Vaccine 19 (2001) 3568 3574

www.elsevier.com/locate/vaccine

Immunogenicity of recombinant class 1 protein from Neisseria

meningitidis refolded into phospholipid vesicles and detergent
Olivia Niebla *, Anabel Alvarez, Alejandro Martn, Ariane Rodrguez, Maite Delgado,
Viviana Falcon, Gerardo Guillen
Di6ision of Vaccines, Centre for Genetic Engineering and Biotechnology, PO Box 6162, A6e 31, Apartado 6162, La Ha6ana 10600, Cuba

Received 9 August 1999; received in revised form 7 December 2000; accepted 19 December 2000

Abstract

The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined
to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as
intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and
detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies
against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS
to reconstitute the P1 protein in a conformation that exposes the immunodominat regions. 2001 Elsevier Science Ltd. All rights
reserved.

Keywords: porA; Liposomes; Detergent

1. Introduction proteins in the outer membrane and it is responsible for

Neisseria meningitidis is a pathogen responsible for
serious invasive disease throughout the world. The cur-
rently available vaccines composed of the capsular
polysaccharides from one or more serogroups are inad-
equate, since they are poorly immunogenic in young
children, and do not provide protection against
serogroup B (MenB) [1]. Group B N. meningitidis is a
major cause of endemic and epidemic meningococcal
disease in the United States and many other countries
[2].
A vaccine based on the group B polysaccharide is
complicated because of its poor immunogenicity, prob-
ably due to structural similarities with human tissue [3].
In attempts to develop vaccines against serogroup B
infection, attention has been focused on the use of
outer membrane proteins (OMP).
Meningococci contain four classes of major OMPs:
class 1, 2/3, 4 and 5 proteins. The class 1 protein (P1) is
a porin constituting one of the two most abundant
* Corresponding author. Tel./fax: + 53-7-214764.
E-mail address: alejandro.martin@cigb.edu.cu (A. Martn).
subtype specificity. Immunisation of mice with outer
membrane vesicles (OMV) induces bactericidal antibod-
ies, which are directed mainly against the class 1
protein [4]. In addition, complete protection has been
obtained in an infant rat passive immunisation model
with 9 anti/class 1 monoclonal antibodies tested with
six serotypes of MenB [5]. Therefore, class 1 proteins
are considered as a potential vaccine candidate for
group B meningococci [6].
The obtention of pure P1 from meningococci repre-
sents a formidable feat, due to its association as hetero-
trimers with class 3 proteins [7]. Furthermore,
high-level heterologous expression of neisserial porins
in the outer membrane of Escherichia coli is detrimental
to the host [8]. However, it has been previously shown
that it is possible to obtain high amounts of recombi-
nant class 1 protein with an N-terminal unprocessed
signal peptide extension by intracellular expression as
inclusion bodies in Bacillus subtilis, followed by refold-
ing with the help of heterologous LPS, phospholipid
vesicles or detergents [912]. Similarly, Christodoulides
et al. [13] demonstrated high level intracellular expres-
sion of a different P1 recombinant in E. coli, followed

