VIRAL IMMUNOLOGYVolume 18, Number 4, 2005© Mary Ann Liebert, Inc.Pp. 657–667

Recombinant Adenovirus Vector Vaccine Induces StrongerCytotoxic T-Cell Responses Than Recombinant VacciniaVirus Vector, Plasmid DNA, or a Combination of These

KEN MAEDA,1,2 KIM WEST,1 DAISUKE HAYASAKA,1 FRANCIS A. ENNIS,1and MASANORI TERAJIMA1

ABSTRACT

The efficiency of prime-boost vaccinations on the induction of T-cell responses to Sin Nombre virusnucleocapsid protein expressed by recombinant vaccinia virus, replication-deficient adenovirus, andplasmid DNA in mice was quantitated by the number of epitope-specific interferon-�–producing Tcells and cytotoxic T-lymphocyte activity induced. In prime-boost immunizations, all combinationsthat included the recombinant adenovirus induced a much higher number of epitope-specific in-terferon-�–producing T cells than did other combinations. A single immunization of the recombi-nant adenovirus was able to induce similarly high levels of epitope-specific interferon-�–producingcells, despite the fact that the recombinant adenovirus produces less amount of the Sin Nombre virusnucleocapsid protein.

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1Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts.2Present address: Department of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.

INTRODUCTION

IT IS VERY IMPORTANT to develop effective vaccinesagainst viral infections by inducing strong and long-

lasting cellular immune responses, especially cytotoxicT-lymphocyte (CTL) responses, (1). CTL responses arealso important for some intracellular bacterial and proto-zoan infections (16,22) and for tumor immunity (17). Re-combinant vaccinia virus (5) and plasmid DNA (7) areefficient vectors for inducing CTL responses (1), andprime-boost immunization strategies using a combinationof these two vectors expressing the same antigen havebeen shown to induce stronger CTL responses (13,15,22).A replication-deficient human type 5 adenovirus has beentested as a vaccine vector and induced strong cellular im-munity (6,19). Recombinant adenoviruses can also beused in prime-boost immunization (4).

As a part of our ongoing research studying the im-munopathogenesis of hantavirus pulmonary syndromecaused by Sin Nombre virus (SNV), we have been map-ping T-cell epitopes recognized by mouse and human CTLsspecific to SNV proteins, as we hypothesize that CD8� CTLspecific for SNV may play a key role in pathogenicity ofSNV to human (9,11,12). To identify mouse CTL epitopeswe immunized C57BL/6J mice with a recombinant vacciniavirus, a plasmid DNA (12), and a recombinant adenovirus(this report) expressing nucleocapsid (N) protein of SNV(Table 1). In the course of these experiments we observedconsiderable differences in the number of interferon (IFN)-�–producing T cells specific to the SNV-N protein. In thisreport we compare the differences in T-cell induction bythese three vectors, including their use in combinations inprime-boost immunizations, using CTL and IFN-� enzyme-linked immunospot (ELISpot) assays.

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MATERIALS AND METHODS

Cells. Target cell lines used in CTL assays were theEL-4 murine lymphoma cell line (H-2b), the MC57Gmurine fibrosarcoma cell line (H-2b), the L929 (H-2k)cell line, an L929 cell line expressing with H-2Db (L-Db), a human T-cell line Jurkat, a Jurkat cell line ex-pressing with H-2Kb (Jurkat-Kb). L-Db and Jurkat-Kb celllines were obtained from Dr. Raymond M. Welsh of theUniversity of Massachusetts Medical School. 293 Cells(ATCC number CRL-1573) were used to generate, prop-agate, and titrate recombinant adenoviruses. CV-1 cells(ATCC number, CCL-70) were used to propagate andtitrate vaccinia viruses.

