doi: 10.1111/j.1365-3083.2006.01829.x
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Differential Capacities of Outer Membrane Proteins
from Neisseria meningitidis B to Prime the Murine
Immune System after Vaccination
F. C. Silva Junior*, C. A. C. Gioia , J. M. Oliveira*, S. C. Cruz*, C. E. Fraschà & L. G. Milagres*
Abstract
*Disciplina de Microbiologia e Imunologia,
Universidade do Estado do Rio de Janeiro, Rio
de Janeiro, RJ, Brazil; Departamento de
Patologia/Microbiologia e Imunologia, Hospital
Universitário, Fundação Universidade Federal
de Rio Grande, Rio Grande, RS, Brazil; and
àFrasch Biologics Consulting, Martinsburg, WV,
USA
Received 9 May 2006; Accepted in revised
form 22 June 2006
Correspondence to: L. Milagres, Universidade
do Estado do Rio de Janeiro, Bl. 28 de
Setembro, 87. Fundos 3 andar, Disciplina de
Microbiologia e Imunologia, CEP 20551030,
Rio de Janeiro, RJ, Brazil. E-mail: lucimar@
uerj.br
Understanding the specificity of antibody response to Neisseria meningitidis
serogroup B (Men B) is a key requirement for the development of an effective
vaccine. This study was designed to investigate the antigen specificity of
murine IgG1 and IgG2b antibodies induced by different primary immunization schedules and the booster dose with the Cuban Men B vaccine. Immunoblotting analyses were performed using outer membrane vesicles (OMV) from
the vaccine strain (B:4,7:P1.19,15). IgG subclasses binding to PorA, PorB and
RmpM were determined by digital scanning of the immunoreactive bands.
Bactericidal antibody response after vaccination was also evaluated. The results
indicated that IgG2b anti-PorA was the main antibody response induced by
two doses of the vaccine. A primary series of three doses was found important
for increasing IgG2b as well as IgG1 to PorB and RmpM. The fourth dose
favoured the recognition of RmpM as detected by the increase of specific IgG1
and IgG2b. IgG subclasses anti-PorA did not change significantly if animals
received two, three or four doses of the vaccine during the primary immunization or after the booster dose for all vaccine groups. The booster response to
PorB and RmpM of groups BC2 and BC3 showed a significant increase in
IgG2b levels compared with the primary response. However, the recall and
the primary response of group BC4 were similar, suggesting a saturated dose–
effect response after four doses of vaccine. The same was seen for bactericidal
antibody response when human complement source was used in the assay.
Introduction
Since the publication of the entire genome of Neisseria
meningitidis at the beginning of this decade, several studies focusing on alternative antigens to compose a new
vaccine against N. meningitidis serogroup B (Men B) have
been described [1]. Despite some encouraging results
[2, 3] it is likely that several years will be needed for the
development of a meningococcal vaccine suitable to control or prevent endemic diseases caused by a diverse population of Men B strains.
Currently, tailor-made outer membrane vesicle (OMV)
vaccines are being used to control outbreaks provoked
by few prevalent phenotypes of Men B [4, 5]. Since
1992, the Cuban Men B vaccine (VA-MENGOC-BC)
has formed part of the Cuban infant vaccination schedule for all infants, and the reported incidence of
meningococcal disease in Cuba has remained extremely
low [6].
At the beginning of the 1990s, millions of Brazilian
children were immunized with two doses of the Cuban
Men B vaccine. Despite a satisfactory vaccine efficacy
(74%) in children aged over 48 months, younger children
were poorly protected [7]. In a recent study [8] of the
immunogenicity of the Cuban vaccine in mice, we demonstrated that the primary antibody response (IgG and
bactericidal antibody levels) showed a positive dose–
effect. There was a significant increase of IgG and bactericidal antibody levels after three doses compared with
two doses and also after the fourth dose compared with
the third dose. However, the bactericidal booster response
of mice primed with four injections of the vaccine indicated a negative dose–effect. The ELISA analysis of IgG subclasses induced by vaccination indicated a predominance of
2007 The Authors
Journal compilation 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 65, 1–7
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F. C. Silva Junior et al.
