immune modulation of human b lymphocytes by gene transfer with recombinant epstein–barr virus...

Journal of Virological Methods 72 (1998) 81–93

Immune modulation of human B lymphocytes by gene transferwith recombinant Epstein–Barr virus amplicons

Fred Wang a,*, David C. Seldin b, Bethany Annis a, Alicja Trocha c,R. Paul Johnson c,d

a Department of Medicine, Har6ard Medical School, Brigham and Women’s Hospital, 181 Longwood A6enue, Boston,MA 02115, USA

b Departments of Medicine and Microbiology, Boston Uni6ersity Medical Center, Boston, MA 02118, USAc Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital, Har6ard Medical School,

Charlestown, MA 02129, USAd Di6ision of Immunology, New England Regional Primate Research Center, Har6ard Medical School, Southborough,

MA 01172, USA

Received 15 October 1997; received in revised form 6 January 1998; accepted 6 January 1998

Abstract

We described previously a novel mode of gene transfer by infection of human B lymphocytes with recombinantEpstein–Barr virus (EBV) amplicons. This system was explored for its potential use in expressing various recombi-nant genes, including the cytokine IL-4, the HIV envelope glycoprotein (gp120) and a suicide and gag gene.Recombinant genes were present as multiple copy episomes and stable, high level recombinant gene expression couldbe detected by antigenic and functional assays. Amplicon-infected B cells secreted high levels of recombinant cytokineand efficiently presented recombinant antigens through classes I and II MHC-restricted antigen processing pathways.Thus, recombinant EBV amplicons can be used to express components of the immune system or heterologous genesfor immune recognition in human B cells. Combining gene transfer with EBV infection may provide uniqueadvantages for in vitro and in vivo gene transfer. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Gene transfer; Epstein–Barr virus; Antigen presentation; Cytotoxic T cell; Cytokine

1. Introduction

Several features of Epstein–Barr virus (EBV)infection make it a useful vector for recombinant

* Corresponding author. Fax: +1 617 5254257; e-mail:[email protected]

0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0166-0934(98)00023-8

F. Wang et al. / Journal of Virological Methods 72 (1998) 81–9382

gene transfer. EBV infects preferentially and im-mortalizes the growth of human B cells andmultiple copies of the viral genome persist in Bcells as circular episomes (for review see Kieff,1996). EBV-infected B lymphoblastoid cell lines(B-LCL) can be generated from an easily acces-sible source, i.e. peripheral blood and can bepropagated indefinitely in vitro and preserve thegenotype of a specific individual for genetic andimmunologic studies. These B cells present anti-gen efficiently by MHC class I- or II-restrictedpathways and can be used to produce secretedproducts such as human monoclonal antibodies(Roome and Reading, 1984).

While gene transfer into EBV-infected B-LCLis desirable, frequently, for a variety of experi-mental designs, the technical aspects of the genetransfer can be technically difficult and limiting.EBV-immortalized B-LCL derived from periph-eral blood B cells are relatively resistant to elec-troporation and do not readily tolerate lowserum conditions required for liposomal trans-fection. A variety of viral vectors can be usedsuccessfully, but may be associated with otherproblems. High level gene expression can beachieved with recombinant vaccinia viruses, buta number of vaccinia virus genes are expressedresulting in shut down of host cell gene expres-sion and subsequent cell death. Similarly, recom-binant adenovirus vectors are associated withexpression of other immunogenic adenoviralproteins and do not efficiently infect B cell lines.Gene transfer into B-LCL can be achieved withrecombinant retroviruses but requires chromoso-mal integration and introduces the possibilitiesof replication competent retroviruses.

Incorporating gene transfer in an EBV-basedviral vector combines B cell immortalization andgene transfer into a single step without introduc-ing additional viral gene products. Advances inEBV genetics permits the introduction of recom-binant genes directly into the EBV genome byhomologous recombination. These techniqueshave been used extensively to study EBV genefunction by introducing mutated EBV genes intothe EBV genome (Cohen et al., 1989; Hammer-schmidt and Sugden, 1989; Lee et al., 1992;Tomkinson and Kieff, 1992). Conceptually, gen-

erating a recombinant EBV with a heterologousgene would be an effective gene transfer vector.However, to date, the only non-EBV genes re-combined into the EBV genome have been thoseencoding resistance to cytotoxic drugs, probablydue to the technical hurdles required to generaterecombinant EBV (Wang et al., 1991; Lee et al.,1992; Shimizu et al., 1996).

