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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.
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 3569
2.2. Bacterial strains and growth conditions 2.6. Expression and purification of the PM82 protein
The E. coli strain W3110 (thyA36 deoc2 IN(rrnD- E. coli W3110 cells, containing the plasmid pM82,
rrnE)1 rph pyrE lF [15] was grown in Luria Bertani were grown overnight in LB at 37C, then diluted 1:100
medium (LB) overnight at 37C. The meningococcal in minimal medium (M9) supplemented with 1% w/v
strain B385 (B4:P1.19,15) was isolated from a patient casein hydrolysate, 1% w/v glucose and 50 mg/ml of
with meningococcal disease and obtained from the Fin- ampicillin. The cells were grown 12 h at 37C, with 750
lay Institute for Sera and Vaccines (Havana, Cuba). rpm and regulated pH 6.9 in a 1.5 l fermenter. Expres-
Strain H355 (B15: P1.15) was kindly provided by Dr. sion of PM82 was assessed by sodium dodecyl sulphate-
Mark Achtman from the Max-Planck-lnstitut fur polyacrylamide gel electrophoresis [22] (SDS-PAGE),
Molekulare Genetik (Berlin, Germany). Strain followed by Western blotting [23] using Mab CB-Nm.1.
H355DP1 (B.15.NT) was constructed by inactivating a The culture was centrifuged and the cells were washed
cloned copy of the porA gene in plasmid pILM28 [14] at a ratio of 0.1 g of biomass per ml of TE (10 mM
by insertion of a chloramphenicol resistance cassette Trishydroxymethyl amino methane, 1 mM ethylenedi-
from pCAT19 [16], and introducing this copy by elec- amine tetracetic acid, pH 8.0) and disrupted by
troporation [17] into the strain H355. Proper replace- sonication.
ment of the wild type porA copy was verified by After centrifugation, the insoluble inclusion bodies
Southern blotting and examination of OMP profiles in were washed essentially as described by Nurminen et al.
SDS-PAGE (Data not shown). [9], but following the washing scheme described in
All meningococcal strains were grown in Brain Heart
Infusion Agar (Oxoid, UK) overnight at 37C in a 5% Table 1
Washing steps of the insoluble fraction
CO2 atmosphere, with addition of chloramphenicol to 5
mg/ml as needed. Washing step Solution
1 TE Tris 10 mM EDTA
2.3. Monoclonal antibodies and outer membrane 6esicle
1 mM, pH 8
preparation 2 TE
MgCl2 0.8 mol/l
CB-Nm.1 [18] is a monoclonal antibody specific for a NaCl 0.1 mol/l
linear epitope in PorA of the meningococcal strain NP-40 0.5% v/v
3 TE
B385 (B:4:P1.19,15). It was a kind gift from Dr. C.
4 TE
Nazabal (CIGB, La Habana, Cuba). OMV were Urea 2M
purified from meningococcal strain B385 as described 5 TE
[19].
3570 O. Niebla et al. / Vaccine 19 (2001) 35683574
2.8. Antisera
2.9. ELISA
After gel filtration the recombinant polypeptide was 2.10. Bactericidal assay
refolded by dialysis against TE containing 0.1% w/v
SDS (PM82-SDS). The protein content was determined The bactericidal assays were done as described [29].
by the amidoblack 10B method [24]. Twofold dilutions of the sera were assayed with an
The reconstitution of PM82 into phospholipid vesi- inoculum of 80100 colony-forming units per well, in
cles was performed essentially as described [25]. Briefly, the presence of 25% human complement.
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 2.11. Opsonization and phagocytosis measurement
added to a phospholipid PC (phosphatidylcholine from
soy bean) detergent film and mixed gently until the film Meningococcal strains B385 and H355DP1 were
was dissolved. The detergent was finally removed from killed by 70% v/v ethanol at room temperature for 1 h
the micellar mixture by gel filtration on a Sephadex and labelled with FITC [30]. As effector cells, we used
G-50 column equilibrated with PBS (phosphate buffer human polymorphonuclear leukocytes (PMNs) from
saline pH 7.2 containing 0.01% w/v thiomersal). The heparinized blood of a healthy donor. The red cells
resulting fractions were analysed by SDS-PAGE and were lysed during 5 min at room temperature with a
phospholipid content [26], and those containing both solution containing 8.3 mg/ml NH4Cl, 1 mg/ml
proteins and lipids (PM82-PC) were further examined NaHCO3 and 0.08 mg/ml EDTA, pH 6.8. Then, the
by electron microscopy after negative staining to solution was centrifuged and leukocytes were washed
confirm that vesicles had been formed [27] (Fig. 1). twice with Hanks Balanced Salt Solution and BSA
O. Niebla et al. / Vaccine 19 (2001) 35683574 3571
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 ent, to the mean value of bactericidal titres obtained in
immediate precipitation of the protein. However, step- the PM82-PC group (1:27). In all cases, there was no
wise removal of urea by gel filtration to CB and its detectable killing by preimmune sera.
