1079

Sialylation and Human Neutrophil Killing of Group C Neisseria meningitidis Michele M. Estabrook, Norman C. Christopher, J. McLeod Grifliss, Carol J. Baker, and Robert E. Mandrell

Center for Immunochemistry. VA Medical Center, Departments of Pediatrics and Laboratory Medicine, University of California, San Francisco; Department ofPediatrics. Case Western Reserve University School ofMedicine, Cleveland, Ohio; Department ofPediatrics. Microbiology, and Immunology, Baylor College ofMedicine, Houston, Texas

Acute otitis media (AOM) is one of the more common illnesses of childhood, but isolation of meningococci from middle-ear fluid is rare. In two large series describing 487 children undergoing tympanocentesis for AOM, Neisseria meningitidis (group Y) was isolated only once from middleear fluid [1, 2]. The immunologic characterization of meningococci causing unusual infections may provide information on the pathogenesis of and host response to the more invasive N. meningitidis disease. Few individuals with N. meningitidis nasopharyngeal colonization become ill. In those who do, bloodborne dissemination usually leads to a severe systemic illness that can be fulminant and rapidly fatal. Bactericidal antibodies that initiate complement-dependent immune lysis are of prime importance in blocking dissemination of N. meningitidis from the nasopharynx [3, 4]. The role of neutrophil killing in host defense against the meningococcus is less well known but was examined by Ross et al. [5], who found that serogroups Band 29E but not A, C, Y, or W 135 were phagocytosed and killed by neutrophils in the presence of pooled normal human serum depleted of C8. We describe a 5-year-old boy with serogroup C meningo-

Received 17 March 1992; revised 8 June 1992. Presented in part: American Federation for Clinical Research, Seattle. May 1991 (abstract N-41-0024). Informed consent was obtained from parents. Paper number 45 from the Center for Immunochemistry. Financial support: Thrasher Research Fund (2802-0) and Department of Veterans Affairs. Reprints or correspondence: Dr. Michele Estabrook. San Francisco General Hospital. 1001 Potrero Ave.• 6-E-6, San Francisco, CA 94110. The Journal of Infectious Diseases 1992;166:1079-88 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6605-0018$01.00

coccal AOM who did not develop disseminated disease. To understand why, we characterized his immune response and, the structural and antigenic properties of the cell surface of the infecting meningococcal strain, including its ability to sialylate its lipooligosaccharide (LOS) [6]. We found that the child's acute serum had no bactericidal activity against his infecting organism. His strain, however, was extremely susceptible to killing by neutrophils in the presence of active complement. We also found that the strain was unusual in that it had relatively little endogenous LOS sialylation, suggesting an association between sialylation and susceptibility to killing by neutrophils. To further investigate this, we examined the LOS sialylation characteristics and susceptibility to killing by neutrophils of serogroup C N. meningitidis strains isolated before and during a focal epidemic in Houston.

Case Report A 5-year-old white boy presented with a 2-day history of cough, fever, and mild nausea and a l-day history of severe left otalgia. Physical examination revealed a child in mild distress related to the otalgia; he did not appear systemically ill. His temperature was 38.1 oC and other vital signs were normal. There was no rash or physical findings suggestive of invasive bacterial disease. Otoscopic examination confirmed the presence of left AOM with effusion. At presentation, the white blood cell count was 19,370jmm 3 with 67% neutrophils, 6% band forms, 24% lymphocytes, and 3%monocytes. The platelet count was 355,000jmm 3 , and the hematocrit was 40%. As part of an ongoing study of AOM, diagnostic tympanocentesis of the left middle-ear effusion was done as previously described [7]. The middle-ear fluid was cultured and bacteria isolated and identified by standard bacteriologic methods. Acute serum was obtained at this visit. The child was given an oral antibiotic and sent home.

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

This study describes the association of lipooligosaccharide (LOS) and capsule sialylation with the survival of 25 serogroup C meningococcal strains in phagocytosis assays. Eleven strains isolated from children were of diverse protein serotypes or were nontypeable; 14 were serotype 2b:Pl.2 and were isolated from children during or immediately after a focal epidemic in Texas. Degree of endogenous LOS sialylation and amount of sialic acid capsule were associated with each other and with susceptibility to killing by neutrophils for the non-2b:P1.2 strains. The 2b:P1.2 strains as a group had significantly greater survival in the presence of neutrophils than did the non-2b:P1.2 strains. The susceptibility of these strains to killing by neutrophils was not associated with endogenous LOS sialylation or amount of capsule. These data suggest that many virulent strains evade neutrophil killing, either by sialylation or another mechanism. Evasion of neutrophil killing might enhance a strain's epidemic potential.

1080

Estabrook et al.

The next day, the culture of the middle-ear exudate yielded a group C strain of N. meningitidis (designated strain 15700), and the patient was asked to return to the clinic. At that time, he was afebrile and appeared clinically well. He denied otalgia, and otoscopic examination of the left tympanic membrane revealed a perforation and purulent drainage. A culture of blood yielded no organisms. He was treated with oral penicillin for 10 days, and he and his family received rifampicin prophylaxis. The patient had complete clinical and otoscopic clearing of the AOM. Convalescent serum was obtained 21 days after his initial presentation. All sera were stored frozen at either -20° or -70°C.

