Neisseria gonorrhoeae IgA1 proteases share epitopes recognized by neutralizing antibodies

Vaccine, Vol. 13, No. 13, pp. 1213-1219, 1995 Cowriaht 0 1995 Elsevier Science Ltd Printed ;n &eat Britain. All rights reserved 0264-410X/95 $iO+O.OO

0264-410X(95)00057-7

Neisseria gonorrhoeae IgAl proteases share epitopes recognized by neutralizing antibodies Hans Lomholt”,

Inga Lind’f and Mogens

Kilian*$

The antigenic diversity among IgAl proteases of 61 Neisseria gonorrhoeae strains isolated during a period of 23 years and on four continents was examined in enzyme neutralization assays employing rabbit antisera raised against selected IgAl proteases. The antigenic analyses were compared with results of iga gene-region RFLPpatterns and enzyme cleavage speciJicity for substrate IgAI. Type 1 IgAl proteases were antigenically uniform while six direrent antigenic types were detected among type 2 enzymes. Extensive cross-reactions of antibodies against the d#erent antigenic types suggested only minor d$erences in relevant epitopes. Epitopes previously found to be common to all Neisseria meningitidis IgAl proteases were also shared by all N. gonorrhoeae IgAl proteases in the collection. Human sera from patients with gonorrhoea showed broadly cross-reactive neutralizing activity at titers comparable to those of sera from immunized rabbits. In conclusion, N. gonorrhoeae ZgAl proteases show a remarkable lack of diversity of epitopes recognized by enzyme-neutralizing antibodies. If future studies confirm that cleavage of IgAI is an important step in gonococcal infections, Neisseria IgAl proteases may be attractive vaccine candidates. Keywords: Nei,sseriu gonorrhorrre; gonorrhoea;

vaccine;

IgA 1 protease;

Neisseria gonorrhoeae employs an array of complex genetic strategies to generate antigenic variation and diversity of pili, opacity proteins (PII), and lipooligosaccharides’. Combined with the lack of a virulenceassociated capsule this antigenic diversity has hampered the development of an efficacious vaccine against gonococcal infections. A gonococcus pilus vaccine brought to field trial failed to show protection’, though all individuals developed cross-reactive antibodies against a fragment of pilin important for gonococcal attachment’. Likewise, vaccines based on the major outer porin, PI, which shows only inter-strain variation, have been unsuccessful’. One of the putative virulence factors of N. gonorrhoeae is the IgAl protease (for review see Refs 6-8). By cleaving specific peptide bonds in the hinge region of human IgAl the enzyme separates antigen-binding monomeric Fab,, fragments from secondary effector functions associated with the Fc part of the molecule’. In vitro studies indicate that this cleavage interferes with IgA-mediated inhibition of adherence’“.’ ’ and

*Institute of Medical Microbiology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark. TWHO Collaborating Centre for Reference and Research in Gonococci, Statens Seruminstitute, Copenhagen, Denmark. fCorresponding author. (Received 25 October 1994; accepted 22 March 1995)

mucosal

immunity

potentially allows the pathogen to mask surface epitopes with monomeric Fab,, fragments’2p14. Patients with gonococcal urethritis and cervicitis respond with locally g reduced S-IgA antibodies to gonococcal antigens”,’ and the response may include neutralizing antibodies to the gonococcal IgAl protease, both in secretions and in serum”.“. Vaginal washings from women with gonorrhoea cleaved IgAl into fragments identical to those produced by gonococcal IgAl protease, hereby providing evidence of in vivo activity”. The antigenic diversity among IgAl proteases of several bacterial species has been previously studied with enzyme-neutralizing antibodies raised in rabbits. More than 30 antigenic types were found among Haemophilus injuenzae IgA proteases- “J’ in spite of a high degree of overall homology of the corresponding IgAl protease genes (iga)22.23. In contrast, only five highly crossreactive types were identified among Neisseria nzeningitidis IgAl proteases2”. The antigenic diversity of gonococcal IgAl proteases and their antigenic relationship to meningococcal IgAl proteases have not been systematically investigated. IgAl protease genes from several strains of N. gonorrhoeae have been cloned and mapped with restriction endonucleases or by partial or complete sequencing’5-30. Evidence that horizontal genetic exchanges occur in vivo between gonococcal iga genes has been presented” suggesting a mechanism for the generation of antigenic and genetic heterogeneity in the population. In a study