0264-410X/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S0264-410X(01)00008-1
 O. Niebla et al. / Vaccine 19 (2001) 35683574
by refolding with liposomes to produce bactericidal
antibodies.
Our group has previously cloned and expressed intra-
cellularly in E. coli the porA gene from N. meningitidis
strain B385 as a fusion protein to the first 45 amino
acids of the P64k antigen [14]. Since the influence of
this fusion partner on the antigenicity of the class 1
protein epitopes is unknown, this work examines the
possibility of eliciting bactericidal antibodies in mice
against this recombinant protein, using detergent and
phospholipid vesicles for its refolding.
2. Materials and methods
2.1. Reagents and biochemicals
All the chemicals used were obtained from Merck
(Germany) and BDH (UK). The restriction and modifi-
cation enzymes, as well as the primers used for PCR
amplification were from Heber Biotec (Cuba).
2.2. Bacterial strains and growth conditions
The E. coli strain W3110 (thyA36 deoc2 IN(rrnD-
rrnE)1 rph pyrE lF [15] was grown in Luria Bertani
medium (LB) overnight at 37C. The meningococcal
strain B385 (B4:P1.19,15) was isolated from a patient
with meningococcal disease and obtained from the Fin-
lay Institute for Sera and Vaccines (Havana, Cuba).
Strain H355 (B15: P1.15) was kindly provided by Dr.
Mark Achtman from the Max-Planck-lnstitut fur
Molekulare Genetik (Berlin, Germany). Strain
H355DP1 (B.15.NT) was constructed by inactivating a
cloned copy of the porA gene in plasmid pILM28 [14]
by insertion of a chloramphenicol resistance cassette
from pCAT19 [16], and introducing this copy by elec-
troporation [17] into the strain H355. Proper replace-
ment of the wild type porA copy was verified by
Southern blotting and examination of OMP profiles in
SDS-PAGE (Data not shown).
All meningococcal strains were grown in Brain Heart
Infusion Agar (Oxoid, UK) overnight at 37C in a 5%
CO2 atmosphere, with addition of chloramphenicol to 5
mg/ml as needed.
2.3. Monoclonal antibodies and outer membrane 6esicle
preparation
CB-Nm.1 [18] is a monoclonal antibody specific for a
linear epitope in PorA of the meningococcal strain
B385 (B:4:P1.19,15). It was a kind gift from Dr. C.
Nazabal (CIGB, La Habana, Cuba). OMV were
purified from meningococcal strain B385 as described
[19].
3569
2.4. Animals
Balb/c female mice (68 weeks old) were supplied by
Centro Nacional de Produccion de Animales de Labo-
ratorio (CENPALAB), Santiago de las Vegas, Habana,
Cuba. They were housed under controlled lighting (12 h
light, 12 h dark) at 22C, and were provided with water
and fed 1001EMO commercial chow ad libitum. The
chow was obtained from CENPALAB.
2.5. Construction of the expression plasmid pM82
Plasmid pM82 codes for a fusion polypeptide (PM82)
comprising the mature class 1 protein from N. meningi-
tidis B385 joined through its N-terminus to the first 45
amino acids of the neisserial P64k protein (134 bp from
the lpdA gene) [20], under the control of the tryptophan
promoter. For its construction plasmid pILM28 was
digested with Xbal and BamHl, and the band coding for
the mature porA sequence was used to replace the opc
coding sequence in pM80 [21].
2.6. Expression and purification of the PM82 protein
E. coli W3110 cells, containing the plasmid pM82,
were grown overnight in LB at 37C, then diluted 1:100
in minimal medium (M9) supplemented with 1% w/v
casein hydrolysate, 1% w/v glucose and 50 mg/ml of
ampicillin. The cells were grown 12 h at 37C, with 750
rpm and regulated pH 6.9 in a 1.5 l fermenter. Expres-
sion of PM82 was assessed by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis [22] (SDS-PAGE),
followed by Western blotting [23] using Mab CB-Nm.1.
The culture was centrifuged and the cells were washed
at a ratio of 0.1 g of biomass per ml of TE (10 mM
Trishydroxymethyl amino methane, 1 mM ethylenedi-
amine tetracetic acid, pH 8.0) and disrupted by
sonication.
After centrifugation, the insoluble inclusion bodies
were washed essentially as described by Nurminen et al.
[9], but following the washing scheme described in
Table 1
Washing steps of the insoluble fraction