An alternative, simpler approach for EBVbased gene transfer is to use an EBV-based am-plicon, or defective genome, which can be repli-cated and packaged into infectious viralparticles. When the EBV DNA elements, re-quired for episomal maintenance, initiation ofviral DNA replication and cleavage and packag-ing of viral DNA, are combined into a singleplasmid, this amplicon plasmid can be repli-cated, packaged into capsids and released as anenveloped virion (Hammerschmidt and Sugden,1989; Wang et al., 1991). Gene transfer by re-combinant EBV amplicons will target, specifi-cally, human B lymphocytes, persist inimmortalized B cells along with the wild typeEBV and introduce multiple copies of the re-combinant gene into B cells. This technique canbe used to transfer non-EBV genes in order tocomplement genetic deficiencies in B-LCL fromselected patient populations (Banerjee et al.,1995; Wang et al., 1995; Sun et al., 1996). Weexamined whether this technique can be em-ployed to enhance the utility of EBV-infected Bcells for antigen presentation and production ofsecreted cell products.

2. Methods

2.1. Plasmids

The amplicon plasmid, pAmp, contains theEBV lytic origin of replication (ori-lyt; SacI toTaqI fragment from B95-8 EBV BamHI HDNA) (Hammerschmidt and Sugden, 1988), theEBV latent origin of replication (ori-p; SacII toSphI fragment of B95-8 EBV BamHI C DNA)(Yates et al., 1984), the terminal repeats (BamHIto EcoRI fragment of B95-8 EBV BamHI NHet

DNA with deletion of the MluI fragment con-

F. Wang et al. / Journal of Virological Methods 72 (1998) 81–93 83

taining the LMP1 coding region) and a hy-gromycin phosphotransferase gene under thecontrol of a herpes simplex virus ICP4 promoterin a PUC18 plasmid. A human IL-4 cDNA(provided by B. Morrison (Morrison and Leder,1992)) was cloned under the control of an SV40early promoter (pSG5, Stratagene) and theSV40-IL-4 cDNA fragment was cloned intopAMP. The SV40 promoter and poly A alonewere cloned into pAMP for the vector control.

The BSA plasmid (Wang et al., 1995) is simi-lar to the pAMP plasmid except for substitutionof a SV40 early promoter driven hygromycinphosphotransferase gene in a Bluescript vector(Stratagene). A genomic HIV fragment contain-ing the coding region for gp160 (pIIIenv3-1,provided by Dr Joseph Sodroski (Sodroski etal., 1986)) was cloned under control of an SV40early promoter and inserted into the BSAplasmid to construct the gp120 amplicon vector,BSA-gp120. An HIV gag open readingframe was derived from the pDAB72 plasmid(Reagent obtained through the AIDS Researchand Reference Reagent Program, Divisionof AIDS, NIAID and NIH: pDAB72 fromDr Susan Erickson-Viitanen (Erickson-Viitanenet al., 1989)). A hygromycin phosphotransferasegene fused with a HSV thymidine kinasegene (tgCMV/HyTK (Lupton et al., 1991))was cloned under control of an SV40 early pro-moter and was used to replace the hygromycinphosphotransferase gene in the BSA-hyg/tkplasmid.

2.2. Cell lines

The EBV-producing cell lines B95-8 (Miller etal., 1972) and P3HR-1 (Rabson et al., 1982) areinfected with an immortalization-competent andimmortalization-defective EBV strain, respec-tively. Louckes are EBV-negative BurkittLymphoma cells and B-LCL were derived byB95-8 EBV infection of peripheral blood B cellsfrom normal individuals. All cell lines weremaintained in RPMI 1640 with 10% fetal bovineserum.

2.3. Transfection, 6irus induction and 6iralinfections

B95-8 and P3HR-1 cells were transfected byelectroporation with 30 mg of circular DNA.One day after transfection, cells were plated at10000 cells per well in microtiter plates andgrown in the presence of 400 mg/ml hygromycin(Calbiochem). Hygromycin-resistant cells wereexpanded and induced for lytic EBV infectionby exposure to 20 ng/ml phorbol ester and 3mM butyrate for 3–5 days. Cell supernatantswere harvested after 5 days and filtered througha 0.45 u filter. For viral infections, 0.5–1.0 mil-lion Louckes or LCL cells were infected with1–3 cc of filtered supernatants for 1 h at 37°C.Cells were then washed three times with Hank’sbuffered saline and cultured in 1 cc of media for3 days. Supernatants were then harvested andassayed for IL-4 activity.

