sera of the same LPS type with all reactions between LPS unrelated strains and sera. Strain-specific reactions were excluded. The median values of LPS type related and unrelated titres were both 512, indicating that there was no correlation between LPS serotype and titre in the immunofluorescence test. Several studies have been conducted with the objective of determining a specific antibody response during infection with Moraxella catarrhalis (4, 5, 11-13). Chapman et al. (13) showed that most patients with pulmonary infections caused by MoraxeUa catarrhalis developed convalescent phase IgG antibodies which had bactericidal activity. However, in serological studies of respiratory infections where whole bacterial cells were used as antigen, the titre rise in paired sera was very variable, only a minority of the patients having a two-fold or greater increase (4, 5). This divergence in the antibody responses
during infection could be due to differences in the immune response to Moraxella catarrhalis in different individuals, but it could also be due to differences in the predominant antigens in different strains. Gram-negative bacteria, in particular enteric pathogens, display great variation in the antigens of their cell surfaces. Likewise in Ilaemophitus influenzae extensive variations in antigenic specificity of outer membrane proteins as well as LPS determinants occur. For the preparation of antigens for determination of antibody responses during infections it is therefore necessary to investigate possible antigenic diversity of the different bacterial species. Although some investigations have indicated homogeneity in numbers and molecular mass of major outer membrane proteins of Moraxella catarrhalis as demonstrated by SDS-PAGE, antigenic differences have been demonstrated and differences in LPS antigens have also been found (7, 10). Our results show that all nine strains produced a marked antibody response in rabbits to antigens expressed on the surface of Moraxella catarrhalis. Each serum, except one, was capable of reacting at high titres with antigens from all nine strains tested. This must be due to the presence of one or more antigens common to the species on the outer membrane of different strains of Moraxella catarrhalis. Since there was no correlation between the LPS serotype and titre in the immunofluorescence test we conclude that other surfaceexposed antigens, probably outer membrane proteins, predominate in the reaction with antibodies raised against whole cell antigens. We have shown in an EIA using LPS as antigen that the antisera contain antibodies against the LPS (unpublished results). The lack of correlation of titres with the LPS serotype could possibly be ex-
922
plained by p o o r presentation of the antigenic d e t e r m i n a n t s of LPS on the bacterial surface. However, as shown by Murphy and Vaneechoutte et al. (9, 10) efficient absorption of antibodies to LPS is easily obtained with whole bacterial cells, indicating surface exposure of these antigens. In addition to the antigens c o m m o n to the different strains, M o r a x e l l a catarrhalis seems to carry antigens that are unique for each strain since with most strains higher titres were obtained when testing sera with their h o m o l o g o u s strain than when sera were tested against other strains. This is not unique to M o r a x e l l a catarrhalis, since strain-specific and i m m u n o d o m i n a n t surface epitopes have been d e m o n s t r a t e d in Haemophilus influenzae outer membrane proteins (14, 15). In the immunofluorescence test only surface-exposed antigens will react with their corresponding antibody. As shown by Murphy and Bartos (9) not all outer m e m b r a n e proteins possessed surface-exposed antigenic determinants, as d e m o n s t r a t e d by i m m u n o b l o t ting experiments using rabbit antisera a b s o r b e d with whole bacterial cells. Two of the O M P s could be shown to vary in antigenic properties, some strains failing to absorb antibodies to surface-exposed antigens. It might thus be possible that the strain variations in titres in the immunofluorescence test with different sera depend on variations in O M P antigens. However, one must then assume that the variable O M P antigens display several specificities in addition to those d e m o n strated by M u r p h y and Bartos (9). Our s t u d y has shown that although strain differences exist, there is a basic similarity in surface antigens giving high cross-reactivity a m o n g the nine M o r a x e l l a catarrhalis strains used.
References
1. Doern GV: Branhamella catarrhalis - an emerging human pathogen. Diagnostic Microbiology and Infectious Disease 1986, 4: 191-201. 2. lngvarsson L, Lundgren K, Ursing J: The bacterial flora in the nasopharynx in healthy children. Acta Otolaryngologica 1982, 386, Supplement: 94-96. 3. Ejlertsen T: Pharyngeal carriage of Moraxella (Branhamella) catarrhalis in healthy adults. European Journal of Clinical Microbiology & Infectious Diseases 1991, 10: 89. 4. Leinoncn M, Luotonen J, Herva E, M~ikelfi H: Preliminary serologic evidence for a pathogenic role of Branhamella catarrhalis. Journal of Infectious Diseases 1981, 144: 570-574.
