JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1988, p. 1543-1548
Copyright C 1988, American Society for Microbiology
Antibody Responses to Serogroup B Meningococcal Outer
Membrane Antigens after Vaccination and Infection
EINAR ROSENQVIST,1* STIG HARTHUG,2 L. ODDVAR FR0HOLM,' E. ARNE H0IBY,1 KJELL B0VRE,3
ANDWENDELL D. ZOLLINGER4
National Institute ofPublic Healthl and Norwegian Defence Microbiological Laboratory,2 N-0462 Oslo 4, and
Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Rikshospitalet, N-0027 Oslo j,3 Norway,
and Walter Reed Army Institute ofResearch, Washington, D.C. 200124
Received 4 February 1988/Accepted 12 May 1988
Antibody responses of adult volunteers given a vaccine containing meningococcal capsular polysaccharides
(serogroups A, C, Y, and W-135) noncovalently complexed with serotype 2b:P1.2 and 15:P1.16 outer
membrane proteins have been studied. Sera were analyzed by enzyme-linked immunosorbent assay methods
for immunoglobulin G (IgG), IgM, and IgA antibodies and for bactericidal activities against the homologous
strains. The vaccination was performed as a double-blind experiment with 47 volunteers, of whom 23 received
the protein-polysaccharide vaccine and 24 received the control preparation containing the polysaccharides
only. Ten additional persons volunteered for the protein-polysaccharide vaccine. Before vaccination, carriers
of meningococci had significantly higher levels of specific IgG and IgA and also higher bactericidal activities
than noncarriers. At 2 weeks postvaccination we found significant IgG and bactericidal antibody responses
against both the 2b:P1.2 and 15:P1.16 strains in about 70% of the protein-polysaccharide vaccinees. The
immune response induced by disease was compared with that induced by vaccination by analyzing paired sera
from 13 survivors of serogroup B serotype 15 meningococcal disease. We found that the mean specific IgG level
in acute-phase sera was lower than average in prevaccination sera from the vaccinees but similar to that of
healthy noncarriers before vaccination. The convalescent-phase sera showed IgG responses similar to those of
the vaccinees, but the IgM response to disease was significantly higher than after vaccination. The immune
response to disease caused by serogroup B serotype 15 meningococci was found by enzyme-linked immuno-
sorbent assay analysis to be about the same with outer-membrane antigens from a serotype 2b strain as it was
with antigens from a serotype 15 strain.
The incidence of meningococcal disease (MCd) has re-
mained at epidemic levels in parts of Norway for over 10
years. In the country as a whole, the incidence has varied
between 5.6 and 9.0 cases per 100,000 people per year, with
the highest incidence (26.3) in northern Norway in 1975 (1,
2). There have been no signs of decreasing incidence of
disease in the country as a whole, and the need for a
caused by serogroup B organisms, most often those carrying
the serotype:subtype combinations 15:P1.16, NT:P1.16, or
15: - (10). At the moment, no vaccine against group B
meningococcal disease is available, but immunization trials
in animals and humans with outer membrane proteins from
serotypes 2a and 2b complexed with capsular polysaccha-
rides have provided encouraging results (3, 4, 8, 19, 21, 23).
We present results from a clinical vaccination trial in
Norway where human volunteers have been immunized with
a new vaccine against serogroup B meningococci, and we
compare the serological responses of the vaccinees with
those of MCd patients. The vaccine was composed of outer
membrane proteins from both serotype 15:P1.16 and sero-
type 2b:P1.2 meningococci, noncovalently complexed with
A, C, Y, and W-135 capsular polysaccharides. The lipopoly-
saccharide content in this vaccine was very low compared
with that in other experimental vaccines against group B
is obvious. The epidemic
MATERIALS AND METHODS
Vaccinees. A total of47 Norwegian soldiers (43 male and 4
female) volunteered for the vaccination trial. Of this total, 23
received one injection of the protein-polysaccharide vac-
cine, and 24 received the corresponding polysaccharide
control preparation in a double-blind manner. In addition,
five medical students and five laboratory personnel volun-
teered for the vaccine and were included in the study. Nose
and throat specimens were collected from all the vaccinees
weekly to detect carriage of meningococci and related bac-
teria. This sampling started 1 week before vaccination and
ended 3 weeks after. A carrier is defined here as a person
from whom meningococci ofany serogroup or serotype were
isolated during this period. The serum samples studied were
collected just before and 16 days and 6 weeks after vaccina-
Patients. Paired serum samples from 13 patients surviving
serogroup B MCd were analyzed along with the vaccinee
serum. The meningococci isolated from 10 of the patients
were classified as serotype B:15:P1.16; the remaining organ-
isms were serotypes B:15:P1.15, B:15:-, and B:NT:P1.16.
