Antibodies to Meningococcal Class 1 Outer Membrane Proteins in

0 downloads 7 Views 1MB Size Report
Titertek polyvinyl chloride (PVC) flat-bottomed microplates. (Flow) were sensitized as ... Plates were then washed, incubated with conjugated anti- mouse IgG, and .... The initial experiments for the studywere carried out with the anti-P1.2 MAbs ...

Vol. 60, No. 11

INFECrION AND IMMUNITY, Nov. 1992, p. 4510-4516

0019-9567/92/114510-07$02.00/0 Copyright X) 1992, American Society for Microbiology

Antibodies to Meningococcal Class 1 Outer Membrane Proteins in South African Complement-Deficient and Complement-Sufficient Subjects A. ORREN,1* R. E. WARREN,2 P. C. POTrER,3 A. M. JONES,1 P. J. LACHMANN,' AND J. T. POOLMAN4 Molecular Immunopathology Unit, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH,1 and Public Health Laboratories, Addenbrooke's Hospital, Cambridge CB2 2QW,2 United Kingdom; Department of Clinical Science and Immunology, University of Cape Town, Cape Town, South Africa; and Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands4 Received 11 May 1992/Accepted 29 July 1992

Inhibition assays were used to investigate human serum antibodies to the meningococcal class 1 outer membrane proteins. We adapted the whole-cell enzyme-linked immunosorbent assay technique to determine the ability of sera to inhibit the binding of murine subtyping monoclonal antibodies. Serum samples from 33 South African subjects with a deficiency in the sixth component of complement as well as serum samples from various groups of complement-sufficient subjects were investigated. Subjects were subdivided according to whether they were (i) convalescent from Neisseria meningitidis infections, (ii) nonconvalescent, or (iii) controls. Preliminary subtyping investigations had shown that P1.2 was present on 36% of meningococcal clinical isolates from Cape Province, South Africa. Assays with the anti-P1.2 antibodies showed the presence of high antibody levels in many deficient sera and moderately elevated levels in some sera from the complementsufficient convalescent patients. P1.2, P1.4, P1.15, and P1.16 are epitopes situated on loop 4 of the class 1 outer membrane proteins, whereas P1.7 is on loop 1. Inhibition assays showed that human sera that inhibited binding by P1.2 monoclonal antibodies tended to inhibit the other monoclonal antibodies directed to loop 4 epitopes. This suggests that the epitopes recognized by the human antibodies are not exactly the same as the epitopes recognized by the murine monoclonal antibodies and raises the possibility of the importance of other epitopes.

likely components of a meningococcal vaccine (16, 18). The class 1 proteins are porin proteins present on almost all meningococcal isolates (14), and antigenic diversity is limited and allows subtyping classification (2). Cloning and sequencing the genes for the class 1 proteins (3) have led to deduction of the amino acid sequences of four different strains (11, 18). Models for class 1 and other porins predict eight surface-exposed regions or loops, of which loops 1 and 4 are the most accessible in class 1 (18). Differences in the amino acid sequences between subtypes are mostly confined to the two variable regions coding for these loops. Epitope analysis with the relevant murine monoclonal antibodies (MAbs) showed that the anti-P1.7 MAb reacted with peptides from loop 1 of the P1.7 strain and the anti-P1.16 and anti-P1.15 MAbs reacted with peptides from loop 4 of the relevant strains (11). The epitope for an anti-P1.2 MAb has now been ascribed to loop 4 (18). The epitope for P1.4 is also believed to be within loop 4, since many P1.4 strains also express the loop 1 P1.7 epitope. Murine MAbs are tools developed to recognize subtype differences, and their specificities will not necessarily be reflected by human anti-class 1 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies combined with electroblotting (19) have shown the presence of anti-class 1 antibodies in immune sera, but this method does not allow any determination of epitope specificity. To address this problem, Mandrell and Zollinger (10) developed an inhibition enzyme-linked immunosorbent assay (ELISA) and showed that the majority of serum samples from convalescent patients were able to inhibit the binding of appropriate subtyping MAbs to OMP preparations from the patient's own infecting meningococcal strains. Neverthe-

