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Vaccine 26 (2008) 5778–5783

Contents lists available at ScienceDirect

Vaccine

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roduction of biologically active CXC chemokines by Lactococcus lactis:valuation of its potential as a novel mucosal vaccine adjuvant

aima G. Cortes-Pereza,b, Luis F. da Costa Medinaa, Francois Lefèvreb,hilippe Langellaa, Luis G. Bermúdez-Humarána,∗

INRA 0910, 78352 Jouy-en-Josas, FranceINRA 0892, 78352 Jouy-en-Josas, France

r t i c l e i n f o

rticle history:eceived 15 July 2008eceived in revised form 18 August 2008ccepted 19 August 2008vailable online 13 September 2008

eywords:

a b s t r a c t

Chemokines have been described as essential mediators in leukocytes migration to inflammatory sitesand to secondary lymphoid organs. Mig and IP-10 are two CXC chemokines that recruit mononuclearcells in vivo and inhibit angiogenesis. In addition to their chemotactic roles, Mig and IP-10 have alsoan important role in the adaptative immune response. In this study, we asked whether a food-gradebacterium, Lactococcus lactis, is able to produce a fusion protein comprising Mig and IP-10 (Mig::IP-10).The activity of the recombinant Mig::IP-10 produced by the genetically engineered L. lactis (LL-Mig::IP-10)

hemokinesdjuvantactococcus lactisig

P-10ucosal vaccines

was confirmed in a murine spleen cells chemotaxis assay. Moreover, the adjuvant properties of LL-Mig::IP-10 strain were evaluated in mice by the co-expression of a model antigen, the human papillomavirus type16 E7 protein. Our data show that LL-Mig::IP-10 can produce a genetic fusion of Mig::IP-10 biologicallyactive. This recombinant strain represents a potential candidate for the development of new strategies formucosal vaccination.

© 2008 Elsevier Ltd. All rights reserved.

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. Introduction

Chemokines are a group of small secreted proteins that inducenflammatory response by orchestrating the selective migrationnd activation of leukocytes [1]. Mig and IP-10 are two structurallyelated CXC chemokines, markedly induced by IFN-�. They sharehe same receptor (CXCR3) and are chemotactic for activated T-ymphocytes and NK cells [2–6]. In addition to their pronouncedhemotactic activities, both have been shown to inhibit angiogen-sis and thus exhibit anti-tumoral properties. For example, IP-10an generate a T-lymphocyte-dependent anti-tumor response byecruiting other lymphocytes, neutrophils, and monocytes and Mignhibits neovascularization in vivo [7,8].

Escherichia coli is the most extensively used bacterial host toroduce recombinant proteins. However, most eukaryotic proteins

re typically obtained as insoluble, misfolded inclusion bodies thateed solubilization and refolding. Lactococcus lactis is a Gram-ositive lactic acid bacterium widely used for the production ofeterologous proteins of vaccinal and therapeutic interest [9,10]. It

∗ Corresponding author. Tel.: +33 134 65 24 63; fax: +33 134 65 24 62.E-mail address: luis.bermudez@jouy.inra.fr (L.G. Bermúdez-Humarán).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.08.044

as also been demonstrated that L. lactis can efficiently produce andecrete molecules in a soluble and bioactive form [11–17]. Further-ore, L. lactis is a food-grade bacterium that could be used to deliver

roteins at the mucosal level [9,10]. It thus represents a potentialandidate for the production of biologically active molecules, suchs Mig and IP-10 chemokines.

In this study, we tested whether L. lactis is able to produce ausion protein comprising IP-10 and Mig (Mig::IP-10) biologicallyctive. Furthermore, we tested whether this chimeric protein pro-uced by genetically engineered L. lactis has adjuvant activitieshen co-delivered with a vaccinal antigen.

