comparative immunogenicity analysis of modified vaccinia ankara vectors expressing native or...

Vaccine 22 (2004) 3917–3928

Comparative immunogenicity analysis of modified vacciniaAnkara vectors expressing native or modified forms

of hepatitis C virus E1 and E2 glycoproteins

Jean-Daniel Abrahama, Nourredine Himoudib, François Kiena, Jean-Luc Berlandc,Audrey Codrana, Birke Bartoschd, Thomas Baumerte, Glaucia Paranhos-Baccalac,

Catherine Schustera,∗, Geneviève Inchauspéb, Marie Paule Kienyaa INSERM U544—Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France

b UMR 2142 CNRS/BioMérieux—ENS LYON, 46 allée d’Italie, 69364 Lyon Cedex 07, Francec UMR 2142 CNRS/BioMérieux—CERVI, 21 Av. Tony Garnier, 69365 Lyon Cedex 07, France

d INSERM U412—IFR 128—ENS LYON, 69364 Lyon Cedex 07, Francee Department of Medicine II, University of Freiburg, Freiburg, Germany

Received 3 July 2003; received in revised form 7 April 2004; accepted 9 April 2004Available online 12 May 2004

Abstract

We have evaluated in C57/Bl6 and HLA-A2.1 transgenic mice the immunogenicity of three MVA vectors expressing either native HCVE1E2 polyprotein, truncated and secreted E1 (E′1311) and E2 (E′2661) proteins, or a chimeric E1E2 heterodimer presented at the plasmamembrane. Immunization induced mainly a Th1 response in HLA-A2.1 transgenic mice while a Th2-type response was detected in C57/Bl6mice. Comparison of the three vectors shows an increase in the humoral response when antigens are secreted or membrane bound, andslightly in the cellular response when antigens are exposed on the cell surface.© 2004 Elsevier Ltd. All rights reserved.

Keywords:HCV; MVA; Immunization; Humoral and cellular responses

1. Introduction

HCV is a single-stranded RNA (+) virus belonging tothe Flaviviridae family, encoding a polyprotein precursorwhich is cleaved by both host cell and viral proteases intostructural (core, E1, E2, p7) and non structural (NS2, NS3,NS4A, NS4B, NS5A, NS5B) proteins[1]. HCV is believedto infect 200 millions people worldwide and represents themajor cause of cirrhosis and hepatocellular carcinoma, as80% of infection become chronic. It is thought that the re-maining 20% corresponding to spontaneous clearance arecorrelated with a vigorous, durable and polyclonal cellularresponse[2]. In particular, Thimme et al. have shown thatthe drop in viremia in HCV-infected patients is associatedwith an increased IFN-� response[3], what was confirmedrecently in chimpanzee studies[4]. Nevertheless, an effec-

∗ Corresponding author. Tel.:+33-3-90-24-37-41;fax: +33-3-90-24-37-23.

E-mail address:[email protected] (C. Schuster).

tive humoral response can be protective against HCV infec-tion. In particular, it has been shown that specific antibodiestargeted to hypervariable region 1 (HVR-1) of E2 are neu-tralizing [5–7].

HCV E1 and E2 glycoproteins are type I integral trans-membrane proteins[8,9]. E2 interacts with E1 to form astable noncovalently-linked heterodimer or heterogeneousdisulfide-linked aggregates[10,11], which are believed toresult from a non productive folding pathway. Immunolo-calization studies and glycans analyses have shown thatHCV glycoproteins are located in the endoplasmic retic-ulum (ER) [12,13]. Deletion of the transmembrane (TM)domains of E1 and E2 has shown that these truncated formsof HCV glycoproteins are secreted into the extracellularmedium[14,15]. In addition, studies of chimeric proteins inwhich the TM domains were exchanged for correspondingdomains of proteins transported to the plasma membranehave shown that these TM domains play a major role in theER localization of the glycoprotein complex[16,17]and areinvolved in the assembly of noncovalent E1E2 heterodimers

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2004.04.005

3918 J.-D. Abraham et al. / Vaccine 22 (2004) 3917–3928

[15,18,19]. These chimeric proteins are moreover able tomigrate to the plasma membrane, but fail to heterodimerize.The addition of the Fos and Jun leucine zipper dimer-ization motifs have been reported to allow the assemblyand secretion of soluble recombinant HLA-DR2 molecules[20]. Based on these results, we used these leucine zip-per dimerization peptides to assemble on the cell surfacean heterodimeric HCV E1-VSVGTM-(AlaGly)2-JunB andE2-RVGTM-(AlaGly)2-Fra2 glycoproteins complex (Kienet al., in preparation).

Modified Vaccinia Virus Ankara (MVA) is a highly atten-uated strain of vaccinia virus (VV) obtained following over500 passages of the Ankara VV isolate in Chicken EmbryoFibroblasts (CEF)[21]. It has been shown to be drasticallyattenuated when tested in several animal models. In addition,no significant side-effects have been observed when MVAwas administrated to about 150 thousand healthy individuals[22]. Replication-deficient MVA is therefore established asan exceptionnally safe viral vector and has been used widelyin Germany, France and the USA under laboratory biosafetylevel 1 conditions[23]. MVA replication-deficiency is as-sociated with six major deletions that occured during thepassages in CEF cells[24]. Sequences or restriction sites lo-cated at the vicinity of these deletions can be used to clonerecombinants cDNA in order to use MVA as a gene transfervector [25]. Many prototype vaccines based on MVA havebeen engineered following this principle[26,27]. This vec-tor thus presents all the advantages required for the designof a vaccination tool.

