FEMS Microbiology Letters 18 (1983) 189-195
Published by Elsevier Science Publishers
189
Antibody response against outer membrane components of
Haemophilus influenzae type b strains in patients with meningitis
Loek van Alphen, Tineke Riemens and H.C. Zanen
Laboratorium voor de Gezondheidsleer, University of Amsterdam, Mauritskade 57, NL- 1092 AD Amsterdam, The Netherlands
Received 15 December 1982
Accepted 17 January 1983
1. I N T R O D U C T I O N
Haemophilus influenzae type b is the major cause
of bacterial meningitis in children between 6
months and 4 years of age [1,2]. Since the number
of strains which are resistant to antimicrobial
agents is increasing [3] and since patients run a
serious risk of obtaining neurological sequelae [4],
a vaccine against H. influenzae type b is desired.
The available capsular polysaccharide vaccine is
incapable of eliciting protective antibody levels in
children, who in fact run the highest risk for
meningitis [5]. Therefore, increasing attention is
payed to the immunogenic potential of other
surface-exposed bacterial antigens, especially outer
m e m b r a n e proteins and lipopolysaccharides.
Serum antibodies against these antigens can protect animals from experimental infection with H.
influenzae [6-8] and facilitate opsonisation [9] and
complement mediated bacteriolysis of H. influenzae type b strains [8-10] in vitro. Moreover, monoclonal antibodies against an outer membrane protein of M r 39000 are bactericidal in vitro and
protective in rats infected with the homologous H.
influenzae type b strain [11,12].
Recently, Gulik et al. [13] reported that m a n y
outer membrane proteins of H. influenzae type b
cells were immunoprecipitated by sera from patients with systemic diseases and by sera from
infant and adult rats [14,15]. Since the immunoprecipitation method has the disadvantage
that various outer membrane components may
coprecipitate with outer membrane antigen to
which specific antibodies are bound due to interactions between these components [16], we used
the gel-immuno-radio-assay (GIRA), developed in
our laboratory [17]. In this method the outer membrane components are separated before they are
allowed to react with antibodies. In this paper we
want to demonstrate that the majority of the acute
and convalescent phase sera of patients reacted
with various components and that the antibodies
in the acute phase sera are likely derived from
previous contacts with related antigens.
This work was presented at the Fourth International Conference on Immunity and Immunization
in Cerebrospinal Meningitis, Siena, Italy, November 1981.
2. M A T E R I A L S A N D M E T H O D S
2.1. Bacterial strains and growth conditions
Ten strains of H. infiuenzae isolated from the
cerebrospinal fluid of patients with meningitis were
sent to us by the bacteriological laboratories in
The Netherlands. These strains had a type b capsular polysaccharide. This was determined with
coagglutination using Staphylococcus aureus cells
coated with type-specific antisera as described before [18]. H. influenzae b strains with the various
0378/1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies
190
outer membrane subtypes occurring in The
Netherlands that were used for reference are: strain
760705 (subtype 1), 770233 (subtype la), 810297
(subtype lb), 770235 (subtype 2) and 770277 I
(subtype 3) [27] and the non-typable strains 1481,
509 and 1439 isolated from sputum cultures.
Cells were stored at - 7 0 ° C in proteose peptone (Difco) supplemented with 15% glycerol and
cultured overnight under vigorous shaking in Brain
heart infusion broth (Difco) supplemented with 10
/~g/ml N A D and 10/zg/ml haemine.
2.2. Cell envelopes and cell envelope components
Cell envelopes were obtained after ultrasonic
desintegration of cells by differential centrifugation as described before [19], except that EDTA
was omitted from the buffers. Lipopolysaccharide
was isolated according to the hot phenol/water
extraction method developed by Westphal et al.
[20] and purified type b capsular polysaccharide
was used for reference purposes (gift of Dr. R.
Tiesjema, Rijksinstituut voor de Volksgezondheid,
Bilthoven, The Netherlands).