The first set of serum samples was drawn during the first 2
days after hospitalization, and the second set of samples was
drawn 1 week to 2 months later (average, 22.5 days).
Vaccine. Meningococcal capsular polysaccharides were
supplied from Connaught Laboratories, Swiftwater, Pa., as
the commercially available vaccine meningococcal polysac-
charide vaccine, groups A, C, Y, and W-135 combined
(ACYW polysaccharide vaccine). Multiple vials of vaccine
were reconstituted, pooled, and used as a bulk product in the
preparation of the experimental vaccine. Members of the
Vol. 26, No. 8
ROSENQVIST ET AL.
67 K __
30 K _i
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electropho-
resis analyses of the outer-membrane preparations used as antigens
in ELISA. Lanes:
weights; b, 15:P1.16 (strain 44/76);
protein-polysaccharide vaccine. Molecular standards in kilodaltons
(K) are indicated on the left; serotypes and subtypes are indicated
on the right.
a, standard proteins with known molecular
c, 2b:P1.2 (strain 8047); d,
control group received 50 ,ug of each polysaccharide. Outer-
membrane proteins from the meningococcal strains 8047 (B:
2b:P1.2) and 44/76 (B:15:P1.16) were prepared and purified
by use ofthe polar-ion detergent Empigen BB (19). One dose
ofthe vaccine contained 52 ,ug each of serotypes 2b:P1.2 and
15:P1.16 outer membrane proteins, 40 ,ug each of the four
nucleic acid. In addition, the vaccine contained 4.5 mg of
lactose per dose and was reconstituted in 0.85% saline.
About 50% of the polysaccharide was bound to protein as
estimated by the elution profile on Sepharose CL-4B com-
pared with that of the pure polysaccharide mixture.
ELISA antigens. To prepare antigens for enzyme-linked
immunosorbent assay (ELISA), cultures of Neisseria men-
p.g of lipopolysaccharide, and 1.2 ,ug of
ingitidis (strains 8047 and 44/76) were grown overnight at
33°C in 1.4 liters of dialyzed tryptic soy broth (Difco Labo-
ratories) in 2.8-liter Fernbach flasks on a rotatory shaker at
120 rpm. Outer-membrane vesicles were prepared by extrac-
tion of the wet cell pellet for 2 h at 45°C with 5 ml of 0.2 M
lithium chloride-0.1 M sodium acetate buffer (pH 5.8) per g
of cells (5, 12). The protein concentrations were determined
by the Lowry technique, and sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis analyses ofthe products were
performed. The carbohydrate and lipid contents were ana-
lyzed by gas-liquid chromatography.
ELISA technique. ELISA analyses were performed in
triplicate on microdilution plates as described previously
human immunoglobulin G (IgG), IgM, and IgA antibodies.