Washing step Solution
1 TE Tris 10 mM EDTA
1 mM, pH 8
2 TE
MgCl2 0.8 mol/l
NaCl 0.1 mol/l
NP-40 0.5% v/v
3 TE
4 TE
Urea 2M
5 TE
3570 O. Niebla et al. / Vaccine 19 (2001) 35683574
Fig. 1. Electron microscopy of the negative stained of PM82-PC
vesicles obtained by gel filtration. The length of the bar is 200 nm.
Table 1. The insoluble material containing PM82 was
collected by centrifugation at 18000g at 4C for 20 min,
and solubilised in carbonate bicarbonate buffer, pH 10
containing 8 M urea (CB).
PM82 was then gel filtrated (diluted slowly 1:2 in
carbonate bicarbonate buffer) in a Sephadex G-25 (12
mm 20 cm) column equilibrated with CB. A second
chromatography step was performed in TE containing
0.02% w/v thiomersal w/v at a flow rate of 1 ml/min.
2.7. Reconstitution of PM82
After gel filtration the recombinant polypeptide was
refolded by dialysis against TE containing 0.1% w/v
SDS (PM82-SDS). The protein content was determined
by the amidoblack 10B method [24].
The reconstitution of PM82 into phospholipid vesi-
cles was performed essentially as described [25]. Briefly,
PM82-SDS was heated to 100C for 5 min and diluted
in 2% w/v OG (n-octyl b D-glucopyranoside), followed
by incubation at room temperature for 3 h. It was then
added to a phospholipid PC (phosphatidylcholine from
soy bean) detergent film and mixed gently until the film
was dissolved. The detergent was finally removed from
the micellar mixture by gel filtration on a Sephadex
G-50 column equilibrated with PBS (phosphate buffer
saline pH 7.2 containing 0.01% w/v thiomersal). The
resulting fractions were analysed by SDS-PAGE and
phospholipid content [26], and those containing both
proteins and lipids (PM82-PC) were further examined
by electron microscopy after negative staining to
confirm that vesicles had been formed [27] (Fig. 1).
2.8. Antisera
Antisera against PM82 protein were obtained by
immunising two groups of mice with PM82-SDS and
PM82-PC, respectively. Balb/c mice were immunised
subcutaneously with 10 mg of recombinant protein ad-
sorbed to 40 mg of aluminium hydroxide/mg of protein
and boosted twice with the same immunogen. The
preimmune sera were collected and then we obtained
the immune sera one week after each immunisation.
2.9. ELISA
The sera after the third dose were assayed by ELISA
against OMV from strain B4:P1.19,15 as described [28].
Briefly, Costar plates (high binding) were coated with
10 mg/ml of N. meningitidis OMV, in 0.05 M carbonate
buffer pH 9.6 at 37C for 3 h. The plates were washed
with PBS containing 0.05% v/v Tween-20 (PBSt) and
allowed to react with polyclonal antibodies of different
extractions, diluted 1:100 and then serially diluted 1:2
in PBSt. The plates were washed again and were incu-
bated with anti-mouse immunoglobulin-horseradish
peroxidase conjugate (Amersham, UK), diluted 1:1000
in PBSt for 1 h. Finally, the plates were washed with
PBSt and incubated with the chromogenic substrate
o-phenylendiamine dihydrochloride (0.5 mg/ml) and
0.03% v/v hydrogen peroxide for 10 min. The reaction
was stopped with H2SO4 2.5 N and absorbance was
measured at 492 nm in a TITERTEK Multiscan
MC340 spectrophotometer. ELISA titers for each
serum were determined as the maximum dilution dou-
bling the absorbance value of the preimmune serum of
each animal.
2.10. Bactericidal assay
The bactericidal assays were done as described [29].
Twofold dilutions of the sera were assayed with an
inoculum of 80100 colony-forming units per well, in
the presence of 25% human complement.
2.11. Opsonization and phagocytosis measurement
Meningococcal strains B385 and H355DP1 were
killed by 70% v/v ethanol at room temperature for 1 h
and labelled with FITC [30]. As effector cells, we used
human polymorphonuclear leukocytes (PMNs) from
heparinized blood of a healthy donor. The red cells
were lysed during 5 min at room temperature with a
solution containing 8.3 mg/ml NH4Cl, 1 mg/ml
NaHCO3 and 0.08 mg/ml EDTA, pH 6.8. Then, the
solution was centrifuged and leukocytes were washed
twice with Hanks Balanced Salt Solution and BSA
 O. Niebla et al. / Vaccine 19 (2001) 35683574 3571

