INFECTION AND IMMUNITY, Aug. 1994, p. 3178-3183 Vol. 62, No. 80019-9567/94/$04.00+0Copyright © 1994, American Society for Microbiology

Antigenic Relationships among Immunoglobulin Al Proteasesfrom Haemophilus, Neisseria, and Streptococcus Species

HANS LOMHOLT1' 2 AND MOGENS KILIAN"*Institute of Medical Microbiology, University ofAarhus, DK-8000 Aarhus C,' and

Statens Seruminstitut, DK-2300 Copenhagen,2 Denmark

Received 3 February 1994/Returned for modification 31 March 1994/Accepted 10 May 1994

To investigate the antigenic variation and relationships of immunoglobulin Al (IgAl) proteases amongdifferent species and genera, we examined a comprehensive collection of serine type and metallo-type IgAlproteases and corresponding antisera in enzyme neutralization assays. Sharing of neutralizing epitopes ofmetallo-type IgAl proteases from Streptococcus pneumoniae, Streptococcus sanguis, Streptococcus mitis, andStreptococcus oralis and of serine type IgAl proteases from Haemophilus and pathogenic Neisseria species wasextremely limited. A number of limited to strong cross-reactions in such epitopes were found among serine typeIgAl proteases released by members of the genera Haemophilus and Neisseria, reflecting the common origin oftheir iga gene. However, the relatively limited prevalence of shared "neutralizing" epitopes of IgAl proteasesfrom the two genera indicates that they rarely induce immunity to each other. In contrast, extensive sharingof neutralizing epitopes was found between N. meningitidis and N. gonorrhoeae IgAl proteases, making thempotentially attractive vaccine components. Among metallo-type IgAl proteases, several pneumococcal pro-teases were found to induce neutralizing antibodies to IgAl proteases of oral streptococci whereas the oppositewas not the case.

Several bacterial mucosal pathogens produce proteaseshighly specific for the hinge region of the heavy chain of humanimmunoglobulin Al (IgAl). These pathogens include Neisseriameningitidis, Neisseria gonorrhoeae, Haemophilus influenzae,Streptococcus pneumoniae, and Ureaplasma urealyticum. Inaddition, some members of the oral and nasopharyngealmicroflora belonging to the genera Streptococcus, Prevotella,and Capnocytophaga produce IgAl protease. IgAl is theprincipal immunoglobulin isotype protecting the human upperairways (13). Bacterial IgAl protease activity eliminates the Fc(or Fc2* SC in the case of secretory IgAl [S-IgAl]) effectorpart of the IgAl molecule from monomeric Fab fragments withretained antigen-binding capacity (1, 23).

Molecular characterization reveals that bacteria of differentgenera have, through convergent evolution, developed IgAlproteases that work by completely different catalytic principlesyet are functionally identical. Streptococcal species producezinc-binding metalloproteases (8), Neisseria and Haemophilusspecies produce serine type IgAl proteases (2), and Prevotellaspecies produce cysteine type IgAl proteases (26).

IgAl proteases from Haemophilus and Neisseria strains maydisplay two different cleavage specificities. Type 1 proteasescleave one of several prolyl-seryl bonds, and type 2 proteasescleave one of several prolyl-threonyl bonds within a duplicatedoctapeptide in the al hinge (for reviews, see references 15, 27,and 30). In addition to this difference in cleavage specificity,the serine IgAl proteases of H. influenzae and N. meningitidisshow remarkable antigenic heterogeneity as revealed by en-zyme neutralization assays. More than 30 antigenic types withlimited cross-reactive properties have been defined among H.influenzae IgAl proteases, whereas somewhat less variationexists among N. meningitidis proteases (16, 21, 22). IgAl

* Corresponding author. Mailing address: Institute of Medical Mi-crobiology, University of Aarhus, The Bartholin Building, DK-8000Aarhus C, Denmark, Phone: 45 8942 1735. Fax: 45 8619 6128.

proteases released by oral streptococci display very limitedantigenic heterogeneity (35). The reason for the differentdegree of antigenic variation among IgAl proteases of thevarious species is not clear.