As an internal antibody standard, a twofold dilution series of
a positive postvaccination serum was used in all experi-
ments. The mean value of the observed optical density was
transformed to arbitrary units per milliliter by a sigmoidal
standard curve (logit-log transformation) calculated from the
values of the reference serum (14). Initially,
samples were analyzed in a 1:200 dilution. Samples with
optical density values of .0.9 maximum optical density were
further diluted and reanalyzed. The amounts of IgG, IgM,
and IgA specific against the two antigens in the positive
reference serum were later analyzed by solid-phase radioim-
munoassay (20). This gave IgG:IgM:IgA ratios of 20:1:1 and
12.5:1.5:1 for the 15:P1.16 and 2b:P1.2 antigens, respec-
tively. The IgG, IgM, and IgA values obtained by ELISA
were scaled according to these results. One scaled unit
corresponds approximately to 1 ,ug of specific antibody per
Bactericidal assay. The bactericidal activities of the vacci-
nation sera were analyzed as described by Frasch and
Robbins (7) in duplicate against the two vaccine strains. A
normal blood donor with very low levels of meningococcal
antibodies, as determined by ELISA, and no bactericidal
activity against the strains investigated served as a comple-
TABLE 1. ELISA antibody level of vaccinees and patients as a function of time after vaccination or onset of meningococcal disease
Antibody levels in':
"For vaccinees, sera 1, 2, and 3 were taken 0, 2 and 6 weeks after vaccination, respectively. For patients, serum 1 was taken between days 0 and 2 and serum
2 was taken between days 9 and 65 (average, 23 days) after hospitalization.
bPolysacc., persons vaccinated with ACYW polysaccharide vaccine (n=23); polysacc-prot., persons vaccinated with polysaccharide-protein vaccine
ACYW2bl5-2 (n= 33); and patients, persons with group B serotype 15 meningococcal disease (n = 13).
rGeometric mean units per milliliter. One unit corresponds to approximately 1 ,ug of specific antibody. Ranges are means + one standard error of the mean.
J. CLIN. MICROBIOL.
ANTIBODY RESPONSES TO MENINGOCOCCAL ANTIGENS
TABLE 2. Geometric mean bactericidal titers against two
different serogroup B N. meningitidis strains as a
function of time after vaccination
Geometric mean titer at:
aPolysacc., ACYW polysaccharide vaccine; polysacc-protein, polysaccha-
ride-protein vaccine ACYW2b15-2.
ment source. Titers are given as the highest serum dilution
resulting in >50% killing of the inoculum.
Statistical methods. The statistical analyses of the data
were performed by use of the program package SPSSIPC+
(SPSS Inc., Chicago, Ill.). The ELISA results were trans-
formed to logarithmic values to calculate the geometrical
means. This gave an approximately normal distribution of
the data and allowed us to use standard statistical tests. The
significance levels of differences between groups were ex-
amined with the Student t test on the log-transformed data.
Antigen analyses. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis analyses of the ELISA antigens are
shown in Fig. 1. In strain 44/76, the class 1 (serosubtype
P1.16) and class 3 (serotype 15) outer membrane proteins
predominated, with only small amounts of class 5 protein.
The antigen from strain 8047 contained mainly class 1
(serosubtype P1.2) and class 2 (serotype 2b) proteins but also
a greater amount of class 5 protein. The relative amount of
class 5 protein in the ELISA antigen from strain 8047 was
higher than in the vaccine (Fig. 1). The ratios of lipopoly-
saccharide to protein in the ELISA antigens were about 1:1,
and only traces of sialic acid could be detected by gas-liquid
chromatography. The vaccine contained the same major
proteins as the ELISA antigens, but in addition, some weak
bands corresponding to proteins with molecular sizes of
about 60,000 to 70,000 daltons were seen.
Prevaccination and early-phase sera from patients. Tables 1
and 2 show the levels of antibodies against the antigens 2b:
P1.2 and 15:P1.16 in the groups examined. The observed
differences in prevaccination sera between the polysaccha-
ride-vaccinated control group and the protein-polysaccha-
ride vaccinees were not statistically significant (Student's t
TABLE 3. Influence of meningococcal carriage on ELISA
antibody levels before and after vaccination
ELISA antibody level for antigen (time):
aSee Table 1, footnote c.
bOnly the 33 subjects who received the polysaccharide-protein vaccine
were studied. +, Carriers ofN. meningitidis (n=14); -, noncarriers (n=19).