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13 1213

Antigenic diversity among N. gonorrhoeae proteases: H. Lomholt et al. of 28 gonococcal isolates using RFLP analysis Mulks et al.‘” showed restriction site polymorphism in genes encoding type 2 IgAl proteases, which cleave a prolylethreonyl bond, but only one genotype among genes encoding type 1 IgAl protease, which cleaves a prolylseryl peptide bond in the al hinge region. How this is reflected in antigenic properties of the protease has not been investigated. In this study we compared the antigenic properties and cleavage specificities of IgAl proteases with restriction site polymorphism of the corresponding iga gene regions in a collection of 62 gonococcal isolates assembled to represent a wide diversity in serotype, auxotype, isolation time, and geographic origin. Neutralizing antibody responses in immunized rabbits were furthermore compared with those of patients with gonococcal and meningococcal infection.

MATERIALS

AND

METHODS

Bacterial strains The 62 gonococcal isolates examined in this study were patient isolates maintained in the collection of the WHO collaborating centre for reference and research in gonococci at Statens Seruminstitut, Copenhagen. Of these, 45 were isolated between 1968 and 1991 in Denmark, Greenland, Finland, Senegal, Indonesia and Korea and were selected to represent a wide geographic area and time span. Isolates from Senegal were of auxotypes met , pro - , pro arg-, and zeroj’. Isolates from other areas were not auxotyped. In addition, 17 auxot pe isolated during 1979 in strains of the AHU Denmark were includedX3.j Y Preparation of IgAl protease IgAl proteases were prepared as described by Higerd et d.“’ The activity titer of all IgAl protease preparations was determined by overnight incubation at 37°C of 20 ~1 of serial protease dilutions with 20 ~1 of IgAl substrate (1 mg ml-‘). Cleavage of IgAl was detected by immunoelectrophoresis (IEP)36 and the titer was defined-as the reciprocal of the highest dilution that caused complete cleavage of substrate IgAl. For enzyme neutralization studies all IgAl protease preparations were adjusted to a final titer of 4 in the reaction mixture. IgAl protease cleavage specificity IgAl protease preparations were incubated overnight at 37°C with substrate IgAl (IgAl Fri 1.3 mg ml-’ or IgAl Mor 2.8 mg ml-‘) and cleavage was detected by SDS-PAGE’(j. Fragment sizes were compared with IgAl cleavage fragments produced by gonococcal strains NG74 and RI6 which possess type 1 and type 2 IgA 1 proteases, respectively”. Enzyme-neutralizing antibodies Enzyme-neutralizing antibodies were raised in rabbits and the immunoglobulin fraction of serum was purified by precipitation with ammonium sulphate (25% w/v) as described3’. IgAl protease preparations of the following strains were used for generation of antibodies: four strains of N. gonorrhoeae representing four different iga

1214

Vaccine 1995 Volume 13 Number 13

gene types previously identified”: NC74 (protease cleavage type l), MS11 (protease cleavage type 2), FA514 (protease cleavage type 2), and R16 (protease cleavage type 2); five strains of N. nzeningitidis representing the five IgAl protease inhibition types found in that species14: NK183 (protease cleavage type 1, inhibition type a), HF48 (protease cleavage type 1, inhibition type b), NGC80 (protease cleavage type 1, inhibition type c), NG117 (protease cleavage type 2, inhibition type d), and HF13 (protease cleavage type 2, inhibition type e); two strains of H. in@enzae excreting IgAl proteases that share epitopes recognized by neutralizing antibodies with Neisseria IgAl proteases’*: HK284 (protease cleavage type 2), and HK295 (protease cleavage types 1+3). The enzyme-neutralizing activity of each antibody preparation against the homologous IgAl protease was titrated by incubating one volume of IgAl protease at a final activity titer of 4 with one volume of each of twofold serial dilutions of antibody preparations for 1 h at room temperature. Subsequently, two volumes of substrate IgA (1 mg ml-‘) were added and the mixture was incubated overnight at 37°C. Cleavage of IgAl was detected by IEP and the titer was defined as the reciprocal of the highest dilution of antibodies that caused complete neutralization of the homologous IgAl protease.

