Eur.J.Biochem.

255,4462454(1998)
FEBS1998

TherecombinantKlebsiellapneumoniaeoutermembraneproteinOmpAhas
carrierpropertiesforconjugatedantigenicpeptides
JeanFrancoisHAEUW,IsabelleRAULY,LaurenceZANNA,ChristineLIBON,ChristineANDREONI,ThienNgocNGUYEN,
ThierryBAUSSANT,JeanYvesBONNEFOYandAlainBECK
Centred'ImmunologiePierreFabre,SaintJulienenGenevois,France
(Received9February/30April1998)2EJB980192/3

Klebsiella pneumoniae OmpA, the 40kDa major protein of the outer membrane, was cloned and
expressedinEscherichiacoli.TherecombinantproteinwasproducedintracellularlyinE.coliasinclusion
bodies.FusionofashortpeptidetotheNterminusofnativeP40facilitatedhighlevelexpressionofthe
recombinantprotein.PurifiedrecombinantP40wasanalyzedtoverifypurityandstructuralintegrity.The
molecularmassofpurifiedrecombinantP40determinedbyelectrospraymassspectrometrywas3706 1
Da,inagreementwiththetheoreticalmassdeducedfromtheDNAsequence.Specificproliferationof
recombinantP40primedmurinelymphnodecellsinresponsetorecombinantP40stimulation invitro
indicatedthepresenceofaTcellepitopeonrecombinantP40.Theinductionofhighserumantibodytiters
to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when
chemicallycoupledtorecombinantP40indicatedthattheproteinhadpotentcarrierproperties.
Keywords : bacterial outermembrane protein; recombinantprotein; carrier protein; peptide coupling ;
conjugatevaccine.
protein G of the respiratory
syncytialvirus.

OmpAisoneofthemajorproteinsintheoutermembrane
ofgramnegativebacteria,presentatabout105copies/cell.Itis
believedtooccurinamonomericforminitsnativestate.A
typicalfeatureofOmpAisthatitcanbemodifiedbyheat:the
mobilityof Escherichiacoli OmpAonSDS/PAGEdecreases
whenitisheatedinthepresenceofSDS[1].OmpAishighly
conservedamonggramnegativebacteriaandisthoughttocon
sistoftwodomains.TheNterminus,includingaminoacidresi
dues 12170,formsamembranespanningdomainandcrosses
the outer membrane eight times in antiparallel strands,
leadingtoatypicalamphiphilicbarrel[224].TheCterminal
moiety of the protein is thought to be periplasmic [5]. The
protein seems to be multifunctional. In addition to non
physiological functions, such as serving as a receptor for
phages and colicins [6], it serves as a mediator in Ffactor
dependentconjugation[7].Itisalsorequiredforthestructural
integrityoftheoutermembraneandthegenerationofnormal
cell shape [8]. The capacity to form pores in the bacterial
membrane [9, 10] and confer serum resistance and
pathogenicitytoE.coliK1[11213]hasbeendemonstrated.
Bacterialintegralmembraneproteins,suchasE.coliTraT
[14,15]andthegroupBmeningococcaloutermembranepro
tein 3 [16], are excellent carriers for chemically conjugated
antigens.Theyfunctionalsoascarriersforgeneticallyfused
epi
Correspondence to J.F. Haeuw, Department of Biochemistry,
Centred'ImmunologiePierreFabre,5AvenueNapoleonIII,BP497,
F74164SaintJulienenGenevoisCedex,France
Fax:133450353590.
Email:jean.francois.haeuw@pierre
fabre.comURL:http://www.cipf.com
Abbreviations. CFA, complete Freund adjuvant; ESMS,
electrospray mass spectrometry ; HBA, Nhydroxysuccinimidyl
bromoacetate; Zw 314, zwittergent 314; G protein, attachment

topes:proteinssuchasLamB
[17], PhoE [18], OmpC [19]
andOmpA[20,21]havebeen
demonstrated to mediate
expression of peptides on the
surface of live enterobacterial
vectors. OmpC, the outer
membraneproteincomplexof
Neisseria meningitidis, has
beenshowntobeeffectivein
humansasaconjugatevaccine
with Haemophilus influenzae,
pneumococcal

and
meningococcal capsular
polysaccharides [22225].
Other clinically useful carrier
proteins have been derived
from bacteria: diphteria and
tetanus toxoids are both
successfully used in
conjugated H. influenzae
vaccines to transform the
capsularpolysaccharide,which
is a Tindependent antigen,
into a Tdependent antigen
[26].
Inthispaperwedescribe
theexpressionand
productionin
E. coli and purification of
Klebsiellapneumoniae OmpA
[27]. Using various analytical
criteria, the purity and
structural integrity of the
protein were evaluated. We

demonstratethepresenceofatleastoneTcellepitopeonthe protein.
molecule and its potential use as a carrier protein for
conjugatedpeptides.Theimmunologicalcarrierpropertiesof
the recombinant P40 were evaluated with conjugates ofMATERIALSAND
immunogenicsyntheticpeptidesderivedfromaloopregionof METHODS
the respiratorysyncytialvirus attachment protein [28230].
Bacterial strains and
Results obtained with the recombinantP40peptide
plasmids.
K. pneumoniae I
conjugates;andthedemonstrationofthepresenceofaTcell
epitope onrecombinant P40 provide a basis for the rational 145 (CIPF, Saint Julien en
designofsyntheticvaccineswithrecombinantP40ascarrier Genevois,France)wasusedas

the source of DNA. E. coli

RV308[31]wasusedasahost
forplasmids.PlasmidpRIT28
[32] was used as vector for
cloning and plasmid pVAL,
derived from plasmid
pTrpB1B2[33],wasusedfor
subcloningandexpressionof
theOmpAgene.