posterior change to TE maintained the recombinant These results were in correspondence with the phago-
polypeptid in a soluble form. Storage temperature was cytosis assay depicted in Fig. 5. Immunisation with
also an important variable, since storage at 4C resulted PM82-SDS generated the highest levels of antibodies
in the progressive formation of aggregates. This formu- that could mediate the opsonization of fluorescent bac-
lation, however, was stable at 20C (data not teria and allow their internalisation in PMNs (A),
shown). although the frequency of responders was slightly lower
ELISA was used to determine the titres of antibodies than that obtained in the case of PM82-PC (B). Sera
induced in mice after immunisation with the partially from both groups, when evaluated against the
purified recombinant polypeptide PM82, refolded with H355DP1 strain, which does not express the class 1
either SDS or PC. Fig. 4 shows the reactivity in ELISA protein, showed only a background response (C).
of the individual polyclonal antisera. In both groups PM82-SDS and PM82-PC were able to elicit bacteri-
high levels of antibodies (up to 1:6400) were produced, cidal and opsonic antibodies at high levels. The same
which allowed the recognition of the native class 1 sera were analysed for the antibody isotype (data not
protein in OMVs. There were no statistically significant shown) in an ELISA against the native OMV. The
(P = 0.05) differences, as determined using a Mann response for all sera was essentially of IgG1 subclass.
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 4. Discussion
positive controls and they reacted strongly with the
OMV. The expression at high levels of heterologous proteins
in E. coli as inclusion bodies and their refolding in vitro
3.3. Acti6ity of the antisera with detergents may be an interesting strategy for vac-
cine development when outer membrane bacterial
All individual sera were assayed in a bactericidal test. proteins are used. However, this strategy presents a
Fig. 4 shows the bactericidal activity of the sera against number of drawbacks, such as potentially low yields
strain B385. Although both groups elicited high titres during the refolding process, and the difficulties inher-
of antibodies by ELISA, the frequency of bactericidal ent to scale-up in an industrial setting.
response of the individual animals in the PM82-SDS The class 1 protein is the target for subtype-specific,
group and the PM82-PC was different. The highest bactericidal monoclonal antibodies. The epitopes recog-
frequency (70%) was obtained with PM82-PC. In the nised by these antibodies have been mapped to linear
PM82-SDS group, only 4 mice out of 10 (40%) devel- peptides thought to correspond to the apices of exposed
oped bactericidal antibodies against N. meningitidis, but loops on the native protein.
those exhibited higher titres giving a mean value of The native class 1 protein traverses the membrane
1:191, which is higher, although not statistically differ- forming a trimeric b-barrel structure similar to that of
O. Niebla et al. / Vaccine 19 (2001) 35683574 3573
E. coli porins. Muttilainen et al. have shown by UV CD Several distinct strategies might be employed for
spectroscopy that addition of zwitterionic detergent, refolding porins. One of them is the solubilization of
zwittergent, octylglucoside, octylpolyoxyethylene and the inclusion bodies in a chaotropic solution followed
deoxycholate results in high, moderate or no amount of by slow removal of the chaotropic reagent by dialysis
b-structure, respectively [32]. In their work, there was a or dilution in detergent. We have assayed this method
good correlation between the amount of b-structure employing SDS 0.1%. This denaturing detergent can
and the antibody titre to antigenic epitopes of the produce secondary structure of a helix and random coil
native protein as determined by EIA. The reconstitu- [32]. A b-sheet secondary structure appears not to be
tion of these epitopes is important for eliciting protec- generated as a consequence of the treatment of the
tive immunity [9]. 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.
References
[9] Nurminen M, Butcher S, Idanpaan-Heikkila I, Wahlstrom E, [20] Guillen G, Alvarez A, Silva R, Morera V, Gonzalez S, Musac-
Muttilainen S, Runeberg-Nyman K, et al. The class 1 outer chio A, et al. Expression in Escherichia coli of the lpdA gene,
membrane protein of Neisseria meningitidis produced in Bacillus protein sequence analysis and immunological characterization of
subtilis can give rise to protective immunity. Mol Microbiol the P64k protein from Neisseria meningitidis. Biotechnol Appl
1992;6:2499 506. Biochem 1998;27:189 96.
[10] Muttilainen S, Butcher SJ, Runeberg K, Nurminen M, Idan- [21] Duarte C, Guillen G, Alvarez A, Carpio E, Quintana D, Gomez
paan-Heikkila I, Wahlstrom E, et al. Heterologous production C, et al. System for the expression of heterologous antigens as
of the P1 porin of Neisseria meningitidis in Bacillus subtilis: the fusion proteins. WO 97/26359.
effect of an N-terminal extension on the presentation of native- [22] Laemmli UK. Cleavage of structural proteins during the assem-
like epitopes. Microb Pathogen 1995;18:365 71. bly of the head of bacteriophage T4. Nature 1970;227:680 5.