Materials and Methods

LOS and outer membrane complex (OMe). LOS and OMC antigens were prepared as previously described [21, 22]. Whole cell ELISA. We used MAbs and the whole cell ELISA method of Abdillahi and Poolman [23] to serogroup and protein serotype meningococcal strains. We also used this method to assess the LOS epitope repertoire of strain 15700. A modification of this same method was used to compare the binding ofMAb to group C polysaccharide. Briefly, whole organisms were suspended to an OD of 0.2 at 640 nm in PBS (pH 7.4) and diluted 1:8. This suspension was used to sensitize polyvinyl microtiter wells that were then reacted with MAb to the group C capsule. After incubation with secondary antibody conjugated to alkaline phosphatase, the wells were developed with p-nitrophenyl phosphate (2 mg/ml, in 100 mM bicarbonate buffer [pH 9.5] with 10 mM MgCl 2 ; Sigma, St. Louis) and the absorbance read at 405 nm (2550 EIA Reader; Bio-Rad, Burlingame, CA). The assay was done three times and the mean for the triplicate determinations was used to compare C polysaccharide production among the strains. For most strains the SD of the triplicate estimates was within 15%; variation was somewhat greater for the strains with the highest reactivity. SDS-PAGE analysis and immunoblotting. The proteins and LOS in the purified OMC from meningococcal strain 15700 were separated by SDS-PAGE using a modification of the method of Laemmli [14, 24]. Purified LOS from meningococcal strain 8032 was included as a molecular mass standard for LOS molecules. This strain makes six LOS of determined molecular masses of 5.4, 5.1,4.5,4.0, 3.6, and 3.2 kDa [12, 15]. The SDS-PAGE procedure we used has been recently described [12]; either the Protean II or the Mini-Protean II apparatus (Bio-Rad) was used. The LOS molecules were visualized by silver stain [25], and proteins were stained with Coomassie R-250 (Sigma). The proteins were divided into five classes by molecular weight [26]. We separated the proteins and LOS in the OMC of strain 15700 by SDS-PAGE as described. We used a modification [12] of the immunoblot method of Burnette [27] to analyze them with polyclonal rabbit antibodies to purified R-lO gonococcal pili [28] that were provided by G. Schoolnik (Stanford University, Palo Alto, CA). After being washed with PBS, the blot was incubated in alkaline phosphatase-conjugated goat antibody to rabbit IgG (Sigma). The blot was developed with a solution of 50 mM TRIS-HCl (pH 8.0), 0.1 % naphthol AS-MX phosphate disodium salt (Sigma), and 0.2% fast red TR salt (Sigma). The SDS-PAGE-separated proteins and LOS in the OMC of strain 15700 were also immunoblotted with a 1:60 dilution of either the child's acute or convalescent serum. The immunoblots were then incubated with alkaline phosphatase-conjugated goat antibody to human IgG, IgA, and IgM combined (CALTAG; South San Francisco) and developed in the substrate described above. Endogenous sialylation of LOS. We used neuraminidase to remove endogenous sialic acid from meningococcal LOS and thereby increase expression of the MAb 3Fl1-defined epitope [6, 20]. Polyvinyl microtiter wells were sensitized with whole organisms or OMC (,...., 100 JLg/mL protein) containing LOS. Clostridium perfringens neuraminidase (type V; Sigma) diluted (50 milliunits/ml.) in PBS, pH 6, was added to the wells and

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

N. meningitidis strains. In addition to the middle-ear isolate described above (strain 15700), we examined 24 group C meningococcal strains isolated from children in Houston during 19771981. Six (25%) of the strains were isolated from blood or cerebrospinal fluid (CSF) from children during a period of endemic disease in Houston (1977-1978) [8-10]. Five strains (21 %)were case isolates from an epidemic in an elementary school [11] that occurred in February 1981, and 6 were carrier strains isolated from healthy children in the school during the epidemic [11]. Seven strains (29%) were isolated from blood or CSF from children in Houston who became ill within 3 months of the epidemic (February-April 1981) but who did not attend the elementary school. One additional meningococcal strain (8032) was used in this study and has been described [12, 13]. Culture procedures have also been described [14]. Monoclonal antibodies (MAbs). MAbs specific for capsular polysaccharide of group A, B, C, Y, or W 135 were provided by W. D. Zollinger (Walter Reed Army Institute of Research, Washington, DC). MAbs specific for serotype proteins 2a, 2b, and 2c (class 2), 15 (class 3), and p 1.2 and p 1.16 (class 1) were also provided by W. D. Zollinger. J. T. Pool man (Rijksinstitut voor Volksgezonheid, Bilthoven, Netherlands) provided an additional MAb directed at a class 1 protein (p 1.15). Nine MAbs known to bind neisserial LOS were used to characterize the LOS of strain 15700. Four MAbs were LOS serotypespecific [15]: 2-1-L8 (L8), 1-9C4 (L9), MCA14.2 (L10), and 4C4 (L11). Kim et al. [15] found that MAb D6A bound an epitope that was highly conserved on N. meningitidis group A strains. It was also expressed on some group B, C, and Y strains [15]. MAb 6B7 has been described [16] and appears to bind to an epitope located in the core region of the LOS molecule. Maeland and Wedege [17] described a MAb similar to MAb 9-2L379 that binds meningococci that make the L3, 7, 9 LOS antigenic complex. MAbs 3F 11 and 06B4 bind to many meningococci and gonococci [18, 19] and recognize terminal lactosamine structures on LOS as well as similar structures on glycosphingolipids of human erythrocytes and neutrophils [19, 20]. MAbs 2-1-L8 and 9-2-L379 were provided by W. D. Zollinger, and MCA 14.2 was provided by R. Sugasawara (IGEN, Rockville, MD). MAbs 06B4, 3F 11, 4C4, and 6B7 were provided by M. A. Apicella and M. A. J. Westerink (State University of New York, Buffalo). J. T. Poolman provided MAbs 1-9C4 and D6A.

JID 1992; 166 (November)

JID 1992; 166 (November)

Sialic Acid and Neutrophil Killing

vation through C3 (necessary for complement-dependent phagecytosis) but not through C9 (necessary for complement-mediated bacterial lysis). Heat-inactivated PHS-C8D was used to measure complement-independent neutrophil killing, as fixation ofC3 to the organism does not occur in heated serum [30]. We used the opsonophagocytosis assay of Ross et al. [5]. The PHS-C8D was purchased from Quidel Q (San Diego). Each assay consisted of a reaction mixture containing bacteria (1.25 X 106 cfu), neutrophils (1.25 X 106 ) , and 10% PHS-C8D; a reaction mixture containing bacteria, neutrophils, and heat-inactivated (56°C for 30 min) PHS-C8D; and a reaction mixture containing bacteria and 10% PHS-C8D. Duplicate aliquots were removed, diluted, and each plated twice. Survival was expressed as the percentage of organisms at time 0 that survived to 60 min. Each strain was tested with neutrophils from at least two different donors and the results were averaged. It had been shown previously that meningococci grown in the presence of cytidine monophospho-N-acetylneuraminic acid (CMP-NANA) incorporated more sialic acid into the 4.5-kDa component of their LOS than they did when cultured in the absence ofan exogenous sialylating nucleotide [6]. We assessed the effect ofexogenous sialylation on bacterial killing by neutrophils by growing selected strains in broth that did or did not contain 500 JLg/mL CMP-NANA (Sigma). The organisms were washed twice and used in the opsonophagocytosis assay as described above. The binding of MAb 3F II to LOS on these strains was used to monitor exogenous LOS sialylation. Statistical analysis. Data from groups are expressed as mean ± SD. Differences between groups were analyzed by unpairedsample, two-tailed t test. Differences before and after experimental modifications were analyzed by paired-sample, two-tailed t test. Spearman's rank correlation or simple linear correlation was used to assess the relationship between variables.