CTL lines. CTL lines specific for epitopes with SNVN proteins, NC91-105, NC175-189, NC217-231, orNC331-345 were established using splenocytes frommice immunized with the plasmid DNA, pSNVS, ex-pressing the SNV N protein as previously reported (12).These CTL lines were stimulated in vitro with �-irradi-ated feeder cells pulsed with 5 �g/mL of each peptidevery 2 weeks and maintained in RPMI1640 medium con-taining 10% heat-inactivated fetal bovine serum (FBS),25 U/mL of human interleukin (IL)–2 and 5 � 10�5

mol/L 2-mercaptoethanol (2-ME). Medium was replen-ished twice every week. In CTL assays, CTL lines wereused as effector cells at 7 days after stimulation.

Construction of replication-deficient recombinantadenoviruses. For construction of replication-deficientrecombinant adenoviruses, Adenovirus Expression Vec-tor Kit (Takara Shuzo, Kyoto, Japan) was used follow-ing the manufacturer’s recommendations. The SNV Nprotein cDNA was excised from the plasmid, pSNVS(12), with restriction endonuclease PstI, blunt-ended withKlenow fragment and inserted into the cosmid, pAx-CAwt, at its SwaI site. The cosmid carrying the SNV NcDNA under the control of the strong CAG promoter,pAxCAwt-SNV-N, was packaged using GigapackR IIIXL packaging extract (Stratagene, La Jolla, CA). The 293

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cells were transfected with pAxCAwt-SNV-N or pAx-CAiLacZ carrying the LacZ gene (Takara Shuzo) and re-striction enzyme-digested DNA-TPC (Takara Shuzo) us-ing a Calcium Phosphate Transfection Kit (Invitrogen,Carlsbad, CA). Recombinant adenoviruses, rAd-SNV-Nand rAd-LacZ, were cloned by limiting dilution andgrown in 293 cells for preparation of high-titer virus stocksolutions. Viral titers were determined by the 50% tissueculture infectious dose (TCID50) method in 293 cells. Theinserted SNV N protein cDNA was amplified by poly-merase chain reaction (PCR) using adenovirus genomicDNA as a template and the nucleotide sequence was con-firmed by direct sequencing of the PCR product. The nu-cleotide sequence of the SNV N cDNA in this recombi-nant adenovirus was identical to those in Vac-SNV N andpSNVS. All sequencing reaction was performed by theNucleic Acid Facility at the University of MassachusettsMedical School. The expression of the SNV N protein inrAd-SNV-N–infected HeLa, 293, and MC57G cells wasconfirmed by Western blotting analysis (Fig. 4). Recom-binant adenoviruses were subcultured less than five gen-erations to avoid acquiring the ability to replicate throughhomologous recombination in 293 cells.

To confirm that SNV N protein was expressed and pre-sented on the antigen presenting cells infected with rAd-SNV-N, EL-4 cells or MC57 G cells infected with rAd-SNV-N were used as target cells in CTL assays usingSNV N-specific CTL lines as effectors (data not shown).We also used rAd-SNV-N to stimulate bulk culturesplenocytes from Vac-SNV N–immunized mice to con-firm that splenocytes infected with rAd-SNV-N were ableto stimulate SNV N-specific CTLs (data not shown). Inthese assays rAd-LacZ was used as a negative control.

Recombinant vaccinia virus. The recombinant vac-cinia virus expressing SNV N protein, Vac-SNV-N, waspreviously described (9). CV-1 cells (ATCC number CCL-70) were used to propagate and to titrate the vaccinia virus.

Plasmid DNA. The plasmid, pSNVS, carrying the SNVN cDNA under the control of human cytomegalovirus im-

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TABLE 1. PEPTIDES USED IN THIS STUDY

Peptide Amino acid sequencec MHC class I restriction

NC91-105a 91KEKSSLRYGNVLDVN105 (H-2Kb)d

NC175-189a 175RHLYVSMPTAQSTMK189 H-2Db

NC211-225b 211KARNIISPVMGVIGF225 H-2Kb

NC217-231a 217SPVMGVIGFSFFVKD231 H-2Kb

NC331-345a 331FAILQDMRNTIMASK345 Unknown

aPreviously reported (12).bMinimal CTL epitope was identified in this study.cBoxes indicate the minimal epitopes identified in the 15mer peptide.dMHC class I restriction has not been experimentally determined. The minimal epitope “SSLRYGNV” has a typical H-2Kb

binding motif (12).