2.3. Sera of patients and controls
Acute phase sera from the children with
meningitis were obtained within 6 days after
hospitalization started and convalescent phase sera
after 14-30 days. Sera of 52 children between 0
and 48 months of age without any known episode
of H. influenzae were provided by Dr. K.W.
Slaterus (Department of Virology, Academic
Hospital, Amsterdam).
For the adsorption of sera, cells of H. influenzae
were harvested from liquid culture in Brain heart
infusion broth (see above) by centrifugation (10
rain at 10000 x g) and suspended in the sera in
1 : 1 (v/v) ratio of packed cells over serum. The
suspension was incubated for 2 h at 37°C and the
(adsorbed) sera were recovered by ultracentrifugation (30 min at 100000 × g) as being the supernatant.
2.4. Analysis of the antibodies against cell envelope
components of H. influenzae (Gel-immuno-radio-assay (GIRA))
Basically, the method as described by Poolman
and Zanen was used [17a]. Cell envelopes were
solubilized in sample buffer (0.0625 M Tris-HCl
(pH 6.8), 2% (w/v) sodium dodecyl sulphate (SDS,
Serva art. 20760), 5% (v/v) 2-mercaptoethanol,
10% (v/v) glycerol, 0.01% (w/v) bromphenol blue),
heated for 10 min at 100°C and applied to a 3-ram
10% SDS polyacrylamide gel. The electrophoresis
was performed as described by Lugtenberg et al.
[19] modified in that a constant current of 50 mA
was used and that the length of the running gel
was 5 cm. Subsequently, the gels were mounted on
holders and frozen with liquid nitrogen; they were
cut longitudinally at - 28°C into 50/~m slices with
a Bright 5030 microtome (Bright Instruments
Comp., U.K.) as described before [17]. The remaining part of the gel (about 1 mm) was stained
for (lipo)polysaccharides with AgNO 3 [21] and
after that for proteins with Coomassie Brilliant
Blue [22]. After storage of the slices overnight in
ethanol/acetic acid, 6/1 (v/v) they were preincubated with phosphate buffered saline (PBS), pH
7.4, supplemented with 0.05% Tween-80 and 3%
bovine serum albumin (essentially globulin-free
BSA) for 30 min at room temperature. After rinsing with H 2 0 the slices were incubated with sera
of patients (1 : 3 diluted in PBS (pH 7.4), supplemented with 0.5% BSA, 0.1% Tween-80 and 0.1%
Nonidet P40 (NP 40) for 90 min at 37°C, followed
by washing with PBS (pH 7.4), 0.05% Tween-80
(3-times 20 min)). Bound antibodies were detected
by subsequent incubation with [12sI]protein A (106
dpm/slice of 5 x 5 cm 2) in PBS (pH 7.4), 0.5%
BSA, 0.1% Tween-80, 0.1% NP 40 for 1½ h at
room temperature, followed by extensive washings
(at least 5-times 20 min) in PBS (pH 7.4), 0.1%
Tween-80, 0.1% NP 40 and autoradiography as
described previously [17]. Protein A from S. aureus
(Sigma) was labelled by the chloramine T method
[23].
3. RESULTS
3.1. Resolution of outer membrane proteins, lipopolysaccharides and capsular polysaccharide by
SDS-polyacrylamide gel electrophoresis
The cell envelope protein patterns of the H.
191
influenzae
Fig. 1. Gel-immuno-radio-assay of serum antibodies of
meningitis patient 800201 against cell envelope components of
the homologous H. influenzae type b strain: (A) Coomassie
Brilliant Blue staining of proteins; (B) silvernitrate staining of
isolated lipopolysaccharide; (C) of polysaccharide (20 ~g per
slot); (D) autoradiogram of slices of this SDS-polyacrylamide
gel after incubation with sera of the patient obtained at day 1,
4, 20 and 29 after hospitalization, as indicated and subsequent
labelling of the bound antibodies with [L25I]protein A. Mr-value
markers and the indication for the major outer membrane
,proteins are positioned on the left.