Inhibition typing All gonococcal IgAl proteases from the Neisseria reference laboratory strains were tested against the 11 antibody preparations described above. For this screening each antibody preparation against Neisseria IgAl proteases was adjusted to a homologous inhibition titer of 16 while the two antibody preparations against H. influenzue IgAl proteases were used in undiluted form. Subsequently, a more detailed discrimination between antigenic types was obtained by titrating the reference antibody preparations (unadjusted) against one representative IgAl protease of each antigenic type identified by the initial screening. The preparation of antibodies raised against N. meningitidis HF13 IgAl protease was titrated against all proteases in the collection. This antibody preparation has previously been shown to neutralize all meningococcal IgAl proteases at high titer?. In order to examine possible differences in the neutralizing antibody response of individual rabbits to HF13 IgAl protease immunoglobulins from 3 rabbits immunized with this IgAl protease were titrated against 9 selected Neisseriu IgAl proteases representing different inhibition types.

ELISA assay for human neutralizing antibodies Paired samples of serum from a 5-year-old boy suffering from group B meningococcal meningitis obtained at the day of hospitalization and 4 weeks later, respectively, were kindly provided by Dr H. Kgythy, National Public Health Institute, Helsinki, Finland. The IgAl protease of the patient isolate showed type 1 cleavage specificity. In addition, 9 sera from patients with gonorrhoea and the corresponding N. gonorrhoeae isolates were examined.

Antigenic diversity among N. gonorrhoeae proteases: The ELISA assay was essentially as described39. In short, adjusted IgAl protease, patient serum, and substrate IgA (IgA Fri 10 pug ml-‘) were mixed and incubated as described above for IEP. Sixfold diluted sample was then transferred to an ELISA well coated with rabbit anti-mouse immunoglobulin (1:2000, DAKO, Glostrup, Denmark) and a second layer of monoclonal anti Fc, (1:400, DAKO). Intact IgAl molecules in the sample were detected after incubation and subsequent washing by addition of peroxidase-conjugated anti kappa light chain (1: 1000, DAKO). After addition of OPD the optical density at 492 nm was measured. RFLP analysis of igu gene region

Chromosomal DNA was prepared as described”. Restriction endonuclease Alul was used for digestion according to the manufacturer’s instructions (Boehringer Mannheim GMBH, Germany). After electrophoresis in 1% agarose gels DNA was transferred to cellulose nitrate membranes by Southern blotting”‘. The probe used covered the iga gene of meningococcal strain HF13 (Lomholt et al., manuscript in preparation) corresponding to the secreted IgAl protease. The probe was labelled with “P by nick translation4’. Hybridization and wash of filters were performed using stringent conditions3’ and RFLP patterns were visualized by autoradiography. RESULTS IgAl protease activity and cleavage specificity

Of the 62 gonococcal isolates included 61 showed IgAl protease activity. Sixteen and 45 isolates cleaved at the type 1 and type 2 positions, respectively. All strains with type 1 activity and the negative strain belonged to the AHU auxotype. IgAl protease inhibition typing

Evidence of common epitopes were found by the initial screening of the 61 gonococcal IgAl proteases for neutralization by each of the nine Neisseria reference antibody preparations adjusted to homologous titer 16 and the two undiluted H. influenzae antibody preparations. However, the IgAl proteases showed differences in the pattern of inhibition by the antibody preparations. One isolate representing each variant was chosen for further titration of neutralization by all antibody preparations. Differences of three or more doubling dilutions were considered significant for discrimination of antigenic types. Accordingly, seven inhibition types could be defined based on different reactions with a few of the antibody preparations, thus only separated by minor differences. The cleavage type 1 IgAl proteases were antigenically uniform while six inhibition types were defined among the cleavage type 2 proteases (Table I). Five of the nine Neisseria antibody preparations showed neutralizing activity against all representative IgAl proteases. The two H. injuenzae antibody preparations inhibited representative IgAl proteases of all cleavage type 2 inhibition types. One antibody preparation raised against N. meningiridis HF13 IgAl protease and previously shown to recognize neutralizing epitopes shared by all meningo-