Considerable homology between IgAl protease genes (iga)of N. meningitidis and N. gonorrhoeae has been observed, andboth show partial homology with that of H. influenzae (19).Comparison of iga gene sequences from selected strains of H.influenzae and N. gonorrhoeae, furthermore, revealed thatcertain regions are highly variable and display a mosaic-likestructure (10, 33). In an otherwise highly variable part, strikinghomology in the sequences of two H. influenzae and onegonococcal iga gene was found (33). These observations indi-cate that transfer and homologous recombination of iga geneshave taken place not only within these species but also betweenmembers of the two genera. Furthermore, all three bacterialspecies that produce serine type IgAl protease are naturallytransformation competent, and two copies of the specificuptake signal for homologous DNA have been found sur-rounding the iga gene (31, 33).

It is not known to what extent the genetic recombinationbetween species is reflected in the antigenic properties of theIgAl proteases. Of particular interest in this respect areepitopes involved in antibody-induced neutralization of theproteases. Antibodies to IgAl proteases conceivably are im-portant parts of the complex host-parasite relations involvingIgAl protease-producing bacteria in their natural habitat (3, 7,14, 18). To study this factor, we have used enzyme-neutralizingantibodies raised in rabbits to characterize and examine theantigenic relationship among IgAl proteases of different spe-cies and genera. Our study included representative strainsfrom most of the known antigenic types among serine IgAlproteases secreted by H. influenzae, N. meningitidis, and N.gonorrhoeae. Also, representative metallo-type IgAl proteasesfrom S. pneumoniae, S. sanguis, S. mitis, and S. oralis wereincluded.

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ANTIGENIC RELATIONSHIPS AMONG BACTERIAL IgAl PROTEASES 3179

MATERIALS AND METHODS

Bacterial isolates. Twenty strains representing bacterialspecies secreting serine type IgAl proteases were included: 11strains of H. influenzae representing all four antigenic types ofIgAl protease found among type b isolates (32) and seven ofthe antigenic types of IgAl protease found among otherisolates (16), 5 strains of N. meningitidis representing the 5different IgAl protease inhibition types (21), and 4 strains ofN. gonorrhoeae representing IgAl proteases encoded by differ-ent iga gene types (10). Details of the strains are given in Table1. In addition, 55 strains representing bacterial species thatsecrete metallo-type IgAl proteases were included: 51 strainsof S. pneumoniae, 2 strains of S. sanguis representing the twoIgAl protease inhibition types of this species (35), and 1 straineach of S. oralis and S. mitis (35). Details about the oralstreptococci are given in Results. On the basis of preliminaryscreening with neutralizing antibodies (unpublished data), the51 S. pneumoniae isolates included were chosen to representthe antigenic diversity found among IgAl proteases of the nineserotypes most frequently isolated from infected children, i.e.,serotypes 1 (six isolates), 3 (five isolates), 6A (four isolates), 6B(seven isolates), 7F (four isolates), 14 (six isolates), 18C (nineisolates), 19F (four isolates), and 23F (six isolates). Detailsabout 16 pneumococcal strains used for immunization ofrabbits are given in Table 2.

IgAl protease preparations. IgAl proteases from H. influ-enzae, N. meningitidis, and N. gonorrhoeae strains were pre-pared by the method described by Higerd et al. (12). IgAlproteases from S. pneumoniae, S. sanguis, S. mitis, and S. oralisstrains were made from supernatants of 24-h Todd-Hewittbroth cultures. The supernatants were precipitated by 60%saturation with ammonium sulfate (pH 7.2), and the precipi-tate was redissolved in PBS buffer (0.01 M phosphate-bufferedsaline [pH 7.4], 0.04% NaN3). For the enzyme neutralizationstudies, all proteases were adjusted to an activity level two toeight times that causing complete cleavage of 0.5 mg ofsubstrate IgAl per ml (final concentration) in a reactionmixture containing 1 volume each of enzyme and substratewhen incubated overnight at 37°C. Cleavage was detected byimmunoelectrophoresis as previously described (24). The exacttiter of each protease preparation was taken into account inthe calculation of the inhibition titer.