. Materials and methods

.1. Bacterial strains, plasmids, and DNA manipulations

E. coli was grown in Luria Bertani medium at 37 ◦C with vigoroushaking and L. lactis was grown in M17 (Oxoid, France) supple-ented with 0.5% glucose (GM17) at 30 ◦C without shaking. Plasmid

onstructions were established in E. coli by heat-shock [18] or in L.actis by electroporation [19]. Recombinant clones were selectedy the addition of chloramphenicol at 10 �g/ml. DNA manipu-ations were essentially performed as described previously [18].olymerase chain reaction (PCR) (PerkinElmer Cetus apparatus,

N.G. Cortes-Perez et al. / Vaccine 26 (2008) 5778–5783 5779

Fig. 1. Schematic representation of pSEC–Mig::IP-10 plasmid and expression of Mig::IP-10 by L. lactis. (A) A 605 bp DNA fragment encoding mature Mig::IP-10 was fused inf , deriv

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rame with a DNA fragment containing the Usp45 signal peptide (SEC–Mig::IP-10)

xpression is controlled by the nisin-inducible promoter (PnisA) and harbors the Uster) [20] for clone stability. The pSEC–Mig::IP-10 carries the pC194 chloramphenicoor 1 h. The anti-IP-10 monoclonal antibody detected a band in total protein extract

orwalk, CT, USA) was performed using High Fidelity PCR enzymeix (Fermentas, France) and PCR sequences were confirmed by

equencing (MWG-Genomic Company, Germany).

.2. Construction of a plasmid for controlled Mig::IP-10roduction in L. lactis

In order to produce murine Mig and IP-10 CXC chemokines in. lactis (Fig. 1A), pSEC–Mig::IP-10 plasmid was constructed. Theusion gene Mig::IP-10 was PCR amplified from pBLAST–mMig::IP-0 plasmid (InvivoGen, France). In this vector, murine Mig genewithout the stop codon) is fused to murine IP-10 mature sequenceevoid of its signal peptide (SP) by a ten-amino acid elas-ic linker (VPGVGVPGVG), which allows proper folding of theusion protein. Briefly, a 605 bp DNA fragment encoding mature

ig::IP-10 was PCR amplified from pBLAST–mMig::IP-10 plasmidith primers: NsiI–Mig::IP-10 (5′-CCAATGCATCAACCCTAGTGATAA-GAATGCACG-3′) for the coding strand and SpeI–Mig::IP-10

5′-GGACTAGTCCAGTTAAGGAGCCCTTTTAGACC-3′) for the comple-entary strand. Primer NsiI–Mig::IP-10 was designed to delete the

rst 21 codons in the Mig::IP-10 coding sequence. These codonsncode the Mig SP that is replaced by the lactococcal SPUSP45.he PCR product was subcloned into a pCR-TOPO kit (Invitrogen,arlsbad, CA) resulting in pCR:TOPO-Mig::IP-10. Afterwards, theNA fragment encoding mature Mig::IP-10 was obtained fromCR:TOPO-Mig::IP-10 with NsiI/SpeI and cloned into purified back-one isolated from NsiI–SpeI-cut pSEC-E7 vector [20], resulting inSEC–Mig::IP-10 (Fig. 1A). This vector was established into NZ9000train (a MG1363 L. lactis derivative carrying chromosomal inte-rated nisRK genes, [21] to obtain LL-Mig::IP-10 strain. We useds a negative control, a NZ9000 strain transformed with the pSECmpty vector (LL).

.3. Inducible expression of Mig::IP-10

Bacterial strains were grown until OD600 = 0.4–0.6 and induc-ion with 10 ng/ml of nisin (Sigma) for 1 h was performed. Proteinxtractions and immunoblotting assays were performed as previ-

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ed from the predominant L. lactis-secreted protein [40] . In this vector, Mig::IP-10

bosome binding site (RBS) and the rho-independent trpA transcription terminatortance marker (cm). (B) Strain LL-Mig::IP-10 was grown and induced with 10 ng/mlLL-Mig::IP-10 cultures with an apparent molecular mass of ∼18 kDa.

usly described [20]. Antibodies used were mouse anti-IP-10 (G-15,anta Cruz Biotechnology, Inc.) and human anti-Mig (H-40, Santaruz Biotechnology, Inc.).

.4. Animals

Specific pathogen-free C57BL/6 mice (females, 6–8 weeks of age;harles River Breeding Laboratories) were housed in a pathogen-

ree isolator under sterile conditions with water and fed ad libitumn the animal facilities of the Unité d’Ecologie et de Physiologie duystème Digestif at the National Institute of Agricultural ResearchJouy-en-Josas, France). All experiments were performed accordingo protocols in accordance with institutional guidelines.