In this study, we analyze the modulation of the cellularand humoral immune responses induced by MVA vectorsexpressing either the native E1E2 polyprotein, secreted E1and E2 proteins, or a membrane bound E1E2 heterodimer.C57/Bl6 mice were used for analyses of proliferative andhumoral responses, and transgenic HLA-A2.1 mice wereused for analyses of specific cytotoxic and Th1 responses.

2. Materials and methods

2.1. HCV sequences

E1 and E2 sequences were amplified by PCR from aHCV-JA strain (1b genotype) partial cDNA kindly providedby Dr. Kunitada Shimotohno (Laboratory of Human TumorViruses, Department of Viral Oncology, The Institute forVirus Research, Kyoto University, Kyoto 606-8937, Japan)[28]. Recombinant viruses expressing the E1E2 polyproteinwere engineered so that the E1E2 sequence is placed underthe control of a single promoter.

2.2. Recombinant MVA construction

Plasmid pIV250 for homologous recombination at a sitecorresponding to MVA genome deletion II is based on

plasmid pTG6018 (TRANSGENE S.A., France) contain-ing the flanking sequences (BRG2 and BRD2) surroundingthe so-called deletion II of MVA. TheEscherichia coligpt (xanthine-guanine phosphoribosyltransferase) selectiongene [29] downstream of the early-late VV pH5R pro-moter (obtained from plasmid pTG9997; TRANSGENES.A., France) was used to facilitate selection of recom-binant viruses. Plasmids containing PCR amplified geneswere sequenced for verification of the absence of accidentalmutations.

2.2.1. pIV257The DNA sequence encoding the E1E2 polyprotein and

the preceding E1 signal sequence (aa 170–746) was intro-duced into the MVA transfer plasmid vector (pIV250) down-stream of the early-late VV pH5R promoter, generating plas-mid pIV257.

2.2.2. pIV306Cloned DNA encoding the complete G protein of VSV

(pSVGLI) was a generous gift of J.K. Rose (Yale Uni-versity, New Haven, USA) (Fig. 1A). Modified vacciniaAnkara (MVA) transfer plasmid vectors pTG6018 and 9997,and plasmid pTG9128 containing the rabies G glycopro-tein cDNA were obtained from TRANSGENE S.A., France.Plasmids pLexA408-hJunBZip and pLexA408-hFra2Zip wereused to excise the leucine zipper motifs of human Jun-Band Fos related antigen 2 (Fra2) molecules (M. Dimitrova,unpublished results).

To construct E2-RVGTM-(AlaGly)2-Fra2, a DNA se-quence encoding the ectodomain of E2 and the precedingsignal sequence (aa 371 to 661) was first joined to the ra-bies G protein TM sequence using overlap extension PCR.The resulting E2-RVGTM PCR product was then joinedto the leucine zipper sequence of Fra2 using overlap ex-tension PCR with synthetic oligonucleotides covering the(AlaGly)2 spacer. The E2-RVGTM-(AlaGly)2-Fra2 DNAsequence was introduced into the MVA transfer plasmidvector (pIV250) downstream of the early-late VV pH5Rpromoter, generating plasmid pIV305.

To construct E1-VSVGTM-(AlaGly)2-JunB, a DNA se-quence encoding the ectodomain E1 and the preceding sig-nal sequence (aa 170–311) was first joined to the VesicularStomatitis Virus G protein TM sequence using overlap exten-sion PCR. The resulting E1-VSVGTM PCR product was thenjoined to the leucine zipper sequence of JunB using overlapextension PCR with synthetic oligonucleotides covering the(AlaGly)2 spacer. The E1-VSVGTM-(AlaGly)2-JunB DNAsequence was subsequently introduced into pIV305 down-stream of the early-late VV p7.5 promoter, generating plas-mid pIV306.

2.2.3. pIV316The DNA sequence encoding the E2 ectodomain and the

preceding signal sequence (aa 371–661) was introduced intothe MVA transfer plasmid vector pIV250, downstream of the

J.-D. Abraham et al. / Vaccine 22 (2004) 3917–3928 3919

Fig. 1. (A) Schematic representation of chimeric E1 and E2 proteins expressed by vvIV306. VSVGTM stands for transmembrane domain from the Gprotein from the vesicular stomatitis virus, and RVGTM stands for transmembrane domain from the G protein from the rabies virus. (B) Native andchimeric E1 and E2 glycoproteins detection; vvIV257- and vvIV306-infected Huh-7 cells were lysed at 48 h after infection (MOI 0.1) and intracellularE1and E2 proteins were detected by Western Blot analysis using rabbit anti-E1 and monoclonal anti-E2 (H52, Dubuisson). (C) E1 and E2 labelling at theplasma membrane; vvIV257- and vvIV306-infected Huh-7 cells were labelled at 48h after infection (MOI 0.1) without permeabilization with monoclonalanti-E1 (A4, Dubuisson) or monoclonal anti-E2 (H52, Dubuisson) primary antibodies, followed by FITC-conjugated anti-mouse Fab’ secondary antibody,and analysed by flow cytometry (FACscan, Beckton Dickinson). Results are expressed as percent of positive cells as compared to MVA WT-infectedcells. Only vvIV306-infected cells show intense labelling, revealing expression of both chimeric enveloppe protein at plasma membrane.


early-late VV pH5R promoter, generating plasmid pIV265.The DNA sequence encoding the E1 ectodomain and thepreceding signal sequence (aa 170–311) was introduced intopIV265 downstream of the early-late VV p7.5 promoter,generating plasmid pIV316.