Preparation of antisera. Female rabbits were given subcu-taneous injections every 2 weeks with 0.5 ml of incompleteFreund's adjuvant mixed with 0.5 ml of protease preparation.Test bleedings were made 9 days after the fourth immuniza-tion. When the enzyme-neutralizing titer was satisfactory, theanimals were exsanguinated by heart puncture. Serum wasrecovered and the immunoglobulin fraction was isolated asdescribed by Harboe and Ingild (11) but excluding the final gelfiltration step. The immunoglobulin preparations were storedat 4°C. For each preparation, the titer of inhibition of thehomologous IgAl protease was determined as described be-low.

IgAl protease inhibition. For the examination of cross-reactions, the titer of the enzyme-neutralizing activity of eachrabbit antiserum was determined by mixing 10 ,ul of activity-adjusted protease preparation with 10 ,ul of twofold serialdilutions of antibodies followed by incubation for 1 h inmicrotiter wells at room temperature. Subsequently, 20 ,u1 ofIgAl substrate (1 mg/ml in a mixture of 0.05 M Tris-HCl [pH7.4] and 0.85% NaCl) was added, and the reaction mixture wasincubated overnight at 37°C. Cleavage was detected by immu-noelectrophoresis as described previously (24). The titer ofinhibition was defined as the highest dilution that abolished

cleavage of IgAl substrate multiplied with the activity of theprotease preparation. Adjustment of enzyme activity followedby determination of homologous inhibition titer performed 10times and read blindly for each of three IgAl proteasesrepresenting the species H. infiuenzae, N. meningitidis, and S.pneumoniae revealed standard deviations of 0.7, 0.5, and 0.7doubling dilutions, respectively, for the three IgAl proteases.ELISA for comparing rabbit and human responses to IgAl

protease. Sera from a 5-year-old boy (patient 1) and a 3-year-old girl (patient 2) suffering from H. influenzae type b menin-gitis and the corresponding H. influenzae isolates were kindlyprovided by H. Kaythy, National Public Health Institute,Helsinki, Finland. The sera were collected at the time ofhospitalization and again 4 weeks later.

Titers of neutralizing antibodies in patient sera were deter-mined by an enzyme-linked immunosorbent assay (ELISA)(34) instead of the immunoelectrophoresis used for determi-nation of rabbit antibody titers. Reaction of the non-isotype-specific antiserum used for development of both Fab and Fcfragments of substrate IgA with Igs in the patient sera rendersthis assay less suited for titer determination in human sera.The ELISA was performed essentially as described previ-

ously (34). Activity-adjusted IgAl protease, patient serum, andsubstrate IgAl Fri (10 ,ug/ml) were mixed as described abovefor immunoelectrophoresis. After overnight incubation at37°C, 200 pI of washing buffer (0.5 M NaCl, 1.5 mM KH2PO4,6.5 mM Na2HPO4, 0.15% [wt/vol] Tween 20 [pH 7.2]) wasadded to 40 pI of sample. A 100-pI portion was transferred toan ELISA well coated with rabbit anti-mouse immunoglobulin(1:2,000; DAKO, Glostrup, Denmark) and subsequently withmonoclonal anti-human Fcat (1:400; DAKO) as the secondlayer. After incubation for 2 h and washing, peroxidase-conjugated anti-kappa light chain (1:1,000; DAKO) was usedfor detection of intact IgAl molecules in the sample. The colorthat developed after addition of o-phenylenediamine (Sigma,St. Louis, Mo.) was measured by monitoring the optical densityat 492 nm. For titer determinations the 50% inhibition titerwas determined as the midpoint between complete and nocleavage on the titer graph.

RESULTS

IgAl protease and antibody preparations. Inhibition titersto the homologous IgAl protease of rabbit antibody prepara-tions produced against serine IgAl proteases and pneumococ-cal IgAl proteases are shown in Tables 1 and 2, respectively.Immunization of rabbits with IgAl proteases from oral strep-tococci resulted in the following inhibition titers to the homol-ogous IgAl protease: S. sanguis SK1, 256; S. sanguis SK45, 512;S. mitis SK282, 128; and S. oralis SK10, no detectable response.The lack of inhibiting antibody response to the S. oralis IgAlproteases is consistent with our experience (17).With a few exceptions, all combinations of the 20 serine type

and 20 metallo-type IgAl proteases used for immunization andthe corresponding antibody preparations were tested in en-zyme neutralization assays. Antisera raised against IgAl pro-teases of the four oral streptococci were tested for neutraliza-tion of 43 S. pneumoniae IgAl proteases for a morecomprehensive investigation of possible cross-reactions be-tween IgAl proteases of pathogenic and nonpathogenic strep-tococcal species.

