the recombinant klebsiella pneumoniae outer membrane protein has carriers properties for conjugated...

8/10/2019 The Recombinant Klebsiella Pneumoniae Outer Membrane Protein Has Carriers Properties for Conjugated Antigeni

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Eur. J. Biochem. 255, 4462454 (1998)

FEBS 1998

The recombinantKlebsiella pneumoniaeouter membrane protein OmpA has

carrier properties for conjugated antigenic peptides

Jean-Francois HAEUW, Isabelle RAULY, Laurence ZANNA, Christine LIBON, Christine ANDREONI, Thien Ngoc NGUYEN,Thierry BAUSSANT, Jean-Yves BONNEFOY and Alain BECK

Centre d'Immunologie Pierre Fabre, Saint Julien en Genevois, France

(Received 9 February/30 April 1998) 2EJB 98 0192/3

Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned andexpressed inEscherichia coli.The recombinant protein was produced intracellularly in E. colias inclusionbodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of therecombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. Themolecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of

recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitroindicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titersto a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus whenchemically coupled to recombinant P40 indicated that the protein had potent carrier properties.

Keywords : bacterial outer-membrane protein; recombinant protein; carrier protein; peptide coupling ;

conjugate vaccine.

OmpA is one of the major proteins in the outer membrane

of gram-negative bacteria, present at about 105copies/cell. It isbelieved to occur in a monomeric form in its native state. Atypical feature of OmpA is that it can be modified by heat: themobility of Escherichia coliOmpA on SDS/PAGE decreaseswhen it is heated in the presence of SDS [1]. OmpA is highlyconserved among gram-negative bacteria and is thought to con-sist of two domains. The N terminus, including amino acid resi-

dues 12170, forms a membrane-spanning domain and crossesthe outer membrane eight times in antiparallel -strands,leading to a typical amphiphilic -barrel [224]. The C-terminalmoiety of the protein is thought to be periplasmic [5]. Theprotein seems to be multifunctional. In addition to non-physiological functions, such as serving as a receptor forphages and colicins [6], it serves as a mediator in F-factor-dependent conjugation [7]. It is also required for the structuralintegrity of the outer membrane and the generation of normalcell shape [8]. The capacity to form pores in the bacterial

membrane [9, 10] and confer serum resistance andpathogenicity toE. coliK-1[11213] has been demonstrated.

Bacterial integral membrane proteins, such as E. coliTraT

[14, 15] and the group-B meningococcal outer-membrane pro-tein 3 [16], are excellent carriers for chemically conjugatedanti-gens. They function also as carriers for genetically fusedepi-

Correspondence to J.-F. Haeuw, Department of Biochemistry,Centred'Immunologie Pierre Fabre, 5 Avenue Napoleon III, BP 497,F-74164 Saint Julien en Genevois Cedex, France

Fax: 133 4 50 35 35 90.

E-mail:jean.francois.haeuw@pierre-

fabre.comURL : http://www.cipf.com

Abbreviations. CFA, complete Freund adjuvant; ESMS,electrospray mass spectrometry ; HBA, N-hydroxysuccinimidylbromoacetate; Zw 3-14, zwittergent 3-14; G protein, attachment

protein G of the respiratorysyncytial virus.

topes: proteins such as LamB[17], PhoE [18], OmpC [19]and OmpA [20, 21] have beendemonstrated to mediateexpression of peptides on thesurface of live enterobacterialvectors. OmpC, the outer-membrane protein complex ofNeisseria meningitidis, hasbeen shown to be effective inhumans as a conjugate vaccinewith Haemophilus influenzae,pneumococcal andmeningococcal capsularpolysaccharides [22225].Other clinically useful carrierproteins have been derivedfrom bacteria: diphteria andtetanus toxoids are both

successfully used inconjugated H. influenzaevaccines to transform thecapsular polysaccharide, whichis a T-independent antigen,into a T-dependent antigen[26].

In this paper we describe

the expression and

production in

E. coli and purification ofKlebsiella pneumoniae OmpA[27]. Using various analyticalcriteria, the purity and

structural integ-rity of theprotein were evaluated. We


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demonstrate the presence of at least one T-cell epitope on themolecule and its potential use as a carrier protein forconjugated peptides. The immunolog-ical carrier properties ofthe recombinant P40 were evaluated with conjugates ofimmunogenic synthetic peptides derived from a loop region of

the respiratory-syncytial-virus attachment pro-tein [28230].Results obtained with the recombinant-P40-pep-tideconjugates; and the demonstration of the presence of a T-cellepitope on recombinant P40 provide a basis for the rationaldesign of synthetic vaccines with recombinant P40 as carrier

protein.

MATERIALS AND

METHODS

Bacterial strains and

plasmids. K. pneumoniae I-

145 (CIPF, Saint Julien en

Genevois, France) was used as

the source of DNA. E. coliRV308 [31] was used as a hostfor plasmids. Plasmid pRIT28[32] was used as vector forcloning and plasmid pVAL,derived from plasmidpTrpB1B2 [33], was used forsub-cloning and expression ofthe OmpA gene.