2.4. Il-4 assay

IL-4 activity in supernatants was measured bythe ability to support proliferation of an IL-4responsive cell line. The S2/4 cell line is an IL-3dependent murine cell line which has been trans-fected with a human IL-4 receptor cDNA(Seldin and Leder, 1994). Supernatants wereadded at a final concentration ranging from0.1–10% to 10000 S2/4 cells per well and cul-tured for 3 days. Cell growth was quantified us-ing a colorimetric assay to measure the cellularreduction of 2,3-bis(2-methoxy-4-nitro-5-sul-fophenyl)-5-{(phenylamino)carbonyl}-2H- tetra-zolium hydroxide (XTT) in the presence ofphenazine methosulfate (PMS), as described(Scudiero et al., 1988). Briefly, cells were incu-bated for 4 h at 37°C with 50 m l of a stocksolution containing 1 mg/ml XTT and 25 mMPMS and the absorbance at 450 nm was mea-sured. All samples were measured in triplicate.No viable cells were visible after 3-day culturein control medium lacking IL-4. IL-4 concentra-tions were determined by calibration against astandard curve generated with a W.H.O. Na-tional Biological Standards Board recombinantIL-4 standard.


2.5. Northern blotting

Total cell RNA was prepared using RNAzol.Fifteen mg of RNA was loaded per well andseparated on a formaldehyde agarose gel. TheRNA was transferred to a nylon membrane andhybridized with a 32P-labelled IL-4 cDNA probeor human GAPDH probe.

2.6. In situ lysing gel

In situ lysing gels were run as described byGardella et al. (1984). Briefly, two million viablecells were resuspended in loading buffer contain-ing RNAse and loaded onto a 0.8% agarose gel.An agarose plug containing 2% SDS and 1 mg/mlprotease was formed behind the wells so that cellswere lysed in situ when electrophoresis was ini-tiated. 100 pg of linearized pAMP-IL-4 was di-luted in 2.5 mg/ml salmon sperm DNA andloaded in a separate lane. Gels were run at 15 vfor 4 h to lyse the cells in the wells and then at100 v for 18 h to allow episomes to migrate intothe gel. The gels were transferred to nylon mem-branes and hybridized with a 32P-labelled IL-4cDNA probe.

2.7. Western blotting

Western blotting was performed as previouslydescribed (Wang et al., 1991) using a murinemonoclonal antibody against HIV gp120 (NEA-9301, NEN) or a rabbit antiserum to HIV p25/24gag (Reagent obtained through the AIDS Re-search and Reference Reagent Program, Divisionof AIDS, NIAID and NIH: Antiserum to p25/24gag from Dr Kathelyn Steimer, Chiron Corpora-tion (Steimer et al., 1986).

2.8. Cytotoxic T cell (CTL) effector cells andCTL assays

Isolation and characterization of the CD4+(414.32) and CD8+ (414.27, 414.46 and 18030D23) HIV-1 specific CTL clones has been de-scribed in detail previously (Hammond et al.,1992; Johnson et al., 1994; Walter et al., 1997).The HIV-1 gag-specific CTL line was generated