H. Lomholt et al.

coccal IgAl proteases also neutralized all 61 gonococcal IgAl proteases at titers ranging from 64 to 1024 (homologous titer 512) (Table 2). To examine if the induction of neutralizing antibodies to shared epitopes by HF13 protease was reproducible, three rabbits were immunized with this enzyme. The resulting immunoglobulin preparations were titrated for inhibition of nine N. meningitidis and N. gonorrhoeae reference proteases. The antibodies induced in the three rabbits were directed against common epitopes on seven of the nine proteases while antibodies from two rabbits each showed a comparatively low inhibition titer to one of the IgAl proteases (data not shown). Thus, in general an immune response was induced by the common epitopes. RFLP analysis of the iga gene region

Restriction fragment polymorhism among gonococcal by digestion of chromosomal DNA with the restriction endonuclease Alul and subsequent Southern blotting using a probe corresponding to the region encoding the secreted N. meningitidis IgAl protease. Chromosomal DNA of four isolates could not be digested with Alul. Among the remaining 58 isolates 8 RFLP types designated A-H were defined based on the sizes of 4-6 fragments hybridizing to the probe used. Two and six RFLP patterns were detected among type 1 and type 2 IgAl protease genes, respectively (Table I). Identical iga RFLP type was found for IgAl proteases of several different inhibition types (Table I) and vice versa. In addition, several isolates of distant geographic origin and isolates collected years apart showed the same iga gene region RFLP type. iga genes was analysed

Analysis of human neutralizing IgAl protease

antibodies to Neisseviu

Neutralizing antibodies to Neisseria IgAl proteases in acute phase and convalescent sera from a meningococcal meningitis patient were analyzed by ELISAj9. IEP was not used because the non-isotype-specific serum used to visualize substrate IgAl produced disturbing precipitation lines corresponding to immunoglobulins of the patient serum. It should be noted that in the ELISA assay higher O.D. values correspond to more intact IgAl, i.e. inhibition of IgAl protease activity. The IgAl protease of the patient isolate (not shown) as well as nine selected Neisseria IgAl proteases were neutralized to a higher degree by the convalescent serum than by the acute-phase serum, indicating that broadly crossneutralizing antibodies to Neisseria IgA 1 proteases were induced during the infection. All proteases were neutralized to some extent by acute-phase serum when compared to the buffer controls (Figure I). The possible existence of free antigen or complexes of antigen with antibody in acute-phase serum, was not investigated. Neutralizing activity against the disease-causing isolate was found for 8 of 9 convalescent sera from patients with gonorrhoea. Titers ranged from 16 to 1024 (Tab/e 2). The remaining isolate showed no detectable IgAl protease activity. However, this patient serum, like the other eight human sera, neutralized IgAl proteases representing six of the seven inhibition types defined among N. gonorrboeae IgAl proteases in this study (Table 2). The antibodies found in patient sera reacted

Vaccine 1995 Volume 13 Number 13 1215

1 2 2 2 2 2 2

1 2 3 4 5 6 7

HF48 (2Y

N. meningitidis

NK183 4(26)” NGC80 (2’) NG117 (2Y

HF13 (2Y NG74 (2’) FA514 (2Y

N. gonorrhoeae

strains

MS11 (2Y

R16 (2Y

HK284 (2Y

H. influenzae HK29.5 (2Y

Number of strains

RFLPb

G(3)’ F

;I#

E;;U;,

’

$42,

l(5),

E

F, -(2)

A(g)>B(5), -@I

type

aNumbers in parentheses indicate the inhibition titer against the homologous IgAl protease. ‘Eight RFLP patterns designated A-H. - indicates that chromosomal DNA could not be digested by Alul. For each combination of RFLP pattern and inhibition type the corresponding number of isolates is given in parentheses. “All 61 IgAl proteases were titrated for inhibition by antibodies to HF13 IgAl protease. For each inhibition type titers detected were within the given interval

type

Cleavage type

detected among 61 gonococcal

preparations

IgAl proteases

Inhibition titers by antibody

Inhibition types of N. gonorrboeae

Inhibition

Table 1

Antigenic diversity among N. gonorrhoeae proteases: H. Lomholt et al. IgAl protease neutralization

00 I

1.0

-

o

l

.