2 Specificity of IgG Subclasses after Immunization against B Meningococci
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IgG2b over IgG1 after the primary immunization. However, the increase in the number of vaccine injections
favoured development of IgG1 antibodies. This was
observed after the fourth dose and also after the booster
injection in mice primed with two or four doses of vaccine.
The rational of the present study was to investigate
the antigen specificity of IgG subclasses after different
primary immunization schedules and after the booster
dose. The correlation between specific IgG subclasses and
bactericidal antibody titers was also evaluated.
Overall, the results showed that the predominant
OMP (PorA, PorB and RmpM) from N. meningitidis present in the Men B Cuban vaccine had differing capacities
to prime the immune system. A predominant IgG2b
anti-PorA response was detected after two doses of vaccine. The increase in the number of injections of the vaccine was associated with a marked increase of IgG2b and
IgG1 reacting with PorB and RmpM. Based on the booster IgG subclass and bactericidal antibody responses, this
study showed that administration of more than four doses
of the vaccine did not change the magnitude of the antibody response to Men B.
Materials and methods
Serogroup B meningococcal strain. The Cuban vaccine
strain (Cu385/83) of serotype:serosubtype:immunotype
4,7:P1.19,15:L3,7,9 was used for the preparation of
immunoblot OMV antigens and as the target strain for
the bactericidal antibody assay.
Vaccine and immunization of mice. The Men B Cuban
vaccine (VA-MENGOC-BC), was obtained commercially. Female Swiss mice (5–6 weeks old) in groups of
8–10 were immunized with two (BC2 group), three
(BC3 group) or four (BC4 group) intramuscular injections of the vaccine given 2–3 weeks apart. Each vaccine
injection (100 ll) contained 2 lg of OMP, 2 lg of C
polysaccharide, 400 lg of Al(OH)3 and 0.01% thimerosal as preservative [9]. Mice were bled before each injection, and 14 days after the last injection. Approximately
7 months after the last dose, mice in each study group
received a booster injection of the vaccine. Blood was collected 14 days after the booster dose. The serum samples
were stored at )20 C.
Immunoblot assay. Outer membrane vesicles were prepared by extraction of the wet cell pellet for 2.5 h at
50 C with 5 ml of 0.2 M lithium chloride in a 0.1 M
sodium acetate buffer (pH 5.8) per 1 g of cells [10].
SDS-PAGE and the detection of antibodies by immunoblot were performed as previously described [11]. Each
gel (7·8 cm) was loaded with 80 lg of OMV. After electrotransfer at 250 mA for 2 h, each blot was cut into
strips with about 3 lg of protein per strip. The strips
were blocked (10 mM PBS pH 7.2 plus 3% BSA) and
incubated overnight at room temperature with 1:300
dilutions of sera. A set of sera was incubated in the presence and absence of 0.15% Empigen BB (EBB, Calbiochem, San Diego, CA, USA) for partial renaturation of
OMP epitopes [12]. All blots were incubated for 2 h
with a 1:500 dilution of peroxidade-conjugated rat antimouse IgG1 or IgG2b (Zymed, S. San Francisco, CA,
USA) and stained for 10 min with 3-amino-9-ethylcarbazole and hydrogen peroxide. Monoclonal antibodies
(MoAb) against RmpM (BE12, IgG2b isotype, 1:200
dilution) and subtype P1.15 (F8-7A2/1H11, IgG1 isotype, 1:250,000 dilution) were used to identify RmpM
and PorA, respectively, and as band intensity controls. A
positive post-vaccination pool of serum was used to control IgG1 and IgG2b binding to RmpM. IgG subclasses
binding to PorA, PorB and RmpM were processed after
scanning (Hewlett Packard Scanjet 6100C, Hewlett Packard Company, Palo Alto, CA, USA) the strips under
identical contrast enhancement conditions. An image analysis was performed with the 1D Image analysis software
(Kodak Digital ScienceTM, Eastman Kodak Company,
Rochester, NY, USA). A positive post-vaccination pool
of serum, diluted at 1:300, was used as an internal antibody standard and assigned 100 scan units (U) for IgG2b
anti-PorA. Band densities were recorded in arbitrary scan
units as integrated peak areas between baseline and curve
[12]. Care was taken to employ the same conditions for
incubation, staining and scanning of all strips. Band density analyses were done by two independent observers.