Interrabbit variability in neutralizing response to IgAlprotease. Because only one rabbit was used for immunizationwith each protease, some of the observed cross-reactivities, orlack thereof, might reflect differences in the reaction patternsof individual rabbits and not antigenic properties of the

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ANTIGENIC RELATIONSHIPS AMONG BACTERIAL IgAl PROTEASES 3181

v n 'n n n enzymes. To examine this factor, four rabbits were immunized>cncnsR Z S a a with the same preparation of H. influenzae type b HK368 IgAlao 15,o. -0 C-;O cprotease and three rabbits were immunized with H. influenzae

o CD type b HK393 IgAl protease. The titers of the seven antisera. oo° against 14 different adjusted preparations of H. influenzae IgAl

O 0 proteases were determined. Generally, the responses, deter-eQ CA + + + + ~ :t |mined as neutralizing titers and inhibition patterns, of individ-

A + + ual rabbits were similar, and differences were found to beCD t 8 + + within a range of three twofold dilutions (data not shown).

Intergenus IgAl protease cross-inhibition. Very limited0 3 cross-inhibition was observed between streptococcal metallo-o, type IgAl proteases and serine IgAl proteases from Hae-

O Z o s _3Hmophilus and Neisseria species. Only one antiserum to an S.

+ + pneumoniae IgAl protease neutralized four Neisseria proteasesc + > < mat low titers (between 1 and 4; data not shown). Among serine

o mQ > s )IgAl proteases, a number of cross-inhibitions were foundco =.2- between Haemophilus and Neisseria IgAl proteases with titers

+ El + ranging from close to the homologous reaction to considerably<CL+ _ > U lower (Table 1). All high-titer cross-inhibitions among serine

D c° ° IgAl proteases involved the IgAl protease of H. influenzae0o.,:s° HK295 or the corresponding antibody preparation. In addi-00 Ition, antibodies to H. influenzae HK284 IgAl protease neutral-

ized all type 2 Neisseria IgAl proteases at midlevel titers and a

single type 1 meningococcal protease at low titer (Table 1).aI,,|+ 0t (> Interspecies cross-inhibition. Extensive cross-inhibition be-

rAot°-eDtween IgAl proteases of the two species N. meningitidis and N.0 O a a

0 gonorrhoeae was detected. Antibodies against IgAl proteases0~~~~~~_3z : ° from strains NK183, HF48, HF13, FA514, and R16 showed0~~~~~O + C)0 neutralizing activity against all meningococcal and gonococcal

IgAl proteases included. Generally, the titers of neutralizationwere highest to proteases of the same cleavage specificity asthe one used for immunization (Table 1).Among the Streptococcus species, more limited interspecies

+ cross-inhibition was found. Thus, none of the antisera to IgAlIIII proteases from oral streptococci inhibited any of the 43 S.

0- oo 9 : _,pneumoniae IgAl proteases tested. Conversely, 10 of the 16antisera to S. pneumoniae IgAl proteases inhibited one or

o_.No °more of the IgAl proteases from oral streptococci (Table 2).0 + *- w Notably, antiserum to the IgAl protease of one of the serotype

+ 0So, 18C strains (726/91) inhibited the S. oralis IgAl protease at a00o > ¢:> titer of 32, equivalent to the homologous reaction.