by stimulation of PBMC from an HIV-1 seropos-itive subject (010-115i) with an autologousCD4+ T cell clone or B-LCL sensitized with theHLA-A2-restricted peptide SLYNTVATL corre-sponding to amino acids 77–85 of the HIV-1 p17gag protein (Johnson et al., 1991) as describedpreviously (Johnson et al., 1992). Stimulation ofPBMC from subject 115i with the autologous,amplicon-infected B-LCL, was carried out by in-cubating irradiated (10000 rads) B-LCL withPBMC at an effector:stimulator ratio of 1:1 inRPMI with 10% FCS and adding IL-2 (providedby Maurice Gately, Hoffman LaRoche) to a con-centration of 20–40 units/ml on day 4. Stimulatedeffector cells were then tested for CTL activityafter 10–14 days. CTL activity was determinedusing standard chromium release assays consistingof B-LCL target cells infected with either the EBVamplicon or recombinant vaccinia viruses orpreincubated with synthetic HIV-1 peptides. Vac-cinia-infected targets were prepared by incubating2.5–5×106 B-LCL in log-phase growth with re-combinant vaccinia at 1–5 pfu/cell for 16 h at37°C. Recombinant vaccinia used for these studiesincluded vAbt141 (encoding the HIV-1 p55 gagprotein, kindly provided by D. Panicali and G.Mazarra, Therion Biologics, Cambridge, MA) orthe control VSC8, encoding the b-galactosidasegene (provided by Dr Pat Earl, NIH). Target cellswere then labeled with 100 mCi of Na2(51CrO4)(New England Nuclear, N. Billerica, MA) for45–60 min and washed three times with RPMIsupplemented with 10% FCS. Peptide-coatedtargets used for analysis of CD8+ CTL wereobtained by incubating 2–3×106 B-LCL withpeptide at 100 mg/ml for 60 min during 51Crlabeling or by adding peptides at the indicatedconcentrations to 51Cr-labeled targets for 50–60min in 96 well plates prior to the addition ofeffector cells. Peptide-sensitized targets used foranalysis of CD4+ CTL were prepared by incu-bating 1×106 B-LCL with 100 mg/ml of peptideovernight prior to the 51Cr-labeling. Peptides usedfor these assays include the HLA-A2-restrictedp17 gag epitope SLYNTVATL (Johnson et al.,1991; Tsomides et al., 1994), the HLA-A3-re-stricted gp120 epitope TVYYGVPVWK (Johnsonet al., 1994) and the MHC class II-restricted


gp120 peptide ITQACPKVSFEPIPHYCAPAG-FAI (Johnson et al., 1994). Cytolytic activity wasdetermined in a standard 51Cr-release assay (John-son et al., 1991) using U-bottom microtiter platescontaining 104 targets per well. Plates were incu-bated in a humidified incubator at 37°C or for4–5 h (CD8+ CTL clones) or 5–6 h (CD4+CTL clones). All assays were performed in dupli-cate. Supernatants were then harvested andcounted on a Cobra Gamma Counter (PackardInstrument Company, Meriden, CT) and percentlysis determined from the formula: 100×{(experimental release–spontaneous release)/(maximum release–spontaneous release)}.Maximum release was determined by lysis oftargets in detergent (1% Triton X-100, Sigma).Spontaneous release was B30% of maximal re-lease for all reported assays.

3. Results

3.1. Gene transfer of IL-4 by amplicon infection

Multiple DNA elements were combined into asingle plasmid to construct the amplicon vectors,pAmp and BSA (Fig. 1). An SV40 early promoter

driving an IL-4 cDNA, HIV envelope glyco-protein, HIV gag, or a hygromycin/thymidine ki-nase fusion gene were included in variousderivatives to provide for expression of recombi-nant genes. The vector control amplicon plasmidcontained the SV40 early promoter cassette alone.

The amplicon plasmids are replicated and pack-aged into infectious amplicons when transfectedinto EBV-infected cell lines which are capable ofreplicating EBV. Stable transfectants with thepAMP-IL-4 or pAMP vector control plasmidwere derived in two EBV-infected cell lines, B95-8and P3HR-1, commonly used to produce hightiter EBV. The B95-8 EBV strain is the prototypeEBV which efficiently immortalizes B cells in vitro(Miller et al., 1972). The P3HR-1 cell line is asubclone of the Jijoye cell line, originally derivedfrom an EBV-infected Burkitt’s lymphoma. Thiscell line is noteworthy because the P3HR-1 EBVDNA has undergone a spontaneous deletion ofEBV DNA including all of the transformation-es-sential EBNA-2 gene (Rabson et al., 1982), (Kinget al., 1982), (Bornkamm et al., 1982). Thus, EBVfrom the P3HR-1 cell line is incapable of immor-talizing B lymphocytes (Miller et al., 1974).