Results showing a discordance higher than 15% were reanalysed. The mean scan units were taken as the final result.
Interassay variability of IgG subclasses binding to
OMP. The coefficient of variation (CV) of MoAb P1.15
(control for IgG1) and the positive control serum (IgG1
anti-RmpM) were 12.9% (n ¼ 10) and 18.8% (n ¼ 12)
respectively. The controls for IgG2b reactions were
MoAb anti-RmpM and the IgG2b anti-RmpM reaction
of the positive control serum, which gave a CV of 19.6%
(n ¼ 7) and 16.1% (n ¼ 6) respectively. These results
demonstrate good assay reproducibility, and are similar
to that found by Wedege et al. [12].
Bactericidal assay. Serum bactericidal antibodies were
measured as previously described [8]. Briefly, the final
reaction mixture contained 25 ll of diluted test serum
previously heat inactivated at 56 C for 30 min, 12.5 ll
of guinea-pig serum that lacked detectable intrinsic bactericidal activity diluted at 1:2, and 12.5 ll of log-phase
meningococci (about 5 · 103 cfu/ml) grown on Tryptic
Soy Broth (Difco, Detroit, MA, USA) solidified with
1.5% (w/v) Noble agar (Difco) and containing 1% (v/v)
horse serum. For groups BC2 and BC4, bactericidal tests
were also done using human serum diluted at 1:2
(12.5 ll) as the complement source. This serum was negative for bacterial killing. The bactericidal reaction was
carried out at 37 C for 30 min. The CFU per well were
determined with the aid of a stereoscopic microscope
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F. C. Silva Junior et al.
Specificity of IgG Subclasses after Immunization against B Meningococci 3
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(·40). The bactericidal titer was defined as the reciprocal
of the serum dilution (before addition of complement
and bacteria) causing ‡50% killing and recorded as the
log2 titer. A value of 1 was assigned to each titer of <2;
thus, log2 1 ¼ 0. The positive control for each assay consisted of a pool of post-vaccination mouse serum with
previously determined bactericidal titer. The negative
control consisted of the complement source in the
absence of test serum.
Statistical analyses. The significance levels of differences
between groups were examined by paired or unpaired
t-test (parametric tests). Wilcoxon matched pairs test and
Mann–Whitney test were used as non-parametric tests in
a few cases. Correlations between bactericidal titers and
IgG binding were assessed by Pearson’s r coefficient.
These analyses were performed with a GraphPad-Prism
software, version 4.02 (Graphpad Software, San Diego,
CA, USA). P < 0.05 was taken as significant.
Results
Specificity of antibodies induced by two doses of the vaccine
(group BC2)
The results described below for groups BC2 and BC4
were similar in two independent experiments. The data
presented below represent the experiment with a greater
number of animals.
Table 1 shows the geometric mean (GM) of IgG1 and
IgG2b towards PorA, PorB and RmpM (strain Cu385/
83) measured after primary and booster immunizations.
Overall, there was a predominant IgG2b response after
primary immunization. In spite of the fact that the
amounts of the various antigens in the OMV are different
and that their transfer to the nitrocellulose paper during
electrotransfer may be dissimilar, our results indicated
that IgG2b antibodies induced by two doses of vaccine
were mainly (P < 0.05) directed to PorA (GM of
Table 1 Geometric mean (95% confidence
interval) of IgG1 and IgG2b to PorA, PorB
and RmpM of Strain Cu385 in serum of
mice obtained 14 days after two injections
(group BC2), three injections (group BC3) or
four injections (group BC4) of the Neisseria
meningitidis serogroup B vaccine and 14 days
after the booster dose given 7 months after
the last dose.