Amnong the 47 military recruits in the trial, we found that
25 carried meningococci, whereas only 2 of the 10 other
volunteers were carriers. The carriers were evenly distrib-
uted between the vaccine and the control groups. When
carriers were compared with noncarriers (Table 3), we found
significantly higher values for IgG against both serotype 15
and serotype 2b antigens in the carriers (P = 0.007 and 0.009;
Student's t tests). This also applied to bactericidal antibodies
(P = 0.006 and 0.01) (Table 4). A similar difference was not
demonstrated for IgM and IgA.
The acute-phase serum samples from the meningococcal
patients showed lower levels of IgG specific against both
antigens than the samples from vaccinees who were carriers
(Tables 1 and 3), and their geometric mean IgG levels were
close to those of the noncarriers. For IgM and IgA, the
differences between patients and the carriers were small.
Postvaccination and convalescent-phase sera. Two weeks
after vaccination, a significant increase in mean IgG antibod-
ies to both ELISA outer-membrane vesicle antigens, as well
as in bactericidal activity against the corresponding men-
ingococcal strains, was found in the group given the poly-
saccharide-protein vaccine (Tables 1 and 2). With both
strains, at least a doubling in bactericidal titer was shown in
about 70% (23 of 33) of the vaccinees. In ELISA studies,
82% (27 of 33) showed at least a doubling of IgG antibodies
with the 2b:P1.2 antigen, and 70% (23 of 33) showed a similar
result with the 15:P1.16 antigen. For those who received the
vaccine, the mean fold rise of IgG was about 11 times with
both the 15:P1.16 and 2b:P1.2 antigens. The IgA levels also
increased significantly (3 to 4 times), whereas the IgM
response proved poor. In the control group, which was given
only the ACYW polysaccharides, a slight increase in mean
antibody activities was observed, but the differences be-
tween week 6 and week O were not statistically significant.
The increase in antibody levels after protein-polysaccha-
ride vaccination was substantially higher among carriers
than noncarriers. Two weeks after vaccination, the mean
IgG level in the serum samples taken from the vaccinated
carriers was about two to three times that found in the serum
taken from the noncarriers (Table 3). The mean IgG level in
the serum taken from vaccinated noncarriers was then found
to be about twice that of the serum taken from the carriers
before vaccination, and it was four to six times higherthan in
the acute-phase sera from the patients (Tables 1 and 3).
Apparently, the vaccination had no effect on the carrier
state. Table 5 shows the importance of preimmunization
antibodies for bactericidal activity. The increase in bacteri-
cidal titer after vaccination was substantially higher for the
group with demonstrable antibodies before vaccination than
for the group without such antibodies. The mean titer ratios
were, however, about the same for the two groups. Of the 57
healthy adults in this study, 32 (56%) had no measurable
bactericidal activity against the two vaccine strains before
TABLE 4. Influence of meningococcal carnage on serum
bactericidal activity before and after vaccination
Bactericidal titer for strain and serotype (time):
aOnly the 33 subjectswho received thepolysaccaride-protein vaccine were
studied. +, Carriers of N. meningitidis (n=14); -, noncarriers (n=19).
VOL. 26, 1988
ROSENQVIST ET AL.
TABLE 5. Geometric mean bactericidal titers of pre- and
postvaccination serum samples from protein-polysaccharide
vaccinees without and with detectable prevaccination
Titer of serum from vaccinees:
vaccination. Of the 33 vaccinees who received the protein-
polysaccharide vaccine, 20 subjects were without prevacci-
nation bactericidal titers. Of these, seven (35%) and eight
(40%) vaccinees did not respond to vaccination with in-
creased bactericidal activity against the serotype 2b or 15
strains, respectively. Of the 13 vaccinees who had prevac-
cination bactericidal titers, two (15%) did not respond sig-
nificantly to the serotype 2b strain and 3 (23%) did not
respond significantly to the serotype 15 strain.
oO o,~~ 'o
J. CLIN. MICROBIOL.
Six weeks after vaccination, we observed a decline in
antibody levels among the protein-polysaccharide vacci-
nees. Thé geometric mean IgG antibody level, determined
by ELISA, was reduced about 25% compared with the peak
value at 2 weeks. However, the mean IgG level still re-
mained about four times higher than before vaccination.