[11] Idanpaan-Heikkila I, Muttilainen S, Wahlstrom E, Saarinen L, [23] Towbin H, Staehelin T, Golden J. Electrophoretic transfer of
Leinonen M, Sarvas M, et al. The antibody response to a proteins from polyacrylamide gel to nitro-cellulose sheets. Proce-
prototype liposome vaccine containing Neisseria meningitidis dure and some applications. Proc Natl Acad Sci USA
outer membrane protein P1 produced in Bacillus subtilis. Vaccine 1979;76:4350 4.
1995;13:1501 8. [24] Shefield JB, Graff DM, Li HP. A solid-phase method for the
[12] Idanpaan-Heikkila I, Wahlstrom E, Muttilainen S, Nurminen quantitation of protein in presence of the SDS and other inter-
M, Kayhty H, Sarvas M, et al. Immunization with meningococ- fering substances. Anal Biochem 1987;166:49 54.
cal class 1 outer membrane protein produced in Bacillus subtilis [25] Muttilainen S, Idanpaan-Heikkila I, Wahlstrom E, Nurminen
and reconstituted in the presence of Zwittergent or Triton X-100.
M, Makela PH, Sarvas M. The Neisseria meningitidis outer
Vaccine 1996;14:886 91.
membrane protein P1 produced in Bacillus subtilis and reconsti-
[13] Christodoulides M, Brooks JL, Rattue E, Heckels JE. Immu-
tuted into phospholipid vesicles elicits antibodies to native P1
nization with recombinant class 1 outer-membrane protein from
epitopes. Microb Pathogen 1995;18:423 36.
Neisseria meningitidis: influence of liposomes and adjuvants on
[26] Fiske C, Subarrow Y. The colorimetric determination of phos-
antibody avidity, recognition of native protein and the induction
phorous. J Biol Chem 1925;66:375.
of a bactericidal immune response against meningococci. Micro-
[27] Hayat MA, Miller SE. Negative staining. New York: McGraw-
biology 1998;144:3027 37.
Hill, 1990.
[14] Guillen G, Alvarez A, Niebla O, Silva R, Gonzalez S, Musac-
[28] Rosenqvist E, Hiby EA, Wedege E, Kosecek B, Achtman M.
chio A, et al. Cloning and expression of the porA gene of N.
meningitidis strain B: 4: P1.15 in E. coli. Preliminary characteri- The 5C protein of N. meningitidis is highly immunogenic in
zation of the recombinant polypeptide. Acta Biotechnol humans and stimulates bactericidal antibodies. J Infect Dis
1996;16:165 73. 1993;167:1065.
[15] Hill CW, Harnish BW. Transposition of chromosomal segment [29] Hiby EA, Rosenqvist E, Froholm LO, Bjune G, Feiring B,
bounded by redundant rRNA genes in Escherichia coli. J Bacte- Nokleby H, et al. Bactericidal antibodies after vaccination with
riol 1982;149:449 57. the norwegian meningococcal vaccine. NIPH Annals
[16] Claiborne W. An improved chloramphenicol resistance gene 1991;14:147 56.
cassette for site-directed marker replacement mutagenesis. Bio- [30] Sjursen H, Halstensen A, Naess A, Srnes S, Solberg CO. Flow
technology 1992;12:154 302. cytometric assay for the measurement of serum opsonins to
[17] Duenas S, Pajon R, Nogueiras EC, Delgado M, Martn A. Neisseria meningitidis serogroup B, serotype 15. J Immun Meth
Electroporation of Neisseria meningitidis with plasmid DNA. 1989;116:235 43.
Biotecnologia Aplicada 1998;15:247 9. [31] Menendez T, Alonso LM, Perez M, Pajon R, Silva R. Influence
[18] Cruz S, Fernandez ME, del Valle J, Nazabal C, Ohlin M, of the length of the P64k protein N-terminus stabilizing peptide
Gavilondo J. Monoclonal antibodies against P1, P3 and 31-kDa on the expression in E. coli of the fused TbpB from Neisseria
outer membrane proteins of Neisseria meningitidis B:4: P1.15. meningitidis. Biotecnologia Aplicada 1998;15:254 7.
Biotecnologia Aplicada 1993;10:89. [32] Susanna M. Refolded class 1 outer membrane protein of Neisse-
[19] Frash CE, Gotschlich EC. An outer membrane protein of Neis- ria meningitidis produced in Bacillus subtilis: an experimental
seria meningitidis group B responsible for serotype. J Exp Med vaccine against group B meningococci, Publications of National
1974;140:87 101. Public Health Institute, Helsinki. KTL A14/1995.