PorA
PorB
RmpM
1
3
4
Figure 1 Immunoblots showing IgG1 binding to PorA (lane 1) and
IgG2b binding to RmpM (lane 2) of Neisseria meningitidis Cu385/83
(B:4,7:P1.19,15). Lanes 3 and 4 represent IgG2b binding of two postbooster sera from group BC2. Scan units for serum 1 to PorA, PorB and
RmpM were 45, 73 and 53 respectively (lane 3); for serum 2 scan units
to PorA, PorB and RmpM were 70, 80 and 115 respectively (lane 4).
55.5 U). Eighty per cent of mice had IgG2b levels
higher than 40 U (which means a distinct band with
medium intensity, Fig. 1 – lane 3) to PorA, while only
30% responded with similar antibody levels to PorB
(GM of 6.8 U). Half of the mice did not show IgG2b
binding towards RmpM and most others (40%) had a
positive, but low response to this protein.
IgG1 and IgG2b reactions induced by primary
immunization were similar for PorB and RmpM showing
a low response (GM of 3.5–7.1 U). In opposition to the
strong IgG2b to PorA, this protein was barely recognized
by IgG1.
The booster dose, given 7 months after the primary
series, broadened the immune response and induced a
IgG1
Group BC2
PorA
PorB
RmpM
Group BC3
PorA
PorB
RmpM
Group BC4
PorA
PorB
RmpM
2
2 doses
1.4 (0.8–2.5)
3.5 (1.3–9.6)
7.1 (2.3–21.7)
3 doses
5.9 (1.1–32.5)
25.5 (6.5–100.2) 19.6 (3.6–105.6) 4 doses
6.8 (2.0–23.6)
37.3 (12.7–109.4)
93.9 (52.6–167.9)à
IgG2b
IgG1
Booster dose
5.6 (2.2–14.2)
7.2 (1.4–37.0)
18.3 (4.1–81.1)
Booster dose
42.7 (11.6–157.3) 18.1 (6.2–52.7) 19.8 (3.7–105.9)
64.0 (37.7–108.5)
33.2 (8.6–128.6) 52.3 (36.02–75.9)
Booster dose
45.4 (19.2–107.0) 8.2 (2.5–26.6)
95.5 (65.7–139.0)à 25.8 (7.4–89.7)
83.1 (44.2–156.2) 92.5 (55.8–153.4)à
55.5 (30.4–101.5)
6.8 ( 2.0–22.9)
4.2 (1.3–13.7)
IgG2b
44.0 (16.2–119.2)
49.4 (21.4–113.7)*
32.6 (11.8–90.1)*
53.2 (32.6–86.8)
93.6 (62.9–139.4)*
92.6 (61.3–140.1)
52.7 (30.7–90.3)
46.2 (15.2–140.9)
116.0 (95.5–141.0)
*P < 0.05 compared with the primary antibody response
P < 0.05 compared with the primary or booster antibody response of group BC2
àP < 0.05 compared with the primary or booster antibody response of group BC3.
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4 Specificity of IgG Subclasses after Immunization against B Meningococci
F. C. Silva Junior et al.
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Bactericidal titer (log2)
A
Group BC2
8
Group BC3
Group BC4
7
6
5
4
3
2
1
B 8
Group BC2
Bo
os
te
r
do
se
s
4
Bo
os
te
r
do
se
s
st e
r
3
2
Bo
o
do
se
s
0
Group BC4
7
6
5
4
3
2
1
te
os
Bo
se
do
r
s
r
os
te
4
2
do
se
s
0
Bo
Bactericidal titer (log2)
dose was needed to increase the IgG2b response to PorB
and RmpM, but not to PorA. Two doses of the vaccine
were not sufficient to induce a bactericidal antibody titer
higher than 2 (log2) in most mice. Nevertheless, it was
efficient to induce a long-lasting immune memory that
was activated 7 months later as evaluated by the recall
antibody response (with human complement).