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Haeuw et al. (Eur. J. Biochem. 255) 447

Reagents. Glutaraldehyde was obtained from

Merck. N-Hy-droxysuccinimidyl bromoacetate

(HBA) was obtained from Pierce. Zwittergent 3-14 (Zw 3-14) was purchased from Sigma.Tetanus toxoid was purchased from SBL VaccinAB. Macro-Prep chromatography media wereobtained from Bio-Rad. Su-perose 12 columnwas from Pharmacia Biotech AB.

Peptides. Peptides G1C(SICSNNPTCWAISK) and G1

(SIDSNNPTOWAISKC) were purchased fromNeosystem La-boratoire. They correspond toamino acid residues 1742187 of the attachmentprotein (G protein) of the sub-group-A respira-

tory syncytial virus. Peptide G1C contains 14residues. Cys186 found in native G protein wasreplaced by a serine residue. Thus peptide G1C

presents a single disulfide bridge. Peptide G1contains 15 residues. Cys186 was also replacedby serine, and Cys176 and Cys182 were replacedby aspartic acid and orni-thine, respectively, toprovide a lactame bridge potentially mim-ickingthe native disulfide bridge [34]. A C-terminalcysteine was added for coupling purposes.

Cloning and expression of K. pneumoniae

OmpA in E. coli. The K. pneumoniae gene

coding for OmpA was identified and sequenced

previously from bacterial genomic DNA by di-rect chromosomal sequencing as described [27].The gene en-coding recombinant P40 wassubcloned in an expression vector,pVALOmpAKpn. The resulting vector, pVALP40,encodes, under the control of the tryptophanpromoter, K. pneumoniaeOmpA with a 9-amino-acid sequence derived from the trpoperon leader

sequence (L peptide) added to its N-terminal end.E. coliRV308 cells transformed with pVALP40

were grown overnight at 37 C in shake flasks

containing 20 ml tryptic soy broth (Difco) sup-plemented with yeast extract and tetracyclin (8g/ml). Trypto-phan promoter was blocked byaddition of 100 g/ml trypto-phan. This overnightculture was used as an inoculum for 2-lfermentations (Chemap CF3000, Alfa Laval).

The culture was diluted to anA580of about 1withthe same medium. Gene ex-pression was inducedat mid-log phase by the addition of 2-indolacrylic acid to 25 g/ml. 5 h after induction, cellswere harvested by centrifugation at 10003g for

10 min at 4C.

Purification of recombinant K.

pneumoniae OmpA. The pellet was suspendedin 25 mM Tris/HCl, pH 8.5, and cells were lyzedby sonication on ice. Inclusion bodies wereobtained by centrifugation at 100003gfor 25 min

at 4C and washing in 25 mM Tris/HCl, pH 8.5,5 mM MgCl2, followed by centrifuga-tion at

100003gfor 25 min at 4 C. Washed inclusionbodies were suspended in 25 mM Tris/HCl, pH8.5, 7 M urea and 10 mM dithiothreitol and

incubated for 2 h at 37C under gentlestirring.Insoluble material was removed by centrifugation

at 10 0003g for15 min at 4C.13 vol. 25 mMTris/HCl, pH 8.5, 150 mM NaCl and 0.1%

(mass/vol.) Zw 3-14 were added to the clear

supernatant. The solution wasincubated overnight at roomtemperature under gentle stirring.After centrifugation at 10 0003g for

15 min at 4C, the supernatant was

dialyzed over-night at 4C against 25mM Tris/HCl, pH 8.5, 0.1%(mass/vol.) Zw 3-14 and applied to aMacroPrep High Q column equili-

brated in the same buffer at 15 cm/h.Absorbance was monitored at 280nm. Proteins were eluted at 60 cm/hwith a linear gradient from 0 to 0.5 MNaCl in 25 mM Tris/HCl, pH 8.5,0.1% (mass/ vol.) Zw 3-14. Fractionscontaining recombinant P40 werepooled and concentrated byultrafiltration with a YM 10 filter(Amicon cell). Concentratedrecombinant P40 was dialyzed

overnight at 4C against 20 mMsodium citrate, pH 3.0, 0.1%(mass/vol.) Zw 3-14 to reduce the salt

concentration, and applied to aMacroPrep High S columnequilibrated in the same buffer at 60cm/h. Bound recombinant P40 waseluted with a linear gradient from 0 to1 M NaCl. Recombinant P40fractions were pooled andconcentrated as described previously.Purified re-combinant P40 was stored

at 220 C. Protein concentrations of

the intermediate and purified fractions

were determined by the Lowry

method using BSA as the standard.

Analytical characterization of

purified recombinant P40.