0

O 3 3 c,, Comparison between rabbit and human neutralizing re-

c + + + sponse to IgAl protease. To examine if results obtained by> + + x ; w immunizing rabbits with IgAl protease may be used as aA+ 000

05 t-) % irelevant model for a human response to colonization orCD e t.infection, we made comparisons between the serum responseo. +-~, - of two H. influenzae meningitis patients and the serum re-

&+ I.- ;sponse of two rabbits immunized with IgAl proteases from the0.~~~~~0o in respective patient isolates. Acute- and convalescent-phase sera

' e 0 were compared for each patient to test if responses were

mQ _ a oinduced by the infecting strain or by previously colonizingcu.I $:-n cD strains. Three H. influenzae IgAl proteases representing the

antigenic and genetic diversity known to occur in phylogenetic0 division I of the H. influenzae population (29, 32) were chosen

+ -~ as reference proteases. For both patients, a rise in inhibition0. + *titer against the homologous and two of the reference pro-B t teases was found, whereas the undiluted serum showed less0o N ~~~~~~~~~than50% inhibition of the third protease HK393 (Table 3).

The rabbit sera showed inhibition titers to the IgAl protease ofllll00-, the patient isolates within two doubling dilutions of the

CD acorresponding patient serum response. After adjustment of

o e neutralizing titers of rabbit and human sera against the IgAl.| + + -> C protease of the corresponding patient isolate, an almost com-

+ + + plete accordance between their inhibition potential against the+ " four proteases was found (Table 3).

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3182 LOMHOLT AND KILIAN

TABLE 3. Comparison between human and rabbit responsesto IgAl protease

Inhibition titer of antisera from:

IgAl Patient 1 Patient 2protease Rabbit Rabbit

Acute Convales- la Acute Convales- 2aphase cent phase phase cent phase

Patient isolate 2 56 56 _b 20 20HK368 20 17 5 10HK715 1 11 3 4 4HK393 -

a Rabbits 1 and 2 were immunized with IgAl protease secreted by H.influenzae isolates from patients 1 and 2, respectively. The homologous inhibitiontiter of the rabbit antiserum was adjusted to that of the respective patient serum.The titers against HK368, HK715, and HK393 IgAl proteases were adjustedaccordingly.b_, less than 50% inhibition.

DISCUSSION

Neutralizing antibodies against IgAl proteases conceivablyplay a role in the complex host relationships of H. influenzae,N. meningitidis, N. gonorrhoeae, S. pneumoniae, and some ofthe oral streptococci. Supporting this concept is our recentfinding that consecutively replacing clones of H. influenzaeexpress IgAl protease not inhibited by antibodies induced bypreviously colonizing clones (22). Theoretically, antibodiesinduced by IgAl protease may therefore have an impact on thesusceptibility of a host to subsequent colonization or infectionwith clones of the same or other species expressing cross-reactive IgAl proteases.

This study employed sera of hyperimmunized rabbits toexamine cross-reactions of IgAl proteases from Haemophilus,Neisseria, and Streptococcus species. The finding that individualrabbits responded similarly to neutralizing epitopes on H.influenzae IgAl proteases indicates that the different patternsof cross-reactivities observed are not due to idiosyncrasiesamong the immunized rabbits. Moreover, comparisons withthe response of two meningitis patients support the notion thatthe reactions observed with rabbit antisera adequately mirrorthe situation in humans infected or colonized by these bacteria.More high-titer antisera than the ones used in this study mighthave revealed further cases of limited cross-inhibition amongthe proteases, which, however, may not be relevant in vivo.Although functionally identical, the IgAl proteases exam-

ined in the study belong to two separate classes of proteaseswith different catalytic mechanisms and no common ancestor,as revealed also by the lack of detectable homology betweenrepresentatives of iga genes of the two classes (8). Accordingly,neutralizing epitopes shared between the serine type andmetallo-type IgAl proteases were extremely limited. A com-mon motif in the substrate-binding site might have created thesporadic antigenic similarities observed, because both types ofIgAl protease are post-proline endopeptidases.

In contrast, the serine type IgAl proteases, which areevolutionarily related, as reflected in a high degree of sequencehomology within the iga genes, showed significant cross-reactions suggestive of epitope identity or similarity, whichwere most pronounced between meningococcal and gonococ-cal enzymes. On the basis of the common origin of the iga geneof Haemophilus and Neisseria species (19), as well as evidenceof intergenus recombination of iga genes (33), cross-reactionsmight have been expected. However, these were limited, andevidence of close similarity was revealed only by the reciprocalinhibitions involving strain HK295. Interestingly, this H. influ-

enzae strain has previously shown both type 1 and type 2protease activity, but the molecular basis of this phenomenonis unknown (16).