Figure 2 Mean (log2) of bactericidal antibodies to Cu385 strain
(B:4,7:P1.19,15) induced by primary and booster immunization of
groups BC2, BC3 and BC4. (A) Guinea-pig complement, (B) human
complement.
significant IgG2b response to PorB (GM of 49.4 U) and
RmpM (GM of 32.6 U). The IgG2b anti-PorA did not
change significantly (GM of 44.0 U) in comparison with
the response after two doses (GM of 55.5 U). The booster
IgG1 response to each protein studied was similar to the
primary response.
The bactericidal titers of mice from group BC2 are
shown in Fig. 2A and B, left. We found significant differences of bactericidal titers when guinea-pig or human
complement was used. Fig. 2A, shows that the booster
antibody response (log2 mean of 1.7) and the primary
antibody response (log2 mean of 1.0) of group BC2 were
similar when guinea-pig complement was used in the
assay. A similar proportion of mice responded with bactericidal titers >2 (log2 titer) after the primary (22%) and
booster doses (33%).
However, using human complement source (Fig. 2B)
a significant increase (mean of 1.6-fold) in antibodies was
evident after the booster dose (log2 mean titer of 3.7).
30% and 70% of animals, respectively, following the primary and the booster dose, had bactericidal titers >2
(log2 titer). We did not detect any significant correlation
between bactericidal antibody titers and IgG subclasses
directed to PorA, PorB or RmpM.
In summary, IgG2b anti-PorA was the major antibody
response induced by two doses of the vaccine. A booster
Specificity of antibodies induced by three doses of the
vaccine (group BC3)
As shown in Table 1, the third dose of the vaccine
induced a marked IgG1 and IgG2b reaction to PorB and
RmpM in addition to the IgG2b response to PorA.
Group BC3 had a significant higher IgG1 response to
PorB (GM of 25.5 U) and RmpM (GM of 19.8 U) when
compared with group BC2.
Similarly, a higher IgG2b response to RmpM (GM of
33.2; P < 0.05) and PorB (GM of 19.8; P > 0.05) was
detected after three doses of the vaccine when compared
with two doses. About 60% of mice demonstrated IgG2b
levels higher than 40 U towards PorB and RmpM. It is
noteworthy that the IgG2b levels to PorA (GM of
42.7 U) were similar to the levels induced by two injections (GM of 55.5 U) of the vaccine (Table 1).
In contrast to the remarkable difference in IgG subclasses binding to PorA, there were no significant differences between IgG1 and IgG2b levels to PorB and
RmpM after the primary immunization.
A marked increase of 5.7-fold (P < 0.05) and 3.6-fold
(P ¼ 0.08) in the IgG2b anti-PorB (GM of 93.6 U) and
anti-RmpM (GM of 92.6 U), respectively, was seen after
the booster dose compared with the primary response.
Nonetheless, the IgG2b levels to PorA did not change
significantly after the booster dose (GM of 53.2 U) compared with the primary response (GM of 42.7 U). IgG2b
levels anti-PorB and anti-RmpM higher than 100 U
(very strong reactivity, Fig. 1 – lane 4) were detected in
60% and 50%, respectively, of mice after the booster
dose. Only two (20%) animals had the same pattern of
response against PorA.
The booster IgG1 response to PorB (GM of 64.0 U)
and RmpM (GM of 52.3 U) showed an increase of threefold (P > 0.05) compared with the primary response.
IgG1 levels higher than 40 U towards PorB and RmpM
were detected in about 80% of mice. Only 40% of animals had such IgG1 levels to PorA (GM of 18.1 U).
Because of the small volume of sera, the bactericidal
tests of this vaccine group were done using guinea-pig
complement only. As can be seen in Fig. 2A, three doses
of vaccine (group BC3) induced a higher (log2 mean of
4.4; P < 0.05) bactericidal antibody response than two
doses (group BC2; log2 mean of 1.0). The recall bactericidal antibody response (log2 mean of 5.7) of group BC3
was higher, but not statistically different from the
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Journal compilation 2007 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 65, 1–7
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Specificity of IgG Subclasses after Immunization against B Meningococci 5
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primary response (log2 mean of 4.4). 75% and 90% of
mice showed a bactericidal titer higher than 2 (log2 titer)
after the third and booster doses respectively. There was
no significant correlation between bactericidal antibody
titers and IgG subclasses directed to PorA, PorB or
RmpM.