Results Serogroup and protein serotype of strains. Strain 15700 bound only the group C-specific capsular polysaccharide MAb, confirming that it was ofthis serogroup. It did not bind MAbs specific for class I (p 1.15, P 1.16, and p 1.2), class 2 (2a, 2b, and 2c), or class 3 (15) serotype and serosubtype antigens. The 24 group C meningococcal strains isolated from children in Houston were also serotyped. The 6 endemic case strains were of diverse protein serotypes or were non typeable; none were type 2b:P 1.2. The 5 strains isolated from a focal outbreak of disseminated meningococcal disease in an elementary school were all serotype 2b:P 1.2. Six carrier strains were isolated from healthy children present in the elementary school at the time of the outbreak, and 3 were 2b:P 1.2. Seven case strains were isolated within 3 months of the outbreak from Houston children who did not attend the elementary school; 6 were 2b:P 1.2. LOS. LOS MAbs 06B4, 9-2-L379, and 3Fll bound strongly to the LOS of strain 15700, indicating that this strain had the acceptor molecule for sialic acid. MAb 6B7

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

incubated for 2 h at 37°C. PBS was added to duplicate wells as a negative control. The wells were then washed and treated with 0.1 % bovine serum albumin (BSA) in PBS to block nonspecific binding. The wells were incubated first in MAb 3FII and then in either goat anti-mouse IgM conjugated with alkaline phosphatase or goat anti-mouse IgM labeled with 1251. Wells with all reagents except the MAb showed no binding of the secondary antibody to enzyme-treated LOS. To rank the relative degree of endogenous sialylation of each strain, we expressed the MAb 3F II binding before treatment as a percentage of the binding after neuraminidase treatment. Smaller percentages reflect greater degree of endogenous sialylation. The results of at least two assays were averaged. C polysaccharide ELISA. We used the ELISA method of Jarvis et al. [29] to compare the amount of antibody (IgG, IgA, and IgM combined) to the meningococcal group C capsule in the child's acute and convalescent sera. In addition, we compared the relative contributions of IgG, IgA, and IgM to the group C capsular antibody response in his convalescent serum. Serum bactericidal assay. A standard bactericidal assay was used to assess the ability of the child's acute and convalescent sera to kill his infecting strain. Because sera were not processed or stored to preserve endogenous complement, they were heated at 56°C for 30 min and exogenous complement was added. This consisted of hypogammaglobulinemic serum (HGS, previously obtained from an adult with acquired hypogammaglobulinemia) that contained 95% pure and were used within 1-2 h of isolation. Bacterial killing by neutrophils. We measured bacterial killing by neutrophils using pooled normal human serum that was depleted of C8 (PHS-C8D) and thus allowed complement acti-

1081

Estabrook et at.

1082

kDa ....

66

.... 43

....

31

....

14

1 2 3 Figure 1. SDS-PAGE-separated outer membrane comple x of N. meningitidis 15700. Lipooligosaccharides (LOS) are visualized with silver stain; proteins are stained with Coomassie R-250 . Lane I, N. meningitidis 8032 LOS used as molecular mass markers; lane 2, 15700; lane 3, protein molecular mass markers. The six LOS bands of strain 8032 have molecular masses of 5.4, 5.1, 4.5, 4.0, 3.6, and 3.2 kDa. N. meningitidis outer membrane complex proteins are divided into five classes by molecular mass: class I, 44.047.0 ; class 2, 40.0-42.0; class 3, 37.0-39.0; class 4,33.0-34.0; and class 5, 26.0-30.0 kDa.

the bacteria with neuraminidase significantly increased the binding ofMAb 3FII (P < .001) for all 25 strains taken as a group (figure 2). Three strains were heavily sialylated, as judged by the fact that they bound < 10%as much MAb 3F II before neuraminidase treatment as after desialylation. Three strains had relatively little LOS sialylation (>80%epitope expression before treatment), including strain 15700, which expressed 81%of the MAb 3FII-defined epitope before desialylation. The other strains were variously sialylated (33%-75%). No significant difference in the degree of endogenous sialylation was found between serotype 2b:P 1.2 strains and those of all other serotypes combined (50% ± 15% vs. 57% ± 32%, P = .49). Except for one heavily sialylated strain, the 2b:P1.2 strains were much more homogeneous in their degree of endogenous LOS sialylation than the non-2b:PI.2 strains. Immune response. We used an ELISA to compare the child's acute and convalescent antibody response to the group C capsule . He had almost no antibody directed at the group C capsule in his acute serum; such antibody increased markedly in his convalescent serum . During convalescence, his capsular antibody response included all major immunoglobulin isotypes (IgG, IgA, and IgM). The immunoblots of the SDS-PAGE-separated OMC of strain 15700 with the patient's acute and convalescent sera showed that antibody in his acute serum bound weakly to LOS molecules and several protein bands . His convalescent serum had a marked increase in antibody that bound to LOS, class 5 protein, and a protein that was -26.0 kDa (probably H.8). His convalescent serum also contained new antibody that bound the pilus.