Endotoxin determination wasperformed by gas-chromato-graphicanalysis of 3-hydroxytetradecanoicacid content and with Limulus

amebocyte lysate assay. N-terminalsequencing was performed with anApplied Biosystems 470A gas-phasese-quencer equipped with a 120-Aon-line phenylthiohydantoin an-alyzer. The molecular mass wasdetermined by SDS/PAGE,electrospray mass spectrometry(ESMS) and gel-filtration analy-sis.For molecular-mass determination,ESMS analyses were carried out on atriple quadrupole VG-BioQ massspectrometer equipped with apneumatically assisted electrospray

source (Fi-sons Bio-Q, VG-BioTech)after elimination of the detergent Zw3-14. The protein was precipitated byaddition of 20 vol. cold ethanol

(220C). After vortexing, the mixture

was allowed to stand at 220 C for 1h, then centrifuged. The pellet wasdis-solved in 1% (by vol.) formicacid, and the precipitation pro-cedurewas repeated twice beforefreeze/drying of the sample. Foranalysis, the samples were dissolvedin 5 l formic acid and diluted with 45l propanol/methanol/water (4 :4: 1,

by vol). 10 l of this solution wereintroduced into the electrospray ionsource at a flow rate of 3 l/min.Calibration was performed usingmultiply charged ions from a separateintroduction of horse heart myoglobin(16 951.5 Da). Molecular masses aregiven as average values based on theatomic masses of the ele-ments(C512.011, H51.00794, N514.0067,O515,9994, S5 32.06). Gel-filtrationanalyses were undertaken using aSuperose 12 column (Pharmacia)equilibrated with NaCl/Pi(20 mM so-

dium phosphate, pH 7, 0.15 M NaCl)containing 0.1% Zw 3-14. Thecolumn was calibrated using dextranblue (2000 kDa),catalase (232 kDa),BSA (66 kDa), ovalbumin (43 kDa)and chymotrypsinogen (25 kDa).100-l samples (1 mg/ml) were in-jected.

Preparation of glutaraldehyde

conjugates. Peptide G1C wascoupled to recombinant P40 and BSAby using a modifica-tion of theprocedure of Avrameas and Ternynck[35]. Proteins, 5 ml at 2 mg/ml in 0.1

M sodium carbonate pH 9.0, werereacted at 4C under constant gentle


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stirring with a solution of peptide (2.5 ml at 1mg/ml in the same buffer) in the presence ofglutaraldehyde at 0.0185% (mass/vol.). After 4days, glutaralde-hyde was added to 0.037 %(mass/vol.). Reaction was stopped after 5 dayswith addition of lysine to 3 mM. Conjugates were

dialyzed overnight at 4 C against 0.1M sodiumphosphate pH 7.0. Peptide G1C was coupled torecombinant P40 in the presence of 4% SDS(mass/vol.) and 0.1% Zw 3-14 (mass/vol.). SDSwas subsequently eliminated by precipitation at 4

C with addition of 0.01 vol. 2 M KCl and

centrifugation 10 0003gfor 10 min at 4C. Thisstep was repeated until no precipitation oc-

curred. Conjugates were conserved at

4C after filtration with a 0.22-mfilter.

Conjugation of peptide G1 to

recombinant P40, tetanus toxoid and

BSA. Proteins were activated by

bromoacetylation with a method that

employs HBA [36]. Solutions of 5 mg

of protein in 1 ml of 0.1 M sodium

phosphate pH 7.0 were treated with 50

l HBA dissolved in dimethylformamide

by using an HBA/protein ratio of 1.2: 1,

3.3 : 1and 2 : 1(by mass) for recom-

binant P40, tetanus toxoid and BSA.

The solutions were incu-bated for 1h at

room temperature under gentle agitation

before desalting on PD10 columns ;

proteins were eluted with 0.1M sodium

phosphate pH 7.0. 500-l fractions were

collected, and bromoacetylated proteins

were located by absorbance at 280 nm.

2.5 mg peptide were added to the

proteins [protein/peptide ratio (by mass)

2.5: 1] and the solutions were saturated

with argon before incubation for 2 h at

room temperature under constantstirring. Conjugates were dialyzed

overnight against 0.1M so-


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448 Haeuw et al. (Eur. J. Biochem. 255)

dium phosphate pH 7.0 and

were conserved at 4C after

filtra-tion with a 0.22-m filter.

0.1% Zw 3-14 (mass/vol.) waspre-sent throughout coupling of

the peptide to recombinant P40.

SDS/PAGE and

immunoblot analyses.

Intermediate frac-tions of

partially purified protein,purified protein, and conju-gates were analyzed bySDS/PAGE according to themethod of Laemmli [37] using12% acrylamide gels and theMini Protean II gel system(Bio-Rad) at 200 V for 45260min. All samples contained 5%2-mercaptoethanol and 2 %SDS and were incu-bated at

100 C for 15 min prior toelectrophoresis unless speci-fied otherwise. Proteins were

visualized by staining withCoo-massie brilliant blue. Heatmodifiability of purifiedrecombinant P40 wasdetermined by subjectingsamples to SDS/PAGE aftersolubilization in sample buffer

at room temperature or 100 Cfor 15 min. For immunoblotanalysis of recombinant P40,pro-teins were separated by12% SDS/PAGE andelectroblotted ontopoly(vinyldifluoride)

membranes (Immobilon-Pmembrane, Millipore) in thepresence of 2% SDS for 45min. After transfer,immunoblotting was performedwith a fast blot developer sys-tem (Pierce), with 1%(mass/vol.) BSA as a blockingagent throughout and a 1026

dilution of rabbit anti-P40polyclonal se-rum or 1:2000dilution of mouse anti-P40mAb prepared in house, a 1:1000 dilution of biotinylated

goat anti-rabbit or mouse IgGserum as secondary antibody,and a streptavidin-al-kaline-phosphatase conjugate.