Production of infectious amplicons was testedfor by the ability to transfer IL-4 expression tohuman B cells after exposure to cell free superna-tants. B95 or P3HR-1 cells stably transfected withthe pAMP-IL-4 or vector control plasmid weretreated with phorbol esters and butyrate to induceviral replication. Filtered, viral supernatants har-vested after 3–5 days were used to infect Louckescells, an EBV-negative human B lymphoma cellline. To test whether IL-4 gene expression hadbeen transferred by infection with recombinantamplicons, supernatants from the infectedLouckes cells were assayed for IL-4 activity usingan IL-4 responsive cell line (Seldin and Leder,1994). Louckes cells infected with IL-4 ampliconsproduced from three different B95-8 clones pro-duced 150–190 ng/ml of IL-4 in the 3-day super-natants. Virus from P3HR-1 cells transfected withthe IL-4 amplicon plasmid induced similar IL-4levels after acute infection of Louckes cells (Table1). Uninfected Louckes cells (data not shown) andLouckes cells infected with virus from the B95-8cell lines transfected with the vector control am-

Fig. 1. Map of representative amplicon plasmid. The lytic(Ori-lyt) and latent (Ori-p) origins of replication and thepackaging and cleavage signals in the terminal repeats (TR) ofEBV DNA are shown. A HSV ICP4 thymidine kinase pro-moter (pAMP) or SV-40 early promoter (BSA) driven hy-gromycin phosphotransferase gene provided a positiveselection resistance marker. SV40 early promoter driven IL-4,HIV gp120, or HIV gag genes were included for recombinantgene expression.


Table 1IL-4 produced by acute infection with IL-4 amplicon

Amount of IL-4 in supernatants of 3 day infected cells (ng/ml)Virus

LCLLouckes

Exp. c1 Exp. c2 Exp. c1 Exp. c2

B95 vector c1 ND0 ND ND0 00 0B95 vector c2

160B95 IL-4 c1 ND ND NDND 190B95 IL-4 c2 170170150 190190 170B95 IL-4 c3

110P3 IL-4 c2 100 140 190P3 IL-4 c3 390 360 440 660

plicon plasmid (Table 1) produced no detectableIL-4 after 3 days. IL-4 expression could also betransferred by amplicon infection to two B-LCLderived from normal peripheral blood (Table 1,LCL c1 and c2). For comparison, transienttransfection of COS cells with an SV40 drivenIL-4 expression vector produced 7 day superna-tants with 400–1100 ng/ml of IL-4 (data notshown).

The ability to transfer stable gene expression byamplicon infection was tested by infecting humanB cells with recombinant amplicon and selectingfor hygromycin resistance. Hygromycin-resistantclones were derived from Louckes cells infectedwith vector control or IL-4 recombinant ampli-cons produced from P3HR-1 transfected cells.Clones infected with the IL-4 amplicon produced140–380 ng/ml of IL-4 in the cell culture superna-tant (Table 2; Louckes P3/IL-4 c1, c2). Super-natants from cell clones infected stably withvector control amplicons contained no detectableIL-4 whether harvested after 5 or 10 days inculture (Table 2; Louckes P3/vec c5, c6). Sincevector control or IL-4 amplicons are produced inconjunction with larger amounts of P3HR-1 EBV,all hygromycin resistant Louckes clones were alsocoinfected with P3HR-1 EBV as expected whenanalyzed on immunoblot (data not shown).

Recombinant amplicons could also stably infectB-LCL derived from peripheral blood of normalindividuals. An established B-LCL (LCL c3)was superinfected with vector control or IL-4

amplicons made in B95-8 cells and hygromycin-resistant cell lines were selected. The B-LCL sta-bly infected with the IL-4 amplicon producedsignificantly higher IL-4 levels of :1100 ng/ml(Table 2; LCL 3/IL-4).

Northern blot analysis of these stably infectedclones was performed to confirm specificity of theIL-4 activity detected by functional assay. Asshown in Figs. 2 and 3, IL-4 mRNA was detectedonly in the three IL-4 producing clones. Thedifferences in mRNA size were likely due to alter-native polyadenylation sites present within theplasmid, e.g. polyadenylation sites within the IL-4cDNA, SV40 expression vector or cryptic siteswithin the plasmid.