-

1.5

•

c

CII

E i;

and the LOS L9 serotype-specific MAb, 1-9C4, bound weakly. Neither the L8-, LlO-, nor Lll-specific MAbs nor D6A bound to 15700 LOS. SDS-PAGE. Figure I is a silver-stained gel of the LOS of strain 15700 with the six LOS bands of strain 8032 included as molecular mass markers. Strain 15700 made two major LOS molecules of -4.5 and -4.8 kDa. The protein profile of strain 15700 is also shown. The binding of polyclonal rabbit neisserial pilus antibodies to a 20-kDa band provided evidence that strain 15700 was piliated. Endogenous sialylation. We next examined the degree of endogenous LOS sialylation of strain 15700 and the 24 group C meningococcal strains isolated from children. Each strain made the MAb 3Fll-defined sialic acid acceptor LOS molecule. Despite variation in the degree of endogenous LOS sialylation for individual strains with repetition of the assay, the ranking of the strains by their degree of'sialylation remained remarkably constant. As expected, treatment of

... ...

•

CII

I-

0

1.0

- .•

.'.

•

CII



's

:J

aD

i: CIl l:! CIl a.

60

en

100

Gi

80

~:)

60

>

III

C

Non 2b:Pl.2

• •

40

•

GI

...U

GI

Q.

20

o

o

o

20

40

•

•

•

60

•

• •

80

100

Percent 3Fll Epltope Expression Before Neuraminidase Treatment

160

.'"'"

140

Gi

> 120

'> li

III

C GI

U

~

2b:Pl.2

'"

'" ••

100 80

'"

60

•

• •

40

Q.

20

•

••

0 0

20

40

60

80

100

Percent 3Fll Epllope Expression Before Neuraminidase Treatment

Figure 4. Percentage survival in phagocytic killing assay versus degree ofendogenous lipooligosaccharide (LOS) sialylation . Bacterial survival in presence of neutrophils and pooled human serum depleted ofC8 was measured at 60 min . Neuraminidase treatment desialylates LOS and increases monoclonal antibody (MAb) 3FII binding . For each strain, amount of its binding ofMAb 3FII before neuraminidase treatment was expressed as percentage of its binding ofMAb after treatment. Smaller percentages reflect greater degree of endogenous sialylation . Upper panel: group C N. meningitidis strains (including strain 15700) that were not protein serotype 2b:PI.2. 0 , strain remained susceptible to killing by neutrophils despite being heavily sialylated. Spearman's rank correlation was used to assess relationship between variables in upper panel. Lower panel : group C N. meningitidis strains that were serotype 2b:PI.2. t::., epidemic strains. Encircled strains became resistant to killing by neutrophils after growth in cytidine monophospho-Nacetylneuraminic acid.

40 20

o Donor 1

•

o o

Donor 2

Neutrophils + PHs-caD Neutrophlls + Inactivated PHS-CaD PHs-caD

Figure 3. Phagocytic killing assays of N. meningitidis 15700 (middle-ear isolate). Survival was determined at 60 min. PHS-C8D, pooled human serum depleted ofC8. Percentage survival of> 100% reflected bacterial growth during phagocytic assay.

non-2b:P1.2 strains than for the 2b:P1.2 strains (31% ± 27% survival vs. 83% ± 48% survival, P = .006). Figure 4 shows the relationship between the survival of non-2b :P1.2 organisms (including strain 15700) and their degree of endogenous LOS sialylation. Decreasing survival was associated with decreasing endogenous LOS sialylation. Spearman's rank correlation coefficient with correction for ties was -.75 (P = .025). The 4 non-2b :P1.2 strains that had not disseminated (3 carrier and 15700) were among the most sensitive to killing (survival :;;20%) and among the least sialylated. One strain that was isolated from a 16-year-old remained susceptible to killing by neutrophils despite being heavily sialylated. The strain with the highest amount of LOS sialylation sur-

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

Using a complement-mediated bactericidal assay, we determined that the child's acute serum could not kill his infecting meningococcal strain 15700 (0.3% ± 1.0%). This strain was partly killed (24 .8% ± 15.7%) in the HGS that was used as a complement source, but this killing was blocked by the child's acute serum . In contrast, strain 15700 was completely killed (99.8% ± 0.3 %) in the child's convalescent serum. Killing ofendogenously sialylated meningococci by neutrophi/so The absence of bactericidal antibodies in the child's acute serum suggested that opsonophagocytosis could have prevented dissemination of the infection through the circulation. Therefore, we examined the ability of neutrophils to kill strain 15700. As a comparison, 24 group C meningococcal strains isolated from children in Houston were tested for their sensitivity to killing by neutrophils. The results were grouped by strains that were protein serotype non-2b:PI .2 (n = II) and strains that were 2b:P1.2 (n = 14). Some strains that were not killed by neutrophils continued to show bacterial growth during the phagocytic assays resulting in a value for percentage survival that was> 100%. In the presence ofPHS-C80, strain 15700 was almost completely killed by neutrophils at 60 min (figure 3). Organisms were not killed by PHS-C80 alone or by neutrophils in heatinactivated PHS-C80. These results indicated that strain 15700 was very sensitive to complement-dependent killing by neutrophils. The percentage survival at 60 min in the presence of neutrophils and active PHS-C80 was significantly less for the

1083

1084

Estabrook et al.

Table 1. Survival of group C meningococcal strains grown with and without exogenous cytidine monophospho-N-acetylneuraminie acid (CMP-NANA)in the presenceofneutrophils and active pooled normal human serum depleted ofC8.

Binding' ofMAb 3FII to strain grown

% survival

of strain grown

Without CMP-NANA

With CMP-NANA

Without CMP-NANA

With CMP-NANA

NT NT Pl.l5 NT NT Pl.l5 2b:P1.2 2b:P1.2 2b:PI.2 2b:PI.2

1.14 1.04 0.81 0.83 1.06 0.56 0.66 0.07 0.48 0.47

0.11 0.15 0.13 0.08 0.40 0.06 0.12 0.02 0.08 0.03

6 5 32

II

58

6

5

0 60 8 5 79 13

12

I

97 96 6

55

1081

NOTE. MAb. monoclonal antibody; NT. non typeable. , As measured by Of) at 405 nrn byELISA. 1 Bacterial growth occurred during assay.