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+

0.5

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0

-

0

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cl

0

+

+

0 0

0

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0

+

+

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+

+

+ +

Figure 1 Neutralization of nine selected Neisseria IgAl proteases by acute and reconvalescent phase sera from a patient with meningococcal meningitis. Inhibition was measured by ELBA. High O.D. values correspond to high levels of intact substrate IgAl, i.e. low IgAl protease activity. IgAl proteases are listed along the X-axis and O.D. values are given on the Y-axis. Closed circles indicate values obtained with reconvalescent serum, open circles indicate values for acute phase serum, and crosses indicate buffer controls (no IgAl substrate)

Table 2 Inhibition of IgAl protease by human serum from patients with gonorrhoea

Patient isolate

Auxotype

427 451F 442 303 578 1081F 313F 1082F 1296

AHUAHUAHUProProProProto Proto Proto

This

Homologous inhibition titer 24 2” -= 2’O ;: 24 28 28

Inhibition titer of patient serum against IgAl proteases of indicated inhibition types 1

2

3

4

6

7

;:

$

;I:

$

$

28 2’O

$

;:

;+z

;;

;:

;:

;:

26 2’O

26 2”

26 2”

25 2’0

2’ 2’O

25

26

29

2g

28

29

patient isolate showed no IgAl

protease activity

with common epitopes and showed neutralizing at titers comparable to those observed in sera from immunized rabbits.

DISCUSSION Recurrent gonococcal infections show a high incidence and prevalence among members of certain high-risk groups42. If cleavage of IgAl is an important factor in the pathogenesis of gonococcal infection then recurrent infection could be due to inadequate induction of IgAl protease-neutralizing antibodies in local secretions. Alternatively, reinfection might be possible as a result of antigenic heterogeneity of IgAl proteases as is the case with several other gonococcal antigens. The latter possibility was examined in this study of the antigenic and genetic diversity of IgAl proteases in a collection comprising 62 N. gonorrhoeae strains from six countries on four continents and spanning 23 years in isolation time.

The degree of iga gene sequence polymorphism found in this study by RFLP analysis is in good agreement with that reported by Mulks et af.29 based on analysis of 28 gonococcal isolates. Both studies found very limited or no polymorphism among iga genes encoding proteases with type 1 cleavage specificity. This was also reflected in shared antigenic properties of type 1 IgAl proteases. Recent studies have concluded that gonococci show a panmictic population structure, i.e. random association between alleles or linkage equilibrium, as a result of extensive recombination43. However, this study and the comprehensive studies published by Mulks and coworkers4 demonstrate that gonococcal type 1 IgAl protease activity is strongly associated not only with the requirements for arginine, hypoxanthine, and uracil (AHU-) but also with the protein IA-l and IA-2 serovars, and with the Dam methylation phenotype (Dam+). This suggests either that these isolates represent a genetically separate phylogenetic lineage or that isolates with this phenotype belong to a single rapidly spreading genotype. The fact that the apparent linkage disequilibration of these alleles may be detected in AHU isolates spanning more than 30 years in isolation time45 supports the former explanation and suggest that the associations are due to a recombination barrier, similar to what may be suggested for the group A meningococcal population. The iga gene encoding type 2 IgAl protease, on the other hand, shows restriction site polymorphism, and this antigenically heterogeneous IgAl protease type shows no association with other phenotypic traits in agreement with a panmictic structure of this part of the population. The low degree of antigenic diversity observed among gonococcal IgAl proteases is in contrast to the extensive diversity among the closely related IgAl proteases of H. inj&enzae20. It is, furthermore, in contrast to the antigenic diversity of several gonococcal surface antigens such as Pl. opa proteins, pili, and LOS’. Opposed to the genes encoding these surface molecules the iga gene is a single-copy gene and lacks mechanisms for intra-strain genetic variation. Although there is evidence for recombination between gonococcal iga genes”’ it apparently has resulted in a very limited antigenic diversity. The quantity and quality of neutralizing serum antibodies in rabbits immunized with gonococcal IgAl proteases appeared to correspond well to the response in sera of patients with gonorrhoea. It is not known if the patient antibodies were induced by the actual infecting gonococcal strain or by previously encountered Neisseria strains. However, the finding that convalescent serum from a child with meningococcal meningitis showed higher titers of neutralizing antibodies against nine reference IgAl proteases than the corresponding acute-phase serum suggest that broadly cross-reacting neutralizing antibodies to Neisseria IgAl proteases may be induced or boosted in humans during a single infection. Likewise, high titered responses have been observed in humans also as a response to asymptomatic meningococcal carriaged6. When incubated with substrate (IgAl), vaginal washings from women colonized by N. gonorrhoeae or with clinical gonorrhoea yielded cleavage fragments indicative of IgAl protease activity”. However, the presence of neutralizing antibodies in vaginal secretions of these