Eur. J. Clin. Microbiol. Infect. Dis.
5. Black AJ, Wilson TS: Immunoglobulin G serological response to Branhamella catarrhalis in patients with acute bronchopulmonary infections. Journal of Clinical Pathology 1988, 41: 329-333. 6. Goldblatt D, Seymour ND, Levinsky R J, Turner MW:
An enzyme-linked immunosorbent assay for the determination of human IgG subclass antibodies directed against Branhamella catarrhalis. Journal of Immunological Methods 1990, 128: 219-225. 7. Murphy TF: The surface of Branhamella catarrhalis: a systematic approach to the surface antigens of an emerging pathogen. Pediatric Infectious Diseases Journal 1989, 8: 75-77. 8. Murphy TF: Studies of the outer membrane proteins of Branhamella catarrhalis. American Journal of Medicine 1990, 88, Supplement 5A: 41-45. 9. Murphy TF, Bartos LC: Surface-exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis. Infection and Immunity 1989, 57: 2938-2941. 10. Vaneechout/e M, Verschraegen G, Claeys G, van den Abeele A: Serological typing of Branhamella catarrhalis strains on the basis of lipopolysaccharide an-
tigens. Journal of Clinical Microbiology 1990, 28: 182187. 11. Eliasson !: Serological identification of Branhamella catarrhalis. Serological evidence for infection. Drugs 1986; 31: 7-10. 12. Chi DS, Verghese A, Moore C, Hamati F, Berk SL: Antibody response to P-protein in patients with Branhamella catarrhalis infections. American Journal of
Medicine 1990, Supplement 5A: 25-27. 13. Chapman A J, Musher DM, Jonsson S, Clarridge JE, Wallace R J: Development of bactericidal antibody during Branhamella catarrhalis infection. Journal of
Infectious Diseases 1985, 151: 878-882. 14. Groeneveld K, van AIphen L, Voorler C, Eijk PP, Jansen HM: Antigenic drift of Haemophilus influenzae in patients with chronic obstructive pulmonary disease. Infection and Immunity 1989, 57: 3038-3044. 15. Haase EM, Campagnari AA, Sarwar J, Shero M, Wirlh
M, Cumming CU, Murphy TF: Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae. Infection and Immunity 1991, 59: 1278-1284.
919
I
IIII
II
Variability of Surface-Exposed Antigens of Different Strains of Moraxella catarrhalis I. J 6 n s s o n l , , T. H o l m e 2, A. K r o o k 1, M. Rahrnan 2, M. Thor6n 2
For serological
diagnosis of infection
with
Moraxella (Branhamella) catarrhalis it is important to determine if there is variability of antigenic properties among different strains. Cross-reactions of nine strains were investigated by an immunofluorescence test using sera from immunized rabbits. All titres but one were 1:256 or higher, the highest being 1:4096. Thus a high degree of antigenic similarity was demonstrated among different strains of Moraxella catarrhalis. However, the homologous titres of six sera were 2 to 16 times higher than the titres for other strains indicating strain variations in antigenic properties of some surface components. There was no correlation between lipopolysaccharide type and titre in the immunofluorescence test.
Moraxella catarrhalis has been increasingly recognized as a major pathogen in childhood otitis media and a frequent cause of maxillary sinusitis. It may also cause respiratory disease in patients with chronic bronchitis and in immunoCOmpromised hosts (1). The isolation of Moraxella catarrhalis in clinical samples is, however, not sufficient evidence of a role as pathogen in respiratory disease. It is found in nasopharyngeal samples from healthy children in frequencies up to 45 % and is also found, although at much lower rates, in healthy adults (2, 3). Therefore, serological evidence of infection with Moraxella catarrhaliv is of great importance.