Neisseria meningitidis infections frequently result in lifethreatening meningitis and/or bacteremia. In immunocompetent individuals, the disease seldom occurs more than once. Goldschneider et al. (8) presented convincing evidence of the role of bactericidal antibodies in preventing the development of the disease, although the role of opsonic antibodies in protection against meningococcal disease requires further investigation (6). The situation in individuals who have defects in the complement system, particularly of the terminal complement pathway, is somewhat different. These individuals frequently suffer recurrent infections, and the role that antibodies play both in eliminating the infections and in protecting subjects from further infections is still uncertain. In the Western Cape Province, South Africa, meningococcal infections are endemic, with an incidence of approximately 20 cases per 100,000 population per year, rising to about 100 cases per 100,000 per year in epidemic periods (15). A relatively large number of individuals with a genetic deficiency of the sixth complement component (C6D) have been diagnosed within this area, and many of the affected subjects suffered recurrent meningococcal infections (13). Preliminary data showed that some patients did develop antibodies to meningococcal outer membrane proteins (OMP), but the specificity of these antibodies was not investigated (15). Studies of serum antibodies in American complement-deficient subjects also confirmed the presence of potentially bactericidal and opsonic antibodies (4). Class 1 meningococcal OMP are currently regarded as *

Corresponding author. 4510

VOL. 60, 1992

HUMAN ANTIBODIES TO CLASS 1 MENINGOCOCCAL PROTEINS

TABLE 1. MAbs used, subtyping antigens they recognize, and loop locations of these antigens MAb

Antigen

Loop

MN14C11.6

P1.7

1

MN2B6G MN4C5E MN2OB9.34 MN3C5C

P1.2 P1.2 P1.4

MN5C11G

P1.16

4 4 4 4 4

P1.15

less, inhibition of a murine bactericidal MAb gives limited information, and the critical question is still which antibodies, directed at which epitopes, are biologically relevant? Preliminary epidemiological investigations of Cape meningococcal isolates indicated that class 1 subtype P1.2 was present on 36% of strains and represented the most prevalent subtype recovered from both complement-sufficient and -deficient individuals (12). Of the other antigens studied, P1.15 was found on 15% of isolates, whereas P1.16, P1.7, and P1.4 were rare and found in approximately 2, 2, and 0% of the isolates, respectively. We investigated the human response to natural infection by determining the development of antibodies that would inhibit the binding of the anti-P1.2 loop 4 murine MAbs. Because P1.4 is also situated within loop 4, an anti-P1.4 MAb was used in order to investigate the degree of specificity of the inhibition. The study also included inhibition assays with MAbs directed against P1.15, P1.16, and P1.7. MATERIALS AND METHODS Antibody assays. The antibody assays were a modification of the inhibition ELISA described by Mandrell and Zollinger (10). The anti-class 1 murine MAbs used were produced at the Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands. Table 1 lists the MAbs and the loop locations of the class 1 antigens that they recognize. Titertek polyvinyl chloride (PVC) flat-bottomed microplates (Flow) were sensitized as for the whole-cell ELISA (1). Suspensions (100 ,u/well; optical density at 660 nm [OD660] of 0.05) in phosphate-buffered saline (PBS) of heat-killed (56°C for 45 min) meningococci of appropriate subtypes were added, and plates were left to dry overnight at 37°C. Plates were stored at room temperature, and Tween 80 (0.025%) in tap water was used to wash the plates three times just prior to use and also during the assays. The concentrations of MAbs to be used in the inhibition assays were determined by pretitration. For these titrations, antibodies were diluted with PBS containing 0.5% bovine serum albumin (BSA) and 0.01% Tween 80 (BSA-Tween); 100 pl of each dilution was added to the sensitized wells and incubated for 1 h at 37°C. Plates were washed, and 100 i±l of peroxidase-conjugated anti-mouse immunoglobulin G (IgG; Sigma, St. Louis, Mo.), diluted 1:1,000 in BSA-Tween, was added. After reincubation for 1 h at 37°C, the plates were washed, and color was developed with freshly prepared tetramethylbenzidine (Sigma) (90 ,ug/ml) in 110 ,M sodium acetate (pH 5.5) with 0.025% H202 (30%). The reaction was stopped with 100 pl of 2 M H2SO4, and the OD was read at 450 nm on a Microplate reader (Bio-Rad). Antibody dilutions producing just less than maximum (plateau) antibody binding to the organisms were used as the final MAb concentration for the inhibition assays.