In serum samples collected at any time, there was a
significant positive correlation between the antibody re-
sponses against the two strains. Two weeks after vaccina-
tion, the correlation coefficient (r) of the ELISA IgG anti-
body levels between the two outer membrane vesicle
antigens was -0.8 (Fig. 2a). The correlation between the
bactericidal assays with the two strains was somewhat lower
(r = 0.6 to 0.7) (Fig. 2b). Six weeks after vaccination, we
found that of those subjects who had received the protein-
against the serotype 15 strain but not against the serotype 2b
strain; the reverse situation was observed in six vaccinees.
The correlation coefficients between bactericidal titers and
IgG ELISA values with the corresponding antigens were
also relatively high (r
only a weak positive correlation was observed between
bactericidal titers and IgA. No correlation was demonstrated
between bactericidal activity and IgM.
0.7 to 0.8) (Fig. 2c and d), whereas
Log IgG against 15:PI.16
Log Bactericidal titer against 44/76
Log IgG against 2b:PI.2
o o o.--
Log IgG against 15:P1.16
FIG. 2. Correlations between ELISA IgG values against 15:P1.16 and 2b:P1.2 antigens (r
meningococcal strains 44/76 and 8047 (r=0.69) (b), ELISA IgG values and bactericidal titers against strain 8047 (2b:P1.2) (r=0.78) (c), and
ELISA IgG values and bactericidal titers against strain 44/76 (14:P1.16) (r = 0.81) (d). All serum samples were collected 2 weeks after
vaccination with the protein-polysaccharide vaccine. Least-squares regression lines are shown.
= 0.82) (a), bactericidal titers against
ANTIBODY RESPONSES TO MENINGOCOCCAL ANTIGENS
The convalescent-phase sera from the patients, collected
from 9 to 65 days (average, 22.5 days) after hospitalization,
also showed a substantial increase in antibodies compared
with their acute-phase sera. The highest response was with
IgG against the homologous serotype (15:P1.16). Although
no patient was known to have been infected with meningo-
cocci of serotype 2a or 2b, we also observed a strong
increase in IgG antibodies against the 2b:P1.2 antigen. In
contrast to the protein vaccinees, the convalescent group
showed a distinct IgM increase, in particular to the serotype
15:P1.16 antigen. Also, the IgA response was higher in
patients than in vaccinees. One patient had negligible anti-
body response as determined by ELISA. Three months
later, he suffered a new MCd caused by the same serotype
(B:15:P1.16), still without developing a significant immune
The protein-polysaccharide vaccine induced significant
immune responses against both the 2b:P1.2 and the 15:P1.16
meningococcal strains in about 70% of the vaccinees, judged
from both the ELISA results and the bactericidal assay. In a
previous vaccination trial with a B:2a vaccine, we observed
85% seroconversion with both ELISA and bactericidal assay
(9). The B:2a vaccine was, however, given twice with a
4-week interval between inoculations, indicating that revac-
cination is necessary to give a satisfactory response. It is
also possible that the higher lipopolysaccharide content in
the B:2a vaccine, or differences in minor protein composi-
tion, could be of importance. Two vaccine doses and an
adjuvant should probably be tried to increase the serocon-
version rate. Experiments in mice and humans with alumi-
num hydroxide and aluminum phosphate as adjuvants have
been promising (8, 15, 17).
The observation that about 50% of the recruits were
carriers of meningococci is similar to our findings in a
previous study in another Norwegian military camp (9). The
mean prevaccination and postvaccination bactericidal titers
and IgG antibody levels were significantly higher for the
carrier group than for the noncarriers, although most carrier
isolates were nongroupable and nontypable (Tables 3 and 4).