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mediate-early/enhancer promoter (12) was purified usingEndoFree Plasmid Giga Kit (Qiagen, Valencia, CA) andthen dissolved in 0.85% saline solution at a concentrationof 1 mg/mL.

Peptides. Peptides were synthesized at the ProteinChemistry Core Facility at the University of Massachu-setts Medical School using an automated Rainin Sym-phony peptide synthesizer (Table 1).

Immunization of mice with recombinant aden-ovirus. Male C57BL/6J mice (H-2b) (4–6 weeks old)were purchased from Jackson Laboratories (Bar Harbor,ME). Mice were immunized with the recombinant aden-ovirus (rAd-SNV-N) at indicated virus titers, and spleno-cytes were collected 21 days after immunization. All micewere kept in the Animal Facility in the University ofMassachusetts Medical School, which is regulated byAWA-1995, PHS-1986, and MA140-1985 and followsthe AAALAC-1965 guidelines. Anesthesia provided byketamine/xylazine and cervical dislocation consistentwith the recommendations of the Panel on Euthanasia ofthe American Veterinary Medical Association were used.

Prime-boost immunization. Prime-boost immuniza-tion were performed using pSNVS, Vac-SNV-N, rAd-SNV-N, and phosohate-buffered saline (PBS) (Table 3).Vac-SNV-N and rAd-SNV-N were dissolved in PBS at1.0 � 108 PFU/mL and 1.0 � 108 TCID50/mL, respec-tively, and inoculated intraperitoneally into C57BL/6Jmice in 200 �L per mouse. For plasmid injection, 50 �Lof plasmid solution (1 mg/mL) was intramuscularly in-jected into each rear quadriceps.

Preparation of splenocytes. Freshly isolated spleno-cytes were treated with 1 � ACK buffer (0.829% (W/V)of NH4Cl, 0.1% (W/V) of KHCO3, and 0.0072% (W/V)of EDTA-Na2), suspended in RPMI 1640 medium with10% FBS and then used for ELISpot and CTL assays.

In vitro stimulation. The freshly isolated splenocyteswere stimulated with 10 �g/mL of each peptide inRPMI1640 medium containing 10% FBS, 25 U/mL ofhuman IL-2, and 5 � 10�5 mol/L 2-ME. Bulk culture51Cr release assay was performed at day 7 of culture.

51Cr release assay. Target cells were labeled with 0.25mCi of 51Cr for 60 min at 37°C. After labeling, the cellswere washed three times and then resuspended at 4 �104/mL in RPMI 1640 containing 10% FBS. Effectorcells were added to 2 � 103 target cells pulsed with 10�g/mL of peptide or 2 � 103 recombinant adenovirus-in-fected cells per milliliter in a 96-well, round-bottom plateat various effector-to-target ratios (E:T ratio). When re-combinant adenovirus was used to infect target cells, 1/10of the freshly isolated splenocytes were infected withrAd-SNV-N or rAd-LacZ at 37°C for 1 h, washed threetimes, and mixed with the remaining 9/10 of splenocytes.Mixed splenocytes were incubated at 37°C for 7 days andused as effector cells in CTL assay. Plates were incu-

RECOMBINANT ADENOVIRUS VECTOR VACCINE AND CTL RESPONSES

bated for 4.5 h at 37°C, supernatants were harvested (Ska-tron Instruments, Sterling, VA), and specific lysis wascalculated as ([experimental release-spontaneous re-lease]/[maximal release � spontaneous release]) � 100(%). All experiments were performed at least twice. Spe-cific lysis higher than 20% was considered positive.Spontaneous lysis was �15% in all assays.