2.3. Recombinant MVA vectors production, purificationand titration

Generation of vvIV257, vvIV306 and vvIV316 was per-formed by homologous recombination in primary CEF[30]between pIV257, pIV306 or pIV316 transfer plasmids andMVAN33 vaccinia virus strain (TRANSGENE S.A., France)referred as to MVA WT. Recombinant MVA vectors wereisolated and amplified by successive passages on CEF with aselection cocktail as described previously[29]. After clear-ing of the cell lysate, viruses were purified by two ultracen-trifugation steps (14,000 rpm, 2 h) on 36% sucrose cushionsfollowed by two ultracentrifugations (14,000 rpm, 2 h) on20–40% sucrose gradients, and stored in Tris–HCl 10 mMpH 8.0 buffer at−20◦C. Virus titers are expressed as plaqueforming units per ml (pfu/ml).

2.4. Recombinant adenovirus construction, productionand titration

Recombinant adenoviral genomes were generated as in-fectious plasmids by homologous recombination inE. colias described previously[31]. The two plasmids (pTG 13387and pTG 6624) containing the adenoviral genome werekindly provided by TRANSGENE S.A. In brief, a sequencefrom the HCV-JA strain corresponding to the E1E2 polypro-tein and the preceding E1 signal sequence (aa 170–746) wasinserted into the adenoviral shuttle plasmid (pTG 13387)containing a CMV-driven expression cassette surrounded byadenoviral sequences (nt 1–458 and nt 3511–5788) to allowhomologous recombination with the adenoviral sequencesof the backbone vector (pTG 6624)[31]. The resultingfull-length viral genome (pIV 248) contains a deletionin the adenoviral E3 genes (nucleotides 28,592–30,470),whereas the E1 region (nucleotides 459–3511) is replacedby the expression cassette containing, from 5′ to 3′, theCMV immediate-early enhancer/promoter, a chimeric hu-man b-globin/IgG intron, the HCV E1E2 sequence, and thebovine growth hormone polyadenylation signal. Recombi-nant adenoviruses were generated by transfection into the293 complementation cell line of the corresponding plas-mids restricted byPacI digestion. Virus propagation andamplification were performed by successive passages on293 cells. Recombinant adenovirus (AdIV248) purificationand titration by indirect immunofluorescence detection ofthe viral DNA binding protein (DBP) were carried out asdescribed previously[32]: after clearing of the cell lysate,the viruses were purified by a two-rounds CsCl densitycentrifugation. Purified viruses were stored in 1 M sucrose,10 mM Tris–HCl, pH 8.5, 1 mM MgCl2, 150 mM NaCl,

and their titration was performed by detection of the DBPat 18 h after 293 cells infection, using purified a72K B6-8hybridoma supernatant provided by TRANSGENE S.A.[33]. Ad(b-gal), an adenovirus allowing the production ofb-galactosidase, was provided by TRANSGENE S.A. andused as control. Virus titers are expressed as InfectiousUnits per ml (IU/ml).

2.5. Animals

C57/Bl6 mice (IFFA-CREDO) were used for analysisof E1 and E2 specific proliferative immune responses andfor detection of anti-E1 and anti-E2 antibodies. HDD micetransgenic for HLA-A2.1 (A0201) monochain histocompat-ibility class I molecule and deficient for both H-2Db andmurine b2-microglobulin (b2m) were used for ELISPOTsand CTL analyses. The H-2Db and mouse b2m genes ofthese mice have been disrupted by homologous recombina-tion [34]. Six to eight weeks-old female mice were used foreach experiment. Mice were hosted in appropriate animalcare facilities and handled following international guidelinesrequired for experiments with animals.

2.6. Proliferative response analyses

Proliferation of mice splenocytes was assessed by theuptake of [3H]methyl-thymidine (Amersham, TRK 120).Splenocytes were seeded in U-bottom 96-wells plate at105 cells/well after reticulocytes lysis in NH4Cl 0.15 M;EDTA 0.1mM; KHCO3 1 mM; pH 7.2 buffer, and culturedin different conditions: either in the presence of 100 ng/mlmurine IL2 (Sigma) or 10�g/ml ConA (Sigma C) for thepositive control; in culture medium composed of RPMIwith glutamax (Invitrogen) supplemented with 10% fœtalcalf serum (Life Technologies), 1mM sodium pyruvate(Sigma), 1× non-essential amino acids (Life Technologies)and 5× 10−5 M b-mercaptoethanol (Life Technologies)for the negative control; or in the presence of recombinantadenoviruses (Ad b-gal as negative control or AdIV248expressing HCV E1E2 polyprotein) at a multiplicity of in-fection (MOI) of 1000. After 96 h incubation, cells werepulsed with 1�Ci/well [3H]methyl-thymidine for 18 h. Af-ter three cycles of freezing and thawing, radioactivity wascollected on glass fiber filters (Wallac) with a microcellharvester (Skatron) and quantified with a liquid scintil-lation counter (Betaplate, Pharmacia). Experiments wereperformed in triplicate. Specific proliferation is expressedas the ratio of the mean splenocytes proliferation with ac-tivation (adenovirus infection) on the mean proliferationwithout activation (culture medium).