.5. In vitro chemotaxis assay

The chemoattractive properties of the fusion protein Mig::IP-10roduced by recombinant L. lactis were evaluated in chemotaxisxperiments [22–24] using splenocytes isolated from mice in a 24-ell Transwell plates (5-�m pore filters; Corning Costar). Briefly,

00 �l of a cellular suspension of splenocytes (1 × 107 cells/ml) inPMI supplemented with 10% of fetal calf serum (FCS) was placedn the top of the filter and 400 �l of either supernatant samplesrom induced cultures of LL or LL-Mig::IP-10 strain was placed inhe lower wells. As controls, we added either supplemented RPMIr supplemented RPMI plus 400 �l of FCS 100%. The plates werencubated for 3 h at 37 ◦C and 5% CO2. Cells that had migratedhrough the membrane were harvested from the lower wells andounted under a microscope. The results are expressed as the meanalue of duplicates ±S.D. In some experiments, migrating cells werearvested and counted for 60 s using fluorescence-activated cellorting (FACS) analysis. Data on transmigrated cells from duplicateells are expressed as number migrated cells (total events) ±S.D.

.6. Administration of live L. lactis strain co-producing Mig::IP-10nd E7 antigen to mice

To construct a recombinant L. lactis strain co-expressing Mig::IP-0 and E7 antigen, we introduced a second plasmid encoding a cell

5 Vaccine 26 (2008) 5778–5783

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Fig. 2. (A) Chemoattractive properties of the fusion protein Mig::IP-10 produced byrecombinant L. lactis. Enhanced migratory response of murine splenocytes towardsupernatants of LL-Mig::IP-10 cultures compared with LL control. Data are shownas mean (n = 3); bars, ±S.D. (B) Production levels of IFN-� from spleen cells in miceimmunized with LL-Mig::IP-10/E7. Five C57BL/6 mice were intranasally immunizedotsr

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all-anchored form of human papillomavirus type 16 (HPV-16) E7ntigen [25] into the LL-Mig::IP-10 and the resulting recombinanttrain, LL-Mig::IP-10/E7, was selected in GM17 containing chloram-henicol (10 �g/ml) and erythromycin (2.5 �g/ml). Live bacterial

nocula of both LL-E7 and LL-Mig::IP-10/E7 strains were preparednd induced as previously described [20]. Briefly, cellular pelletsere harvested by centrifugation at 3000 × g at 4 ◦C and washed

hree times with sterile PBS. The pellet was suspended in PBS to anal concentration of 1 × 109 CFU. Groups of C57BL/6 mice (5 miceer group) were immunized intranasally with 1 × 109 CFU fromach induced recombinant lactococci strain suspended in 10 �l ofBS (5 �l was administered with a micropipette into each nostril)n days 0, 14, and 28. Control mice received identical quantities ofL strain.

.7. Sample collection

Seven days after the last immunization (day 35) sera, bron-hoalveolar lavages (BAL) and gastrointestinal lavage (GAL) wereollected. Blood samples were obtained from the retro-orbitalenous plexus, centrifuged and sera stored at −20 ◦C until fur-her analysis. Mice were sacrificed by vertebral dislocation and BALnd GAL fluids were collected in sterile conditions as previouslyescribed [26].

.8. Evaluation of the humoral and systemic immune response

Detection of IgG and IgA E7-specific antibodies were performeds previously described [26]. For the determination of E7-specific-cells, mice were sacrificed 1 week after the last immunizationnd splenocytes isolated. Single-cell suspension of splenocytes1 × 106 cells/well) were stimulated overnight with a combinationf two E7 peptides: E749–57 [27] and E730–67 [28] and 0.5 ng/mlIL-2 (R&D Systems, England) in a 24-well plate in 1 ml of RPMI640 supplemented with 10% FCS at 37 ◦C under 5% CO2. After4 h, cells were harvested, washed, and plated at a concentra-ion of 2.5 × 105 cells/well in a 96-well microplate coated with

onoclonal-specific mouse IFN-� Ab. Measurement of E7-specificcells were allowed to dry before counting the individual spotsith a dissecting scope.