ELISpot assay for single-cell IFN-� secretion.ELISpot assays were performed according to the manu-facturer’s protocol (Mabtech AB, Nacka Strand, Swe-den). Briefly, 96-well Multiscreen-IP plates (Millipore,Bedford, MA) were coated with 15 �g of rat anti-mouseIFN-� monoclonal antibody (AN-18) per milliliterovernight at 4°C. Freshly isolated splenocytes were thenincubated with or without 4 �g/mL of peptide or 5 �g/mLof ConA at 37°C for 2 h and added to the precoatedELISpot plates at 2.5 � 105 cells/well in RPMI 1640 con-taining 10% FBS. Plates were incubated for 18–20 h at37°C. Biotinylated rat anti-mouse IFN-� monoclonal an-tibody (R4-6A2) was added and incubated for 2 h at roomtemperature, followed by the addition of streptavidin-horseradish peroxidase for 1–2 h at room temperature.Spots were stained with Vector NovaRED™ Substrate kitfor peroxidase (Vector Laboratories, Burlingame, CA).The precursor frequency was calculated as ([number ofspots in experimentall well � number of spots inmedium-control well) / total number of cells per well).Experiments were performed in triplicate.

In ELISpot assays using splenocytes from rAd-SNV-N–immunized mice, some peptides, especially NC175-189 and NC211-225, induced too many IFN-�–produc-ing cells to count. Therefore we modified the ELISpotassay as described above. In brief, splenocytes from nor-mal age-matched C57BL/6J mice were also collected atthe same time and used as feeder cells. Splenocytes(2.5 � 104) from immunized mice were mixed with2.25 � 105 splenocytes from normal C57BL/6J mice,pulsed with each peptide, and then plated to one well forELISpot assay. In preliminary experiments, we mixedsplenocytes from immunized mice with splenocytes fromnonimmunized mice at various ratios to confirm the lin-earity between the number of IFN-�–producing cells andthe dilution ratio.

Identification of a minimal CTL epitope within thepeptide NC211-225 and determination of its MHCclass I restriction. C57BL/6J (H-2b) mice were immu-nized intraperitoneally twice with rAd-SNV-N and theisolated splenocytes were analyzed for the identificationof minimal epitope in ELISpot and CTL assays using trun-cated peptides of peptide NC211-225. For identificationof MHC class I restriction, CTL assays were carried outusing the cell lines L-Db, Jurkat, and Jurkat Kb-1 pulsedwith each peptide as target cells. These methods were pre-viously described (12).

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Western blotting. Whole cell lysates were preparedfrom EL-4 cells infected with the recombinant vacciniavirus or the recombinant adenovirus at the indicated mul-tiplicity of infection (MOI) for overnight, separated in a10% sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS-PAGE) and transferred onto a Poly-Screen PVDF Transfer Membrane (Perkin Elmer LifeSciences, Boston, MA) in a Trans-Blot ElectrophoreticTransfer Cell (Bio-Rad Laboratories, Hercules, CA) aspreviously described (20). Nonspecific binding wasblocked by incubating the membrane in 5% nonfat drymilk (Stop & Shop, Boston, MA) in PBS-Tween 20 so-lution (PBS-T). Primary antibody, rabbit anti-SNV serum(kindly provided by Drs. Patrick C. Stockton and ThomasG. Ksiazek) (23) was used at a 1:2000 dilution in 1%bovine serum albumin (BSA) in PBS-T, and a secondaryantibody, goat anti-rabbit IgG antibody conjugated withhorseradish peroxidase (Kirkegaard & Perry Laborato-ries, Gaithersburg, MD), was used at 1:1500 dilution in1% BSA in PBS-T. Bound antibodies were detected by

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a Western Lightning Chemiluminescence Reagent Plus(Perkin Elmer Life Sciences) according to the manufac-turer’s instruction. After washing, the membrane wasstained by SimpleBlue SafeStain (Invitrogen) to visual-ize proteins according to the manufacturer’s instructions.

Statistical analyses. The Student t test and linear re-gression analysis were performed using Microsoft® Ex-cel 2002. Values of p � 0.05 were considered statisti-cally significant.