Cells infected stably with recombinant ampli-con were also analyzed by in situ lysing gel elec-

Table 2IL-4 produced by cell lines stably infected with IL-4 amplicon

Amount of IL-4 in supernatantsCell lines(ng/ml)

10 day harvest5 day harvest

0 0LCL c31100LCL c3/IL-4 1200

Louckes P3 c5 0 000Louckes P3 c6

180Louckes P3/IL-4 140c1

Louckes P3/IL-4 200380c2


Fig. 2. Northern blot analysis and episomal detection of IL-4in B cells stably infected with an IL-4 recombinant amplicon.(A) 15 mg of total RNA from a B-LCL (LCL c3), the samecell line stably infected with the IL-4 amplicon (LCL/IL-4),Louckes cells infected with vector control amplicon andP3HR-1 EBV (Louckes P3/vec c5 and c6) and Louckescells stably infected with IL-4 amplicon (Louckes P3/IL-4 c1and c2) were loaded in each lane. The blot was hybridizedsequentially with a 32P labelled IL-4 cDNA probe andGAPDH probe as a control. (B) Episomal DNA was detectedby loading two million cells into each lane of an in situ lysinggel (Gardella et al., 1984). Southern blot of the in situ lysinggels were hybridized with a 32P labelled IL-4 cDNA probe. 100pg of linearized IL-4 amplicon plasmid (11.4 kb) was loaded asa control.

a radiolabelled IL-4 DNA probe indicated thatthe recombinant IL-4 amplicon was present as alarge episome in these cells (Fig. 2B). The ampli-con episomes migrated with the same relative sizeas wild type EBV episomes (data not shown).Thus, stable high level expression of a recombi-nant cell product such as the cytokine IL-4 can beachieved after gene transfer by infection with arecombinant amplicon.

3.2. EBV amplicon-infected cells present foreignantigens by both MHC classes I- andII-restricted pathways

B-LCL are particularly useful since they canserve as a permanent source of efficient antigenpresenting cells with an HLA genetic backgrounddefined by the individual donating the peripheralblood B cells. To test whether amplicons might beused to transfer recombinant genes for antigenpresentation, the HIV envelope glycoprotein,gp120, was cloned into an EBV amplicon. Vectorcontrol and gp120 amplicons were used to infectthe Louckes B cell line or B-LCL (IB4 and AR)and hygromycin-resistant cell lines were obtained.As shown in Fig. 3A, these amplicon-infected celllines contained the gp120 in episomal form.Gp120 was expressed in the cell lysates of thegp120 amplicon infected cells and in the cell freesupernatant as shown by Western blotting (Fig.3B). Gp120 expression was also present on the cellsurface as demonstrated by cell surface immu-nofluorescence (data not shown).

The ability for amplicon-infected cells topresent recombinant antigen to cytotoxic T cellswas tested by using CD8+ HIV gp120-specificcytotoxic T cell clones restricted by the HLA-A3allele shared by the AR cell line. As shown in Fig.4A, three different clones of gp120 amplicon-in-fected AR cells were efficiently lysed by the CTLclone at 10:1 to 1:1 effector to target ratios, whilethere was essentially no lysis of the vector controlclones. Vector control clones could be efficientlykilled if exogenous peptide was provided.

In order to test if the amplicon-infected cellscould present recombinant antigen through bothMHC classes I and II antigen processing path-ways, recombinant amplicons were used to infect

trophoresis (Gardella et al., 1984) to determine ifthe recombinant genes were maintained as anindependent episome. Linear and episomal viralDNA are able to migrate into the gel with pro-longed electrophoresis, while the chromosomalDNA remains within the well. Hybridization with


Fig. 3. DNA, RNA and protein expression of HIV envelope in B cells stably infected with an HIV envelope recombinant amplicon.(A) B-LCL (IB4, AR) or EBV negative B lymphoma cells (Lo) were stably infected with vector control or gp120 amplicons andanalyzed for episomal amplicons by in situ lysing gel. (B) Gp120 expression in cell lysates and in the supernatants were analyzedby Western blotting with a gp120 specific monoclonal antibody.

the 414 B-LCL for which both CD8+ , MHCclass I-restricted and CD4+ , MHC class II-re-stricted T cell clones were available (Hammond etal., 1992; Johnson et al., 1994). Again the CD8+ ,class I restricted CTL clone killed the gp120 am-plicon-infected cell lines much more effectivelythan the vector control clones (Fig. 4B, left). Inaddition, the gp120 amplicon-infected cell lineswere killed by the CD4+ , class II-restricted CTLclone more efficiently than the vector control cellline (Fig. 4B, right). The CD4+ CTL clone usedfor these studies had lower total killing activitythan the CD8+ CTL, as evidenced by the re-duced lysis of the peptide-sensitized targets (Fig.4B, left versus right). Lower levels of specific lysisby CD4+ CTL clones as compared with CD8+CTL clones have been observed in previous stud-ies (Hammond et al., 1992; Johnson et al., 1994).However, even with this lower total cytolytic ac-tivity, the gp120-infected cell line was killed by theCD4+ CTL clone as efficiently or better than the

peptide sensitized cells or vector control infectedtargets. These studies indicate that recombinantantigen transferred by amplicon infection was pre-sented appropriately through both class I and IIantigen processing pathways.