2.7. Synthetic peptides

Synthetic peptides were synthesized and purified to >95% purity (Neosystem, Strasbourg, France). Peptides


were dissolved in DMSO (20�l/mg) and subsequently di-luted in distilled water to a final concentration of 1mM.All peptides were derived from the HCV-JA sequence;from E1: TIRRHVDLLV (aa 257–266) referred to as TIR;from E2 glycoprotein: RLWHYPCTI (aa 614–622) referredto as RLW.

2.8. ELISPOTs

Four weeks after the first immunization, IFN-� releas-ing cells were quantified by cytokine-specific enzyme-linkedimmunospot assay (ELISPOT) as previously described[35].Briefly, splenocytes from individual mice were isolated andplated directly at a concentration of(3–5) × 105 cell/well.Cells were cultured in complete culture medium and 10 U/mlrecombinant IL-2 (Pedro-Tech EC Ltd.). Splenocytes werecultivated for 48 h either in culture medium (negative con-trol), with 10�g/ml of selected peptide in the presence ofIL-2 (5 U/ml), or with 5�g/ml of ConA (positive control)in triplicate wells. Wells were washed three times with PBS0.05% Tween and PBS before a 2h incubation with biotiny-lated anti-murine IFN-� (dilution 1/500, Pharmingen). Afterwashing, wells were incubated for 1 h with extravidin-PALconjugate (dilution 1/6000 in PBS-BSA 1%, Sigma). Enzy-matic activity was revealed using the BIO-RAD kit: 100�lof AP color reagent A and 100�l AP color reagent B weremixed in 25× AP color development buffer (400�l added to9.6 ml distillated water) (BIO-RAD). After the appearance ofspots (10 min max), the reaction was then stopped by incuba-tion with water, the plates air-dried and spots counted (Spot,KS Zweiss, Carl Zweiss Vision, GmbH, Germany). Back-ground level was determined in wells containing splenocytesin medium only. The number of peptide-specific spots wasobtained by substracting the background from the numberof spots appearing after HLA-A2.1-peptide stimulation. Re-sults are shown as the mean value obtained for triplicatewells.

2.9. CTL analyses

CTL analyses were performed as previously described[36]. Briefly, on day 7, peptide stimulated spleen cells wereused as effectors in a CTL assay. Specific cytolytic ac-tivity was tested in a standard51Cr release assay againstEL-4S3−Rob HHD target cells pulsed or not with 10�Mof selected peptide. After a 4 h incubation period,51Cr re-lease was measured using a�-Cobra II counter (Packard,Rungis, France). Spontaneous and maximum release weredetermined from wells containing either medium alone orlysis buffer (1N, HCl). The percent specific cytotoxicity wascalculated by the formula: [(release in assay with peptide−spontaneous release with peptide)/(maximum release withpeptide− spontaneous release with peptide)]− [(release inassay without peptide− spontaneous release without pep-tide)/(maximum release without peptide− spontaneous re-

lease without peptide)]× 100. For each Effector/Target (E/T)ratio, results are expressed as mean of duplicates.

2.10. Anti-HCV envelope antibodies assay

Blood samples from C57BL/6 (H-2Db) were collectedfrom cardiac punction after 4 weeks from the first immu-nization and analyzed for HCV E1 and E2 antibodies byenzyme-linked immunosorbent assay (ELISA). Mice immu-nized with MVA WT were used to calculate the cut-off value,obtained by adding three times the standard deviation to themean value. Purified recombinant E1 protein produced inE.coli and secreted E2 glycoprotein from supernatant of CHOcell lines, genotype 1b, were used as solid-phase ligands inELISA. The recombinant antigens were coated at 4�g/mlfor E1 protein and 1�g/ml for E2 protein onto 96-well mi-crotiter plates in 50 mM sodium carbonate buffer, pH 9.6,overnight at room temperature. First, serum samples fromMVA WT-, vvIV257-, vvIV306- and vvIV316-immunizedmice were screened at a dilution of 1:100 for the presenceof total IgG against HCV E1 and E2 recombinant antigensby the EIA protocol described below. Positive sera werethen serially diluted to obtain end-point titers. Horseradishperoxidase (HPR)-labeled goat anti-mouse IgG antibody(1:10,000) (Promega, Madison,WI, USA) was used as theprobe to determine IgG end-point titers.. Sera were diluted at1:100 in dilution buffer (50 mM sodium carbonate, 100 mMsodium sulfate, 0.05% Tween 20, 10% of heat-inactivatedgoat serum, pH 9.0) and plated at 100�l/well. Diluted serawere allowed to interact with solid-phase antigen at 37◦Cfor 1 h, and isotype-specific secondary anti-mouse anti-bodies (Interchim, Montluçon, France) were used for IgGsubclass determination. Monoclonal antibodies 17D1C11,7G12A8, 14C12F5 (bioMerieux, France) and MAB1312(Chemicon, Temecula, CA, USA) were used as control forisotype 1, 2a, 2b and 3, respectively.