For the inhibition assays, human

serum

samples

were

4511

diluted with BSA-Tween and heat inactivated at 56°C for 30 min. Samples were added, in duplicate, to appropriately sensitized plates to give final serum dilutions of 0.1, 0.05, and 0.025 in a total reaction volume of 100 ,ul. The plates were incubated for 30 min at 37°C prior to addition of the MAb, and the incubation was continued for another 60 min. Plates were then washed, incubated with conjugated antimouse IgG, and processed for color development as described above. The percent inhibition for each sample was calculated from the OD readings minus the appropriate blanks from the following formula: [(OD of wells with monoclonal only OD of wells with sample and MAb)/OD of wells with MAb only] x 100. Results are expressed either as percent inhibition or in units of antibody compared with a serum with high antibody levels. This serum, from subject N, produced high inhibition of binding of all the MAbs except the anti-P1.7 MAb (see Fig. 2), but it was only used as a standard for calculation of human antibody levels from the inhibition of the anti-P1.2 MAb MN2B6G. The standard was arbitrarily ascribed as having 100 U of anti-class 1 antibody, and levels in the other sera were determined relative to this standard serum. A standard curve was set up for every plate, with four duplicate dilutions of the standard serum. The duplicate results for the dilution of the unknown that produced inhibition that fell within the midrange of the standard curve were used for calculating the units of antibody present in the unknown. This calculation automatically adjusted the results for interplate variation and adjusted for the nonlinear inhibition curves at high antibody concentrations (see Fig. 1). A control serum that was obtained from an individual with no history of clinical meningococcal infection and that produced low inhibition was also run on each plate. Subjects. The subjects investigated were divided into eight groups according to their serum complement activity and clinical and epidemiological criteria (Table 2). All C6D subjects were discovered by meningococcal disease in themselves or in an immediate family member. The absence of the sixth complement component was diagnosed by means of a functional hemolytic assay described previously (9, 13). The C6D subjects were subdivided into convalescent, nonconvalescent (no clinical infection within the past year), and never clinically infected (groups 1, 2, and 3, respectively; Table 2). As meningococcal infection tends to occur at a later age in complement-deficient than in normal individuals (13), and as age may be related to immune responsiveness (15), we excluded data for all children who were less than 5 years old. Convalescence was taken as the period from 14 to 65 days after admission for the acute episode. This wide period was used for both the deficient group 1 patients and the sufficient group 5 patients. It was necessary because the deficient subjects were usually referred to the study after discharge from the hospital. Approximately 85% of the C6D subjects and the complement-sufficient group 5 and group 6 subjects were Cape coloreds (of mixed caucasoid, southeast Asian, and southern African [Hottentot] extraction); however, a few were Xhosa-speaking black Africans. The group of control African blood donors (group 7) were all black Africans, and the group of healthy British laboratory workers (group 8) were all caucasoid. For the purposes of the statistical analyses, any individual subject was investigated only once, and any C6D patient for whom we had a convalescent sample was investigated in group 1. Laboratory diagnosis of meningococcal disease was made by cerebrospinal fluid or blood culture and occasionally by microscopy only. The complement-deficient patients were

4512

IN-FECT. IMMUN.

ORREN ET AL. TABLE 2. Criteria used for categorizing subjectsa

Range

Group (no.)

Complement status

Age range (yr)

Meningococcal infection

Comments

anti-class 14 antibody levels (units)

14-65 days postadmission Infection >1 yr previously

7-398 (48) 8-237 (18)

5-58 (mean, 16.7)

Convalescent Previous, nonconvalescent No history of infection Family members of C6D subjects Convalescent

14-65 days postadmission

2-81 (16)

5-32 (mean, 14.7)

Noninfected control

5-15 (8)

>18

Noninfected control

21-41

Noninfected control

Comparable noninfected group Black African blood donors British laboratory workers

1 (19) 2 (9)

C6D C6D

7-31 (mean, 20.1) 14-32 (mean, 20.2)

3 (4) 4 (29)

C6D Complement sufficient Complement sufficient Complement sufficient

7-21 (mean, 14) 7-52

5 (20) 6 (13) 7 (13) 8 (10)

Complement sufficient Complement sufficient

(median) of

13-42 (20) 4-191 (22)

2-83 (20) 8-13 (11)

a The mean ages are shown when the ages of all members of the group were known. The range and median of the anti-class 14 antibody levels are also shown.