0.2% w/v (HBSS-BSA), and finally the concentration

was adjusted to 5106 cell/ml.
The phagocytic assay was performed as follows: Ten
microlitre of FITC-meningococci, adjusted to 5 108
cell/ml in HBSS-BSA, and 50 ml of sera previously
diluted in HBSS-BSA were incubated at room tempera-
ture for 30 min with shaking to allow opsonization.
Then 5 ml of human serum (used as complement source
to permit the phagocytosis by the effector cells, human
PMNs) were added and incubated at 37C for 8 min.
Finally, 50 ml of human PMNs were added and incu-
bated at 37C for 8 min, with continuous agitation.
PMNs are able of internalise the bacteria opsonized
with murine sera and human complement bound to
Fig. 2. Electrophoretic analysis of the expression of PM82 protein in
them. These human complement factors can bind to E. coli strain: lane 1, molecular weight markers; lane 2, total cellular
complement receptors on human PMNs. The reaction protein of the E. coli strain W3110; lane 3, E. coli strain W3110
was stopped by placing the samples in an ice bath. A expressing the PM82 protein.
Flow Cytometer (Ortho Diagnostics System) with an
argon laser was used for all measurements. We set up a 3.2. Characterisation and immunogenicity of PM82
gate on the PMNs region, easily discriminated from
monocytes and lymphocytes. About 1000 effector cells After cell disruption, PM82 was obtained in the
were counted in each sample. The results were given as insoluble fraction of the E. coli cells, probably forming
percentage of positive effector cells (fluorescent cells) inclusion bodies. This feature allowed us to purify the
within the gate, i.e. fluorescence above the negative recombinant polypeptide, free from the host contami-
control consisting of meningococci, complement source nants by successive washing of the insoluble fraction
and effector cells. with detergents and chaotropic agents. As result, the
recombinant protein was obtained with approximately
76% purity, as determined from densitometric scans of
Coomassie Blue-stained gels.
3. Results
PM82 inclusion bodies required 8 M urea at extreme
pH values to be completely solubilised. Removal of
3.1. Expression of the P64k-class 1 fusion protein

To achieve high expression levels of the class 1

protein in E. coli, the porA gene coding for the subtype
P1.19,15 was cloned fused to the 134 bp from the 5% end
of the lpdA gene under the control of the tryptophan
promoter and the T4 gene 32 terminator, as described
in Section 2. Stabilisation of heterologous protein ex-
pression by using N-terminal fusions with this peptide
has been demonstrated previously [31]. Using this strat-
egy to express the porA gene fused to an N-terminal
fragment of the P64k protein, we were able to express
the meningococcal class 1 porin in E. coli at levels of up
to 30% of total bacterial cellular proteins. The expected
size of the fusion protein was 46 kDa as shown in Fig.
2.
The identity of the recombinant protein was confi-
rmed by Western blotting with the monoclonal anti-
body (Mab) CB-Nm.1, which is specific for P1 subtype
19 and shows bactericidal activity against strains of this
subtype. This Mab recognised the natural protein in Fig. 3. Evaluation of the identity of PM82 protein with monoclonal
OMVs and the recombinant protein PM82, as shown in antibody CB-Nm.01 against meningococcal class 1 protein subtype
19: lane 1, molecular weight markers; lane 2, OMV of the N.
Fig. 3. The higher molecular mass observed for the meningitidis strain B:4:P1.19,15 (B385); lane 3, OMV of B:2a:P1.2
recombinant protein is due to the addition of the (B16B6); lane 4, OMV of B:15:P1.7,16 (H44/76); lane 5, E. coli strain
N-terminal fragment of the P64k protein. W3110 expressing the PM82 protein.
3572 O. Niebla et al. / Vaccine 19 (2001) 35683574

Fig. 4. Bactericidal and antibody titers of sera against PM82 refolded in SDS 0.1% and PC. The bactericidal titer was expressed as the maximum
dilution yielding 50% killing, and ELISA titers as the maximum dilution doubling the absorbance of each preimmune serum. The coating antigen
was an OMV preparation from strain B385. A1,, A10 sera obtained after the third immunization with PM82-SDS; B1,, B10 sera obtained
after the third immunization with PM82-PC; C + , Pooled sera obtained after immunisation with OMV from B385.