Determination of the

peptide-protein conjugate

molar ra-tios. The degree of

reaction was determined byamino acid analysis. Amino

acid composition of theconjugates were deter-minedusing the Waters Pico-TagHPLC system (MilliporeCorp.). Hydrolysates of

recombinant P40 andconjugates were performed by

treating 10 g in 6 M

HCl vapor at 160C for2 h. Amino acids wereanalyzed as theirphenylthiohydantoinderivatives. Forglutaraldehydeconjugates, thepeptide/protein ratio ofthe conjugates was

determined with themethod de-scribed byBriand et al. [38], basedon the determination ofthe difference in aminoacid compositionbetween the conjugateand the carrier protein.

For G1-containingconjugates, the degreeof reaction wasdetermined byquantifying the amountof S-

carboxymethylcysteinecalculated fromcomparison of its inte-grated value with aknown amount of S-carboxymethylcysteineintroduced into theamino acid standardsolution [39].

Lymphoproliferati

on assays. Six-week-

old female BALB/ c

mice purchased fromIFFA CREDO were

used for the experi-ments. Groups of 10mice were immunizedwith 100 g/mouserecombinant P40 orwith NaCl/Pi incomplete Freundadjuvant (CFA ; Sigma-Aldrich)subcutaneously. Tendays after immuni-zation, inguinal andsubaortic lymph nodeswere removed and

pooled withinexperimental groups. Acell suspension waspre-pared by teasing thenodes apart. Cultureswere performed intriplicate in RPMI 1640(Gibco) containing 50U/ml penicillin, 50g/ml streptomycin,0.25 M glutamine and10% fetal calf se-rum.The cells were washedand suspended to give

106cells/ml. Cells werestimulated in vitro by

incubating 43105

cells/well in 96 round-bottom plates (Costar)with variousconcentrations ofrecombinant P40.Backgroundproliferation wasmeasured with cellsderived from NaCl/Pi-

immunized mice.Controls were per-formed using cells inculture medium only orstimulated withconcanavalin A.Methyl[3H]thymidine(1 Ci) (Amersham)was added to each wellafter various times ofculture, and the plateswere incubated for anadditional 18 h.Cultures were

harvested onto filtersusing a semi-automaticharvester (Skatron) andthe incorporated[3H]thymidine wasdetermined using aPackard 1900CA-Tricarb beta counter(Packard Instruments).Resultswere calculatedfrom the geometricmeans of triplicatecultures. Thestimulation index were

calculated as theexperimental amount ofradioactivity/basalamount of radioactivity.


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CD4 and CD8 subsets wereisolated from cells by negativeselection, using a Dynabeadmethod (Dynal). Cells weremixed with Dynabeads (basedon the streptavidin-biotininteraction), coated withmouse-specific anti-CD4 oranti-CD8 (Pharmingen) and leftfor 30 min at room temperatureunder constant agitation. Cells

attached to Dynabeads wereseparated with a magnet. Thecells remaining in suspensionwere counted and used inprolifer-ation assays asdescribed previously. Thepurity of the enrichedpopulations was assessed bystaining the cells with anti-CD4and anti-CD8 Ig, followed byflow cytometry using aFACScan (Becton Dickinson).

Immunization with

conjugates and determination

of pep-tide-specific humoralimmune responses. Six-week-

oldBALB/c mice were used for

the experiments. Groups of fivemice were immunized twice bysubcutaneous injection of pep-tide (10 g/mouse) in thepresence of CFA, and peptide(10 g/ mouse) conjugated torecombinant P40 or tetanustoxoid in ab-sence of adjuvantor NaCl/Pi as control, 20 daysapart. 14 days after the lastimmunization, mice were bled

from the retro-or-bital venousplexus. ELISA were performedessentially as de-scribed [40].96-well plates (Immulon 2,Dynatech) were coated

overnight at 4C with 100 l

BSA-G1 or BSA-G1C (2g/ml) in sodium carbonate pH9.8. Non-specific binding wasblocked with 0.5 % gelatin(Serva). Serum samples wereserially diluted and the plateswere incubated for 2 h at roomtemperature, fol-lowed by

extensive washing. Peroxidase-conjugated goat anti-mouse

IgG (Pierce) was addedto each well for 1 h at

37 C. After washing,

100 l 3,3,5,5-tetramethylbenzidine(KPL) were added toeach well. The reactionwas stopped 10 minlater by the addition of1 M H2SO4.Absorbance at 450 nmwas deter-mined usinga Labsystems IEMSreader (Labsystems).Titers were defined asthe reciprocal of theserum dilution whichgave an A450 of greaterthan 2 standarddeviations above anegative-control serum.The final titer wascalculated from thegeometric means oftriplicate dilutions.

Statistical

analyses. Statistical

analyses were

performed using an

analysis of variancewith P ,0.05 using theStatgraphic program(Manugistics).