Haeuwetal.(Eur.J.Biochem.255)

447

supernatant. The solution was

Reagents.Glutaraldehydewasobtainedfromincubated overnight at room
Merck. NHydroxysuccinimidyl bromoacetatetemperature under gentle stirring.
(HBA)wasobtainedfromPierce.Zwittergent3 Aftercentrifugationat 100003g for
14 (Zw 314) was purchased from Sigma.15 min at 4C, the supernatant was
TetanustoxoidwaspurchasedfromSBLVaccin dialyzedovernightat4Cagainst25
AB. MacroPrep chromatography media weremM Tris/HCl, pH 8.5, 0.1%
obtained from BioRad. Superose 12 column(mass/vol.)Zw314andappliedtoa
MacroPrep High Q column equili
wasfromPharmaciaBiotechAB.
Peptides.
Peptides

G1Cbratedinthesamebufferat15cm/h.
(SICSNNPTCWAISK)

and

G1Absorbance was monitored at 280

(SIDSNNPTOWAISKC) were purchased fromnm.Proteinswereelutedat60cm/h
withalineargradientfrom0to0.5M
Neosystem Laboratoire. They correspond to
NaCl in 25 mM Tris/HCl, pH 8.5,
aminoacidresidues 1742187oftheattachment
0.1%(mass/vol.)Zw314.Fractions
protein (Gprotein)of thesubgroupArespira containing recombinant P40 were
torysyncytialvirus.PeptideG1Ccontains 14pooled and concentrated by
residues.Cys186foundinnativeGproteinwas ultrafiltration with a YM 10 filter
replacedbyaserineresidue.ThuspeptideG1C(Amicon cell). Concentrated
presents a single disulfide bridge. Peptide G1recombinant P40 was dialyzed
contains 15residues.Cys186wasalsoreplacedovernight at 4C against 20 mM
byserine,andCys176andCys182werereplacedsodium citrate, pH 3.0, 0.1%
by aspartic acid andornithine,respectively,to (mass/vol.)Zw314toreducethesalt
providealactamebridgepotentiallymimickingconcentration, and applied to a
the native disulfide bridge [34]. A CterminalMacroPrep High S column
equilibratedinthesamebufferat60
cysteinewasaddedforcouplingpurposes.
Cloningandexpressionof K.pneumoniaecm/h. Bound recombinant P40 was
OmpA in E. coli. The K. pneumoniae geneelutedwithalineargradientfrom0to
codingforOmpAwasidentified andsequenced1 M NaCl. Recombinant P40
fractions were pooled and
previouslyfrombacterialgenomicDNAbydi
concentratedasdescribedpreviously.
rectchromosomalsequencingasdescribed[27].
PurifiedrecombinantP40wasstored
The gene encoding recombinant P40 was
at220C.Proteinconcentrationsof
subcloned in an expression vector,
pVALOmpAKpn.Theresultingvector,pVALP40,
encodes, under the control of the tryptophan
promoter,K.pneumoniaeOmpAwitha9amino
acidsequencederivedfromthetrpoperonleader
sequence(Lpeptide)addedtoitsNterminalend.
E.coli RV308cellstransformedwithpVALP40
weregrownovernightat37 Cinshakeflasks
containing20mltrypticsoybroth(Difco)sup
plemented with yeast extract and tetracyclin (8
g/ml). Tryptophan promoter was blocked by
additionof100g/mltryptophan.Thisovernight
culture was used as an inoculum for 2l
fermentations (Chemap CF3000, Alfa Laval).
TheculturewasdilutedtoanA580ofabout1with
thesamemedium.Geneexpressionwasinduced
at midlog phase by the addition of 2indol
acrylicacidto25g/ml.5hafterinduction,cells
wereharvestedbycentrifugationat 10003g for
10minat4C.
Purification of recombinant
K.
pneumoniae OmpA. The pelletwassuspended
in25mMTris/HCl,pH8.5,andcellswerelyzed
by sonication on ice. Inclusion bodies were
obtainedbycentrifugationat100003gfor25min
at4Candwashingin25mMTris/HCl,pH8.5,
5 mM MgCl2, followed by centrifugation at
100003g for25minat4 C.Washedinclusion
bodiesweresuspendedin25mMTris/HCl,pH
8.5, 7 M urea and 10 mM dithiothreitol and
incubatedfor2hat37 Cundergentle stirring.
Insolublematerialwasremovedbycentrifugation
at 100003g for 15minat4C. 13vol.25mM
Tris/HCl, pH 8.5, 150 mM NaCl and 0.1%
(mass/vol.) Zw 314 were added to the clear