Carriage may thus induce antibody responses directed
against both B:15 and B:2b strains. This observation is in
close agreement with the report of Reller et al. (13) that
colonization of the nasopharynxes of adults with nongroup-
able meningococci, which very rarely cause disease, can be
a potent stimulus to the production of bactericidal antibod-
ies, not only against the carrier strain but also against
selected disease-causing strains. Goldschneider et al. (11)
found that 87% of men colonized with group B or C
organisms developed bactericidal antibodies to one or more
heterologous strains of pathogenic meningococci. These
observations support the role of the carrier state in develop-
ing and maintaining natural immunity to meningococcal
For evaluation of vaccine efficiency, it seems particularly
important to study the immune response in subjects without
measurable antibodies before vaccination. Although the
proportion ofsuch vaccinees who responded was lower than
that in the whole study, we found that 60 to 65% of them
seroconverted after one injection of the vaccine.
With the bactericidal assay with human complement used
in this study, the correlations between ELISA IgG and the
corresponding bactericidal titers (Fig. 2c and d) were signif-
icantly better than those observed previously with baby
rabbit complement (12). This is in agreement with the report
of Zollinger and Mandrell (22).
Because the response against the individual antigen com-
ponents has not been quantified separately, a comparison of
the immunogenicity of the two serotype antigens must be
interpreted with care. However, both pre- and postimmuni-
zation sera from the vaccinees showed higher IgG antibody
levels with the 2b:P1.2 antigen than with the 15:P1.16
antigen. We found similar results with the bactericidal assay.
These observations may indicate that (i) the serotype 15:
P1.16 antigens are weaker immunogens than the 2b:P1.2
antigens in this vaccine, (ii) the 15:P1.16 antigens studied by
ELISA are less antigenic than 2b:P1.2 antigens, or (iii) the
15:P1.16 strain used in the bactericidal assay is more resis-
tant to antibody-complement attack than the 2b:P1.2 strain.
The ELISA results indicate that infection with group B
serotype 15 meningococci induced about the same IgG
antibody levels as vaccination with a combined protein-
polysaccharide vaccine. However, the high total antibody
responses observed in patients and carriers could have been
due in part to lipopolysaccharide and other antigens which
were components in the ELISA antigens and not present in
the vaccine. Serum samples taken from MCd patients, all
except one of whom were infected with serotype 15 strains,
showed strong responses in ELISA with both the 2b:PI.2
and 15:PI.16 antigen preparations. We do not know whether
this is mainly due to immunological cross-reactions with
common determinants on the serotype and subtype proteins,
or whether the cross-reaction is due to other common
antigens. Immunoblotting experiments with postvaccination
sera from the B:2a vaccination trial have demonstrated
cross-reactions within the class 1 and 5 proteins in both
serotypes 15:P1.16 and 2a:P1.2 (E. Wedege, personal com-
Six weeks after vaccination, the IgG antibody levels and
bactericidal titers had declined 25% from the peak value
observed after 2 weeks. This is in agreement with our results
from the B:2a vaccination trial (16). Preliminary data from
ELISA indicate that 6 months after vaccination, the mean
IgG level was reduced to about one-third of the peak value
but still remained about two to three times higher than the
prevaccination level. Although the antibody titers have
declined, it is quite possible that the immune system has
been primed with induction of memory cells which may be
rapidly activated upon a new infection. The results obtained
with serum taken from the carriers may support this. Exper-
imental data show that immunological memory, as measured
by the degree of antibody response to a challenge dose of
vaccine, progressively increases with time to a maximum at
6 months and thereafter tends to persist at a plateau (18).
However, long-term follow-up of the vaccine response must
be performed to assess the duration of the antibodies and
corresponding protection. Such studies will probably be
complicated by the influence of new carriage of meningo-
cocci in the vaccinees during the follow-up period.
We gratefully acknowledge C. E. Frasch, U.S. Food and Drug
Administration, for use of his computer program for logit-log
transformations of the ELISA data; K. Bryn, National Institute of
Public Health, for gas-liquid chromatography analyses of the anti-
gens; and E. Wedege, National Institute of Public Health, for
immunoblotting experiments and helpful discussions.
VOL. 26, 1988
1548 Download full-text
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