RESULTS

Purification of

recombinant P40. The

expression vectorpVALP40 wasdesigned such that itencoded, under thecontrol of thetryptophan promoter,the K. pneumoniaeOmpA protein with a 9-amino-acid sequencederived from the trpoperon leader sequencefused to its N-terminalend. Recombinant P40was overexpressed inE. coli as an

intracellular protein ininclusion bodies. Theinclusion bodies weresolubilized in 7 M ureain the presence of 10mM dithiothreitol

within 2 h at 37 C, andrenaturation wasachieved by diluting the

mixture 13-fold in thepresence of the

detergent Zw 3-14(0.1%, mass/vol.).Since re-combinant P40is a membrane protein,the presence of a de-tergent was essentialfor completerenaturation. Zw 3-14was chosen from 15detergents testedbecause it had thegreatest ca-pacity tofacilitate thesolubilization and

renaturation of recom-binant P40 (data notshown). Completerefolding was still ob-tained after anovernight dialysisagainst a 25 mMTris/HCl pH 8.5, 0.1%Zw 3-14. Refoldingwas assessed bySDS/PAGE (data notshown). The yield ofrecombinant P40 thusprepared was about 300

mg/l culture.This protein was

purified by a two-stepion-exchange-chro-matography procedure.Zw 3-14 wasmaintained at 0.1%(mass/ vol.) throughoutthe purification process.The first step involvedanion-exchangechromatography. Therenatured dialyzate waschromatographed on a

MacroPrep High Qcolumn equilibrated


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Fig. 1. Analysis of purified

recombinant P40 and

recombinant P40-G1C and G1

conjugates by SDS/PAGE and

immunoblotting. (A) Coomassie-

stained gel. Lane M, molecular-mass markers; lane 1, puri-fiedrecombinant P40; lane 2,recombinant-P40-G1Cconjugate ; lane 3, bromoacetylatedrecombinant P40; lane 4,

recombinant P40-G1 con-jugate.(B) After electrophoresis andblotting onto a poly(vinyldifluo-

ride) membrane, blotted proteins(100 ng) were probed with a mouseanti-(natural P40) mAb, and boundantibodies were detected with abio-tinylated rabbit anti-(mouseIgG) serum followed by astreptavidin-alka-line-phosphataseconjugate. Lane M, molecular-mass markers; lane 1, heat-denatured purified recombinantP40; lane 2, non-denatured purifiedrecombinant P40.

Table 1. Purification of

recombinant P40 from renaturedextract.

185 mg renatured extract was usedfor the purification as described inMaterials and Methods.

Purification step

Renatured extractMacroPrep High QMacroPrep High S

with 25 mM Tris/HCl pH 8.5,

0.1% (mass/vol.) Zw 3-14. Re-combinant P40 eluted early inthe NaCl gradient. The

recombi-nant-P40-containing fractionsfrom the Q columnwere pooled,concentrated andchromatographed afteran overnight dialysisagainst a 20 mMsodium citrate pH 3.0,

0.1% (mass/vol.) Zw 3-14 on a MacroPrepHigh S columnequilibrated with thesame buffer.Recombinant P40eluted at approximately500 mM NaCl.Fractions containingrecombinant P40 werepooled and concen-trated by ultrafiltration.The final yield of thisprocess was around

40% (Table 1).

Characterization of

the purified

recombinant P40. The

puri-fied recombinantP40 was characterizedby different analyticalmethods to evaluate itspurity and examine itsprimary structure. Bythe purificationprocedure described,

recombinant P40 wasover 95% pure asshown by SDS/PAGE(Fig. 1A). On the basisof gas-chromatographyanalysis of the 3-hydroxytetradecanoicacid content, and sincethe lipopolysaccharideof E. coli K-12 i s arough form in whichthe O-antigen ismissing [41], thepurified

Fig. 2. Analysis of purified

recombinant P40 protein

by gel filtration.

Experimental details aredescribed in the text underMaterials and Meth-ods.Elution positions of thestandards are indicated byarrows: 1, dextran blue(2000 kDa) ; 2, BSA (66kDa); 3, ovalbumin (43kDa); 4,chymotrypsinogen (25kDa).

protein contained lessthan 0.1%lipopolysaccharide.This value correlatedwell with the endotoxincontent determinedwith the Limulusamebocyte lysate assay(data not shown).

Gel filtration of thepurified recombinantP40 preparation on

Superose 12 columnshowed a major peakcorresponding to arecombinant P40monomer (Fig. 2). Aminor peak elutedearlier, andcorresponded to adimer of the protein.This dimer form of themolecule was detectedby SDS/PAGE andwestern blot analysisonly with non-

denatured samples (Fig.1B).

When purified


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recombinant P40 was evaluatedby SDS/ PAGE andimmunoblotting, it showed thesame heat-modifiabil-ityproperties as native P40purified from K. pneumoniaeina similar fashion (data notshown). The apparentmolecular mass of the non-denatured protein (native form;Fig. 1B) is lower than that of

the protein incubated at 100Cfor 15 min (dena-tured form;Fig. 1B).

N-terminal sequenceanalysis showed that a singlesequence was detected,MKAIFVLNAA, whichcorresponded exactly to thefirst 10 amino acids at the Nterminus of recombinant P40 aspredicted from the DNAsequence (EMBL accession

number AJ000998).

For ESMS analysisthe detergent Zw 3-14,which is incom-patiblewith subsequentspectrometric analysis,was eliminated. Theprotein was precipitatedby addition of coldethanol. Following theremoval of detergent,

mass measurement ofthe purifiedrecombinant P40 wasperformed by ESMSafter solubi-lization informic acid prior toanalysis (Fig. 3). Themeasured mass, 37

0616 2 Da, was inaccordance with thetheoretical massdeduced from the DNA

sequence (37 059 Da).