urea by dialysis or gel filtration in TE resulted in
immediate precipitation of the protein. However, step-
wise removal of urea by gel filtration to CB and its
posterior change to TE maintained the recombinant
polypeptid in a soluble form. Storage temperature was
also an important variable, since storage at 4C resulted
in the progressive formation of aggregates. This formu-
lation, however, was stable at 20C (data not
shown).
ELISA was used to determine the titres of antibodies
induced in mice after immunisation with the partially
purified recombinant polypeptide PM82, refolded with
either SDS or PC. Fig. 4 shows the reactivity in ELISA
of the individual polyclonal antisera. In both groups
high levels of antibodies (up to 1:6400) were produced,
which allowed the recognition of the native class 1
protein in OMVs. There were no statistically significant
(P = 0.05) differences, as determined using a Mann
Whitney U test, between the PM82-SDS and PMB2-PC
groups. The monoclonal antibody CB-Nm.1 and the
polyclonal antibodies against B385 OMV were used as
positive controls and they reacted strongly with the
OMV.
3.3. Acti6ity of the antisera
All individual sera were assayed in a bactericidal test.
Fig. 4 shows the bactericidal activity of the sera against
strain B385. Although both groups elicited high titres
of antibodies by ELISA, the frequency of bactericidal
response of the individual animals in the PM82-SDS
group and the PM82-PC was different. The highest
frequency (70%) was obtained with PM82-PC. In the
PM82-SDS group, only 4 mice out of 10 (40%) devel-
oped bactericidal antibodies against N. meningitidis, but
those exhibited higher titres giving a mean value of
1:191, which is higher, although not statistically differ-
ent, to the mean value of bactericidal titres obtained in
the PM82-PC group (1:27). In all cases, there was no
detectable killing by preimmune sera.
These results were in correspondence with the phago-
cytosis assay depicted in Fig. 5. Immunisation with
PM82-SDS generated the highest levels of antibodies
that could mediate the opsonization of fluorescent bac-
teria and allow their internalisation in PMNs (A),
although the frequency of responders was slightly lower
than that obtained in the case of PM82-PC (B). Sera
from both groups, when evaluated against the
H355DP1 strain, which does not express the class 1
protein, showed only a background response (C).
PM82-SDS and PM82-PC were able to elicit bacteri-
cidal and opsonic antibodies at high levels. The same
sera were analysed for the antibody isotype (data not
shown) in an ELISA against the native OMV. The
response for all sera was essentially of IgG1 subclass.
4. Discussion
The expression at high levels of heterologous proteins
in E. coli as inclusion bodies and their refolding in vitro
with detergents may be an interesting strategy for vac-
cine development when outer membrane bacterial
proteins are used. However, this strategy presents a
number of drawbacks, such as potentially low yields
during the refolding process, and the difficulties inher-
ent to scale-up in an industrial setting.
The class 1 protein is the target for subtype-specific,
bactericidal monoclonal antibodies. The epitopes recog-
nised by these antibodies have been mapped to linear
peptides thought to correspond to the apices of exposed
loops on the native protein.
The native class 1 protein traverses the membrane
forming a trimeric b-barrel structure similar to that of
 O. Niebla et al. / Vaccine 19 (2001) 35683574
E. coli porins. Muttilainen et al. have shown by UV CD
spectroscopy that addition of zwitterionic detergent,
zwittergent, octylglucoside, octylpolyoxyethylene and
deoxycholate results in high, moderate or no amount of
b-structure, respectively [32]. In their work, there was a
good correlation between the amount of b-structure
and the antibody titre to antigenic epitopes of the
native protein as determined by EIA. The reconstitu-
tion of these epitopes is important for eliciting protec-
tive immunity [9].
Fig. 5. Phagocytosis of fluorescent N. meningitidis. Mab anti-P1.19
was used as positive control. The percentage of fluorescent cells
means the percentage of fluorescent PMNs from the total PMNs
counted in the set gate. In A and B sections only the positive sera
were plotted: A, Sera from group PM82-SDS; B, Sera from group
PM82-PC; C, Evaluation of four different sera against the mutant
strain H355DP1.
3573
Several distinct strategies might be employed for
refolding porins. One of them is the solubilization of
the inclusion bodies in a chaotropic solution followed
by slow removal of the chaotropic reagent by dialysis
or dilution in detergent. We have assayed this method
employing SDS 0.1%. This denaturing detergent can
produce secondary structure of a helix and random coil
[32]. A b-sheet secondary structure appears not to be
generated as a consequence of the treatment of the
recombinant class 1 protein with chaotropic agents;
however, we have been able to obtain bactericidal and
opsonic antibodies in this work. These results could be
ascribed to a moderate protein refolding in this solution
with a low detergent content, or to complete exposition
of the immunodominant regions, allowing eliciting
functional antibodies to immunodominant epitopes.
Our results lend credence to the class 1 protein as a
viable vaccine candidate, support the alternative of the
vaccine candidate based on the class 1 protein, owing to
the advantages of expression of a meningococcal OMP
in non-pathogenic E. coli strains. Future research
should be focused in making useful constructions of
recombinant chimeric class 1 proteins that include dif-
ferent P1 subtypes, and their refolding at low detergent
concentration.
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