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1995 Volume 13 Number 13 1217

Antigenic diversity among N. gonorrhoeae proteases: H. Lomholt et al. subjects or their previous encounter with gonococci or meningococci is not known. Neutralizing antibodies to IgAl protease seem to be induced in serum during gonorrhoea17 but, presently, no information is available on the induction or persistence of neutralizing responses to gonococcal IgAl protease in urogenital secretions which may be more relevant to natural infection. Several recent studies indicate that mucosal immune responses in the female genital tract are weakd7 which, in itself, may limit the selection for antigenic diversity among gonococcal IgAl proteases. Furthermore, it is not known what titers of neutralizing antibodies are relevant in viva on mucosal surfaces during gonococcal infection. Even minor antigenic differences may be sufficient ‘to render the IgAl protease unaffected by low levels of neutralizing antibodies. In addition, the extreme diversity of gonococcal surface antigens may limit the pre-existence of host IgAl antibodies to a particular gonococcal strain. An efficacious vaccine against gonococcal infection should probably include several components involved in different steps during gonococcal infection. This is emphasized by the finding that even less variable surface antigens such as Pl and HS, which are considered vaccine candidates, are very heterogeneously expressed on the bacterial surface”‘. IgAl protease may be a potential candidate for inclusion in such a vaccine as all Neisseria IgAl proteases share epitopes inducing neutralizing antibodies.

9

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11

12

13

14

15

16

17

ia

ACKNOWLEDGEMENTS This study was supported by grant S-12-9505 from the Danish Medical Research Council. We thank Dr Knud Poulsen for the HF13 iga gene fragment used as a hybridization probe and Ella Brandt for excellent technical assistance.

19

20

21

REFERENCES Meyer, T.F., Gibbs, C.P. and Haas, R. Variation and control of protein expression in Neisseria. Annu. Rev. Microbial. 1990, 44, 451-477 Boslego, J.W., Tramont, E.C., Chung, R.C. et al. Efficacy trial of a parenteral gonococcal pilus vaccine in men. Vaccine 1991, 9, 154-l 62 Johnson, SC., Chung, R.C.Y., Deal, C.D. ef al. Human immunization with Pgh 3-2 gonococcal pilus result in cross-reactive antibody to the cyanogen bromide fragment-2 of pilin. J. Infect. Dis. 1990,163,128-134 Wetzler, L.M., Blake, MS., Barry, K. and Gotschlich, EC. Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants. J. Infect. Dis. 1992, 166, 551-555 Plaut, A.G., Gilbert, J.V., Artenstein, MS. and Capra, J.D. Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science 1975,190, 1103-1105 Plaut, A.G. The IgAl proteases of pathogenic bacteria. Annu. Rev. Microbial. 1983, 37, 603-622 Mulks, M.H. Microbial IgA proteases. In: Bacterial Enzymes and Virulence (Ed. Holder, I.A.). CRC Press, Boca Raton, FL, 1985, pp. at-104 Kilian, M. and Reinholdt, J. Interference with IgA defence mechanisms by extracellular bacterial enzymes. In: Medical Microbiology (Eds Easmon, C.S.F. and Jeljaszewicz, J.). Academic Press, London, 1986, pp. 173-208