RESULTS

Immunization of mice with recombinant adenovirus,rAd-SNV-N, and identification of a new CTL epitope.A replication-deficient recombinant adenovirus serotype 5expressing the SNV N protein was constructed and desig-nated as rAd-SNV-N. As a control, rAd-LacZ was alsoconstructed according to the manufacturer’s protocol.

C57BL/6J mice were intraperitoneally immunized

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TABLE 2. IDENTIFICATION OF A MINIMAL EPITOPE IN THE PEPTIDE NC211-225

IFN-�–producing cellsExp Peptide Sequence % Specific lysisa,c per 106 splenocytesb,c

1 NC205-219 LFPAQVKARNIISPV 14.6 0NC211-225 KARNIISPVMGVIGF 21.6 465NC217-231 KARNIISPVMGVIGFSFFVKD 20.6 93NC217-229 KARNIISPVMGVIGFSFFV 27.8 132NC217-227 KARNIISPVMGVIGFSF 35.9 320NC217-226 KARNIISPVMGVIGFS 27.9 191NC218-227 KARNIIsPVMGVIGFSF 17.6 69NC218-226 KARNIIsPVMGVIGFS 13.1 7NC218-225 KARNIIsPVMGVIGF 9.4 25NC215-223 KAR IISPVMGVI 6.0 8NC216-223 KAR IISPVMGVI 7.8 11Medium 7.0 0

2 NC211-225 KARNIISPVMGVIGF 21.4 140NC217-231 KARNIISPVMGVIGFSFFVKD 33.7 56NC217-227 KARNIISPVMGVIGFSF 30.9 123NC217-226 KARNIISPVMGVIGFS 21.9 117NC217-225 KARNIISPVMGVIGF 21.5 133NC217-224 KARNIISPVMGVIG 28.5 7NC216-223 KAR IISPVMGVI 14.8 0NC218-225 KARNIIsPVMGVIGF 18.5 3Medium 9.5 0

aMice were intraperitoneally immunized twice with 2.0 � 107 TCID50/200 �L of rAd-SNV-N. Splenocytes were collected andstimulated with 10 �g/mL of each peptide. After 7 days of in vitro stimulation, CTL assays were carried out using EL-4 cells pulsedwith 1 �g/mL of each peptide as target cells. The E:T ratio was 100�1.

bELISpot assays using 4 �g/mL of each peptide were carried out using freshly isolated splenocytes.cUnderlines indicate the specific lysis of target cells ( �20%) and the significantly high number of IFN-�–producing cells

specific for the peptides (over 50 cells per 106 splenocytes).Exp, experiment; boxes indicate the minimal epitopes.

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with rAd-SNV-N. Splenocytes were collected at day 21postimmunization and used to measure the number ofIFN-�–producing cells specific for CTL epitopes in anELISpot assay. In this experiment we included 15merpeptides from SNV N protein, which had not been rec-ognized by SNV N–specific CTL in previous experi-ments (12). One of these peptides, NC211-225, inducedIFN-�–producing cells, suggesting that this peptide mightcontain a previously unidentified CTL epitope. We usedbulk culture CTL assays and ELISpot assays with truncated peptides to identify a minimal epitope withinpeptide NC211-225. The results indicated that a 9-merpeptide, 217SPVMGVIGF225, was a minimal epitope(Table 2). CTL assays using an L929 cell line express-ing H-2Db and a Jurkat cell line expressing H-2Kb as target cells showed that the epitope 217SPVMGVIGF225

was restricted by H-2Kb (data not shown). The peptideNC 217-231, which overlaps with the NC211-225, alsocontained this 9–amino acid sequence (Table 1), but theminimal epitope recognized by a CTL line specific for the NC 217-231 was 218PVMGVIGFS226, not217SPVMGVIGF225 (12).