In conclusion, three doses of the vaccine were needed
to increase IgG1 and IgG2b to PorB and RmpM. The
booster dose of vaccine in group BC3 induced a relevant
increase of IgG2b antibodies to PorB and RmpM. In
contrast, the primary and recall IgG1 and IgG2b
responses to PorA were similar. This immunization
schedule was efficient to induce a bactericidal antibody
response in the majority of mice.
Specificity of antibodies induced by four doses of the
vaccine (group BC4)
Immunoblotting of sera collected after four doses (Table
1) of the vaccine clearly showed a marked IgG1 reaction
to RmpM (GM of 93.9 U) as well as IgG2b binding to
PorB (GM of 95.5 U) and to RmpM (GM of 83.1 U).
Notably, the IgG2b anti-PorB and IgG1 anti-RmpM was
approximately four times (P < 0.05) higher for group
BC4 compared with that for group BC3. IgG2b levels
towards RmpM induced by four doses of vaccine were
also higher, but not statistically different, in comparison
with group BC3. IgG subclasses binding to PorA was
not altered if animals had received three or four injections of the vaccine.
Notably, the booster dose provoked a decrease (P ¼
0.06) of 0.48-fold in the GM of IgG2b anti-PorB compared with four doses of the vaccine. Nonetheless, the
proportion (60%) of mice with IgG2b levels around
100 U was similar after the primary and booster dose.
IgG2b response to PorA and RmpM did not change significantly after the booster dose. The IgG1 recall and primary response were similar for all protein studied.
A marked IgG2b and a less pronounced IgG1 reaction
to high molecular (70–108 kDa) weight proteins, especially a 70-kDa protein, were seen mainly after four doses
and the booster dose in group BC4. A variable reactivity
of IgG subclasses to low molecular weight proteins
(28–30 kDa) was detected after different immunization
schedules.
In terms of bactericidal antibody response, Fig. 2A,
right, shows that antibody titers peaked in mice immunized four times (group BC4) during the primary immunization. A significant decrease of antibody levels was seen
after the booster dose. However, when using human complement (Fig. 2B), we did not see any change in titers
comparing the primary (log2 mean of 6.0) and booster
response (log2 mean of 6.2) of group BC4. A significant
correlation (r ¼ 0.65) was detected between IgG2b antiPorA and bactericidal titers after the booster dose.
In conclusion, four doses of the vaccine favoured the
development of IgG1 to RmpM and IgG2b to PorB
and RmpM. Nonetheless, the booster dose negatively
influenced the recognition of PorB by IgG2b. IgG subclasses against PorA did not change significantly if
animals received two, three or four doses of the vaccine
during the primary immunization. Four doses of vaccine
induced a significantly higher bactericidal antibody
response than two or three doses. Depending on the complement source the booster response of group BC4 was
lower (guinea-pig complement) or similar (human complement) to the primary response.
Discussion
In a previous study of the immunogenicity of the VAMENGOC-BC vaccine, we showed a dominant murine
IgG2b response to OMV from the vaccine strain as detected by an ELISA. In addition, specific IgG1 development
was associated with the number of injections of the vaccine [8].
Data obtained in this study indicated that two doses
of the Cuban Men B vaccine induced a predominant
IgG2b response to PorA. Thus, this was an indication
that one dose of the vaccine was important to prime the
immune system to especially recognize PorA of N. meningitidis. Mouse IgG2b are preferentially induced by a Th1
immune response, but rather little is known about factors
that direct class switching in vivo [13, 14]. Recently published data [15] demonstrated that a specific epitope of
MSP2 protein of Plasmodium falciparum induced a T-celldependent class switching to IgG2b in immunized mice
and strongly implicated IL-10 and INF-c in the process.