vived poorly in active PHS-C8D without neutrophils and could not be evaluated for this type of killing. Thirteen of 14 2b :P 1.2 case and carrier strains isolated in Houston during a 3-month period were sialylated endogenously to a similar degree, but these strains had very different abilities to survive in the presence of neutrophils (0-150% survival, figure 4). Seven strains were not susceptible to killing by neutrophils and even grew during the assay . This group included 4 of the 5 epidemic strains. An eighth strain was relatively resistant, with 85% survival. For the 2b:P 1.2 strains as a group, survival in the presence ofneutrophils and active PHS-C8D was not associated with endogenous LOS sialylation. Killing of exogenously sialylated meningococci by neutrophils. We next evaluated the effect of exogenous LOS sialylation on bacterial sensitivity to killing by neutrophils. Mean survival at 60 min in the presence of neutrophils and PHSC8D was not significantly different when 6 non-2b:PI.2 strains (including strain 15700) were grown with and without exogenous CMP-NANA (table I). Survival without CMP-NANA was 18% ± 23% and with CMP-NANA was 31% ± 38% (P = .1). The mean binding of MAb 3F II as measured by absorbance in the ELISA was significantly decreased by growth in the presence ofCMP-NANA (0.90 ± 0.21 A 405 vs. 0.15 ± 0.12 A 405 , P < .00 I). We also tested 4 2b:P1.2 strains that were sensitive to killing by neutrophils for their ability to survive this type of killing after they were grown in the presence of CMPNANA. Although the mean survival at 60 min in the presence of neutrophils and PHS-C8D was also not significantly different for the strains grown without and with exogenous

CMP-NANA (26% ± 35% vs, 66% ± 46%, P = .28),2 strains had a dramatic increase in survival when they were grown in CMP-NANA (table I) . The mean binding ofMAb 3FII was also significantly reduced by growth in CMP-NANA (0.42 ± 0.25 A 405 vs. 0.06 ± 0.05 A 40 5 , P = .04). These data suggested that only certain 2b:P 1.2 strains were made more resistant to killing by neutrophils when the strains were grown in CMP-NANA. Although the 2b:PI .2 strains were homogeneous in their degree of endogenous LOS sialylation, they were not homogeneous in their sensitivity to killing by neutrophils in active PHS-C8D (figure 4). Eight of the 2b:P 1.2 strains were resistant to killing by neutrophils independent ofLOS sialylation. Two other 2b:PI .2 strains were extremely susceptible to killing by neutrophils but became resistant when grown in exogenous CMP-NANA (table I) . It was apparent that most 2b:P 1.2 strains were resistant to killing by neutrophils or became so when grown in the presence of exogenous CMPNANA, but a subset of the 2b:P 1.2 strains appeared to show a positive correlation between survival and endogenous sialylation that was similar to that of the non-2b:P 1.2 strains. Killing of meningococci in heat-inactivated PHS-C8D by neutrophils. We also found that 4 (16%) of the 25 group C strains that we examined were susceptible to killing by neutrophils in heat-inactivated PHS-C8D (figure 5). These

15 0 31

1 S 0 29

160

160

140

140

as>

a; 120 >

120

.~ 100

.~ 100

::I

::I

Vl

80

Vl

80

E 60

E 60

ii; 40 c..

a,

"u

"ii;u

20

40 20

Donor 1

Donor 3

Donor 5

Donor 1

15 02 1

Dono r 3

80 26

160 140 (; 120

>

~ 100 ::I

Vl

80

E 60 !! 40

" "

c..

20 0

Donor 1

•

Donor 4

Donor 2

Donor 4

Neutr ophil s + PHS-CSD

o

Neu troph ils + Inactivated PH S-CSO

o

PHS-C8D

Figure 5. Phagocytic killing assays of 4 N. meningitidis strains found to be susceptible to complement-dependent and -independent killing by neutrophils. Survival was determined at 60 min. PHS-C80, pooled human serum depleted of C8.

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

Serotype

JID 1992; 166 (November)

lID 1992; 166 (November)

Sialic Acid and Neutrophil Killing

Discussion This study describes the effect of LOS and capsule sialylation on the survival of serogroup C meningococcal strains in phagocytosis assays with human neutrophils. The impetus for this study was provided by a preliminary study of a serogroup C meningococcal strain (15700) causing AOM in a child. Complement-dependent serum bactericidal activity usually blocks dissemination of N. meningitidis from the nasopharynx [3, 4]. The infection remained localized in this child even though his acute serum contained almost no antibody specific for his infecting strain and did not kill the strain in a complement-mediated bactericidal assay. During convalescence, the child developed a vigorous serum antibody response that was similar to that reported after disseminated meningococcal disease [17]. A systemic immune response during convalescence from AOM has been well documented, particularly with non typeable HaemophiIus influenzae [31-33]. We hypothesized that the meningococcus isolated from this child's middle ear did not disseminate because the child was able to clear the strain by a mechanism other than complement-mediated lysis and that structural and antigenic characteristics ofthe organism might have made such clearance possible. Most meningococcal disease in the United States is caused

160

as>

~

::I

Non 2b:P1.2

140 120

•

100

(f)

80

'EII)

60

()

~

D.

40 20

•

0 0.5

0.6

• ••

• 0.7

•

•

til

0.8 A

1.1

0.9

405

Binding of C Polysaccharide MAb

2b:P1.2

160

as> ·E ::I

6.

• •

140 120 100

(f)

• 6.

60

••

()

~

D.

40 20

•

0 0.5

•

6.

80

'EII)

6.

6.

0.6

0.7

0.8 A

•

• 0.9

1.1

405

Binding of C Polysaccharide MAb

Figure 6. Percentage survival in phagocytic killing assay versus binding of monoclonal antibody (MAb) to group C polysaccharide of whole organisms as measured by ELISA (A 40 S) ' Bacterial survival in presence ofneutrophils and pooled human serum depleted ofC8 was measured at 60 min for group C N. meningitidis strains (including 15700) that were not serotype 2b:P 1.2 (upper panel) or were 2b:Pl.2 (lower panel). SD for values at either end ofC polysaccharide production did not overlap. 0 (upper panel), same strain designated by 0 in figure 4 (upper panel). l:::. (lower panel), epidemic strains.

by group B or C organisms. Protein class 2/3 serotypes 2 or 15 account for much of the group Band C disease in North America and Europe [34,35]. While strain 15700 was not of these protein serotypes, we found that 28% of the 18 Houston group C strains that caused disseminated disease were similarly nontypeable with the same MAbs. This percentage was even higher for the case strains isolated during a period of endemic disease, with 4 of 6 being nontypeable. Pili are important in adherence of meningococci to nasopharyngeal mucosa [36], and strain 15700 was piliated. The strain was also encapsulated, on the basis of the binding of an anticapsular MAb. Thus, we cannot explain the failure of this strain to disseminate in the absence of serum bactericidal activity on the lack of important surface molecules implicated in pathogenicity. Another important surface antigen on meningococci, LOS, has been characterized with various anti-LOS MAbs.