Comparison of CTL induced by prime-boost vac-cination strategies using three different vectors, a re-combinant vaccinia virus, a recombinant adenovirus,and a plasmid DNA. To compare CTL induced byprime-boost immunization, Vac-SNV-N, rAd-SNV-N,

RECOMBINANT ADENOVIRUS VECTOR VACCINE AND CTL RESPONSES

and pSNVS were used to immunize C57BL/6 mice withvarious prime-boost combinations (Table 3). We used2.0 � 107 TCID50/200 �L of rAd-SNV-N, as highertiters of the rAd did not increase the number of IFN-�–producing cells in ELISpot assays in the preliminaryexperiments (data not shown). ELISpot assays were car-ried out using five different peptides containing CTL epi-topes (Table 1) (Fig. 1). The result showed that all vac-cination groups immunized with a combination thatincluded one dose of the rAd-SNV-N (Fig. 1A) possessedan approximately 30-fold higher number of IFN-�–pro-ducing splenocytes specific for the SNV N peptides thanthose immunized with a combination that did not includethe rAd-SNV-N (Fig. 1B) (comparison between the com-binations including one dose of the rAd-SNV-N and thecombinations not including the rAd-SNV-N by a two-tailed Student t test, p � 0.001).

Unexpectedly, the hierarchy of epitope specific CTLresponses was different among the combinations. For ex-ample, in six of seven combinations containing the rAd-SNV-N, the IFN-�–producing cells specific for theNC211-225 were highest in number among five peptides(Fig. 1A). On the other hand, in five of seven combina-tions not containing rAd-SNV-N, which induced a totalof �20 IFN-�–producing cells/106 splenocytes, cells spe-cific for the NC175-189 peptide were highest in numberof the five peptides (Fig. 1B).

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TABLE 3. PROTOCOL FOR PRIME-BOOST IMMUNIZATION OF C57BL/6J MICE

MouseGroup no. 0 wk 2 wk 4 wk 6 wk 8 wk

PBS-PBS 2 PBSa PBSPBS-pSNVS 2 PBS pSNVS pSNVSPBS-rVV 2 PBS Vac-SNV-NPBS-rAd 2 PBS rAd-SNV-NpSNVS-PBS 2 pSNVSb pSNVS PBSpSNVS-pSNVS 2 pSNVS pSNVS pSNVS pSNVSpSNVS-rVV 2 pSNVS pSNVS Vac-SNV-NpSNVS-rAd 2 pSNVS pSNVS rAd-SNV-N SamplerVV-PBS 2 Vac-SNV-Nc PBS collectionrVV-pSNVS 2 Vac-SNV-N pSNVS pSNVSrVV-rVV 2 Vac-SNV-N Vac-SNV-NrVV-rAd 2 Vac-SNV-N rAd-SNV-NrAd-PBS 1 rAd-SNV-Nd PBSrAd-pSNVS 2 rAd-SNV-N pSNVS pSNVSrAd-rVV 2 rAd-SNV-N Vac-SNV-NrAd-rAd 2 rAd-SNV-N rAd-SNV-N

aMice were intraperitoneally immunized with 200 �L of PBS.bMice were intramuscularly immunized with 100 �g/100 �L of pSNVS.cMice were intraperitoneally immunized with 2.0 � 107 PFU/200 �L of Vac-SNV-N.dMice were intraperitoneally immunized with 2.0 � 107 TCID50/200 �L of rAd-SNV-N.

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CTL activities specific for the NC211-225 peptide werealso compared using splenocytes stimulated in vitro with10 �g/mL of peptide and peptide-pulsed EL-4 cells as tar-get cells. The results show that NC211-225–pulsed EL-4cells were specifically killed by in vitro stimulated spleno-cytes from groups “rVV-rAd”, “rAd-PBS”, “rAd-rVV”,and “rAd-rAd”, but killing was not detectable usingsplenocytes from groups “rVV-PBS”, “rVV-pSNVS”,“rVV-rVV” or “rAd-pSNVS” (Fig. 2). Specific cytotoxicactivities correlated with the numbers of IFN-�–produc-ing cells measured by ELISpot assays (r � 0.95 (whenspecific lysis at E:T ratio of 100:1 was used), 0.96 (50:1),0.98 (25:1), and 0.98 (12.5:1), p � 0.001 by linear re-gression analysis).