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Kilian, M., Mestecky, J. and Russell, M.W. Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases. Microbial. Rev. 1988, 52, 296-303 Mulks, M.H., Plaut, A.G. and Lamm, M. Gonococcal IgA protease reduces inhibition of bacterial adherance by human secretory IgA. In: Genetics and lmmunobiology of Pathogenic Neisseria (Eds Normark, S. and Danielsson, D.). University of Umea, Umea, 1980, pp. 217-220 Reinholdt, J. and Kilian, M. Interference of IgA protease with the effect of secretory IgA on adherence of oral streptococci to saliva-coated hydroxyapatite. J. Dent. Res. 1987, 66, 492497 Kilian, M. and Reinholdt, j. A hypothetical model for the development of invasive infection due to IgAl proteaseproducing bacteria. Adv. Exp. Med. Biol. 1987, 2168, 12611269 Russell, M.W., Reinholdt, J. and Kilian, M. Anti-inflammatory activity of human IgA antibodies and their Fab alpha fragments: inhibition of IgG-mediated complement activation. Eur. J. Immunol. 1989, 19, 2243-2249 Jarvis, G.A. and Griffiss, J.M. Human IgAl blockade of IgGinitiated lysis of Neisseria meningitidis is a function of antigenbinding fragment binding to the polysaccharide capsule. J. Immunol. 1991, 147, 1962-l 967 Kearns, D.H., O’Reilly, R.J., Lee, L. and Welch, B.G. Secretory IgA antibodies in the urethral exudate of men with uncomplicated urethritis due to Neisseria gonorrhoeae. J. Infect. Dis. 1973, 127, 99-101 O’Reilly, R.J., Lee, L. and Welch, B.G. Secretory IgA antibody responses to Neisseria gonorrhoeae in the genital secretions of infected females. J. Infect. Dis. 1976, 133, 113-l 25 Gilbert, J.V., Plaut, A.G., Longmaid, B. and Lamm, M.E. Inhibition of microbial IgA proteases by human secretory IgA and serum. MO/. Immunol. 1983, 20, 1039-1049 Kobayashi. K., Fujiyama, Y., Hagiwara, K. and Kondoh, H. Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzymeneutralizing antibodies in both serum and secretory IgA, and also in serum IgG. Microbial. Immunol. 1987, 31, 1097-1106 Blake, M., Holmes, K.K. and Swanson, J. Studies on gonococcus infection. XVII. IgAl-cleaving protease in vaginal washings from women with gonorrhoea. J. Infect. Dis. 1979, 139, 89-92 Kilian, M., Thomsen, B., Petersen, T.E. and Bleeg, H. Molecular biology of Haemophilus influenzae IgAl proteases. Mol. Immuf70l. i983, 20, i 051-1058 Lomholt. H., van Alphen, L. and Kilian. M. Antigenic variation of IgAl proteases among sequential isolates of Haemophilus influenzae from healthy children and patients with chronic obstructive pulmonary disease. Infect. Immun. 1993, 61, 45754581 Poulsen, K.. Hjorth, J.P. and Kilian, M. Limited diversity of the immunoglobulin Al protease gene (iga) among Haemophi/us influenzae serotype b strains. Infect. Immun. 1988, 56, 987-992 Poulsen, K.. Reinholdt, J. and Kilian, M. A comparative genetic study of serologically distinct Haemophilus influenzae type 1 immunoglobulin Al proteases. J. Bacterial. 1992, 174, 29132921 Lomholt, H., Poulsen, K., Caugant, D.A. and Kilian, M. Molecular polymorphism and epidemiology of Neisseria meningifidis immunoglobulin Al proteases. Proc. Nat/ Acad. Sci. USA 1992, 89, 2120-2124 Koomey, J.M., Gill, R.E. and Falkow, S. Genetic and biochemical analysis of gonococcal IgAl protease: cloning in Escherichia co/i and construction of mutants of gonococci that fail to produce the activity. Proc. Nat/ Acad. Sci. USA 1982, 79, 7881-7885 Halter, R., Pohlner, J. and Meyer, T.F. IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product. EMBO J. 1984, 3, 1595-1601 Fishman, Y., Bricker, J., Gilbert, V., Plaut, A.G. and Wright, A. Cloning of the type 1 immunoglobulin Al protease from Neisseria qonorrhoeae and secretion of the enzvme from Escherichia co/i. In: The Pathogenic Neisseriae (Ed. Schoolnik, G.K.). American Society of Microbiology, Washington DC., 1985, pp. 164-168