For the detection of an antibody response to Moraxella catarrhalis during infection it is of importance to determine if there is variability of antigens in different strains. In some serological studies whole bacterial cells were used as antigen in enzyme immunoassays. In order to overcome l Department of Infectious Diseases, Huddinge Hospital, Karolinska Institute S-141 86 Huddinge, Sweden. Department of Bacteriology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
errors depending on possible variations in antigens between different strains, a mixture of ten different strains was used (4, 5). In other studies one strain has been considered representative of the species (6) on the basis of the reported similarity in outer membrane proteins of different isolates of Moraxella catarrhalis (7). However, variations in lipopolysaccharide (LPS) as well as outer membrane protein antigens have been demonstrated (8-10). We analysed the variability of antigens exposed on the surface of Moraxella catarrhalis by immunizing rabbits with nine different strains and examining cross-reactivity of the antisera by an immunofluorescence test using whole bacteria as antigen. Materials and Methods, Nine Moraxella catarrhalis strains were used. Four of these were clinical isolates from nasopharyngeal swabs and five were reference strains from the American Type Culture Collection (ATCC 25238) and the Culture Collection of the University of G6teborg, Sweden (CCUG 1497, 3292, 18283, 18284). Strains isolated from nasopharyngeal specimens which were gram-negative diplococci showing a typical colony morphology and a positive oxidase test were provisionally considered as Moraxella catarrhalis. Fermentation of glucose, fructose and maltose and a DNase test was performed to distinguish the isolates from Neisseria species. All isolates were nonfermenters and positive in the DNase test. Antigens for immunization were prepared by culturing strains overnight on horse blood agar plates at 37°C. Bacteria were suspended in saline with 0.5 % (v/v) formalin and incubated for 30 min, washed twice in saline and diluted to OD 0.3 at 600 nm. Nine New Zealand white rabbits were immunized intravenously, each one with a different strain of Moraxella catarrhalis. Two rabbits (immunized with strains CCUG 18283 and 18284) were immunized with 0.5 ml of the bacterial suspension on five occasions over a period of 15 months whereas the other seven rabbits were given 0.5 ml bacterial suspension three times over two months. Rabbits were finally bled 2 to 3 weeks after their last immunization. The antibody response of the rabbits was determined by an enzyme immunoassay (EIA) using whole bacterial cells as antigen. The bacteria were suspended in phosphate buffered saline (PBS) and washed four times. Microtitre plates (M 129 A, Dynatech Laboratories, USA) were coated with 100 ~1 of bacterial suspensions diluted in 0.05 M carbonate buffer, pH 9.6 (OD 0.01 at 600 nm). Plates were incubated overnight at room temperature and then washed four times
920
in saline containing 0.05 % (v/v) Tween 20 (saline-T). Two-fold dilutions of sera were added in 100 ~al amounts to each well. Plates were incubated at 37 °C for 1 h and then rinsed four times in saline-T. Horse radish peroxidase conjugated sheep anti-rabbit immunoglobulin was used in a dilution of 1:4000, 100/al of which was added to each well and incubated for 30 rain at 37 °C followed by washing four times. Finally, 100 ~al of substrate solution (0.1 mg/ml TMB, Merck, Germany) in 0.1 M acetate buffer at pH 6.0 with 0.002 % (v/v) H202 was added to the wells. The reaction was stopped by adding 50 ~ul2 M H2SO4. Absorbance was read at 450 nm in a Titertec Multiscan photometer (Flow Laboratories, UK). Smears for immunofluorescence were prepared by growing strains on horse blood agar plates for 18 h at 37 °C. Bacteria were then transferred to nutrient broth and shaken at 37 °C for 2 h. Bacterial suspensions were centrifuged at 3,000 x g for 5 min and washed twice in PBS. The pellets were diluted in PBS to OD 1.0 at 600 nm. Antigens were coated on eight well multislides washed in ethanol. Ten ~tl of the antigen solution was placed in each well. Smears were allowed to dry at 37 °C and were fixed in acetone for 15 min. They were frozen at -20°C until used. The. slides were thawed at room temperature in a moist chamber, washed twice in PBS for 5 min and dried. Ten/al of serially doubly diluted serum was added to appropriate wells and slides were incubated for 30 min at 37 °C in a chamber, washed twice in PBS for 5 min and let dry. Ten ~1 conjugate (Dakopatts fluorescein conjugated swine anti-rabbit immunoglobulin) was added to the wells, incubated for 20 min at 37 °C in a chamber and again washed twice for 5 min in PBS and dried. Slides were finally mounted under cover glasses with phosphate buffered glycerol (PBS-glycerol 1:10) and kept dark and cool in order not to get bleached. Pre-immune rabbit sera diluted 1:8 were used as negative controls. Smears were read at magnification 1000 times under a Zeiss standard 16 EPIfluorescence microscope. The following criteria for recording the fluorescence were used: 3+ very bright peripheral staining, 2+ bright and clearly visible peripheral staining, 1+ barely visible peripheral staining, 0 no reaction. 2+ and 3+ were considered as positive results. The LPS type was determined by an EIA similar to that described by Vaneechoutte et al. (10), using reference strains ATCC 25238 (type A), F17 (type B), and B3 (type C), kindly supplied by
Eur. J. Clin. Microbiol. Infect. Dis.