Comparison of CTL induced by single immuniza-tion with a recombinant adenovirus or a recombinantvaccinia virus. Because we observed in the prime-boostimmunizations that immunization with combinations in-cluding the adenovirus vector induced a much highernumber of IFN-�–producing cells and stronger CTL ac-tivity with a different epitope hierarchy than combina-tions not including the adenovirus vector, we compared

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the recombinant adenovirus and the recombinant vac-cinia virus in a single immunization protocol. At 21days after immunization, freshly isolated splenocyteswere used in ELISpot assays using the five peptidescontaining CTL epitopes. The results show that immu-nization with rAd-SNV-N alone or Vac-SNV-N aloneinduced a total of approximately 3000 or 100 IFN-�–producing splenocytes respectively specific for thefive peptides (p � 0.05 by the two-tailed Student t test)(Figs. 3A and 3B). We observed a similar pattern ofthe epitope dominance in this single immunization ex-periment. Splenic T cells reactive for the peptideNC211-225 were 55.5–73.7% of the total number ofIFN-�–producing cells specific for these five peptidesin rAd-SNV-N–immunized mice, whereas T cells spe-cific for NC175-189 represented 65.0–70.7% of the to-tal in Vac-SNV-N–immunized mice.

Because all of these experiments were done using15mer peptides, we repeated some of these experimentsusing peptides of minimal epitopes (Table 1). ELISpotassays using peptides of minimal epitopes showed simi-lar results except that the difference in immunodomi-

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FIG. 1. IFN-�–producing cells induced by different immunization protocols. C57BL/6J mice were immunized with variousimmunization protocols listed in Table 3. At 8 weeks, splenocytes were collected, stimulated with 4 �g/mL of each peptide, andadded to each well for ELISpot assay. Experiments were performed in triplicate wells. The precursor frequency was calculatedas ([number of spots in experimental well � number of spots in medium-control well] / total number of cells per well). Data areshown as means of the results from two mice.

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FIG. 2. CTL activities of bulk culture splenocytes frommice immunized by various protocols. C57BL/6J mice wereimmunized with the immunization protocols shown in Table 3.At 8 weeks, splenocytes were collected and stimulated in vitrowith 10 �g/mL of NC211-225 for 7 days. Target cells, EL-4cells, were labeled with 51Cr, pulsed with 10 �g/mL of NC211-225, and mixed with bulk culture splenocytes at various E:Tratios. Experiments were performed in triplicate. Numbers inthe list show IFN-�–producing cells per 106 splenocytes thatwere used for this CTL assay. Open symbols indicate no sig-nificant CTL activity at an E:T ratio of 100:1 (�15%).

nance was not observed between rAd-SNV-N–immu-nized and Vac-SNV N–immunized mice (Figs. 3C and3D). These results suggest that there may be an additionalepitope contained in the peptide NC175-189 that was dif-ferent from the 9mer, 180SMPTAQSTM188, and that wasalso recognized by T cells induced in Vac-SNV N-im-munized mice.

SNV N protein expression from the vectors. Thelevel of the SNV N protein expression was compared be-tween rVV-infected and rAd-infected mouse EL-4 cells,which are commonly used as antigen-presenting cells inmouse CTL assays, by Western blotting using rabbit anti-SNV serum. The cells infected with the rAd-SNV-N atMOI of 10 produced lower amount of the SNV N pro-tein than cells with the Vac-SNV-N at MOI of 1 (Fig. 4)(we used 2.0 � 107 TCID50 of rAd-SNV-N and 2.0 �107 PFU of Vac-SNV-N for immunization).

DISCUSSION

In this study we generated a recombinant adenovirusexpressing the SNV N protein to identify CTL epitopesin the SNV N protein, and we observed differences inCTL induction to CD8� T cell epitopes in the SNV Nprotein using three diferent vectors (adenovirus, vacciniavirus, and plasmid).