theintermediateandpurifiedfractions
were determined by the Lowry
methodusingBSAasthestandard.
Analyticalcharacterizationof
purifiedrecombinantP40.
Endotoxin determination was
performed by gaschromatographic
analysis of 3hydroxytetradecanoic
acid content and with Limulus
amebocyte lysate assay. Nterminal
sequencing was performed with an
Applied Biosystems 470A gasphase
sequencer equipped with a 120A
online phenylthiohydantoin an
alyzer. The molecular mass was
determined by SDS/PAGE,
electrospray mass spectrometry
(ESMS) and gelfiltration analysis.
For molecularmass determination,
ESMSanalyseswerecarriedoutona
triple quadrupole VGBioQ mass
spectrometer equipped with a
pneumatically assisted electrospray
source(FisonsBioQ,VGBioTech)
aftereliminationofthedetergentZw
314.Theproteinwasprecipitatedby
addition of 20 vol. cold ethanol
(220C).Aftervortexing,themixture
wasallowedtostandat220Cfor1
h, then centrifuged. The pellet was
dissolved in 1% (by vol.) formic
acid,andtheprecipitationprocedure
was repeated twice before
freeze/drying of the sample. For
analysis,thesamplesweredissolved
in5lformicacidanddilutedwith45
l propanol/methanol/water (4 :4: 1,
by vol). 10 l of this solution were
introduced into the electrospray ion
source at a flow rate of 3 l/min.
Calibration was performed using
multiplychargedionsfromaseparate
introductionofhorseheartmyoglobin
(16951.5Da).Molecularmassesare
givenasaveragevaluesbasedonthe
atomic masses of the elements
(C512.011, H51.00794, N514.0067,
O515,9994,S5 32.06).Gelfiltration
analyses were undertaken using a
Superose 12 column (Pharmacia)
equilibratedwithNaCl/Pi (20mMso
diumphosphate,pH7,0.15MNaCl)
containing 0.1% Zw 314. The
columnwascalibratedusingdextran
blue(2000kDa), catalase(232kDa),
BSA(66kDa),ovalbumin(43 kDa)
and chymotrypsinogen (25 kDa).
100l samples (1 mg/ml) were in
jected.
Preparation of glutaraldehyde
conjugates. Peptide G1C was
coupledtorecombinantP40andBSA
by using a modification of the
procedureofAvrameasandTernynck
[35].Proteins,5mlat2mg/mlin0.1
M sodium carbonate pH 9.0, were
reactedat4Cunderconstantgentle

stirringwithasolutionofpeptide(2.5mlat 1curred.Conjugateswereconservedat
mg/ml in the same buffer) in the presence of4C after filtration with a 0.22m
glutaraldehyde at 0.0185% (mass/vol.). After 4filter.
days, glutaraldehyde was added to 0.037 %
Conjugation of peptide G1 to
(mass/vol.). Reaction was stopped after 5 daysrecombinantP40,tetanustoxoidand
withadditionoflysineto3mM.Conjugateswere
BSA. Proteins were activated by
dialyzedovernightat4Cagainst0.1Msodium
bromoacetylation with a method that
phosphatepH7.0.PeptideG1Cwascoupledto
recombinant P40 in the presence of 4% SDS employsHBA[36].Solutionsof5mg
(mass/vol.)and0.1%Zw314(mass/vol.).SDSof protein in 1 ml of 0.1 M sodium
wassubsequentlyeliminatedbyprecipitationat4phosphatepH7.0weretreatedwith50
C with addition of 0.01 vol. 2 M KCl andlHBAdissolvedindimethylformamide
centrifugation100003gfor10minat4C.ThisbyusinganHBA/proteinratioof1.2:1,
step was repeated until no precipitation oc 3.3: 1 and2: 1 (bymass)forrecom
binant P40, tetanus toxoid and BSA.

Thesolutionswereincubatedfor1hat
roomtemperatureundergentleagitation
before desalting on PD10 columns ;
proteinswereelutedwith0.1Msodium
phosphatepH7.0.500lfractionswere
collected,andbromoacetylatedproteins
werelocatedbyabsorbanceat280nm.
2.5 mg peptide were added to the
proteins[protein/peptideratio(bymass)
2.5:1]andthesolutionsweresaturated
withargonbeforeincubationfor2hat
room temperature under constant
stirring. Conjugates were dialyzed
overnightagainst0.1Mso

448

Haeuwetal.(Eur.J.Biochem.255)

treating 10 g in 6 M
dium phosphate pH 7.0 and HClvaporat160Cfor
were conserved at 4C after2h.Aminoacidswere
filtrationwitha0.22mfilter.analyzed as their
0.1%Zw314(mass/vol.)wasphenylthiohydantoin

For
presentthroughoutcouplingofderivatives.
glutaraldehyde
thepeptidetorecombinantP40.

the
SDS/PAGE

andconjugates,
peptide/protein ratio of
immunoblot

analyses.
the conjugates was
Intermediate fractions of
determined with the
partially purified protein,
method described by
purified protein, and conju
Briandetal.[38],based
gates were analyzed by
onthedeterminationof
SDS/PAGE according to the
thedifferenceinamino
methodofLaemmli[37]using
acid composition
12% acrylamide gels and the
between the conjugate
Mini Protean II gel system
andthecarrierprotein.
(BioRad)at200Vfor45260
For G1containing
min.Allsamplescontained5%
2mercaptoethanol and 2 %conjugates, the degree
SDS and were incubated atof reaction was
determined

by
100 C for 15 min prior to
quantifyingtheamount
electrophoresis unless speci
of
S
fied otherwise. Proteins were
carboxymethylcysteine
visualized by staining with
calculated

from
Coomassiebrilliantblue.Heat
comparison of its inte
modifiability of purified
grated value with a
recombinant P40 was
known amount of S
determined by subjecting
carboxymethylcysteine
samples to SDS/PAGE after
introduced into the
solubilizationin sample buffer
amino acid standard
atroomtemperatureor 100Csolution[39].
for 15 min. For immunoblot
Lymphoproliferati
analysis of recombinant P40,
on