Antigenic diversity among N. gonorrhoeae proteases: H. Lomholt et al. 28

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Rahr, S., Halter, R., MBller, H., Pohlner, J. and Meyer, T.F. Genetic analysis of neisserial immunoglobulin Al proteases. In: The Pathogenic Neisseriae (Ed. Schoolnik, G.K.). American Society of Microbiology, Washington DC., 1985, pp. 157-163 Mulks, M.H., Simpson, D.A. and Shoberg, R.J. Restriction site polymorphism in genes encoding type 2 but not type 1 gonococcal IgAl proteases. Antonie Van Leeuwenhoek 1987, 53, 471-478 Pohlner, J., Halter, R., Beyreuther, K. and Meyer, T.F. Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 1987, 325, 458-462 Halter, R., Pohlner, J. and Meyer, T.F. Mosaic-like organization of IgA protease genes in Neisseria gonorrhoeae generated by horizontal genetic exchange in viva. EMBO J. 1989, 8, 27372744 Lind, I., Arborio, M., Bentzon, M.W. et al. The epidemiology of Neisseria gonorrhoeae isolates in Dakar, Senegal 1982-l 986: antimicrobial resistance, auxotypes and plasmid profiles. Genitourin. Med. 1991, 87, 107-l 13 Plrdum, L., Buchanan, T.M. and Knapp, J.S. Protein I serotype of serum-resistant versus serum-sensitive Neisseria gonorrhoeae strains. Acta Pathol. Microbial. lmmunol. Stand. Sect. B 1987, 95, l-4 (ardum, L. and Lind, I. Studies on Neisseria yonorrhoeaestrains from test-of-cure specimens. Acta Pathol. Microbial. Immunol. Stand. Sect. B 1982,90,145-152 Higerd, T.B., Virella, G., Cardenas, R., Koistinen, J. and Feit, J.W. New method for obtaining IgA-specific protease. J. Immunol. Meth. 1977, 18. 245-249 Mestecky, J. and Kilian, i. lmmunoglobulin A (IgA). In: Methods in Enzymology. Academic Press, New York, 1985, pp. 37-75 Harboe, N.M.G. and Ingild, A. Immunization, isolation of immunoglobulins and antibody titre determination. Stand. J. lmmunol. 1983, 17, suppl.10, 345-351 Lomholt, H. and Kilian, M. Antigenic relationships among immunoglobulin Al proteases from Haemophilus, Neisseria, and Streptococcus species. Infect. lmmun. 1994, 82, 31783183

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Reinholdt, J. and Kilian, M. Titration of inhibiting antibodies to bacterial IgAl proteases in human serum and secretions. Adv. Exp. Med. Biol. 1995, in press Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, Cold Spring Habor Laboratory Press, 1989 Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. Labeling deoxyribonucleic acid to high specific activity in viva by nicktranslation with DNA polymerase I. J. Mol. Biol. 1977, 113, 237-251 Plummer, F.A. and Brunham, R.C. Gonococcal recidivism, diversity, and ecology. Rev. Infect. Ok. 1987, 9, 846-850 O’Rourke. M. and Soratt. B.G. Further evidence for the nonclonal population str;cture of Neisseria gonorrhoeae: extensive genetic diversity within isolates of the same electrophoretic type. Microbioloov 1994, 140. 1285-l 290 f?ulks, M.H. a% Knapp, J.S. lmmunoglobulin Al protease types of Neisseria gonorrhoeae and their relationship to auxotype and serovar. Infect. lmmun. 1987, 55, 931-936 Knapp, J.S., Mulks, M.H., Lind, I., Short, H.B. and Clark, V.L. Evolution of gonococcal populations in Copenhagen, Denmark, 1928-1979. In: The Pathogenic Neisseriae (Ed. Schoolnik, G.K.). American Society for Microbiology, Washington D.C., 1985, pp. 82-88 Brooks, G.F., Lammel, C.J., Blake, MS., Kusecek, 8. and Achtman, M. Antibodies against IgAl protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis. J. infect. Dis. 1992, 166, 1316 1321 Haneberg, B., Kendall, D., Amerongen, H.M., Apter, F.M., Kraehenbuhl, J-P. and Neutra, M.R. Induction of specific immunoglobulin A in the small intestine, colon-rectum, and vagina measured by a new method for collection of secretions from local mucosal surfaces. Infect. Immun. 1994, 62, 15-23 Robinson, E.N., McGee, Z.A., Buchanan, T.M., Blake, M.S. and Hitchcock, P.J. Probing the surface of Neisseria gonorrhoeae: simultaneous localization of protein I and H.8 antigens. Infect. lmmun. 1987, 55, 1190-l 197

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