.9. Statistical analyses

Statistical analyses were performed using StatsDirect softwareversion 2.6.2). Kruskal–Wallis, all pairwise comparison test wassed to evaluate the difference between means. A value of P < 0.05as considered significant.

. Results and discussion

.1. Construction and characterization of a MIG::IP-10-producing. lactis strain

The ability of L. lactis to produce Mig::IP-10 was examined bymmunoblotting using mouse anti-IP-10 antibody. Total proteinxtracts were prepared from non-induced and induced lacto-

occi cultures. As shown in Fig. 1B, a clear band at the expectedize for mature Mig::IP-10 (∼18 kDa) was observed from inducedL-Mig::IP-10 samples. No Mig::IP-10 signal was detected innduced cultures of the negative control (L. lactis transformed

ith empty vector, LL) (data not shown). These results showhat L. lactis can produce successfully a genetic fusion of Mig::P-10.

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n days 0, 14, and 28 with strains LL, LL-E7, and LL-Mig::IP-10/E7. One week afterhe last immunization (day 35), splenocytes from immunized mice were pooled andtimulated in vitro with E7 peptides for identification of IFN-�-producing T-cells,espectively, by ELISPOT.

.2. Mig::IP-10 produced by recombinant L. lactis is biologicallyctive

To demonstrate that Mig::IP-10 produced by recombinant L.actis is biologically active, a major consideration when express-ng recombinant proteins in a heterologous host, we performedn in vitro chemotaxis test. As observed in Fig. 2A, the migrationssay showed clearly that supernatants from induced LL-Mig::IP-0 cultures, in contrast to those from induced LL cultures,ere able to induce the migration of murine splenocytes, con-rming that recombinant Mig::IP-10 produced by L. lactis isiologically active. As positive control of the experiment, wesed FCS, well known for its chemotactic properties (data nothown).

.3. Intranasal administration of L. lactis co-expressing

ig::IP-10 and HPV-16 E7 protein in mice enhances an

ntigen-specific immune response

In previous studies, we have shown that intranasal adminis-ration of a L. lactis strain expressing a cell wall-anchored form

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f HPV-16 E7 antigen (LL-E7) induce an antigen-specific T-cellesponse in mice [25,26,29]. Moreover, co-administration of LL-7 with either a L. lactis strain secreting interleukin-12 or a L.

actis strain secreting leptin had adjuvant effects, resulting in annhancement of the immune response to the co-delivered anti-en [15,16,30]. Since recent studies have suggested an emergingole of Mig and IP-10 CXC chemokines to enhance adaptive immu-ity [31–33], we evaluated whether intranasal co-administration

n mice of LL-Mig::IP-10 and LL-E7 could induce a higher antigen-pecific immune response than after administration of LL-E7 alone.or this, 3 groups of 5 mice were immunized on days 0, 14 and 28ith 1 × 109 CFU of either LL control strain, LL-E7, or a L. lactis strain

o-expressing Mig::IP-10 and E7 antigen (LL-Mig::IP-10/E7). Oneeek after the last immunization, splenocytes from immunized

nimals were used for detection of IFN-�, a cytokine characteristicf a Th1 type of immune response. As shown in Fig. 2B, immuniza-ion with LL-Mig::IP-10/E7 resulted in a significant enhancement ofFN-� secretion by CD4+ and CD8+ lymphocytes in response to E7-erived peptides [27,28]. These results show that co-administration

n mice with LL-E7 and LL-Mig::IP-10/E7 induces E7-specific T-cellesponses.