Preparation and

characterization of

recombinant P40

conju-gates.

Glutaraldehydecoupling of peptide

G1C to recombinant

P40 had a relativelyhigh coupling

efficiency. 16 peptideswere coupled/molrecombinant P40 for atotal number of 18lysine residues in theprotein. This elevatedratio may be explainedby the denaturationinduced by the use ofthe detergent SDS,which


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Fig. 3. ESMS spectrum of purified recombinant P40. Experimental details are described in Materials and Methods.

would facilitate accessibility to lysine residuesduring the long coupling reaction. SDS/PAGEanalysis of the conjugate showed a diffusebroad band between 40 kDa and 60 kDa,corresponding to species containing differentratios of peptide conjugated to recombinant P40monomer, and other broad bands with apparentmolecular masses, correspond to conjugatescomprising recom-binant P40 multimers (Fig.1A).

Analysis by SDS/PAGE of recombinant

P40-G1 conjugates derived from coupling

using the heterobifunctional reagent HBAshowed relatively broad bands with an averagemolecular mass for each conjugate comparableto that calculated from the cou-pling ratio (Fig.1A). The peptide :protein ratio determined byquantification of the spacer carboxymethylcysteine released by acid hydrolysis was lowerthan that estimated for glutaraldehyderecombinant P40-G1C conjugates, with avalue of 12. Contrary to the glutaraldehydemethod, this thiol-group-specific couplingchemistry did not require a denaturation of therecombinant P40 protein for efficient coupling,and thus we can hypothesize that only theaccessible lysine residues located mainly in theper-iplasmic region of the protein wereactivated by bromoacetyla-tion. A tetanus-

toxoid-G1 conjugate was prepared by meansof the HBA reagent. The peptide/protein ratiowas determined by quantification ofcarboxymethyl cysteine, and was estimated tobe 28. Protein concentrations of the conjugateswere estimated to be around 2.5 mg/ml.

Recombinant P40 expresses a T-cell epitope

able to generate CD4-positive cells. To

investigate whether a T-cell epitope was

expressed on recombinant P40, cells fromlymph nodes of mice immunized withrecombinant P40 or NaCl/P iwere stimulated in

vitro with variousconcentrations of recombinant P40 or culturemedium for 3 days.Stimulation of recombinant-P40-primedcells with recombinant P40induced specificproliferation (Fig. 4A),which reached a maximumwith 1.5 g/ml recombi-nantP40. No significantresponse was observed

when recombi-nant-P40-primed cells werestimulated with culturemedium alone (data notshown). In addition, cellsfrom NaCl/Pi-immu-nizedmice did not proliferate inresponse to recombinantP40, indicating thatrecombinant P40 was not amitogen.

To determine theoptimallymphoproliferation, cells

from mice immunized withrecombinant P40 or NaCl/P iwere stimu-lated in vitrowith 1.5 g/ml recombinantP40 for various times.

The results of these kineticexperiments are shown inFig. 4 B. Optimum T-cellproliferation was obtainedafter 72 h culture in thepresence of 1.5 g/mlrecombinant P40. Thisproliferation persisted after96 h culture. No T-cellproliferation was obtainedwith cells from NaCl/Pi-immunized mice.

To identify whichcellular population wasproliferating in response torecombinant P40, cellsfrom recombinant P40 orNaCl/Pi-immunized micewere depleted of CD81cellsor CD41 cells (Fig. 5).Purity of the populationwas assessed by flowcytometry. CD4-enrichedT-cell population had lessthan 1% in CD81 T-cellcontamination and the

CD8-enriched T-cellpopula-tion had less than1% in CD41 T-cellcontamination (data notshown). No proliferationwas obtained when CD41

cells were removed.Depletion in CD81cells didnot abolish proliferation ofcells from recombinant-P40-immunized mice. Onthe con-trary, the extent ofthis proliferation wasgreater than that ob-tainedwith the total cells (Fig. 5).

These results indicatedthat a T-cell epitope was


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expressed on recombinant P40 and that theCD41T cells were the pre-dominant respondingT-cell phenotype in the total population ofimmune T cells.

Effect of conjugation method on the total

anti-peptide IgG levels. The immunogenicity

of the peptide-protein conjugateswas evaluated

in mice (Fig. 6). For this purpose, mice wereim-munized by subcutaneous injection of

peptide alone, or in the presence of CFA or thedifferent conjugates. When mice wereimmunized with the peptide alone, a detectablebut very low anti-peptide IgG titer wasobserved. However, when mice were

immunized with the peptideconjugated to recombinantP40 in the absence of anadjuvant, irrespective of theconjugation methodemployed, the anti-peptideIgG titer was increasedstrongly. This increase wasgreater when mice wereimmunized with the

recombinant P40-G1

conjugates, the anti-peptideIgG titer being significantlyhigher than the antibodytiter obtained with the

recombinant-P40-G1Cconjugates. Moreover, the

anti-G1 IgG titer inducedby immunization of micewith the recom-binant-P40-

G1 conjugate was similarto the titer obtained afterimmunization of mice with

the tetanus-toxoid-G1conjugate, which was usedas a reference because

tetanus toxoid is alreadyused as a carrier protein in aconjugate vaccine for man[26].