assays. Sixweek
proteins were separated by
old

female BALB/ c
12% SDS/PAGE and
mice
purchased from
electroblotted

onto
IFFA CREDO were
poly(vinyldifluoride)
membranes (ImmobilonPused for the experi
membrane, Millipore) in thements. Groups of 10
presence of 2% SDS for 45 mice were immunized
min. After transfer,with 100 g/mouse
immunoblottingwasperformedrecombinant P40 or
in
withafastblotdevelopersys with NaCl/Pi
tem (Pierce), with 1%complete Freund
(mass/vol.)BSAasablockingadjuvant(CFA;Sigma
agent throughout and a 1026Aldrich)
dilution of rabbit antiP40subcutaneously. Ten
polyclonal serum or 1:2000days after immuni
dilution of mouse antiP40zation, inguinal and
mAb prepared in house, a 1:subaortic lymph nodes
1000 dilution of biotinylatedwere removed and
goat antirabbit or mouse IgG pooled

within
serum as secondary antibody,experimentalgroups.A
and a streptavidinalkalinecell suspension was
phosphataseconjugate.
preparedbyteasingthe
Determination of thenodes apart. Cultures
peptideprotein conjugatewere performed in
molar ratios. The degree oftriplicateinRPMI1640
reaction was determined by(Gibco) containing 50
amino acid analysis. AminoU/ml penicillin, 50
acid composition of theg/ml streptomycin,
conjugates were determined0.25 M glutamine and
using the Waters PicoTag10% fetal calf serum.
HPLC system (MilliporeThe cellswerewashed
Corp.). Hydrolysates ofand suspended to give
recombinant P40 and106cells/ml.Cellswere
conjugates were performed bystimulated in vitro by

incubating 43105
cells/well in 96 round
bottom plates (Costar)
with

various
concentrations of
recombinant P40.
Background
proliferation was
measured with cells
derived from NaCl/Pi
immunized mice.
Controls were per
formed using cells in
culturemediumonlyor
stimulated

with
concanavalin

A.
Methyl[3H]thymidine
(1 Ci) (Amersham)
wasaddedtoeachwell
after various times of
culture, and the plates
were incubated for an
additional
18 h.
Cultures

were
harvested onto filters
usingasemiautomatic
harvester(Skatron)and
the

incorporated
[3H]thymidine was
determined using a
Packard
1900CA
Tricarb beta counter
(Packard Instruments).
Results werecalculated
from the geometric
means of triplicate
cultures.

The
stimulation index were
calculated as the
experimentalamountof
radioactivity/basal
amountofradioactivity.

CD4andCD8subsetswereIgG(Pierce)wasadded
isolatedfromcellsbynegativetoeachwellfor 1 hat
selection, using a Dynabead37 C. After washing,
method (Dynal). Cells were100
l 3,3,5,5
mixed with Dynabeads (basedtetramethylbenzidine
on the streptavidinbiotin(KPL) were added to
interaction), coated witheachwell.Thereaction
mousespecific antiCD4 orwas stopped 10 min
antiCD8(Pharmingen)andleftlaterbytheadditionof
M H2SO4.
for30minatroomtemperature1
under constant agitation. CellsAbsorbance at 450nm
attached to Dynabeads werewas determined using
separated with a magnet. Thea Labsystems IEMS
cells remaining in suspensionreader (Labsystems).
were counted and used inTiters were defined as
proliferation assays asthe reciprocal of the
described previously. Theserum dilution which
purity of the enrichedgavean A450 ofgreater
populations was assessed bythan 2 standard
stainingthecellswithantiCD4deviations above a
and antiCD8 Ig, followed by negativecontrolserum.
flow cytometry using aThe final titer was
FACScan(BectonDickinson). calculated from the
Immunization

withgeometric means of
triplicatedilutions.
conjugatesanddetermination
Statistical
of peptidespecific humoral
analyses.
Statistical
immuneresponses. Sixweek
analyses

were
oldBALB/cmicewereusedfor
performed using an
theexperiments.Groupsoffive
micewereimmunizedtwicebyanalysis of variance
subcutaneous injection ofpepwith P , 0.05usingthe
tide (10 g/mouse) in theStatgraphic program
presence of CFA, and peptide(Manugistics).
(10 g/ mouse) conjugated to
recombinant P40 or tetanus
toxoidinabsenceofadjuvantRESULTS
orNaCl/Pi ascontrol,20days

of
apart. 14 days after the lastPurification
immunization, mice were bled recombinantP40. The
from the retroorbital venousexpression vector
plexus.ELISAwereperformedpVALP40

was
essentially as described [40].designed such that it
96well plates (Immulon 2,encoded, under the
Dynatech) were coatedcontrol of the
overnight at 4C with 100 ltryptophan promoter,
BSAG1 or BSAG1C (2the K. pneumoniae
g/ml)insodiumcarbonatepHOmpAproteinwitha9
9.8. Nonspecific binding wasaminoacid sequence
blocked with 0.5 % gelatinderived from the trp
(Serva). Serum samples wereoperonleadersequence
serially diluted and the plates fused to its Nterminal
wereincubatedfor2hatroom end. Recombinant P40
temperature, followed bywas overexpressed in
extensivewashing.PeroxidaseE. coli
as an
conjugated goat antimouse