treated at several Cape hospitals, and all complementsufficient patients were treated at the Cape Town City Hospital for Infectious Diseases. Serum samples were stored at -80 or -20°C and transported on dry ice. Meningococcal isolates. This study includes results from antibody studies on serum samples taken during convalescence from 40 episodes of meningococcal infection. The complement-deficient patients had nearly all also suffered earlier episodes, but information on the current episode only is included here. The complement-sufficient patients all suffered primary infection. Polysaccharide antigen grouping was performed at the South African Institute of Medical Research for 20 isolates. The results for isolates from 10 complement-deficient subjects were one group A and nine group B, and the results for 10 isolates from the complement-sufficient patients were three group A, three group B, and four group C. Three South African isolates that had been sero- subtyped at the Netherlands Meningococcal Reference Laboratory (B:nt:Pl.2; B:4:P1.15; and B:nt:Pl.16,P1.7) were used to sensitize plates for anti-P1.2, anti-P1.16, anti-P1.7, and antiP1.15 ELISAs, and strain 882066 (B:nt:P1.4), which was received from Bilthoven, was used for the anti-P1.4 assay. Statistical tests. Although results are expressed as units, the level of measurement is only ordinal, and therefore the nonparametric Mann-Whitney U test was used to compare antibody levels (17).

RESULTS The initial experiments for the study were carried out with the anti-P1.2 MAbs MN4C5E and MN2B6G (Table 1) in order to identify sera with anti-class 1 P1.2 antibodies. Forty-eight serum samples representative of subjects in the various groups were screened in this way, and inhibitory activity was found in a number of them. The inhibition patterns produced by three serum samples from one C6D case are illustrated in Fig. 1. The samples were taken 3 weeks, 9 weeks, and 3 years after her second meningococcal infection, and this infection was with a B:nt:P1.2 organism. She was 19 years old at the time of infection. The figure illustrates that the convalescent specimen had strong inhibitory activity for both MAbs, with stronger inhibition for

MN4C5E than for MN2B6G. By 9 weeks postadmission, the inhibitory activity was very much lower and had disappeared 3 years later. Results for the control serum from plates run on different days showed up to 20% inhibition of MAb binding, and therefore up to 20% inhibition was considered as indicating that no relevant antibodies could be detected. The mean (range) inhibition of MN2B6G by the control group 6 sera at concentrations of 0.05 was 10% (5 to 16%). In order to investigate whether the inhibition observed was specific for the binding of the MAbs to P1.2 epitopes, we tested the majority of the 48 serum samples for their ability to inhibit three other MAbs directed at the epitopes P1.4, P1.15, and P1.16 within the loop 4 region of the class 1 OMP and one MAb directed against P1.7, an epitope within the loop 1 region. The results for some of these serum samples, tested at a final serum dilution of 0.05, are shown in Fig. 2. Figure 2A shows the percent inhibition of MAb binding brought about by serum samples from representative group 1 subjects (C6D patients convalescing from meningococcal disease), Fig. 2B shows the results for C6D patients who had either suffered infection more than a year previously (group 2) or had never suffered clinical meningococcal disease (group 3), and Fig. 2C shows graphs for various C6-sufficient individuals, including the group 8 serum used as a negative control in all assays. (Groups are detailed in the legend.) Results for the inhibition of all six MAbs were obtained for the majority of the 48 serum samples, but results are illustrated only for 34 representative sera. The results for the other 14 sera confirm the results illustrated in Fig. 2. With almost all of the positive sera, the highest inhibition was produced in assays with the anti-P1.2 MAb MN4C5E. Inhibition of the other anti-P1.2 antibody, MN2B6G, tended to be very similar to the inhibition of the anti-P1.4 antibody MN2OB9.34, and the correlation coefficient for the results with those two MAbs on the 48 sera at a 0.05 final serum dilution was r2 = 0.83. Human sera capable of inhibiting the anti-P1.2 and anti-P1.4 MAbs also inhibited the anti-P1.16 and anti-P1.15 MAbs, although the degree of inhibition was not as closely correlated. The negative-control result is representative of the group 8 results. The inhibition experiments showed that a number of sera that were able to produce marked inhibition of the MAbs directed against loop 4 epitopes did not significantly inhibit

100

I

-Ak

80

100

A

E

-

0

c

0

4513

HUMAN ANTIBODIES TO CLASS 1 MENINGOCOCCAL PROTEINS

VOL. 60, 1992

U

60 -

7060-

*

40

40 20

20 II

0

A

0.025

0.000

0.050 dilution

I

i

0.075

0.100

B

C

J F H G C6D subjects Group 1

D

MN2B6G/Pl.2 * MN4C5E/P1.2 * MN20B9.34/P1.4

B

i* o_

0C :e

80 -

*

40 0

60

MN3C5C/Pl.15 MN14Cl 1.6/Pl.7 MN5CllG/Pl.16

a

-

20

.0

aRC

L

*

c

100 -

K

E

.