PAG E
type b strains isolated from the
cerebrospinal fluid of the patients with meningitis
resembled each other closely. This is illustrated for
6 strains in Figs. 1 and 2. The major outer membrane proteins a ( M r 47000), c ( M r 40000), d ( M r
37000) for strains 801385, 810029I, 810068 and
810297 (Fig. 2) and M r 36500 for strains 800201
(Fig. 1) and 800821 (Fig. 2), e ( M r 30000) and k
( M r 58 000) dominated. (The nomenclature of the
proteins is adapted from Loeb et al. [24] and L.
van Alphen et al. [27].)
This is characteristic for the majority of the H.
infiuenzae type b strains isolated from patients in
The Netherlands [27]. The pattern of the minor
proteins was more variable, especially in the M r
100 000-150 000 region in the gel.
Silvernitrate staining of these SDS-gels showed
broad bands in the upper and lower part of the
running gel. The lower band was also observed
when purified lipopolysaccharide was applied to
the gel and the upper band had a Rv-value corre-
GIRA
PATIENT: 8 0 1 3 8 5
DAY:
3
12
810029
810068
800821
810297
0
28
18
12
17
Fig. 2. Antibodies in acute and convalescent phase sera of 5 meningitis patients on slices of SDS-gels with cell envelopes of the
homologous strains, as indicated by the patient numbers. Left: protein staining of the gel (PAGE). Right: G I R A of acute and
convalescent phase sera obtained on the days after hospitalization as indicated for each of the 5 patients. In the case of patient 810297
cell envelopes of the homologous strain are indicated by an arrow. Cell envelopes of the heterologous strain 800201 are included in
order to show reaction of the serum with a protein band of M r 130000 which is absent in the homologous strain (*). Abbreviations:
LPS, lipopolysaccharide; PS, polysaccharide capsule.
192
sponding to that of the type b capsular polysaccharide (Fig. 1).
3.2. Antibody response in patients against individual
cell envelope components
The results of the antibodies in 4 subsequent
sera of a 6-month-old child with meningitis are
shown in Fig. 1. In the acute phase serum (day 1)
antibodies were not found against cell envelope
components of the H. influenzae type b strain
isolated from that patient. Low levels of antibodies
appeared on day 4, and on days 20 and 29 strong
reactions were observed in the G I R A with proteins
with Mr-values of 30000, 37000, 41000, 47000,
58000 and 130000 and with lipopolysaccharide.
The M r 37 000 and 47 000 bands were heat-modifiable, since the Mr-values of these bands in the
G I R A were reduced to M r 34000 and 38000,
respectively when cell envelope preparations were
solubilized in sample buffer without heating before they were applied to the SDS-polyacrylamide
gel. They are therefore most likely outer mere-
Table 1
Antibodies in sera of patients with meningitis caused by H.
influenzae type b against cell envelope components in the
GIRA
lmmunogenic cell
envelope component
N u m b e r of
patients with
antibodies in
convalescence a
Major outer m e m b r a n e proteins
a ( M r 47000)
10/10
c ( M r 40000)
-/10
d ( M r 37000)
8/10
e ( M r 30000)
8/10
k ( M r 58000)
8/10
Minor proteins
M r 34000
3/10
M r 60000
2/10
M r 82000
2/10
M~ 105000
10/10
Lipopolysaccharide
10/10
Polysaccharide
7/I 0
N u m b e r with
antibody
response
5/10
-/10
2/10
2/10
3/10
/10
-~10
-/10
2/10
3/10
1/ 10
" Acute phase sera were obtained within 6 days after hospitalization. Patients were between 6 months and 4 years of age
brane proteins d and a, respectively (E. van Alphen, T. Riemens and H.C. Zanen, Abstract XIIIth
ASM meeting, Boston, USA 1982). The band with
M r 30000 is most likely protein e, since it is
insensitive to digestion by trypsin and the band
with M r 58000 is protein k, since the sera also
reacted with Triton-X 100/MgC12 extracts containing proteins a and k only (L. van Alphen et al.,
Abstract X I I I t h International Congress of Microbiology, Boston, USA 1982). Antibodies directed
against minor proteins with M r 41 000 and 130000
were detected. According to its Mr-value the band
with M r 41 000 may be protein b [24]. The sera of
the patient also reacted with the cell envelope
components of the various reference strains with
different outer membrane protein patterns, indicating that the antigens are strongly cross-reactive.