intracellular protein in
inclusion bodies. The
inclusion bodies were
solubilizedin7Murea
in the presence of 10
mM dithiothreitol
within2hat37C,and
renaturation

was
achievedbydilutingthe
mixture 13fold in the
presence of the
detergent Zw 314
(0.1%, mass/vol.).
SincerecombinantP40
isamembraneprotein,
the presence of a de
tergent was essential
for

complete
renaturation. Zw 314
was chosen from 15
detergents tested
because it had the
greatest capacity to
facilitate

the
solubilization and
renaturation of recom
binant P40 (data not
shown). Complete
refolding was still ob
tained after an
overnight dialysis
against a 25 mM
Tris/HClpH8.5,0.1%
Zw 314. Refolding
was assessed by
SDS/PAGE (data not
shown). The yield of
recombinant P40 thus
preparedwasabout300
mg/lculture.
This protein was
purified by a twostep
ionexchangechro
matography procedure.
Zw 314 was
maintained at 0.1%
(mass/vol.)throughout
thepurificationprocess.
The first step involved
anionexchange
chromatography. The
renatureddialyzatewas
chromatographed on a
MacroPrep High Q
columnequilibrated

Haeuwetal.(Eur.J.Biochem.255)

recombinantP40
containing fractions
from the Q column
were

pooled,
concentrated and
chromatographed after
an overnight dialysis
against a 20 mM
sodium citrate pH 3.0,
0.1%(mass/vol.)Zw3
14 on a MacroPrep
High S column
equilibrated with the
same
buffer.
Recombinant P40
elutedatapproximately
500 mM NaCl.
Fractions containing
recombinant P40 were
pooled and concen
tratedbyultrafiltration.
The final yield of this
process was around
40%(Table1).

Fig. 1. Analysis of purified

recombinant P40 andCharacterization of
recombinantP40G1CandG1the

purified
conjugates by SDS/PAGE and
recombinantP40. The
immunoblotting. (A) Coomassie
stained gel. Lane M, molecular purified recombinant
mass markers; lane 1, purifiedP40 was characterized
recombinant P40; lane 2,by different analytical
recombinantP40G1C
methodstoevaluateits
conjugate;lane3,bromoacetylated purity and examine its
recombinant P40; lane 4,primary structure. By
recombinant P40G1 conjugate.the

purification
(B) After electrophoresis and
procedure

described,
blotting onto a poly(vinyldifluo
ride) membrane, blotted proteins recombinant P40 was
(100ng)wereprobedwithamouse over 95% pure as
anti(naturalP40)mAb,andboundshown by SDS/PAGE
antibodies were detected with a(Fig. 1A).Onthebasis
biotinylated rabbit anti(mouseof gaschromatography
IgG) serum followed by aanalysis of the 3
streptavidinalkalinephosphatase
hydroxytetradecanoic
conjugate. Lane M, molecular
mass markers; lane 1, heatacidcontent,andsince
denatured purified recombinantthe lipopolysaccharide
P40;lane2,nondenaturedpurifiedof E. coli K12 is a
recombinantP40.
rough form in which
Table1.Purificationof
recombinantP40fromrenatured
extract.
185mgrenaturedextractwasused
forthepurificationasdescribedin
MaterialsandMethods.
Purificationstep
Renaturedextract
MacroPrepHighQ
MacroPrepHighS

with25mMTris/HClpH8.5,
0.1%(mass/vol.)Zw314.Re
combinant P40 eluted early in
the NaCl gradient. The

the Oantigen is
missing [41], the
purified

449

Fig.2.Analysisofpurified
recombinantP40protein
bygelfiltration.

Experimental details are

describedinthetextunder
Materials and Methods.
Elution positions of the
standardsareindicatedby
arrows: 1, dextran blue
(2000kDa);2,BSA(66
kDa); 3, ovalbumin (43
kDa);

4,
chymotrypsinogen (25
kDa).

protein contained less

than

0.1%
lipopolysaccharide.
This value correlated
wellwiththeendotoxin
content determined
with the Limulus
amebocytelysateassay
(datanotshown).
Gelfiltrationofthe
purified recombinant
P40 preparation on
Superose 12 column
showed a major peak
corresponding to a
recombinant P40
monomer (Fig. 2). A
minor peak eluted
earlier,

and
corresponded to a
dimer of the protein.
Thisdimerformofthe
molecule was detected
by SDS/PAGE and
western blot analysis
only with non
denaturedsamples(Fig.
1B).
When purified

recombinantP40wasevaluatednumberAJ000998).
by SDS/ PAGE and
For ESMS analysis
immunoblotting,itshowedthethedetergentZw314,
same heatmodifiabilitywhich is incompatible
properties as native P40with

subsequent
purifiedfromK.pneumoniaeinspectrometric analysis,
a similar fashion (data notwas eliminated. The
shown). The apparentproteinwasprecipitated
molecular mass of the nonby addition of cold
denaturedprotein(nativeform;ethanol. Following the
Fig. 1B) is lower than thatofremoval of detergent,
theproteinincubatedat 100Cmass measurement of
for 15 min (denatured form;the

purified
Fig.1B).
recombinant P40 was
Nterminal

sequenceperformed by ESMS
analysis showed that a singleafter solubilization in
sequence was detected,formic acid prior to
MKAIFVLNAA,

whichanalysis (Fig. 3). The

corresponded exactly to themeasured mass, 37
first 10 amino acids at the N0616 2 Da, was in
terminusofrecombinantP40asaccordance with the
predicted from the DNAtheoretical

mass
sequence (EMBL accessiondeducedfromtheDNA

sequence(37059Da).
Preparation and
characterization of
recombinant P40
conjugates.
Glutaraldehyde
coupling of peptide
G1C to recombinant
P40 had a relatively
high

coupling
efficiency. 16 peptides
were coupled/mol
recombinant P40 for a
total number of 18
lysine residues in the
protein. This elevated
ratiomaybeexplained
by the denaturation
induced by the use of
the detergent SDS,
which