40 -

0

a

Ml

0

N

M2

20 -

P

Q

R

S

U

T

V

C6D subjects Group 2 and Group 3 100 - C

0. 4 0.000

I

I

I

1

0.025

0.050

0.075

0.100

dilution

80

-

c

60

100 C

80-

20

60-

0

c

°0

.0

-

a

c

d

e

f

g

h

i

j

k

1

Complement sufficient subjects

40-

20*

.

0.000

b

0.025

0.050 dilution

0.075

0.100

FIG. 1. Inhibition-of-binding curves for three serum samples taken from one C6D patient. Whole organisms carrying P1.2 class 1 OMP were used to sensitize plates. The MAbs inhibited were the two anti-pl.2 MAbs MN2B6G (0) and MN4C5E (*). The conditions of the assay and the calculation of percent inhibition are described in the text. (A) Serum sample taken 3 weeks after the onset of the patient's second episode of meningococcal infection (the isolate was B:nt:P1.2). (B) Serum samples taken 9 weeks after the onset of the same episode. (C) Serum sample taken 3 years later; there were no intervening episodes of infection.

the anti-P1.7 MAb and that inhibition patterns for the anti-P1.7 MAb do not necessarily follow those of the other MAbs. The correlation coefficient of inhibition of MN2B6G and MN14C11.6 for the 48 sera was only r2 = 0.42. These results led us to the conclusion that investigations of human antibody inhibition of murine MAbs directed against loop 4 epitopes do not determine only antibodies to the subtyping

FIG. 2. Inhibition of binding of anti-class 1 MAbs by the indicated human sera at a 0.05 dilution. (A) Inhibition by sera from group 1 C6D patients convalescent from a recurrent attack of meningococcal disease. All patients were teenaged or adult. (B) Inhibition by sera from other C6D subjects. Six subjects were group 2 nonconvalescent subjects with a history of infection more than 1 year previously (M, N, 0, P, Q, and R); of these, three (M, N, and P) had had recurrent disease. Four were group 3 with no history of meningococcal infection (S, T, U, and V). Results with two samples from a group 2 patient are illustrated (1 and 2); sample 2 was taken 2 years after sample 1, and the patient suffered no interim clinical meningococcal infection. Serum N was used as a standard for the standardized anti-class 1 ELISAs. (C) Inhibition by sera from complement-sufficient subjects. Three subjects were group 4 complement-sufficient family members of C6D subjects (a, b, and c), and seven subjects were group 5 convalescent patients (d, e, f, g, h, i, and j). k, black African control (group 7); 1, British laboratory worker (group 8) control serum. For the results for all individuals illustrated in Fig. 2, the absence of a bar indicates that inhibition of the relevant MAb was not tested with that particular human serum.

epitopes themselves. The situation and possible variability of the epitopes recognized by the human antibodies are not yet defined, and therefore we will refer to the human antibodies that we investigated by the loose term human anti-class 14 antibodies (with the superscript 4 indicating probable loop 4 region antigens).

4514

ORREN ET AL.

INF-ECT. IMMUN.

1000

lescent patients (group 5) and the African blood donor control group (group 7).

0-

U)

c

a) z

:) 0

10 .0 co

o

U 100

*

£

: Jis 8 *

.w £3 0 v *

.

A

*

48 * _ ; ,P , 10 1 2 3 4 6 0 7 8 5 subject groups FIG. 3. Units of anti-class i4 antibodies in eight groups of EF

* ,*

subjects: ggroup 1, complement component 6-deficient, nonconva(n = 20); lescent (n = 9); group 3, complement component 6-deficient, never clinically iinfected (n = 4); group 4, complement-sufficient family members (of complement-deficient subjects (n = 29); group 5, South African co'mplement-sufficient convalescent subjects (n = 20); group 6, South African controls (n = 13); group 7, black African blood donors (n = 13); Group 8, healthy British laboratory workers (n = 10). Range-s and median antibody levels are given in Table 2. Group 1 subjects had the highest antibody levels; these were significantly higher thaini those found in group 4 (P < 0.01), group 5 (P < 0.01), or group 7 (P < 0.01) subjects and highly significantly higher than those in group 6 and group 8 subjects (P < 0.001) (Mann-Whitney U test).