M. Vaneechoutte. The strains to be tested were grown overnight on blood agar plates at 37 °C. The bacteria were suspended in saline and washed three times. Finally, they were suspended in PBS and the protein content was estimated using the BioRad protein assay (BioRad Laboratories, USA). For absorption of sera, a bacterial suspension at a density corresponding to OD 0.7 at 595 nm in the BioRad assay was used. The suspension (500 ~al)was mixed with 500 lal of serum diluted 1:250 an d kept for 4 h at 37 °C. It was then centrifuged at 5,000 x g and the supernatant collected. Each antiserum was absorbed separately with the homologous strain and the reference strains. For typing, fiat bottom microtitre plates (Immunolon II, Dynatech Laboratories) were coated with LPS (extracted by the phenol/water procedure) from the strains to be tested. Coating was done in carbonate buffer at pH 9.6 and the plates kept overnight at room temperature. After washing in PBS, each LPS was incubated with homologous unabsorbed serum (positive control), homologous absorbed serum (negative control) and antisera absorbed with the reference strains for types A, B and C in paired wells and incubated for 2 h at 37 °C. The serum dilutions used were 1:500 to 1:50,000. Horse radish peroxidase conjugated sheep antirabbit immunoglobulin was then added to each well at a dilution of 1:1,000 and incubated for i h at 37 °C. After washing four times 100 tal of substrate solution (TMB, Merck) in 0.1 M acetate buffer (pH 6.0) with 0.002 % (v/v) H202 was added. The reaction was stopped after 10 min by adding 50 lal 2 M H2SO4. Absorbance was read at 450 nm in a Titertec Multiscan photometer (Flow Laboratories, UK). The absorbance was plotted on a logarithmic scale and the degree of inhibition calculated (10). Results and Discussion. The sera from the immunized rabbits were tested in an EIA using whole bacterial cells as antigen. Despite differences in number of booster doses and time for the immunization, all sera showed the same high antibody titres of 1:25,000 for their homologous strain. Sera from three rabbits obtained before immunization and sera from non-immunized rabbits did not show any antibody activity against strains of Moraxella catarrhatis in the EIA. Immunofluorescence tests for surface antigens were read without difficulty in differentiating between positive and negative smears. The antibody titres, represented as the reciprocal value of the highest serum dilution giving a positive reading in the immunofluorescence tests of the nine sera for the
VoI. 11, 1992
921
Table 1: Cross-reactionswith Moraxella catarrhal&surface-exposedantigensas detected by immunofluorescence.Figures represent the reciprocalvalueof the highestserumdilutiongivinga positivereading.Homologoustitres are underlined.The LPS type was determinedby inhibitionEIA (10).
Antigen
ATCC25238 CCUG1497 CCUG18283 CCUG18284 RS272 CCUG3292 RS 13 RS10 RS26 --
LPS type A A A A A B B C C
Serum no. 1
2
3
4
5
6
7
8
9
2048 512 1024 1024 512 1024 1024 512 256
256 4096 256 256 256 256 128 256 256
1024 512 2048 1024 1024 512 1024 1024 1024
1024 512 1024 2048 512 512 512 512 512
512 512 1024 256 1024 512 512 512 1024
1024 1024 1024 512 1024 1024 512 1024 512
1024 512 512 2048 1024 1024 4096 512 512
512 512 1024 512 2048 512 1024 4096 1024
1024 512 1024 2048 4096 1024 1024 2048 204~
,,,,
nine strains, are shown in Table 1. The heterologous reactions ranged, with one exception, from 256 to 4096. When the sera were tested against their homologous antigens (underlined figures in Table 1) the titres were 1024 in two, 2048 in four and 4096 in three sera. The titres of the CCUG 1497 antiserum against other strains were low but the homologous titre was high indicating that this strain might differ from the other strains in antigenic composition.