450

Haeuwetal.(Eur.J.Biochem.255)

Fig.3.ESMSspectrumofpurifiedrecombinantP40.ExperimentaldetailsaredescribedinMaterialsandMethods.

vitro
with various
concentrations

of
wouldfacilitateaccessibilitytolysineresidues recombinantP40orculture
duringthelongcouplingreaction.SDS/PAGE medium for 3 days.
analysis of the conjugate showed a diffuse Stimulation

of
broad band between 40 kDa and 60 kDa, recombinantP40primed
corresponding to species containing differentcellswithrecombinantP40
ratiosofpeptideconjugatedtorecombinantP40 induced

specific
monomer,andotherbroadbandswithapparentproliferation (Fig. 4A),
molecular masses, correspond to conjugates whichreachedamaximum
comprising recombinant P40 multimers (Fig.with1.5g/mlrecombinant
1A).
P40. No significant
Analysis by SDS/PAGE of recombinantresponse was observed
P40G1 conjugates derived from coupling when recombinantP40
using the heterobifunctional reagent HBA primed cells were
showedrelativelybroadbandswithanaverage stimulated with culture
molecularmassforeachconjugatecomparable medium alone (data not
tothatcalculatedfromthecouplingratio(Fig. shown). In addition, cells
1A).Thepeptide:proteinratiodeterminedbyfrom NaCl/Piimmunized
quantification of the spacer carboxymethylmice did not proliferate in
cysteinereleasedbyacidhydrolysiswaslower response to recombinant
than that estimated for glutaraldehydeP40, indicating that
recombinant P40G1C conjugates, with arecombinantP40wasnota
value of 12. Contrary to the glutaraldehydemitogen.
method, this thiolgroupspecific coupling
To determine the
chemistrydidnotrequireadenaturationofthe optimal
recombinantP40proteinforefficientcoupling,lymphoproliferation, cells
and thus we can hypothesize that only thefrommiceimmunizedwith
accessiblelysineresidueslocatedmainlyintherecombinantP40orNaCl/Pi
periplasmic region of the protein were were stimulated in vitro
activated by bromoacetylation. A tetanuswith 1.5 g/mlrecombinant
toxoidG1 conjugate was prepared by meansP40forvarioustimes.
oftheHBAreagent.Thepeptide/proteinratio
was determined by quantification of
carboxymethylcysteine,andwasestimatedto
be28.Proteinconcentrationsoftheconjugates
wereestimatedtobearound2.5mg/ml.
RecombinantP40expressesaTcellepitope
able to generate CD4positive cells. To
investigate whether a Tcell epitope was
expressed on recombinant P40, cells from
lymph nodes of mice immunized with
recombinantP40orNaCl/Piwerestimulatedin

Theresultsofthesekinetic
experiments are shown in
Fig. 4 B. Optimum Tcell
proliferation was obtained
after 72 h culture in the
presence of 1.5 g/ml
recombinant P40. This
proliferation persisted after
96 h culture. No Tcell
proliferation was obtained
with cells from NaCl/Pi
immunizedmice.
To identify which
cellular population was
proliferatinginresponseto
recombinant P40, cells
from recombinant P40 or
NaCl/Piimmunized mice
weredepletedofCD81cells
or CD41 cells (Fig. 5).
Purity of the population
was assessed by flow
cytometry. CD4enriched
Tcell population had less
than 1% in CD81 Tcell
contamination and the
CD8enriched
Tcell
population had less than
1% in CD41 Tcell
contamination (data not
shown). No proliferation
was obtained when CD41
cells were removed.
DepletioninCD81cellsdid
notabolishproliferationof
cells from recombinant
P40immunized mice. On
thecontrary,theextentof
this proliferation was
greater than that obtained
withthetotalcells(Fig.5).
These results indicated
that a Tcell epitope was

expressed on recombinant P40 and that the immunizedwiththepeptide

CD41Tcellswerethepredominantresponding conjugated to recombinant
Tcell phenotype in the total population of P40 in the absence of an
adjuvant,irrespectiveofthe
immuneTcells.
conjugation

method
employed, the antipeptide
Effect of conjugation method on the total IgG titer was increased
antipeptideIgGlevels. Theimmunogenicitystrongly.Thisincreasewas
ofthepeptideproteinconjugateswasevaluatedgreater when mice were
inmice(Fig.6).Forthispurpose,micewere immunized with the
immunized by subcutaneous injection ofrecombinant P40G1
peptidealone,orinthepresenceofCFAorthe conjugates,theantipeptide
different conjugates. When mice were IgGtiterbeingsignificantly
immunizedwiththepeptidealone,adetectable higher than the antibody
but very low antipeptide IgG titer was titer obtained with the
observed. However, when mice were

recombinantP40G1C
conjugates. Moreover, the
antiG1 IgG titer induced
by immunization of mice
withtherecombinantP40
G1 conjugate was similar
to the titer obtained after
immunizationofmicewith
the tetanustoxoidG1
conjugate,which wasused
as a reference because
tetanus toxoid is already
usedasacarrierproteinina
conjugate vaccine for man
[26].