However, group 1 subjects had only marginally higher levels than group 2 (nionconvalescent, deficient) subjects (P < 0.05). Group S subjects h,ad significantly higher levels than either group 6 or group 8 subjects (P < 0.01), but there was no significant difference between tihe levels in group 5 and group 7 subjects. There were only four subje-cts in group 3, and therefore their results were not analyzed.

In orde1r to investigate the levels of anti-class 14 antibodies in serum samples from the various groups of subjects designated ab4ove, we took the inhibition of the anti-P1.2 MAb MN2B6GJ as being representative. We determined the ability of these sera to inhibit MN2B6G, with the assay being standardiized with serum from a C6D patient. The antibody levels ar e expressed in units, calculated as described in Materials and Methods. The standardizing serum, from subject Nr, as well as inhibiting MN2B6G and MN4C5E, was also able to inhibit the anti-P1.15, -P1.16, and -P1.4 MAbs (Fig. 2B). but for this part of the investigations, we studied only inhilbition of MN2B6G. Figure 3 shows the calculated serum anti-class 14 levels in the varioius groups of subjects. Because the units are derived from per cent inhibition and nonparametric statistics apply (17), we Ihave not calculated means and standard deviations, although ranges and medians are included in Table 2. The C6D conivalescent subjects in group 1 had significantly higher leNrels than other subjects. However, there was a wide range of antibody levels in samples from these subjects. Nonconv,alescent group 2 C6D subjects and subjects who were nonideficient members of the C6D families (group 4) had a wkJe range of antibody levels, and certain individuals had highier antibody levels than those occurring in the complemlent-sufficient convalescent group (group 5). The South Afirican control group 6 and the British control group 8 both hiad very low antibody levels. However, elevated levels we re found among the complement-sufficient conva-

DISCUSSION The meningococcus carries a number of cell surface OMP, and many of these show antigenic variations which allow classification of the organisms. Among these are the class 2 and 3 OMP, which carry the serotype specificities, and the class 1 OMP, which carry the subtype specificities. In addition, there are many other cell surface proteins as well as polysaccharide and lipopolysaccharide antigens, and the situation is thus very complex. The majority of studies of antibody levels following meningococcal infection have looked at responses to polysaccharide or OMP preparations produced from organisms of known group and/or sero- or subtype (7, 15). Such studies have not allowed definition of the specificities of the antibodies determined. Although it is known that complement-deficient patients produce antibodies to OMP preparations following infection, very little is known of the biological role of these antibodies. The study of the functional relevance of antibodies in this situation would be greatly facilitated by determination of the antibody specificities.

We commenced the study with investigations of human anti-P1.2 antibodies, and as shown in Fig. 1, the assays measured infection-related antibodies. However, the results inaigr2dieontraeatse antibodies. caples in Fig. 2 demonstrate that sera containing antibodies capable of inhibiting the anti-P1.2 MAbs were also capable of inhibiting MAbs directed against other loop 4 region antigens. Results obtained with the anti-P1.4 MAb MN2OB9.34 showed a particularly strong correlation with the results obtained with the anti-P1.2 MAbs. Subtyping studies of approximately 140 isolates from the Cape have failed to find any carrying the P1.4 antigen (12), and therefore it appears extremely unlikely that the ability of the sera from the Cape subjects to inhibit the anti-P1.4 MAb arises from antibodies induced by exposure to the P1.4 antigen itself. We consider it probable that the close correlation of the inhibition of the MN2B6G and MN2OB9.34 indicates that human antibodies recognize antigens that are spatially close to, but not identical to, the epitopes recognized by these two MAbs. Obviously, the specificities of these human antibodies may differ in different sera, and it may be that some are induced by infections with other organisms, such as nonpathogenic neisseriae. Estabrook and coworkers (5), using similar inhibition assays, were able to define meningococcal lipooligosaccharide epitopes recognized by human immune sera. It may be that murine and human antilipooligosaccharide antibodies have similar specificities or that the epitopes are easier to define on the smaller lipooligosaccharide molecules. Much important information is still needed about the class 14 epitopes recognized by human antisera, including: (i) what number of different epitopes are recognized by human sera, (ii) what are the amino acid compositions and tertiary structures of these epitopes, and (iii) where on the molecule are they situated. The information already available on the class 1 proteins (11, 18) suggests that variations between different class 1 proteins are found on the tips of the loop 1 and loop 4 regions. The greater part of the eight predicted surface loops, including loop 1 and 4, is, however, constant. Nevertheless, our data do present the possibility that the epitopes recognized by the human antibodies are not necessarily directed to the variable sequences themselves. Antibody binding to nearby regions during preincubation with human sera, as performed in our studies, could sterically