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Fig. 4. T-cell epitope on recombinant P40

molecule. (A) Dose-depen-dent proliferation

of lymph node cells from recombinant P40and NaCl/ Pi-immunized mice. Cells from

recombinant-P40-immunized mice (e) orNaCl/Pi-immunized mice (h) were exposedfor 72 h to various con-centrations of

recombinant P40. They were pulsed with 1Ci [3H]thymi-dine for 18 h. Results areexpressed as means 6SD (n 5 3) and arerepresentative of five experiments. (B)Kinetics of proliferation of lymph node cells

from recombinant-P40-immunized (j) andNaCl/Pi-immu-nized (h) mice. Cells wereexposed for various times to 1.5 g/ml re-combinant P40. They were pulsed with 1Ci[3H]thymidine for 18 h. Results are expressedas means 6SD (n53) and are representativeof three experiments.

These results demonstrated thatrecombinant P40 is a protein carrier forweak immunogenic antigens and that

recombinant-P40-G1 conjugates werethe most immunogenic conjugates andelicited high antibody titers without the

need for an adjuvant.

DISCUSSION

We have constructed an E.colivector that drives the effi-cient expression of arecombinant form of K.

pneumoniaeOmpA, the majorouter-membrane protein of K.pneumoniae. As describedpreviously for other bacterialmembrane proteins [42, 43], anN-terminal extension of nineamino acid residues

Fig. 5. Determination of the

cellular population induced by

recombi-nant P40. Cells from

CD4-depleted, CD8-depleted or

total cells from recombinant-P40-

immunized mice (e) or NaCl/Pi-immunized mice (j) were exposedfor 72 h to 1.5 g/ml recombinantP40. They were pulsed with 1 Ci[3H]thymidine for 18 h. Resultsare expressed as means 6SD (n53) and are representative of twoexperiments.

Fig. 6. Anti-peptide IgG elicited

in mice by different

conjugates.

Mice were immunized twice with

10 g/mouse G1in the presence of

CFA or with 10 g/mouse of G1conjugated to recombinant P40(rP40) or tetanus toxoid (TT). 14days after the last immunization,

sera were collected and anti-peptide IgG measured. Results areexpressed as means 6SD (n 5 3)


12/18

and are representative of two experiments.

was necessary for high-yield expressionof K. pneumoniae OmpA in E. coli.Since E. colihas been shown to be anexcellent system for the expression and

production of foreign geneprod-ucts [44, 45], we chosethis system to avoid thepossibility of post-translationalmodifications, such asglycosylation, which could beinduced in non-bacterialexpression systems. Another

OmpA protein, namelyE. coliOmpA, has been produced suc-cessfully with a bacterialexpression system in Bacillussubtilis [46]. However, sinceB. subtilis is a gram-positivebacteria, the


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partially purified OmpA proteinwas not folded correctly, andthe native conformation wasobtained only in the presence oflipopolysaccharide [47]. Theprotein is expressed andproduced in the cytoplasm ofE. coli as an insoluble fusionform. Strong denaturing

conditions (7 M urea) wererequired to quantitativelyextract the protein from theinclusion bodies. Afterdenaturation, the protein wasrenatured in the presence of adetergent, Zw 3-14, and furtherpurified by two ion-exchange-chromatography steps. Theprotein was determined to begreater than 95% pure whenpurified by this procedure.

Biochemicalcharacterization of the purified

recombinant P40 wasundertaken using a variety ofmethods. Recombinant P40 wasrecognized by a mAb raisedagainst native K. pneumo-niaeOmpA and shared the sameheat-modification properties.ESMS and automated-N-terminal-sequencingexperiments con-firmed thepurity of the protein. Themolecular mass determined byESMS was in completeagreement with the theoreticalmolec-ular mass deduced fromthe DNA sequence.

Since small peptides areusually not immunogenic, toraise antibodies against themthey are generally covalentlyconjugated to carrier proteins.

Our model peptide was a 14-amino-acid pep-tide derivedfrom the G protein of the sub-group-A respiratory syncytialvirus (amino acids 1742187).Since a three-dimen-sional

model of this G protein predictsan important role of the centralregion of the protein, includingresidues 1742187 as aconserved part probablyinvolved in protein-proteininteractions [48], and since thispeptide has been demonstratedto be an im-munodominantviral epitope [28230], twopeptides were synthe-sized,which were coupled by twomethods. Both peptides main-tain the conformation of the

peptide occurring in the naturalprotein. Peptide G1C, forwhich Cys186 was replaced by

a serine residue,presented a singledisulfide bridge

between Cys176 andCys182. This peptidewas coupled using anhomobi-functionalglutaraldehyde reagent.To use thiol-specificcoupling chemistry, a

cysteine residue wasincorporated at the N-terminus of the peptideduring solid-phasesynthesis. All threenatural cys-teineresidues, in positions176, 182 and 186, werereplaced by asparticacid, ornithine andserine residues,respectively. Aspar-ticacid and ornithineresidues were

introduced to achieve acy-clization of thepeptide with a lactambridge linking their side

chains. Peptide G1contained 15 aminoacids and mimicked,via a lactam bridge, theG1C loop.