451

Haeuwetal.(Eur.J.Biochem.255)

needforanadjuvant.
DISCUSSION
WehaveconstructedanE.
colivectorthatdrivestheeffi
cient expression of a
recombinant form of K.
pneumoniae OmpA,themajor
outermembraneproteinof K.
pneumoniae. As described
previously for other bacterial
membraneproteins[42,43],an
Nterminal extension of nine
aminoacidresidues
Fig. 5. Determination of the
cellular population induced by
recombinant P40. Cells from
CD4depleted, CD8depleted or
totalcells from recombinantP40
immunized mice (e) or NaCl/Pi
immunizedmice(j)wereexposed
for72hto 1.5 g/mlrecombinant
P40.Theywerepulsedwith 1 Ci
[3H]thymidine for 18 h. Results
areexpressedasmeans6SD(n5
3) and are representative of two
experiments.

Fig.4.TcellepitopeonrecombinantP40
molecule. (A)Dosedependentproliferation
of lymph node cells from recombinant P40
and NaCl/ Piimmunized mice. Cells from
recombinantP40immunized mice (e) or
NaCl/Piimmunized mice (h) were exposed
for 72 h to various concentrations of
recombinant P40. They were pulsed with 1
Ci [3H]thymidine for 18 h. Results are
expressed as means 6SD (n 5 3) and are
representative of five experiments. (B)
Kineticsofproliferationoflymphnodecells
from recombinantP40immunized (j) and
NaCl/Piimmunized (h) mice. Cells were
exposed for various times to 1.5 g/ml re
combinantP40.Theywerepulsedwith1Ci
[3H]thymidinefor18h.Resultsareexpressed
asmeans6SD(n53)andarerepresentative
ofthreeexperiments.

These results demonstrated that

recombinantP40isaproteincarrierfor
weak immunogenic antigens and that
recombinantP40G1 conjugates were
the most immunogenic conjugates and
elicitedhighantibodytiterswithoutthe

Fig.6.AntipeptideIgGelicited
inmicebydifferent
conjugates.
Micewereimmunizedtwicewith
10g/mouseG1inthepresenceof
CFAorwith 10 g/mouseofG1
conjugated to recombinant P40
(rP40)ortetanustoxoid(TT). 14
days after the last immunization,
sera were collected and anti
peptideIgGmeasured.Resultsare
expressedas means 6SD (n 5 3)

andarerepresentativeoftwoexperiments.

production of foreign gene

products [44, 45], we chose
this system to avoid the
possibilityofposttranslational
wasnecessaryforhighyieldexpressionmodifications, such as
of K. pneumoniae OmpA in E. coli.glycosylation, which could be
Since E.coli hasbeenshowntobean induced in nonbacterial
excellentsystemfortheexpressionandexpression systems. Another

OmpAprotein,namely E.coli
OmpA,hasbeenproducedsuc
cessfully with a bacterial
expression system in Bacillus
subtilis [46]. However, since
B. subtilis is a grampositive
bacteria,the

452

Haeuwetal.(Eur.J.Biochem.255)

a serine residue,
partiallypurifiedOmpAproteinpresented a single

bridge
was not folded correctly, and disulfide
between

Cys176
and
the native conformation was
obtainedonlyinthepresenceof Cys182. This peptide
lipopolysaccharide [47]. Thewas coupled using an
protein is expressed andhomobifunctional
produced in the cytoplasm ofglutaraldehyde reagent.
E. coli as an insoluble fusionTo use thiolspecific
form. Strong denaturingcoupling chemistry, a
conditions (7 M urea) werecysteine residue was
required to quantitativelyincorporated at the N
extract the protein from theterminusofthepeptide
inclusion bodies. Afterduring solidphase
denaturation, the protein wassynthesis. All three
cysteine
renatured in the presence of anatural
detergent,Zw314,andfurtherresidues, in positions
purified by two ionexchange176,182and186,were
chromatography steps. Thereplaced by aspartic
protein was determined to beacid, ornithine and

residues,
greater than 95% pure whenserine
respectively. Aspartic
purifiedbythisprocedure.
acid and ornithine
Biochemical

were
characterizationofthepurifiedresidues
recombinant P40 wasintroducedtoachievea
undertaken using a variety ofcyclization of the
methods.RecombinantP40waspeptide with a lactam
recognized by a mAb raised bridgelinkingtheirside
against native K. pneumoniae chains. Peptide G1
OmpA and shared the samecontained 15 amino
heatmodification properties.acids and mimicked,
ESMS and automatedNviaalactambridge,the
G1Cloop.
terminalsequencing
experiments confirmed the
Glutaraldehyde is
purity of the protein. Thethe most popular bis
molecular mass determined byaldehyde homobi
ESMS was in completefunctional crosslinker
agreement with the theoreticalin use. Primary
molecularmassdeducedfromreactions with proteins
theDNAsequence.
and other amine
Since small peptides arecontaining molecules
usually not immunogenic, towould be expected to
raise antibodies against themproceed through the
they are generally covalentlyformation of Schiff
conjugated to carrier proteins.bases. However other
Our model peptide was a 14crosslinking reactions

feasible.
aminoacid peptide derivedare
fromtheGproteinofthesubGlutaraldehyde in
groupA respiratory syncytialaqueous solution can