Moraxella catarrhalis has been divided into tl~ree serotypes on 'the basis of LPS antigens (10). The LPS serotypes of our strains are listed in Table 1. The serotype was determined in order to find out if there was a correlation between LPS antigen type and titre in the immunofluorescence test. This was tested by comparing the median titres of reactions between strains and sera of the same LPS type with all reactions between LPS unrelated strains and sera. Strain-specific reactions were excluded. The median values of LPS type related and unrelated titres were both 512, indicating that there was no correlation between LPS serotype and titre in the immunofluorescence test. Several studies have been conducted with the objective of determining a specific antibody response during infection with Moraxella catarrhalis (4, 5, 11-13). Chapman et al. (13) showed that most patients with pulmonary infections caused by MoraxeUa catarrhalis developed convalescent phase IgG antibodies which had bactericidal activity. However, in serological studies of respiratory infections where whole bacterial cells were used as antigen, the titre rise in paired sera was very variable, only a minority of the patients having a two-fold or greater increase (4, 5). This divergence in the antibody responses
during infection could be due to differences in the immune response to Moraxella catarrhalis in different individuals, but it could also be due to differences in the predominant antigens in different strains. Gram-negative bacteria, in particular enteric pathogens, display great variation in the antigens of their cell surfaces. Likewise in Ilaemophitus influenzae extensive variations in antigenic specificity of outer membrane proteins as well as LPS determinants occur. For the preparation of antigens for determination of antibody responses during infections it is therefore necessary to investigate possible antigenic diversity of the different bacterial species. Although some investigations have indicated homogeneity in numbers and molecular mass of major outer membrane proteins of Moraxella catarrhalis as demonstrated by SDS-PAGE, antigenic differences have been demonstrated and differences in LPS antigens have also been found (7, 10). Our results show that all nine strains produced a marked antibody response in rabbits to antigens expressed on the surface of Moraxella catarrhalis. Each serum, except one, was capable of reacting at high titres with antigens from all nine strains tested. This must be due to the presence of one or more antigens common to the species on the outer membrane of different strains of Moraxella catarrhalis. Since there was no correlation between the LPS serotype and titre in the immunofluorescence test we conclude that other surfaceexposed antigens, probably outer membrane proteins, predominate in the reaction with antibodies raised against whole cell antigens. We have shown in an EIA using LPS as antigen that the antisera contain antibodies against the LPS (unpublished results). The lack of correlation of titres with the LPS serotype could possibly be ex-
922
plained by p o o r presentation of the antigenic d e t e r m i n a n t s of LPS on the bacterial surface. However, as shown by Murphy and Vaneechoutte et al. (9, 10) efficient absorption of antibodies to LPS is easily obtained with whole bacterial cells, indicating surface exposure of these antigens. In addition to the antigens c o m m o n to the different strains, M o r a x e l l a catarrhalis seems to carry antigens that are unique for each strain since with most strains higher titres were obtained when testing sera with their h o m o l o g o u s strain than when sera were tested against other strains. This is not unique to M o r a x e l l a catarrhalis, since strain-specific and i m m u n o d o m i n a n t surface epitopes have been d e m o n s t r a t e d in Haemophilus influenzae outer membrane proteins (14, 15). In the immunofluorescence test only surface-exposed antigens will react with their corresponding antibody. As shown by Murphy and Bartos (9) not all outer m e m b r a n e proteins possessed surface-exposed antigenic determinants, as d e m o n s t r a t e d by i m m u n o b l o t ting experiments using rabbit antisera a b s o r b e d with whole bacterial cells. Two of the O M P s could be shown to vary in antigenic properties, some strains failing to absorb antibodies to surface-exposed antigens. It might thus be possible that the strain variations in titres in the immunofluorescence test with different sera depend on variations in O M P antigens. However, one must then assume that the variable O M P antigens display several specificities in addition to those d e m o n strated by M u r p h y and Bartos (9). Our s t u d y has shown that although strain differences exist, there is a basic similarity in surface antigens giving high cross-reactivity a m o n g the nine M o r a x e l l a catarrhalis strains used.
References
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