Glutaraldehyde isthe most popular bis-aldehyde homobi-functional cross-linkerin use. Primaryreactions with proteinsand other amine-containing moleculeswould be expected topro-ceed through theformation of Schiffbases. However othercross-linking reactionsare feasible.Glutaraldehyde inaqueous solution canform polymerscontaining points of

unsaturation, and sincesuch unsaturatedpolymers are highlyreactive towardsnucleophiles, especiallyprimary amines, theexact nature, struc-tureand size of peptide-protein conjugatesformed by this methodare difficult todetermine. SDS/PAGEanalyses of glutaraldehyde peptide-

protein conjugatesshowed high-molecu-lar-mass materials

corresponding topolymer moleculescontain-ing differentquantities of peptideand protein. Thus, forsuch conjugates thedetermination of thepeptide/protein molarratio is purelyindicative. In contrast,

HBA [36] is aheterobifunc-tionalcross-linking reagentthat limits the degree ofpolymeriza-tionassociated withhomobifunctionalcross-linkers. With thisreagent, carrier proteinsare firstbromoacetylated, thenreacted with the thiolgroup of cysteine-containing peptides.

The extent of conjugation is assessedby amino acid analysisafter acid hy-drolysis,which liberates 1 molS-carboxymethylcysteine/mol thioether linkagebetween peptide andprotein. The two typesof conjugates containedalmost similarpeptide/protein ratios,with the ratio for the

glutaraldehyderecombinant-P40-

G1C conju-


14/18

gate being higher andexhibiting a value close to 90%occupa-tion of available lysineresidues. Recombinant P40contains 18 lysine residues,seven of which are located inthe membrane domain, and 11in the periplasmic region. Since

the G1 cou-pling protocolcould be considered as a non-

denaturing method, G1 couldbe coupled mainly on the lysineresidues from the periplasmictail, whereas the glutaraldehydemethod could dena-ture thetransmembrane part of themolecule and so enhance thepossible reaction sites.

From an immunologicalpoint of view, the conjugation

of peptide G1 or G1C torecombinant P40 resulted in asignifi-cant enhancement of theimmune response to the peptidein com-parison to responseselicited when the peptide alonewas injected with adjuvant.Moreover, since the anti-peptide IgG titer induced by

recombinant-P40-G1 wassimilar to that induced by

tetanus-toxoid-G1, we canconclude that recombinant P40compares well with tetanustoxoid, a reference carrierprotein, in inducing an antibodyresponse to a peptide coupledto it.

Peptide-protein conjugates

that differ in the conjugationmethod were found to elicitdifferent antibody levels. HBA

re-combinant-P40-G1conjugates induced an anti-peptide IgG titer higher thanthat induced by glutaraldehyderecombinant-P40-G1Cconjugates. This differencecould be explained by a modi-fication of the integrity of the Tepitope by the conjugationmethod [49]. Anotherhypothesis for this difference

could be the differentorientation of the B-cell and T-cell determinants. Theorientation of the twodeterminants can have a

profound effect on themagnitude of theantibody response [50].

We demonstrated aT-cell epitope onrecombinant P40 sincelymph nodes cells fromrecombinant-P40-immunized mice pro-liferated specificallywhen activated with

recombinant P40.Moreover, no in vitroproliferation wasobtained after depletionin CD41 cells inresponse torecombinant P40. It hasbeen re-ported thatTraT, a major outer-membrane proteinisolated from E. colicontained T-cell

epitopes [51]. Anotherouter-membrane

protein, OmpC of N.meningitidis wasmitogenic for Tlympho-cytes [52] andwas used as a carrierfor protein andpolysaccha-rideimmunogens inparenteral vaccinationregimens [53].

Furtherexperiments, using thepepscan technique [54],will help to localize on

recombinant P40 thepeptide sequence(s) re-sponsible for the T-cellepitope. Several studiesrevealed that protectiveimmunity was notdetermined primarilyby the level but ratherby the type of immuneresponse [55].Development of theappropriate T-helpersubset is particularlyimportant since certain

pathogens arecontrolled mosteffectively by either acel-lular T-helper-1-

type or a humoral T-helper-2-type immunere-sponse [56].Moreover, knowledgeof the mechanism ofactiva-tion of antigen-presenting cellsactivation byrecombinant P40 andthe role fo these cells inthe induction of

immune response is ofinterest for the rationaldesign anddevelopment of efficient vaccines usingrecombinant P40 as acarrier [57].

In conclusion, ourstudies revealed nosignificant differencesbetween recombinantand native P40. Thehighly purified re-combinant P40 was

characterizedstructurally. Theimmunologi-cal resultsobtained withrecombinant-P40-peptide conjugatesindicate that thisprotein can be used as acarrier for low-immu-nogenic antigens suchas peptides. Furtherexperiments will bemade to compare theefficacy of this carrier

protein with that of areference proteincarrier, tetanus toxoid,and with peptides andpolysaccharides asantigens.

We thank Drs A. VanDorsselaer and N. Zornfor performing massspectrometry and N-terminal sequenceanalyses, J. F. Depoisier,C. Tar-dieux, F. Derouet

for excellent technicalassistance, and Drs N.Corvaia and U. Power forcritical reviewing of themanuscript.


15/18


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the recombinant klebsiella pneumoniae outer membrane protein has carriers properties for conjugated...

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