Antigenic diversity of the serine type IgAl proteases is aresult of horizontal transfer and homologous recombination ofparts of the iga gene, giving rise to a mosaic-like structure ofparts of the gene (10, 33). If epitopes reacting with neutralizingantibodies are nonlinear, as suggested by sequence compari-sons of iga genes from four H. influenzae strains (33), morethan one area would have to be similar among iga genes ofdifferent genera to result in cross-inhibition. This may explainthe relatively few cross-reactions found in the study betweenIgAl proteases of the two genera. On the basis of inhibitionexperiments involving postinfection human neutralizing anti-bodies, Devenyi et al. (5) recently suggested that the regiondetermining the cleavage type, identified as a stretch of 123amino acids near the amino terminus in the H. influenzae IgAlprotease (9), is part of the epitope with which neutralizingantibodies react. Some of our inhibition data on, in particular,gonococcal IgAl proteases support this conclusion by showingcorrelation with cleavage type, but this is not the generalpattern (Table 1).

Interestingly, some of the pneumococcal IgAl proteasesinduced neutralizing antibodies to IgAl proteases of oralstreptococci, whereas the opposite was not the case. Previousstudies revealed that the human immune system usually re-sponds poorly to IgAl proteases of oral streptococci (7). Thisrelative lack of neutralizing response may explain why anti-genic variation in IgAl proteases of oral streptococci has notevolved. Our finding that certain pneumococcal IgAl pro-teases induce neutralizing antibodies against IgAl proteases oforal streptococci raises the intriguing possibility that infectionwith pneumococci modulates the oral microflora. Similaritiesbetween streptococcal metallo-type IgAl proteases are notreadily detected at the DNA level, because previous studieshave failed to show hybridization between DNA from fourIgAl protease-producing isolates of S. pneumoniae and an S.sanguis iga gene (6). However, in the present study antigenicsimilarities between these IgAl proteases were observed. Thegenetic basis for such similarities among streptococcal metallo-type IgAl proteases is, at present, not known, because only theS. sanguis iga gene sequence has been determined (8).

It is conceivable that the antigenic variation among the IgAlproteases serves an immune escape purpose. Within the pop-ulation of H. influenzae, several antigenically distinct IgAlproteases have evolved. Although noncapsulated H. influenzaestrains belong to the commensal nasopharyngeal flora, partic-ularly during childhood (20), there is evidence for continuousexchange of colonizing clones (36, 37). The successful persis-tence of members of the species in a particular host may be dueto the antigenic variation of IgAl proteases and other coloni-zation factors (22). However, N. meningitidis is usually carriedonly by a minor part of the population (4) and N. gonorrhoeaeis even less frequent and is not part of the normal flora.Pathogenic Neisseia species show extensive genetic diversityand have developed specific genetic mechanisms for antigenicvariation of virulence factors such as pili and certain outermembrane proteins (25); this, in itself, may obviate the needfor IgAl protease. Extensive genetic polymorphism has alsobeen demonstrated in the Neisseria iga genes (21, 28). Never-theless, this study and our previous study (21) revealed asurprising paucity of antigenic variation detected with enzyme-neutralizing antibodies. If IgAl protease is important in theprocess of colonization, the lower carriage rate of pathogenicneisseriae could be due partly to this low level of antigenicdiversity. The relatively limited prevalence of shared "neutral-

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ANTIGENIC RELATIONSHIPS AMONG BACTERIAL IgAl PROTEASES 3183

izing" epitopes among H. influenzae and Neisseria IgAl pro-teases indicates that they rarely induce immunity to each other.The extensive sharing of neutralizing epitopes among the

Neisseria proteases makes them potentially attractive vaccinecomponents. Further studies to identify the actual epitopesinvolved in the enzyme neutralization are required.

ACKNOWLEDGMENTS

We thank Ella Brandt and Birte Esmann for excellent technicalassistance.

This study was supported by grant S12-9505-3 from the DanishMedical Research Council.

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