We also determined the humoral immune response (i.e. E7-pecific IgG and IgA antibodies) induced after the intranasaldministration of the different L. lactis strains. As shown in Fig. 3,

ntranasal administration of LL-E7 and LL-Mig::IP-10/E7 strains ledo higher anti-E7 IgG in serum in comparison with the control LLtrain, but without reaching statistical significance (Fig. 3A). ForAL and GAL samples, we observed that anti-E7 IgG levels wereigher in mice administered with LL-E7 and LL-Mig::IP-10/E7 than

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ig. 3. Anti-E7 IgG antibodies in mice vaccinated with LL-Mig::IP-10/E7. Groups of mice wast immunization anti-E7 IgG antibodies were determined by ELISA in serum (A), BAL (Bbtained with JMP software after comparison of treated groups with the control.

e 26 (2008) 5778–5783 5781

n the control group (Fig. 3B and 3C). Furthermore, anti-E7 IgG levelsrom animals immunized with LL-Mig::IP-10/E7 were substantiallyigher than in mice immunized with the strain expressing only E7ntigen.

For anti-E7 IgA levels in serum, we observed that mice admin-stered with LL-E7 and LL-Mig::IP-10/E7 led to significant highernti-E7 IgA levels than control animals (Fig. 4A and B). Interestingly,nti-E7 IgA levels were substantially higher in animals immu-ized with LL-Mig::IP-10/E7 than mice immunized with the strainxpressing only E7 antigen. For anti-E7 IgA levels in GAL samples,nly mice administered LL-Mig::IP-10/E7 led to significant highernti-E7 IgA levels than LL-E7 and LL strains (Fig. 4A and C). Noignificant differences were observed for anti-E7 IgA levels in BALamples between immunized and control animals, although a weaknhancement was observed in mice treated with LL-E7 and LL-ig::IP-10/E7 (Fig. 4B). We already observed this phenomenon inprevious work [26] and we hypothesize that it could be related

o an early production of mucosal IgA by B cells (residing in orga-ized nasal-associated lymphoid tissues; e.g. BAL samples) and a

ater production of IgA by distant mucosal effector tissues (e.g. GALamples), as previously demonstrated [34].

Altogether, these results suggest that Mig::IP-10 delivered byenetically engineered L. lactis may have interesting immunostim-latory properties in the context of mucosal vaccination. Strikingly,

o weight loss was observed in mice receiving recombinant L.

actis at the end of the experiments and no visible toxicity wasetected after careful examination during autopsy, confirming thathe vector is well tolerated, a prerequisite in the use of a mucosaldjuvant.

ere intranasally immunized with either LL, LL-E7 or LL-Mig::IP-10. Seven days after) and GAL (C) samples. Bars represent the mean standard deviations. The P values

5782 N.G. Cortes-Perez et al. / Vaccine 26 (2008) 5778–5783

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ig. 4. Anti-E7 IgA antibodies in mice vaccinated with LL-Mig::IP-10/E7. Groups of mast immunization anti-E7 IgA antibodies were determined by ELISA in serum (A),btained with JMP software after comparison of treated groups with the control.

. Conclusion

Due to the involvement of chemokines in leukocytes trafficking,hese molecules have become attractive candidates for therapeu-ic uses [1]. Recombinant chemokines are generally produced in E.oli; however when expressed in this host, chemokines are poorlyoluble and accumulate in inclusion bodies [35,36]. Several purifi-ation methods have been described but involve time-consumingefolding, buffer exchange, and purification steps that complicatexpression of these proteins. Here, we describe an elegant approacho produce recombinant Mig and IP-10 CXC chemokines in theood-grade bacterium, L. lactis. This bacterium produces very lowmounts of native exoproteins and is thus a good candidate for het-rologous protein production in different applications ranging fromndustrial production of high-added-value proteins to in vivo use aslive bacterial vector [37,38].

Intranasal administration of food-grade bacteria expressingntigens has been proposed as an alternative vaccination approachor the stimulation of mucosal and systemic immune responses39]. In addition, concomitant delivery of immunomodulatory

olecules with food-grade bacteria expressing vaccinal antigensas been reported to have adjuvant effects [15,16,30]. In this regard,ur study reveals for the first time the potential adjuvant effectf a recombinant fusion protein comprising Mig and IP-10 wheno-delivered with a vaccinal antigen.

cknowledgment

We thank Dr. Nicolas Bertho for useful help in chemotaxis exper-ments.

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ere intranasally immunized with either LL, LL-E7 or LL-Mig::IP-10. Seven days after) and GAL (C) samples. Bars represent the mean standard deviations. The P values

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