VOL. 60, 1992

HUMAN ANTIBODIES TO CLASS 1 MENINGOCOCCAL PROTEINS

hinder the binding of the subtype-specific MAbs. The critical question of whether human antibodies to conserved class 1 OMP epitopes have bactericidal potential still needs to be addressed. P1.7 is located within the loop 1 region, and therefore antibodies that inhibit MAb binding to loop 4 region epitopes are unlikely to inhibit the anti-P1.7 MAb, and this may account for the poor correlation between MN2B6G inhibition and MN14C11.6 inhibition. However, specific antibodies were not purified from the human sera, and whole sera with anti-class 14 antibodies are likely to also contain antibodies directed towards the loop 1 region. We studied anti-class 14 antibodies in a number of sera from C6D and complement-sufficient patients who had suffered meningococcal infection. Figure 3 shows the results of anti-class 1i antibodies in the various groups of subjects. Convalescing C6D patients (group 1) tended to have the highest antibody levels and also had a very wide range of levels. For all except one of the patients, the relevant infection was a recurrent infection, which may explain why levels were significantly (P < 0.01) higher than found in the complement-sufficient convalescing patients (group 5); however, other factors, such as increased time of exposure to the organisms or differences in antigen processing, may play a role. The convalescent complement-sufficient patients (group 5) did have significantly (P < 0.01) raised levels compared with the control subjects (group 6). High levels were found in certain nonconvalescing deficient patients (group 2) and nondeficient immediate-family members (group 4), and this may indicate repeated or recent exposure to antibody-inducing organisms. The inhibition results illustrated in Fig. 2B show that deficient subjects who have already suffered recurrent disease can develop markedly increased antibody activity without the development of clinical disease. The African adult blood donor controls (group 7) had levels that were not statistically different from those of convalescent sufficient patients (group 5). Socioeconomic factors probably led to exposure to organisms that induced the high antibody levels. Unfortunately, we had no serum samples taken prior to the development of infection, but comparing the antibody results for groups 1, 2, and 5 with the low levels found in group 6 strongly suggests that the antibodies were infection induced. The tendency to relapse has been reported for United States terminal complement component-deficient subjects (6), and although the Cape patients did not have overt relapses, some maintained very high antibody levels for several years. This would be most easily explained by continuous or repeated exposure to relevant antigens. The rapid decline in antibodies following recovery of the subject illustrated in Fig. 1 reinforces our concern as to the cause of these high antibody levels. The benzathine penicillin that high-risk susceptible subjects receive as prophylaxis (15) against recurrent clinical disease would not be particularly efficient in eliminating foci of infection from the nasopharynx. It may therefore be wise to use a course of rifampin or ciprofloxacin treatment to eliminate organisms from the subjects and their immediate family. Further investigations are required on a number of aspects of this study. As outlined above, more direct measurements of the specificities of the antibodies are required to determine the important meningococcal epitopes for vaccine production. If the epitopes recognized by human antibodies are indeed less diverse than the subtyping epitopes, this may have important implications for vaccine production. Another question of importance to vaccine production is the duration