B cells expressing recombinant genes could beeffectively used for stimulation of, as well astargets for, immune responses. In order to testwhether amplicon-infected B cells could serve asstimulator cells for CTL, an amplicon expressingthe HIV gag gene was constructed and used toinfect the 115 B-LCL. Although only :50% ofthese cells expressed gag by immunofluorescence(data not shown), the gag amplicon-infected cellswere killed more efficiently than the vector con-trol cells by a CTL line and a CTL clone specificfor a MHC class I-restricted epitope in gag (Fig.5A). The increased lysis of the gag amplicon-in-fected cells after the addition of peptide was con-sistent with gag expression in only a portion ofthe infected cell population, most likely as a result


Fig. 4. CTL killing of B cells infected with the HIV envelope recombinant amplicon. (A) Cell lysis by HIV envelope-specific CD8+T cells. Vector control and gp120 amplicon-infected HLA-A3+ AR cells were used as targets in a standard chromium release assayusing a HLA-A3- restricted CD8+ CTL clone. Addition of the cognate peptides for the different CTL clones are described inSection 2 and served as a positive control for killing at an effector:target ratio of 3:1 (open bars in A and B). (B) Killing by HIVenvelope-specific CD4+ and CD8+ T cells. Stable cell lines expressing the HIV-1 envelope were generated by infecting with thegp120 or vector control amplicon and were then examined for lysis using autologous CD8+ and CD4+ CTL clones.

of incomplete drug selection. Even so, thesegag expressing cells could stimulate specificCTL activity when incubated with peripheralblood T cells from the autologous donor for1 week. Peripheral blood T cells were stimulatedwith autologous gag or gp120 amplicon-infectedB-LCL as stimulator cells. These stimulatedeffector cells efficiently killed the autologousB cells infected with a recombinant vaccinia

virus expressing HIV gag with 37% lysis atan E:T ratio of 40:1 (Fig. 5B). There was nosignificant killing of autologous B cells infectedwith control vaccinia virus and there was nosignificant killing of either target with T cellsstimulated by the gp120 amplicon-infected B-LCL (Fig. 5B). Thus, the amplicon-infected Bcells can be used as both targets and stimulatorsof CTL.


Fig. 5. Stimulation of gag-specific CTL by HIV gag recombinant amplicon infected B cells. (A) 115 B-LCL infected with a vectorcontrol (115 vec) or gag amplicon (115 gag A,B) are killed by a CD8+ T cell line and a CD8+ T cell clone specific for the HLAA2-restricted peptide SLYNTVATL. Addition of the cognate peptide served as a positive control for killing at an effector:targetratio of 5:1 (open bars). (B) Peripheral blood T cells from HIV-infected donor 115i were stimulated with either gag (115 gag B) orgp120 (115 gp120 A) amplicon-infected 115 B-LCL. After 1 week of incubation, gag specific cytotoxic activity was tested in thevarious stimulated T cell populations using control (lac) or gag expressing vaccinia virus (p55 gag)- infected 115 B-LCL as targets.

3.3. Incorporation of a suicide gene into EBVamplicons

Toxin genes or genes that can activate pro-drugs can theoretically be engineered into recom-binant amplicons in order to provide negativeselection. A herpes simplex virus thymidine kinasesuicide gene has been used to confer cell sensitiv-ity to the nucleoside analog, ganciclovir. In order

to test whether this system could be incorporatedeffectively into the recombinant amplicons, a fu-sion protein combining the hygromycin phospho-transferase and HSV thymidine kinase genes wascloned into the BSA amplicon vector. The ARB-LCL was stably infected with recombinantHyg/TK amplicon and tested for sensitivity toganciclovir. Uninfected AR cells proliferated wellin the presence of ganciclovir and toxic effects did


not appear until high ganciclovir levels were used,with an IC50 of nearly 100 uM (Fig. 6). Genetransfer of the suicide gene by amplicon infectionreduced the IC50 of the AR B-LCL to B0.1 uMin two different clones (Fig. 6). Thus, gene transferof a suicide gene by amplicon infection provided foreffective positive and negative selection.