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

strains were all non-2b:Pl.2 and 2 were carrier strains. They represented 4 of the 5 least sialylated strains. Strain 15700 was unusual in that it had relatively little LOS sialylation but was resistant to this type ofkilling. When these 4 strains were grown in the presence of exogenous CMP-NANA, only 1 became resistant to killing by neutrophils in inactivated PHS-C8D, with its survival increasing from 39% to 98% at 60 min. Group C polysaccharide capsule. We used an ELISA to semiquantitatively measure the relative amount of group C polysaccharide capsule made by strain 15700 and the 24 Houston strains. There was no significant difference between the binding of MAb to group C polysaccharide on the non2b:P1.2 strains compared with that to polysaccharide on the 2b:P 1.2 strains (0.84 ± 0.11 A 405 vs. 0.89 ± 0.10 A 405 , P = .21). We found that increased binding of C polysaccharide MAb was associated with increased survival in the presence of neutrophils and active PHS-C8D for the non-2b:P1.2 strains but not for the 2b:P 1.2 strains as a group (figure 6). However, a subgroup ofthe 2b:Pl.2 strains appeared to show a positive correlation similar to that of the non-2b:P1.2 strains. There was a weak, positive correlation (r 2 = .49) between MAb binding to C polysaccharide and the degree of endogenous LOS sialylation for the non-2b:P 1.2 strains, but there was no correlation between MAb binding to C polysaccharide and the amount of endogenous LOS sialylation for the 2b:P1.2 strains (figure 7).

1085

Estabrook et al.

1086

1.2

Non 2b:P1.2

•

D

c(

:::E

0_

CD

a. :s

•

o

0.8

o:!:!

C,c

• • • ••

••

0.6

g

.5 as 0.4 l:D U) >0 0.2 a. 0 20

0

60

40

100

80

Percent 3F11 Epltope Expression Before Neuraminidase Treatment

••••

.. -:

D

c(

0_

:::E CD

a. :s o:!:!

•• •

•

0.8

•

0.6

C,c

g

0.4

.5 as l:D U) >0 0.2 a. 0 0

20

40

60

80

100

Percent 3F11 Epltope Expression Before Neuraminidase Treatment

Figure 7. Binding of monoclonal antibody (MAb) to group C polysaccharide of whole organisms as measured by ELISA (A 40 S ) versus degree of endogenous lipooligosaccharide sialylation for non-2b:PI.2 meningococcal strains including strain 15700 (upper panel) and for 2b:P1.2 meningococcal strains (lower panel). Smaller percentages on x axis reflect greater degree of endogenous sialylation. SD for values at either end ofC polysaccharide production did not overlap. 0 (upper panel), same strain designated by 0 in figures 4 and 6. Simple linear correlation was used to assess relationship between variables in upper panel.

Only three MAbs bound strongly to the LOS ofstrain 15700. Two ofthe MAbs, 3FII and 06B4, bind to a 4.5-kDa component of meningococcal LOS [6, 19, 37] that contains the carbohydrate lacto-V-neotetraose (Gal,B 1-+4GlcNAc,B1-+ 3Gal,BI-+4Glc) [38] and to mammalian glycosphingolipids that contain the trisaccharide Gal,B 1-+4GlcNAc,B1-+ 3Gal [19]. It is probable that the third MAb that bound to strain 15700 LOS (MAb 9-2-L379) also binds to LOS epitopes of structure similar to those that bind MAbs 3F II and 06B4. It also has been shown that the LOS component that binds MAbs 3FII and 06B4 is the LOS that becomes sialylated on gonococci grown in the presence of exogenous CMP-NANA [20, 39] and that becomes sialylated endogenously on meningococcal group Band C strains [6] and some strains of H. influenzae [40]. Groups Band C meningococci can synthesize their own CMP-NANA [41-43] and, therefore, do not require exogenous CMP-NANA for sialylation of LOS.

Treatment of sialylated LOS with neuraminidase increases the expression of the 3F II-defined epitope and is a measure of the degree of sialylation [6, 20]. Similar neuraminidase treatment of strain 15700 indicated that its LOS was minimally sialylated and, compared with 24 other group C meningococcal strains isolated from children, the LOS of strain 15700 was among the least sialylated. It was unlikely that the changes in LOS epitope expression in the group C strains were due to capsule degradation, since the neuraminidase used for these studies (c. perfringens) has minimal activity for the group C polysaccharide capsule [44]. The minimal sialylation of the LOS of strain 15700 correlated with the susceptibility of this strain to complement-dependent killing by neutrophils. This result is in contrast to the results of Ross et al. [5], who reported that 7 case and 8 carrier group C strains survived in the presence of neutrophils and either PHS-C8D or heat-inactivated PHS-C80. This inconsistency led us to investigate further whether LOS sialylation played any role in the survival ofgroup C meningococcal strains in the presence of neutrophils. To investigate this, we used a collection ofgroup C meningococcal strains isolated from children in Houston. Six case strains that were isolated during a period of endemic disease in Houston (1977-1978) [8-10] were of diverse protein serotypes or were nontypeable; none were type 2b:P 1.2. Feigin et al. [II] found that from 1970 to 1980, most meningococcal cases in Houston were caused by serogroup B strains. By January 1981, the predominant serogroup circulating in the community had switched from group B to group C, and a focal epidemic ofgroup C meningococcal disease occurred in a Houston school on 4 February 1981 [II]. We found that all 5 of the available epidemic strains were C:2b:P 1.2. In addition, 3 of 6 carrier strains and 6 of 7 case strains that were isolated in Houston within 3 months of the epidemic were serotype C:2b:P 1.2. The 4 serogroup C non2b:P 1.2 strains isolated during this period probably represented underlying endemic disease. Thus, these results indicate that the epidemic in Houston was caused by C:2b:P 1.2 strains that were prevalent in the community around the time of the epidemic. For endemic group C meningococcal strains (represented in this study by non-2b:PI.2 strains), sensitivity to killing by neutrophils in PHS-C8D appears to be correlated with the degree of endogenous LOS sialylation and the amount of capsular polysaccharide but not with exogenous LOS sialylation. Furthermore, it is possible that a relative lack ofendogenous LOS sialylation might also render some of these strains susceptible to killing by neutrophils when complement levels are altered (e.g., heat-inactivated sera). The degree of endogenous LOS sialylation on the strains appeared to correlate directly with the amount ofsialic acid capsule present on the strains. Although this correlation was not strong, it suggested that the synthesis of these sialylated molecules might