3. Results

3.1. Characterization of the MVA vectors

3.1.1. vvIV257 and vvIV306 express native E1E2polyprotein and modified E1 and E2 glycoproteins at theplasma membrane, respectively

Both vvIV257 and vvIV306 Huh-7 infected cells ex-pressed the E1 and E2 proteins, as demonstrated by WesternBlot analysis (Fig. 1B). E1 was detected using a polyclonalmonospecific rabbit anti-E1 and E2 using monoclonalanti-E2 antibody H52. As expected, this analysis revealsa difference of glycosylation according to the localizationof the proteins. To investigate the subcellular localizationof native and modified E1 and E2 glycoproteins, Huh-7cells were infected at a MOI of 0.1 for 24 h with eitherMVA wt, vvIV257 or vvIV306, and labelled without per-meabilization with either a monoclonal anti-E1 antibody


(A4, Dubuisson) or a monoclonal anti-E2 antibody (H52,Dubuisson) followed by a FITC-conjugated anti-mouse sec-ondary antibody. As shown inFig. 1C, E1 and E2 proteinswere well detected only when Huh-7 cells were infected byvvIV306, indicating that E1-VSVGTM-(AlaGly)2-JunB andE2-RVGTM-(AlaGly)2-Fra2 are efficiently transported to thecell surface, on the contrary of native HCV glycoproteins.Nevertheless, it is noteworthy that even vvIV257-infectedcells can be labelled by anti-E2 antibody (14.44%), whatmay be explained by an escape of E1E2 polyprotein tothe cell surface when the core protein is absent. Indeed,comparative experiments using recombinant adenovirusesor MVAs expressing either the whole CE1E2 or E1E2polyproteins showed a slight detection of E2 protein on nonpermeabilized cells in absence of the core protein (data notshown). This escape explains the production of pseudotypedretroviruses, which are known to bud at plasma membrane,bearing native E1E2 heterodimers[37].

3.1.2. vvIV316 expresses truncated and secreted E1 andE2 glycoproteins

Huh-7 cells were infected at a MOI of 0.1 for 24h witheither vvIV257 or vvIV316. Cell lysates were analyzed byWestern Blot using anti-E1 and anti-E2 antibodies. As shownin Fig. 2A, vvIV316 infected cells express an E2 truncatedpolypeptide which is secreted in the culture supernatant. Thistruncated and secreted form of E2 was recognized by theH52 monoclonal antibody. The same experiment was per-formed to detect a truncated and secreted form of E1 using

Fig. 2. (A) Detection of truncated E′2661 in culture supernatants. vvIV257- and vvIV316-infected cells supernatant (SN257 and SN316) were collected at48 h after infection (MOI 0.1). Ten microliters of supernatant were analyzed by Western Blot using monoclonal anti-E2 antibody (H52, Dubuisson) to detectthe highly glycosylated extracellular form of truncated E′2661. (B) Heterodimerization of chimeric E1 and E2: vvIV257-, vvIV306- and vvIV316- infectedHuh-7 cells lysates were incubated with monoclonal anti-E1 antibody (A4, Dubuisson). Heterodimeric complexes were purified by immunoprecipitation(IP) and native and chimeric E2 proteins were detected by Western Blot analysis (WB) using monoclonal anti-E2 antibody (H52, Dubuisson).

the same polyclonal monospecific rabbit anti-E1 antibodyas inFig. 1A. Unfortunately, we were unable to detect E1in the culture supernatant by this method, which may indi-cate that the epitope recognized by the monospecific rabbitserum is not accessible when E1 is secreted. Indeed, it hasbeen demonstrated that E1 expressed in the absence of E2is misfolded[15]. Furthermore, the lack of detectable E1 inthe supernatant can be also explained by its low migrationto the membrane, as shown by comparing the staining in-tensity of membrane-bound E1 and E2 (Fig. 1C). Neverthe-less, truncated E1 is recognized by monoclonal anti-E1 A4in immunofluorescence assays performed on vvIV316-BHKinfected cells (data not shown). We can therefore concludethat truncated E1 is expressed in the vvIV306-infected cells,and assume that this truncated form is secreted in cell su-pernatants.

3.1.3. vvIV306 allows expression of a E1-VSVTM-JunBand E2-RVGTM-Fra2 heterodimer

Modified E1 and E2 glycoproteins were engineered so thattheir C-terminal domains would allow heterodimerizationthrough formation of a JunB/Fra2 leucine zipper. This prop-erty was verified by coimmunoprecipitation, as shown inFig. 2B. Monoclonal anti-E1 antibody (A4) was used to im-munoprecipitate heterodimers expressed by either vvIV257or vvIV306 infected Huh-7 cells. Detection of the E2 pro-tein was performed using monoclonal anti-E2 antibody(H52). As expected, native E2 was found to coimmuno-precipitate with native E1 (in vvIV257-infected cells), and


truncated E1 and E2 did not interact (in vvIV316-infectedcells). Furthermore, detection of a high molecular weightform of E2-RVGTM-Fra2 on the film confirmed heterodimer-ization of this chimeric glycoprotein with E1-VSVTM-JunB(in vvIV306-infected cells).