Moreover, the antibodies of the serum isolated on
day 29 which reacted with cell envelope components in the G I R A could be adsorbed with cells of
the reference strain 760705.
Similar reactions in the G I R A were observed
with subsequent sera of another patient. The sera
of 8 patients already included antibodies against
cell envelope proteins, lipopolysaccharide and
polysaccharide in the acute phase of the disease.
Fig. 2 shows the results for 5 of these patients. The
specificity of the sera varied from patient to patient, but antibodies against proteins d, e and a
minor protein of M r 105 000 were found in most of
them (Fig. 2 and Table 1). Increases of the amount
of antibodies were observed in 4 patients, especially with respect to protein a (Fig. 2, indicated
by the arrow). These results are summarized in
Table 1.
The serum of patient 810297 had high levels of
antibodies against many proteins, lipopolysaccharide and polysaccharide. The serum also reacted with a minor protein of M r 130000 which
did not occur in the strain isolated from the
cerebrospinal fluid of that patient. Adsorption of
both acute and convalescent phase sera of patients
810068 and 810297 with cells of the homologous
strains resulted in the disappearance of the reactions in the G I R A with the cell envelope components of the homologous and heterologous strains,
except for the band of M r 130 000, which was only
observed in the reaction of the sera of patient
193
PAGE
a
b
1 2 3 4 5 6 7 8
68
o
TYPE" h
b b
CE
b
b NT NT N T
AGE:
CP
CE
(months)
d
AGE:
2
6
e
3
g
f
22
IO
15
48
Fig. 3. Antibodies in sera of 7 control children (0-4 years) against cell envelopes of H. influenzae type b reference strains 760705
(subtype I, lane 1), 770233 (subtype la, lane 2), 770235 (subtype 2, lane 3), 7702771 (subtype 3, lane 3) and the acapsular strains 1481
(lane 6), 509 (lane 7) and 1439 (lane 8). The cytoplasm fraction of strain 760705 is included in lane 5. PAGE, protein staining of the
SDS-gel used for the GIRA. Child a, 801784 (6 months); b, 803722 (3 months); c, 805234 (10 months); d, 802507 (2 months); e,
803318 (22 months); f, 804003 (15 months) and g, 802853 (48 months).
810297 with the heterologous strain 800201 (cf.
Fig. 2). Therefore, it is likely that the preexisting
antibodies were raised against a strain different
from that isolated from the cerebrospinal fluid of
patient 810297. Moreover, adsorption of sera of
patient 810297 with the acapsular strain 1481 resulted in disappearance of the G I R A reactions
except with protein a, indicating that the antibodies in patients' sera might have arisen against a
variety of H. influenzae strains.
3.3. Antibodies in controls
In order to investigate whether antibodies
against cell envelope components of H. influenzae
generally occur in sera of children, we analysed the
serum antibodies of 52 children between 0 and 4
years of age, who did not pass any known H.
influenzae episode. Cell envelopes of the reference
H. influenzae type b strains from cerebrospinal
fluid and 3 acapsular strains from sputum were
used as antigen in the G I R A for these sera. The
results for 7 of them are shown in Fig. 3. In the
sera of 3 / 5 2 children antibodies against cell envelopes were not detected. The other sera contained different amounts of antibodies against
various components. Antibodies against protein a
were found in 20, protein c in 9, protein d in 49,
protein e in 33, protein k in 29, a minor protein of
M r 90000 in 33 and against lipopolysaccharide in
22 sera. Antibodies against protein a were most
frequently found in children younger than 6
months (12/18, compared with 8 / 3 4 in children
194
from 6 months-4 years; the difference is statistically significant ( p < 0 . 0 1 ) ) . Sometimes rather
specific reactions with one or a few protein bands
were observed. In other sera reactions occurred
only with proteins of H. influenzae type b strains
and not with the unencapsulated strains or vice
versa (see the serum reaction of the 2-month old
child in Fig. 3).