Accordingly, based on the responses of the IgG subclasses, the data presented here indicated a tendency to a
Th1 response in animals immunized with two doses of
vaccine. A Th1 response in children immunized with two
doses of the Cuban Men B vaccine has been described
[16]. The possibility that linear epitopes of PorA protein
influenced the T-cell–B-cell interactions and consequently
the immunoglobulin switch to IgG2b anti-PorA needs to
be investigated.
A distinct IgG1 as well as IgG2b reaction to PorB
and RmpM was induced by three doses of vaccine (group
BC3). A continued elevation of such antibodies was
detected during the recall response of group BC3. Based
on this significant switch to IgG1 response, the animal
model used in this study indicated that a Th2 response
to OMP from Men B requires repeated antigen exposures.
Based on the responses of the IgG subclasses induced
by primary immunization schedules of groups BC3 and
BC4, our results suggest the need for two or three doses
of the vaccine to prime the immune system to subsequently respond to PorB and RmpM. This was also
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observed after the booster dose of groups BC2 and BC3.
Of note, the fourth dose of vaccine was of great importance to increase the primary antibody response but it did
not influence the development of a stronger recall antibody response compared with three doses.
PorA and PorB function as porin proteins and are the
most abundant neisserial outer membrane proteins [17].
In the Norwegian Men B vaccine, PorA and PorB are
present in equal amounts and account for about 70% of
the proteins [17]. Assuming that the Cuban and Norwegian vaccines have similar amounts of PorA and PorB,
our data indicate that PorB may be less immunogenic
than PorA in the Cuban vaccine. Similar results were
described using human sera collected after two doses of
the Norwegian OMV vaccine [18]. In addition, a markedly lower T-cell response to PorB compared with PorA
and OMV was detected after two or three doses of the
Norwegian vaccine in humans [17]. Interestingly, our
data indicated that the IgG2b anti-PorA induced by
two, three or four doses of vaccine did not alter significantly. The reasons for this unchanged IgG2b response
to PorA are not known. Future studies may investigate
the biological function and avidity of purified IgG2b
anti-PorA.
Despite the low proportion (10%) of RmpM in the
outer membrane of meningococci, [17] the results reported here indicated that four doses of vaccine induced a
predominant IgG1 response to this protein. In addition,
a stronger IgG2b anti-RmpM was seen after the fourth
dose of the vaccine. The biological function of RmpM
remains unknown. It has been described that mouse
MoAb to RmpM can block bactericidal killing by antiPorA MoAb [19]. Nevertheless, an elegant study by
Rosenqvist et al. [20] showed no blocking effect on bactericidal activity of MoAb anti-RmpM or immunoglobulin
(Ig) anti-RmpM purified from serum of vaccinees. Of
note, the human Ig anti-RmpM preparations were
obtained from sera collected after two doses of the Norwegian Men B vaccine. A previous report found that only
22% of Norwegian vaccinees had strong or intermediate
IgG binding to RmpM [18]. Similarly, in the present
study, two doses of the Cuban Men B vaccine induced a
weak IgG1 and IgG2b response to RmpM. As the avidity of antibodies may increase with time and with repeated antigen exposure, we believe that the blocking effect
of anti-RmpM induced by three and four doses of Men B
vaccines should be investigated.
In conclusion, our data showed that PorA, PorB
and RmpM had different abilities to prime the
immune system and consequently to induce a recall
antibody response. An IgG2b response was quickly
induced by vaccination and its specificities were broadened as the number of vaccine injections was increased.
The IgG1 response to vaccination needed a higher
number of injections and was directed mostly against
RmpM and PorB. The biological significance of these
antibody responses in terms of functional activity and
development of immune memory needs to be further
investigated.
Acknowledgment
Monoclonal antibodies and meningococcal strains were
provided by Adolfo Lutz Institute, Bacteriology Division.
We acknowledge FAPERJ/SR2-UERJ/CAPES and CNPq
for financial support.
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