4. Discussion

These studies demonstrate that a gene transfersystem using infection with recombinant EBVbased vectors can be used to augment many usefulproperties of EBV infected B cells. This genetransfer system uses the biologic properties of theEBV to replicate and package recombinant DNAplasmids into defective virions, or amplicons, capa-ble of infecting human B cells. In these studies wehave used the amplicon to express the cytokineIL-4, the HIV envelope glycoprotein, gp120/41, theHIV gag protein and a suicide gene. We obtainedhigh level gene expression after acute infections orafter long term drug selected infections. MultipleDNA copies introduced by the amplicon maycontribute to the stable, high level expression whichcan provide a ready and immortal source of recom-binant cell products such as cytokines. Cells genet-

ically modified to express various cytokines mayalso stimulate anti-tumor immune responses in vivoand be a clinically useful tool for cancer im-munotherapy. (Tepper et al., 1989; Golumbek etal., 1991; Simons et al., 1997).

EBV-infected B-LCL have also been useful forperpetuating the genotype of an individual donor,as in the study of genetic abnormalities or HLA-re-stricted immune responses. In previous experi-ments, we have used recombinant ampliconsexpressing the TAP1 and TAP2 genes involved withpeptide transport into the endoplasmic reticulum tocomplement a class I mediated antigen processingdefect in B-LCL from patients with insulin depen-dent diabetes mellitus (Wang et al., 1995). Otherinvestigators have used a similar system to comple-ment gene defects in B-LCL from patients withFanconi anemia and Lesch–Nyhan syndrome(Banerjee et al., 1995; Sun et al., 1996). The currentexperiments with the HIV envelope and gagproteins demonstrate that recombinant proteinsexpressed from infecting amplicons are efficientlypresented by MHC classes I and II antigen process-ing pathways and these cells can serve as efficienttargets or stimulators for CTL. These experimentsdemonstrate the potential utility of combiningrecombinant gene transfer with EBV infections invitro.

Gene transfer with recombinant EBV ampliconsmay be advantageous for several reasons. No otheradditional viral genes, such as those typicallyassociated with adenovirus and vaccinia virus vec-tors, are introduced. The combination of genetransfer and B cell immortalization shortens theexperimental design and once generated, there is acontinuously renewing source of cells. Recombi-nant amplicons can be produced from relativelysimple plasmids whereas genetically modifyingEBV by homologous recombination of a het-erologous gene requires relatively difficult cosmidcloning for maximal efficiency.

One gene therapy strategy employs geneticmodification of tumor cells to enhance immuno-genicity with subsequent infusion of the geneti-cally modified tumor cells to potentially enhanceanti-tumor responses in vivo (Baskar, 1996).Whether autologous EBV-infected cells expressingrecombinant genes could be used as in vivo stimu-lator cells remains to be determined. Virtually all

Fig. 6. Ganciclovir sensitivity of hygromycin phosphotrans-ferase/thymidine kinase recombinant amplicon-infected B cells.Proliferation of uninfected AR cells (open circles) and twolines of AR cells stably infected with the HYG/TK amplicons(closed squares and triangles) were measured in a XTT assayafter 5 days of culture in various concentrations of ganciclovir.Results are expressed as the percentage of growth in thepresence of ganciclovir versus the absence of ganciclovir.


adults are persistently infected with EBV for life,have EBV-infected B cells circulating in their pe-ripheral blood, shed EBV in their saliva and havecytotoxic T cells specific for a number of EBVgenes. Potential safety could be increased bylethal irradiation of cells or by incorporation of asuicide gene similar to the hygromycin-thymidinekinase fusion gene used in these studies to elimi-nate the recombinant cells if necessary. EBV-in-fected B cells have provided a valuable tool for invitro antigen presentation and continued advancesin recombinant genetics may provide the means toextend these uses in vivo.

Acknowledgements

We thank Scott Hammond and Robert Sili-ciano for providing the HIV-specific CTL clones414.27, 414.32 and 414.46, Spyros Kalamas forCTL clone 18030 D23, Maurice Gately for dona-tion of IL-2 and Bruce Walker for helping toinitiate these studies. This work was supported bygrants AI 33327 (RPJ) and CA 65319 (FW).

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