Adsorption of the sera of two children with
strains 1481 and 760705 resulted in the disappearance of almost all of the reactions in the
GIRA, indicating that the antibodies in the
children of the control group are directed against
highly cross-reactive antigenic determinants on the
various membrane proteins of H. influenzae.
4. DISCUSSION
Children who suffered from meningitis caused
by H. influenzae type b elicited antibodies against
outer membrane proteins a, d, e, k and a minor
protein of M~. 105000 and against lipopolysaccharide (Figs. 1 and 2, Table 1), indicating that all
the major outer membrane proteins except protein
c and a particular minor protein are the best
immunogens in patients as tested with the GIRA.
The antibodies are mainly directed against
surface-exposed determinants of the proteins, since
they can be adsorbed with viable cells of the
homologous (and heterologous) H. influenzae type
b strains. Protein c might be a poor immunogen or
might be denatured during the procedure. Evidence for the latter possibility was obtained for
the pore proteins of Neisseria meningitis, which are
only strongly immunogenic in their (native) multimeric state [17a]. Gulik et al. [13] also reported
that various outer membrane proteins were inamunogenic in man. Although comparison of our
results with those of Oulik is difficult because of
expected differences in strains [25] and since characteristics of the proteins of the strains examined
by Gulik are lacking, it is likely that the major
proteins with M r 45000 and 39000 correspond
with proteins a and c, respectively. In that case it
can be concluded that Gulik et al. [13] found
antibodies against other proteins than we did. This
can partly be explained by differences in membrane isolation procedures, since LiC1-EDTA extracts of cells as source for outer membrane
material lack protein d (L. van Alphen et al.,
abstract XIII International Congress on Microbiology, Boston, USA 1982) or by differences in the
methods of identifying antibodies against membrane proteins in patients' sera (immunoprecipitation vs. GIRA). Eight out of ten children already
had antibodies in their acute phase sera against
various cell surface-exposed components of the
cell envelope. Similar findings were obtained by
Gulik et al. [13]. In contrast to this we found that
the antibodies were most likely directed against
surface-exposed antigenic determinants. The antibodies may be elicited during previous exposure of
the children to H. influenzae antigens or cross-reactive antigens in other bacteria, or alternatively
they may have arisen during the immune response
to the bacterium causing meningitis before
hospitalization. The antibody specificity of the
sera of patient 810297 does not agree with the
latter idea, since both the acute and convalescent
phase sera included antibodies against a protein
which did not occur in the strain isolated from the
patient (Fig. 2). Moreover, the control group had
antibodies against many components of the cell
envelope of a variety of strains (Fig. 3 and Table 1).
Since several investigators reported that antibodies
against outer membrane proteins and lipopolysaccharides of 14. infiuenzae can enhance bactericidal
and opsonic activity of serum against these bacteria
and can experimentally protect infected rats from
meningitis by H. influenzae type b [6-10], we
concluded that the preexisting antibodies detected
with the GIRA are insufficient to protect a child
against meningitis and that in general the antibody
specificity of sera of patients is similar to that of
the control group.
After preparation of this manuscript we became
aware of a paper by M. Loeb and D. Smith [26],
who also used the GIRA method for the analysis
of antibodies in patients. They detected antibodies
against especially outer membrane proteins a, d
and e in convalescent phase sera of patients, which
is in agreement with our results.
195
ACKNOWLEDGEMENTS
T h e gift o f s e r a b y D r . K . W . S l a t e r u s a n d t h e
bacteriologists in The Netherlands, and the gift of
t y p e b c a p s u l a r p o l y s a c c h a r i d e b y D r . R. T i e s j e m a
are gratefully acknowledged. The critical and
s t i m u l a t i n g d i s c u s s i o n b y D r . J.T. P o o l m a n is
strongly appreciated.
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