polymers
virus (amino acids 1742187).form
Since a threedimensionalcontaining points of
modelofthisGproteinpredictsunsaturation, and since
animportantroleofthecentral such unsaturated
regionoftheprotein,includingpolymers are highly

towards
residues 1742187 as areactive
conserved part probablynucleophiles,especially
involved in proteinproteinprimary amines, the
interactions[48],andsincethisexact nature, structure
peptidehasbeendemonstratedand size of peptide
to be an immunodominantprotein conjugates
viral epitope [28230], twoformedbythismethod
peptides were synthesized,are difficult to
which were coupled by twodetermine. SDS/PAGE

of
methods. Both peptides mainanalyses
tain the conformation of theglutaraldehydepeptide
peptideoccurringinthenaturalprotein conjugates
protein. Peptide G1C, forshowed highmolecu
whichCys186wasreplacedbylarmass materials

corresponding to
polymer molecules
containing different
quantities of peptide
and protein. Thus, for
such conjugates the
determination of the
peptide/protein molar
ratio is purely
indicative. In contrast,
HBA [36] is a
heterobifunctional
crosslinking reagent
thatlimitsthedegreeof
polymerization
associated

with
homobifunctional
crosslinkers.Withthis
reagent,carrierproteins
are

first
bromoacetylated, then
reacted with the thiol
group of cysteine
containing peptides.
The extent of
conjugationisassessed
byaminoacidanalysis
after acid hydrolysis,
which liberates 1 mol
S
carboxymethylcysteine/
mol thioether linkage
between peptide and
protein. The two types
ofconjugatescontained
almost

similar
peptide/protein ratios,
with the ratio for the
glutaraldehyde
recombinantP40
G1Cconju

gate being higher andprofound effect on the

exhibitingavaluecloseto90%magnitude of the
occupationofavailablelysineantibodyresponse[50].
residues. Recombinant P40 We demonstrated a
contains 18 lysine residues,Tcell epitope on
seven of which are located inrecombinant P40 since
themembranedomain,and 11lymphnodescellsfrom
intheperiplasmicregion.SincerecombinantP40
the G1 coupling protocolimmunized mice pro
could be consideredas anon liferated specifically
denaturing method, G1 couldwhen activated with
becoupledmainlyonthelysinerecombinant P40.
residues from the periplasmicMoreover, no in vitro
tail,whereastheglutaraldehydeproliferation was
method could denature theobtainedafterdepletion
transmembrane part of thein CD41 cells in
molecule and so enhance theresponse

to
possiblereactionsites.
recombinantP40.Ithas
From an immunologicalbeen reported that
point of view, the conjugationTraT, a major outer
of peptide G1 or G1C tomembrane protein
recombinant P40 resulted in aisolated from E. coli
significantenhancementofthecontained

Tcell
immuneresponsetothepeptideepitopes [51]. Another
in comparison to responsesoutermembrane
elicitedwhenthepeptidealoneprotein, OmpC of N.
was injected with adjuvant.meningitidis
was
Moreover, since the anti
mitogenic for T
peptide IgG titer induced by
lymphocytes [52] and
recombinantP40G1
waswas used as a carrier
similar to that induced byfor protein and
tetanustoxoidG1, we canpolysaccharide
concludethatrecombinantP40 immunogens

in
compares well with tetanusparenteral vaccination
toxoid, a reference carrier
regimens[53].
protein,ininducinganantibody
Further
response to a peptide coupled
experiments,
using the
toit.
pepscantechnique[54],
Peptideprotein conjugates
willhelptolocalizeon
that differ in the conjugation
recombinant P40 the
method were found to elicit
peptidesequence(s)re
differentantibodylevels.HBA
sponsiblefortheTcell
recombinantP40G1
epitope.Severalstudies
conjugates induced an anti
revealedthatprotective
peptide IgG titer higher than
immunity was not
thatinducedbyglutaraldehyde
determined primarily
recombinantP40G1C
by the level but rather
conjugates. This difference
bythetypeofimmune
couldbeexplainedbyamodi
response

[55].
ficationoftheintegrityoftheT
Development of the
epitope by the conjugation
appropriate Thelper
method [49]. Another
subset is particularly
hypothesis for this difference
important since certain
could be the different
pathogens

are
orientationoftheBcellandT
controlled

most
cell determinants. The
effectively by either a
orientation of the two
cellular Thelper1
determinants can have a

type or a humoral T
helper2type immune
response

[56].
Moreover, knowledge
of the mechanism of
activation of antigen
presenting

cells
activation

by
recombinant P40 and
therolefothesecellsin
the induction of
immune response is of
interestfortherational
design

and
development

of
efficientvaccinesusing
recombinant P40 as a
carrier[57].
In conclusion, our
studies revealed no
significant differences
between recombinant
and native P40. The
highly purified re
combinant P40 was
characterized
structurally.

The
immunological results
obtained

with
recombinantP40
peptide conjugates
indicate that this
proteincanbeusedasa
carrier for lowimmu
nogenic antigens such
as peptides. Further
experiments will be
made to compare the
efficacy of this carrier
protein with that of a
reference protein
carrier, tetanus toxoid,
and with peptides and
polysaccharides as
antigens.
WethankDrsA.Van
Dorsselaer and N. Zorn
for performing mass
spectrometry and N
terminal

sequence
analyses, J. F. Depoisier,
C. Tardieux, F. Derouet
for excellent technical
assistance, and Drs N.
CorvaiaandU.Powerfor
critical reviewing of the
manuscript.

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