3.2. Optimization of the immunization protocol

3.2.1. A second recombinant MVA immunization enhancesanti-E1E2 proliferative immune response in C57/Bl6 mice

A booster injection with a viral vector may have onlya marginal effect on specific immunity, due to a highanti-vector immune response induced after the first immu-nization. We investigated the effect on anti-E1E2 prolifer-ative responses of a boost at 2 weeks after priming with107 pfu of vvIV257 by intra-peritoneal immunization. As

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Fig. 3. Anti-E1E2 proliferative response analysis following various immunization protocols: splenocytes were collected and cultivated either with culturemedium (RPMI), recombinant adenovirus encoding b-galactosidase (Ad beta), or recombinant adenovirus encoding the HCV E1E2 polyprotein (AdE1E2). Results are expressed as the proliferation ratio between incorporated radioactivity when splenocytes are stimulated and incorporated radioactivitywithout activation (RPMI proliferation ratio= 1). All results represent the mean of proliferation obtained from analysis of splenocytes from five miceper group, except for (D) (nine mice per group were immunized). WT correspond to MVA WT-immunized mice. (A) C57/Bl6 mice received 107 pfuof vvIV257 intra-peritoneally either once or twice at a 14 days interval, and were sacrificied either 2 or 4 weeks after the first injection. (B) Anti-E1E2proliferative response according to the delay of the MVA boost: C57/Bl6 mice received twice 107 pfu of vvIV257 intra-peritoneally either at a 2 or a 4weeks interval. (C) C57/Bl6 mice received 107 pfu of vvIV257 twice at 14 days interval, either by scarification (SCA), or by the sub-cutaneous (SC),intra-muscular (I.M.), or intra-peritoneal route (IP), and were sacrified at 4 weeks after the first injection. (D) C57/Bl6 mice received 107 pfu of eithervvIV257, vvIV306 or vvIV316, twice at 14 days interval, by intra-peritoneal route (IP), and were sacrified at 4 weeks after the first injection.

shown in Fig. 3A, which represents results obtained onfive mice for each group, the intensity of the anti-E1E2proliferative response was significantly enhanced by a sec-ond immunization 2 weeks after the first one (black bar).Furthermore, this second injection becomes unefficient re-garding proliferative response when applied 4 weeks afterthe first one (Fig. 3B), demonstrating the induction by thefirst immunization of high anti-vector immune responses.

3.2.2. The intraperitoneal route of immunization allowsthe most effective anti-E1E2 proliferative immune responsein C57/Bl6 mice

In order to determine the best route of injection forour MVA vectors, C57/Bl6 mice were immunized via thesub-cutaneous (SC), intra-muscular (I.M.), intra-peritoneal(IP) route or by scarification (SCA) with two injections


at 2 weeks interval of 107 pfu of vvIV257. As shown inFig. 3C, which represents results obtained in five mice foreach group, only the IP route allowed induction of spe-cific anti-E1E2 proliferative responses. This results can beexplained by MVA tissue distribution after systemic ad-ministration. Indeed, Ramirez et al. showed that spleen andmesenteric lymph nodes were preferentially infected whenC57/Bl6 mice were immunized by IP route[38], whatwould induce a better antigen presentation to lymphocytesand thus induce better proliferative immune responses.

3.3. Comparative immunogenicity analysis of vvIV257,vvIV306 and vvIV316

3.3.1. Immunization with modified proteins does notenhance proliferative immune responses

Nine C57/Bl6 mice were immunized by intra-peritonealinjections twice at 14 days interval with 107 pfu of eithervvIV257, vvIV306 or vvIV316. One month after the firstinjection, mice were sacrificied and splenocytes were ana-lyzed for their anti-E1E2 proliferative properties. As shownin Fig. 3D, vvIV257-induced proliferative response wasequivalent to that induced by vvIV306, which itself wasstronger than vvIV316-induced response. Indeed, statisticalstudies show that vvIV257 group do not differ from vvIV306group (least significant difference test,P = 0.966), andthat both differ from vvIV316 group (P = 0.004 andP =0.003, respectively). Secretion of E1 and E2 glycoproteinsseems therefore to decrease specific proliferative immuneresponse.

Table 1Cytotoxic activity of splenocytes from transgenic HLA-A2 immunized mice

Peptide Vaccine group E/T ratio Responder/tested mice % of specific lysis

TIR MVA WT 33 0/6 –11 0/6 –

vvIV257 33 0/6 –11 0/6 –

vvIV306 33 1/6 7011 1/6 34

vvIV316 33 1/6 5811 1/6 52

DNA CE1E2/ Ad CE1E2 100 0/3 –33 0/3 –

RLW MVA WT 33 0/6 –11 0/6 –

vvIV257 33 5/6 92; 66; 60; 29; 2411 4/6 86; 62; 56; 38

vvIV306 33 5/6 96; 78; 75; 48; 3811 5/6 82; 74; 70; 25; 25

vvIV316 33 1/6 8711 1/6 68

DNA CE1E2/Ad CE1E2 100 3/3 59; 40; 3233 2/3 38; 21

Four weeks after the first immunization, splenocytes were pulsed for 5 days either with TIR (E1-specific peptide) or RLW (E2-specific peptide). EL4cells expressing HLA-A2 molecules binding either TIR or RLW were used as target cells in the CTL assay. HLA-A2 transgenic mice were primed byI.M. injection of 100�g of DNA encoding CE1E2 polyprotein, and boosted 2 weeks later by IP injection of 109 IU recombinant adenovirus expressingthe CE1E2 polyprotein (three mice), or immunized by two IP injections at a 2 weeks interval of 107′pfu of either MVA WT (six mice), vvIV257 (sixmice), vvIV306 (six mice), or vvIV316 (six mice).