4515

of the antibody response. The indications from the data illustrated in Fig. 1 are that high antibody levels are not naturally maintained after recovery, and if the anti-class 14 measured here prove to be protective in normal individuals, it would obviously be beneficial to prolong the response. Unfortunately, we had few such sets of sequential samples, but we hope to extend this section of the study. Further investigations of the role of nonpathogenic neisseriae in inducing protective immunity might also be fruitful. The important question that we have not yet answered in this study is whether the antibodies we detect are either bactericidal or opsonic. Another corollary that we wish to address is the possible role of antibodies of various specificities in protecting individuals with complement deficiency from further meningococcal infection. ACKNOWLEDGMENTS We thank Joan Meadows of Groote Schuur Hospital for assistance with care for the deficient subjects. This work was supported by the Meningitis Trust (UK), The Wellcome Trust, and the South African Medical Research Council. REFERENCES 1. AbdiUlahi, H., and J. T. Poolman. 1987. Typing of group B Neisseria meningitidis with monoclonal antibodies in the whole cell ELISA. FEMS Microbiol. Lett. 48:367-371. 2. Abdillahi, H., and J. T. Poolman. 1988. Definition of meningococcal class 1 OMP subtyping antigens by monoclonal antibodies. FEBS Microbiol. Immunol. 47:139-144. 3. Barlow, A. K., J. E. Heckels, and I. N. Clarke. 1989. The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal proteins. Mol. M.icrobiol. 3:131-139. 4. Densen, P., A. Zaleski, and M. Sanford. 1990. Antibody responses to meningococcal infection in late complement deficient individuals, p. 671-677. In M. Achtman, P. Kohl, C. Marchal, G. Morelli, A. Seilere, and B. Thiesen (ed.), Neisseriae 1990. Walter de Gruyter, Berlin. 5. Estabrook, M. E., R. E. Mandrell, M. A. Apicella, and J. McLeod Griffiss. 1990. Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharides. Infect. Immun. 58:2204-2213. 6. Figueroa, J. E., and P. Densen. 1991. Infectious diseases associated with complement deficiencies. Clin. Microbiol. Rev. 4:359-395. 7. Frasch, C. E., J. M. Zahradnik, L. Y. Wang, L. F. Mocca, and C. M. Tsai. 1988. Antibody response of adults to an aluminum hydroxide-absorbed Neisseria meningitidis serotype 2b proteingroup B polysaccharide vaccine. J. Infect. Dis. 158:710-718. 8. Goldschneider, I., E. Gotschllch, and M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307-1326. 9. Lachmann, P. J., and M. J. Hobart. 1978. Complement technology, p. 5A1-5A23. In D. M. Weir (ed.), Handbook of experimental immunology, 3rd ed. Blackwell Scientific Publications, Oxford. 10. Mandrell, R. E., and W. D. Zollinger. 1989. Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination. Infect. Immun. 57:15901598. 11. McGuiness, B., A. K. Barlow, I. N. Clarke, J. E. Farley, A. Anilionis, J. T. Poolman, and J. E. Heckels. 1990. Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. J. Exp. Med. 171:1871-1882. 12. Orren, A., P. C. Potter, and G. Coetzee. 1990. N. meningitidis infections in South African complement component (C6) deficient patients; studies of methods of prophylaxis and of the organisms isolated, p. 701-706. In M. Achtman, P. Kohl, C. Marchal, G. Morelli, A. Seilere, and B. Thiesen (ed.), Neisser-

4516

ORREN ET AL.

iae 1990. Walter de

Gruyter,

INFECT. IMMUN. Berlin.

13. Orren, A., P. C. Potter, R. Cooper, and E. du Toit. 1987. Deficiency of the sixth component of complement and susceptibility to N. meningitidis infections. Studies in ten families and five isolated cases. Immunology 62:249-253. 14. Poolman, J. T., S. de Marie, and H. C. Zanen. 1980. Variability of low-molecular-weight, heat-modifiable outer membrane proteins of Neisseria meningitidis. Infect. Immun. 30:642-648. 15. Potter, P. C., C. E. Frasch, W. M. J. van der Sande, R. C. Cooper, Y. Patel, and A. Orren. 1990. Prophylaxis against N. meningitidis infections and antibody responses in patients with deficiency of the sixth component of complement. J. Infect. Dis. 161:932-937.

16. Saukkonen, K., H. Abdillahi, J. T. Poolman, and M. Leinonen. 1987. Protective efficacy of monoclonals to class 1 and class 3 outer membrane proteins of Neisseria meningitidis B:15:P1.6 in infant rat infection model: new prospects for vaccine development. Microb. Pathogen. 3:261-267. 17. Siegel, S. 1956. Nonparametric statistics for the behavioral sciences, p. 18-34, 116-127. McGraw-Hill, New York. 18. Van der Ley, P., J. E. Heckels, M. Virji, P. Hoogerhout, and J. T. Poolman. 1991. Topology of outer membrane porins in pathogenic Neisseria spp. Infect. Immun. 59:2963-2971. 19. Wedege, E., and L. 0. Froholm. 1986. Human antibody response to a group B serotype 2a meningococcal vaccine determined by immunoblotting. Infect. Immun. 51:571-578.

Suggest Documents