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

2b:P1.2

1.2

JID 1992;166 (November)

1ID 1992; 166 (November)

Sialic Acid and Neutrophil Killing

transferases may reflect an important mechanism of virulence ofthe 2b:Pl.2 strains during the epidemic. Future studies of the biochemistry of the sialyltransferases (e.g., location, kinetics, acceptor specificity, linkage specificity) should help to clarify whether such a mechanism exists.

Acknowledgment

We thank Ilma Rodriguez for excellent technical assistance.

References 1. Shurin PA, Pelton SI. Donner A. Finkelstein BA, Klein JO. Trimethoprim-sulfamethoxazole compared with ampicillin in the treatment of acute otitis media. J Pediatr 1980;96: 1081-7. 2. Van Hare GF. Shurin PA, Marchant CD, et at. Acute otitis media caused by Branhamella catarrhalis: biology and therapy. Rev Infect Dis 1987;9: 16-27. 3. Goldschneider I. Gotschlich EC, Artenstein MS. Human immunity to the meningococcus. The role of humoral antibodies. J Exp Med 1969; 129: 1307-26. 4. Nicholson A. Lepow IH. Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity. Science 1979;205:298-9. 5. Ross SC, Rosenthal PJ. Berberich HM. Densen P. Killing of Neisseria meningitidis by human neutrophils: implications for normal and complement-deficient individuals. J Infect Dis 1987;155:1266-75. 6. Mandrell RE. Kim JJ. John CM. et at. Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis. J Bacteriol 1991; 173:2823-32. 7. Carlin SA. Marchant CD. Shurin PA, Johnson CE, Murdell-Panek 0, Barenkamp SJ. Early recurrences of otitis media: reinfection or relapse? J Pediatr 1987;110:20-5. 8. Griffiss JM. Brandt BL, Broud DO. Goroff OK, Baker CJ. Immune response ofinfants and children to disseminated infections with Neisseria meningitidis. J Infect Dis 1984;150:71-9. 9. Baker CJ. Griffiss JM. Influence of age on serogroup distribution of endemic meningococcal disease. Pediatrics 1983;71 :923-6. 10. Broud DO. Griffiss JM. Baker CJ. Heterogeneity of serotypes of Neisseria meningitidis that cause endemic disease. J Infect Dis 1979; 140:465-70. II. Feigin RD, Baker CJ. Herwaldt LA, Lampe RM. Mason EO. Whitney SE. Epidemic meningococcal disease in an elementary-school classroom. N Engl J Med 1982;307:1255-7. 12. Estabrook MM. Mandrell RE. Apicella MA, Griffiss JM. Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide. Infect Immun 1990;58:2204-13. 13. Griffiss JM. GoroffDK. Immunological cross-reaction between a naturally occurring galactan. agarose, and an LPS locus for immune lysis of Neisseria meningitidis by human sera. Clin Exp Immunol 1981;43:20-7. 14. Schneider H. Hale TL. Zollinger WD. Seid RC Jr. Hammack CA, Griffiss JM. Heterogeneity of molecular size and antigenic expression within lipooligosaccharides of individual strains of Neisseria gonorrhoeae and Neisseria meningitidis. Infect Immun 1984;45:544-9. 15. Kim JJ. Mandrell RE. Zhen H. Westerink MAJ. Poolman JT, Griffiss JM. Electromorphic characterization and description of conserved epitopes of the lipooligosaccharides of group A Neisseria meningitidis. Infect Immun 1988;56:2631-8. 16. Dudas KC, Apicella MA. Selection and immunochemical analysis of

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

be coregulated on some strains or at least that the regulation of sialylation in these strains is complex. We found surprisingly different results when we examined 14 C:2b:P 1.2 meningococcal strains that were clustered both temporally and geographically in Houston. The 2b:P 1.2 strains were not uniformly resistant to killing by neutrophils and three groups were apparent: (1) strains that were resistant to killing by neutrophils but the resistance did not correlate with either the degree of endogenous sialylation of LOS or capsule; (2) strains that were very sensitive to killing by neutrophils but that survived if the strains had been grown in the presence ofCMP-NANA; and (3) strains whose survival in the presence of neutrophils correlated directly with the degree of endogenous sialylation of LOS, capsule, or both. Of note is the observation that group 1 comprised 6 strains: 4 of the 5 epidemic strains, a carrier strain isolated in the school during the epidemic, and a case strain isolated on the same day as the epidemic from a Houston child who appeared to have no connection with the school. Growth in exogenous CMP-NANA appeared to have little effect on the survival of strains whose survival correlated with the degree of endogenous sialylation. Two 2b:P 1.2 strains that were very sensitive to killing by neutrophils despite moderate levels ofendogenous sialylation became completely resistant to killing when exogenous sialylation occurred. Because the decrease in 3F 11 binding that occurred with growth in the presence of exogenous CMP-NANA did not distinguish between strains that became resistant to killing by neutrophils, exogenous sialylation might reflect the involvement of additional LOS acceptors unrelated to the 3Fll epitope, the involvement of other outer surface structures in the sialylation pathway, and/or modification of specific host glycolipids by the bacterial sialyltransferase. We conclude that killing by neutrophils is an important means of preventing meningococcal dissemination and that the ability to evade such killing is an important part of the pathogenic potential of virulent strains. One mechanism of evasion is the increased endogenous sialylation of meningococcal surfaces. The differences in degree of endogenous sialylation probably reflect differences in the amount or regulation of the synthesis of sialic acid or the bacterial sialyltransferase complex necessary to add endogenous sialic acid to the LOS acceptor or to the capsule in the appropriate linkage. Another as-yet-undefined mechanism was manifested by a group of2b:P 1.2 strains in which resistance to killing by neutrophils was unrelated to endogenous sialylation. However, in preliminary studies we have noted that the specific activity of the sialyltransferases that transfer exogenous NANA to LOS is significantly greater for the 2b:P 1.2 strains compared with that of the non-2b:P1.2 strains. This suggests that the 2b:P 1.2 strains might also scavenge or transfer exogenous CMP-NANA more efficiently in vivo compared with the non-2b:Pl.2 strains. The higher activity ofthe 2b:Pl.2 sialyl-

1087

1088

Estabrook et al.