3.3.2. Secretion of E1 and E2 decreases cytolyticT-lymphocyte response

In order to evaluate the capacity of the MVA vectors to in-duce the proliferation of CD8+ cytotoxic cells, HLA-A2.1transgenic mice were immunized intra-peritoneally twice at2 weeks interval with 107 pfu of either vvIV257, vvIV306or vvIV316 (six mice per group). Two HLA-A2.1-restrictedpeptides, one corresponding to amino-acids 257–266 on theHCV polyprotein sequence (TIR in E1) and the other corre-sponding to amino-acids 614–622 on the HCV polyproteinsequence (RLW in E2), were used to pulse splenocytes fromimmunized mice. Lysis of HLA-A2.1-transfected EL4 cells,bound with either TIR or RLW peptides, was assessed. Asshown inTable 1, no CTL activity was detected on targetcells pulsed with the TIR peptide. Indeed, only one mouseout of six has developed CTL response in vvIV306- andvvIV316- groups, which is not significant. Unlike TIR,RLW-pulsed target cells reveal a significant CTL activityfor splenocytes from mice immunized either with vvIV257or vvIV306. Indeed, almost all mice from those two groupshave developed a RLW specific cytotoxic response (5 out of6). Finally, the same RLW-pulsed target cells do not revealany CTL activity for splenocytes from mice immunizedwith vvIV316, as shown by the number of responder mice(1 out of 6). Secretion of E1 and E2 seems to decrease CTLresponses.

3.3.3. Expression of E1 and E2 at plasma membraneslightly enhances IFN-γ producing cells activation

Production of IFN-� by HLA-A2.1 transgenic micesplenocytes was analyzed. TIR and RLW peptides as well


Fig. 4. Detection of IFN-� releasing lymphocytes by ELISPOT. Two HLA-A2 restricted peptides (TIR and RLW) and purified E2 protein were used todetect IFN-� secreting cells among splenocytes from mice immunized by either DNA CE1E2/Ad CE1E2 (A), vvIV257 (B), vvIV306 (C), or vvIV316(D). An irrelevant peptide (IRR) and purified NS3 protein were used as negative controls. TIR+ RLW is a mix of both TIR and RLW peptides. Foreach group WT correspond to MVA WT-immunized mouse.

as purified E2 protein were used to pulse splenocytes fromimmunized mice and to detect IFN-� producing cells. Asshown by ELISPOT assay (Fig. 4), the three MVA vec-tors induced higher numbers of IFN-� producing cellsthan DNA/Adenovirus prime-boost positive control. BothHLA-A2.1 specific peptides TIR and RLW induced IFN-�secretion by CD8+ lymphocytes, and purified E2 proteininduced IFN-� secretion by Th1 CD4+ lymphocytes. Fur-thermore, vvIV306-immunized mice showed a slightlyenhanced activation of their cellular immune responses.Expression of E1 and E2 at the plasma membrane thusseems to enhance the intensity of the Th1-type cellularresponse.

3.3.4. Secretion of E2 increases specific anti-E2antibodies titers

Sera from C57/Bl6 mice were analyzed for their anti-E1and anti-E2 antibodies titers. As shown inFig. 5A, titers in

vvIV316-immunized mice sera were much higher than thoseobserved with vvIV306-immunized mice sera, themselveshigher than those with vvIV257-immunized mice sera. Wetherefore conclude that secreted E1 and E2 induce the high-est antibodies titers, that native E1E2 polyprotein inducethe least response, and that expression of E1 and E2 at theplasma membrane allows an intermediate stimulation of thehumoral immune system.

3.3.5. MVA vectors induce a predominant IgG1production in C57/Bl6 mice

Anti-E2 antibodies from C57/Bl6 mice were analyzedto detect IgG1, IgG2a, IgG2b or IgG3 antibodies. De-spite the use of recombinant viral vectors, very fewIgG2a were detected as shown inFig. 5B. Most an-tibodies were of the IgG1 and IgG2b isotypes, corre-sponding to the activation of B lymphocytes by Th2-typecytokines.


Fig. 5. (A) Titration of anti-E2 antibodies in C57/Bl6 mice immunizedeither by MVA WT, vvIV257, vvIV306 or vvIV316. Each bar correspondsto a single mouse. (B) Isotyping of anti-E2 antibodies from MVA WT-,vvIV257-, vvIV306- or vvIV316-immunized C57/Bl6 mice sera. Each barcorresponds to a single mouse.

4. Discussion

In an attempt to modulate the intensity or breadth of thetwo arms of the immune response against HCV glycopro-teins and potentially to optimize the immunogenicity of arecombinant MVA expressing E1 and E2, we analyzed theimpact of various subcellular localization of these proteins.Indeed, it has been shown for other proteins that immuno-genicity can be modulated by subcellular localization. Inparticular, increased antibodies titers were reported whenthe antigen was secreted, as demonstrated with plasmids en-coding either native or C-terminally truncated and secretedforms of the HCV Core protein[39]. Thus, this study pro-vides the first analysis of the comparative immunogenicityof recombinant MVA vectors expressing native or delocal-ized E1 and E2 glycoproteins. Both cellular and humoralresponses were evaluated, showing different patterns of im-mune induction according to E1 and E2 subcellular local-ization.