31. Faden H, Krystofik OA. Hong 11. et al. Immune response to ~ontyp­ able Haemophilus influenzae in the general population and among children with otitis media with effusion. Ann Otol Rhinol Laryngol Suppl 1988;97:34-6. 32. Faden H. Bernstein 1. Brodsky L, et al. Otitis media in children. I. The systemic immune response to nontypable Hemophilus influenzae. 1 Infect Dis 1989; 160:999-1004. 33. Murphy TF, Apicella MA. Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev Infect Dis 1987;9: 1-15. 34. Poolman IT. Jonsdottir K, Jones 0, Lind I, Froholm OL, Zanen He. Meningococcal serotypes and serogroup B disease in northwest Europe. Lancet 1986;2:555-7. 35. Frasch CE, Coetzee G, Zahradnik 1M, Wang LY. New developments in meningococcal vaccines. In: Schoolnik GK. ed. The pathogenic Neisseria. Washington, DC: American Society for Microbiology, 1985:633-9. 36. Stephens OS. McGee ZA. Attachment of Neisseria meningitidis to human mucosal surfaces: influence of pili and type of receptor cell. 1 Infect Dis 1981;143:525-32. 37. Kim 11. Mandrell RE. Griffiss 1M. Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class I, 2. and 3 protein epitopes. Infect Immun 1989;57:602-8. 38. lennings Hl. Lugowski C, Ashton F. The structure of an R type oligosaccharide core obtained from some lipopolysaccharides of Neisseria meningitidis. Carbohydr Res 1983; 121:233-41. 39. Parsons Nl, Patel PV, Tan EL, et al. Cytidine 5'-monophospho-N-acetyl neuraminic acid and a low molecular weight factor from human blood cells induce lipopolysaccharide alteration in gonococci when conferring serum resistance to killing by human serum. Microb Pathog 1988;5:303-9. 40. Mandrell RE, McLaughlin R. Abu Kwaik Y. et al. Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids. and some LOS are sialylated. Infect Immun 1992;60: 1322-8. 41. Warren L, Blacklow RS. The biosynthesis ofcytidine 5'-monophosphoN-acetylneuraminic acid by an enzyme from Neisseria meningitidis. 1 Bioi Chern 1962;237:3527-34. 42. Frosch M, Weisgerber C, Meyer TF. Molecular characterization and expression in Escherichia coli of the gene complex encoding the polysaccharide capsule of Neisseria meningitidis group B. Proc Nat! Acad Sci USA 1989;86:1669-73. 43. Blacklow RS, Warren L. Biosynthesis ofsialic acids by Neisseria meningitidis.l Bioi Chem 1962;237:3520-6. 44. Liu TY, Gotschlich EC, Dunne Ft. Jonssen EK. Studies on the meningococcal polysaccharides. II. Composition and chemical properties of the group B and group C polysaccharide. 1 Bioi Chern 1971 ;246:4 703-12.

Downloaded from http://jid.oxfordjournals.org/ at Monash University on June 20, 2015

lipooligosaccharide mutants of Neisseria gonorrhoeae. Infect Immun 1988;56:499-504. 17. Maeland lA. Wedege E. Serum antibodies to cross-reactive Neisseria outer membrane antigens in healthy persons and patients with meningococcal disease. APMIS 1989;97:774-80. 18. Mandrell RE. Schneider H. Apicella MA. Zollinger W. Rice PA. Griffiss 1M. Antigenic and physical diversity of Neisseria gonorrhoeae lipooligosaccharides. Infect Immun 1986;54:63-9. 19. Mandrell RE. Griffiss 1M. Macher BA. Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize cross-reacting antigens on LOS and human erythrocytes. 1 Exp Med 1988;168:107-26. 20. Mandrell RE. Lesse Al, Sugai Jv, et al. In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation. 1 Exp Med 1990; 171:1649-64. 21. Schneider H. Griffiss 1M, Williams GO. Pier GB. Immunological basis of serum resistance of Neisseria gonorrhoeae. 1 Gen Microbiol 1982;128: 13-22. 22. Zollinger WO, Mandrell RE, Griffiss1M. Altieri P. Berman S. Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man. 1 Clin Invest 1979;63:836-47. 23. Abdillahi H. Poolman IT. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol Lett 1987; 48:367-71. 24. Schneider H. Griffiss1M. Mandrell RE, larvis GA. Elaboration of a 3.6 kilodalton lipooligosaccharide, antibody against which is absent from human sera, is associated with serum resistance of Neisseria gonorrhoeae. Infect Immun 1985;50:672-7. 25. Tsai C, Frasch CEo A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119:115-9. 26. Frasch CE, Zollinger WD. Poolman IT. Serotype antigens of Neisseria meningitidis and a proposed scheme for designation ofserotypes. Rev Infect Dis 1985;7:504-10. 27. Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gel to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112:195-203. 28. Robinson EN1, McGee ZA, Kaplan 1, et al. Ultrastructural localization of specific gonococcal macromolecules with antibody-gold sphere immunological probes. Infect Immun 1984;46:361-6. 29. larvis GA. Griffiss 1M. Human IgAI initiates complement-mediated killing of Neisseria meningitidis. 1 Immunol 1989; 143:1703-9. 30. Ross SC, Densen P. Opsonophagocytosis of Neisseria gonorrhoeae: interaction of local and disseminated isolates with complement and neutrophils. 1 Infect Dis 1985; 151:33-41.

JID 1992; 166 (November)