As expected from previous studies[14,15], truncation ofE2 at amino-acid 661 (E’2661) allows its efficient secretionin the extracellular medium, as confirmed by Western Blotanalysis. When fused to a chimeric RVGTM-(AlaGly)2-Fra2sequence, E’2661 is expressed at the plasma membrane, asshown by immunofluorescence and flow cytometry analy-ses. We have not been able to detect the truncated E1 glyco-protein (E′1331) in culture supernatant. Nevertheless, whenfused to a chimeric VSVGTM-(AlaGly)2-JunB sequence,E′1331 can be detected at the plasma membrane, attesting ofits migration through the secretion pathway. We can there-fore assume E’1331 is well secreted.

MVA vector vvIV306 allows the expression of the twochimeric membrane bound envelope proteins, which form aheterodimer at the plasma membrane, as demonstrated bya coimmunoprecipitation assay. This enforced heterodimer-ization is likely to mimic the natural heterodimerizationthrough the TM domains of HCV enveloppe protein, and toensure E1 and E2 conformations close to that of the naturalE1E2 complex conformation.

When injected into C57/Bl6 mice, the three MVA vectorsinduce different patterns of immune reactions. Indeed, wehave shown that secretion of the antigens has a negativeeffect on the intensity of specific anti-E1E2 proliferativeresponse. Likewise, we have observed a decrease in CTLresponses in vvIV316-immunized HLA-A2.1 transgenicmice, demonstrating a deleterious effect of antigen secretionon cellular responses, as compared with native antigens,despite the apparent neutrality of this secretion on the num-ber of peptide-specific IFN-� secreting cells. This decreasein CTL activity is compensated by an increased humoralresponse, as shown by the high anti-E2 titers detected insera from vvIV316-immunized C57/Bl6 mice. Isotyping ofanti-E2 antibodies showed a Th2 orientation with high IgG1production, in agreement with the decrease of proliferativeand cytotoxic cellular responses. Such observations havealready been reported,[39–42]. Unfortunately, no anti-E1antibodies were detected in these sera by ELISA. This neg-ative result may be explained by the conformation of thepurified E1 glycoprotein used for the ELISA. Indeed, theE1 protein used in the ELISA was produced from a pro-caryotic E. coli production system, which does not allowprotein post-translationnal modifications.

vvIV306 vector seems to allow a more potent cellularresponse induction than vvIV257, showing that E1 and E2delocalization to the plasma membrane enhances cellularresponses, according to ELISPOT results. Furthermore,this modification of subcellular localization induced ahigher production of antibodies, as shown by the compar-ison of antibodies titers between sera from vvIV257- andvvIV306-immunized C57/Bl6 mice. Comparable resultswere reported in mice immunized with plasmids encodingintracellular, secreted or membrane bound forms ofTaniaovis 45W antigen: secreted antigen induced higher anti-bodies titers than membrane bound antigen, itself inducinghigher titers than intracellular antigen[43].


Neutralization assays were performed using HCV-likeparticles[44], MLV/HCV [37] and VSV/HCV pseudotypes(Codran et al., in preparation), with different dilutions ofMVA-infected immunized C57/Bl6 mice sera. Althoughhigh anti-E2 antibodies could be detected, especially invvIV316-immunized mice sera, no neutralization activitywas associated with these titers (data not shown). These de-sappointing results should not lead us to think that immuneresponses induced by our vaccines would not protect againstan HCV challenge in an other animal model. Indeed, thecorrelation between in vitro neutralization of pseudotypedor virus-like particles and in vivo protection has not beenyet clearly demonstrated, even in the chimpanzee model[45]. Furthermore, the protocole of immunization used inour study was chosen according to its capacity to induce sig-nificant proliferative responses, and not humoral responses.High titers of IgG1 can be detected in MVA-immunizedC57/Bl6 mice sera, which might not be the optimal IgG sub-class to present neutralizing activities. Indeed, it was shownthat IgG3 hinge region confered enhanced HIV-neutralizingability in comparison with other subclasses[46]. On thecontrary, following separation of the four IgG subclassesby chromatography, the IgG2a fraction exhibited the great-est neutralizing activity towards dengue virus[47]. Manyparameters could be modified in our protocole in order tochange the IgG subclass, as the route[48,49] and the timeof immunization, but also the mice strain[50]. This workremains to be performed in order to induce neutralizingantibodies.

In conclusion to this study, vvIV306 seems to be a promiz-ing vaccine candidate due to its capacity to enhance bothcellular and humoral responses, and remains to be tested ina more relevant animal model.

Acknowledgements

We are grateful to Jean Dubuisson (Institut de Biologie,Lille, France) for kindly providing A4 and H52 monoclonalantibodies, and TRANSGENE S.A. (Strasbourg, France) forgenerously supplying shuttle plasmids to generate recom-binant adenovirus and vaccinia viruses. We thank Chris-tine Thumann, François-Loı̈c Cosset (INSERM U412—IFR128—ENS LYON) and Jean-Pierre Martin for helpful dis-cussions, Marie Parnot, Nicolas Brignon and Stéphane Dori-dot for excellent technical assistance, and Anne Fournillier(UMR 2142 CNRS/BioMérieux—ENS LYON) and Nico-las Meyer (DIM—CHU Strasbourg) for statistical analyses.This work was supported by Association pour la Recherchecontre le Cancer (ARC), European Union (HCVacc Cluster,agreement No. QLK2-CT99-00356) and BioMérieux grants.

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