When C57BL/6J mice (H-2b) were immunized withrAd-SNV-N, IFN-�–producing cells specific for pep-tide NC211-225 were higher in number than those spe-cific for other peptides (Figs. 1 and 3). This peptidecontains the 9mer epitope, 217SPVMGVIGF225, re-stricted by H-2Kb. The peptide NC217-231 also con-tains this 9mer epitope in addition to another H-2Kb–re-stricted CTL epitope, 218PVMGVIGFS226, as wepreviously identified (12). However, IFN-�–producingT cells specific for the peptide NC217-231 were muchlower in number than those for the peptide NC211-225,which was of interest. One explanation may be that be-cause 217SPVMGVIGF225 is located at the C-terminusin the peptide NC211-225 and at the N-terminus in thepeptide NC217-231, the efficiency of the generation ofthe antigenic peptide (217SPVMGVIGF225) from thelonger peptides may be different. Therefore cells incu-bated with NC211-225 may present the peptide217SPVMGVIGF225 more efficiently than those incu-bated with NC217-231, may stimulate more IFN-�–producing cells, and may be better recognized byCTLs specific for 217SPVMGVIGF225. On the contrary,cells incubated with NC217-231 appear to present thispeptide 217SPVMGVIGF225 less well, and were recog-nized mainly by CTLs specific for 218PVMGVIGFS226.We usually do not observe a difference in ELISpot orCTL recognition between 9mer peptides as the mini-mal epitopes and the longer peptides containing theminmal epitopes; however, Draenert et al. recently re-ported that CD8 T-cell responses to an epitope withina longer peptide are best detected when the epitope issituated at the C-terminal end of the longer peptide (8).In an ELISpot assays using minimal epitopes, NC217-225 induced a higher number of IFN-�–producing cellsthan did peptide NC218-226 (Fig. 3, lower two pan-els), supporting our interpretation. Contrary to theELISpot assays, the levels of CTL activities specificfor the NC211-225 and NC217-231 were not different,probably because there was an adequate number of pre-cursor CTLs specific for either epitope to be amplifiedin vitro to show specific lysis after several days in bulkculture (Table 2).

Various prime-boost vaccination strategies using rAd-SNV-N, Vac-SNV-N, and pSNVS were analyzed (Table3), and cellular immune responses were compared inELISpot and CTL assays (Figs. 1 and 2). The combina-tions of Vac-SNV-N and pSNVS induced higher num-bers of IFN-�–producing cells than repeated immuniza-tions with the same vaccine vector (“pSNVS-rVV” and“rVV-pSNVS” versus “pSNVS-pSNVS” and “rVV-rVV”). When rAd-SNV-N was used for prime-boost im-munizations, any of the combinations that contained therAd-SNV-N induced a much higher number of IFN-

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FIG. 3. Comparison of the number of IFN-�–producing cells induced by single immunization with recombinant adenovirusor vaccinia virus. C57BL/6J mice were immunized with 2.0 � 107 TCID50/200 �L of rAd-SNV-N (A and C) or 2.0 � 107

PFU/200 �L of Vac-SNV-N (B and D). At 3 weeks, splenocytes were collected from three mice in each group, stimulated with4 �g/mL of each 15mer peptide (top two panels) or with 4 �g/mL of each minimal epitope peptide (C and D), and added to eachwell for ELISpot assay. Experiments were performed in triplicate. The precursor frequency was calculated as ([number of spotsin experimental well � number of spots in medium-control well] / total number of cells per well). Numbers in the y-axis vary inthe panels.

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�–producing cells than those not containing rAd-SNV-N(Fig. 1). In addition, even single immunization with therecombinant adenovirus was as efficient as heterologousprime-boost immunizations that contained the recombi-nant adenovirus (Figs. 1 and 3). In rhesus monkeys a re-combinant adenovirus expressing an HIV-1 gag gene wasmore immunogenic for a cellular immune response thana recombinant vaccinia virus and a plasmid DNA ex-pressing the same antigen (6,19).

The level of the SNV N protein expression was lowerfrom the recombinant adenovirus than that from the re-combinant vaccinia virus in EL-4 cells, which arecommonly used as antigen-presenting cells in mouseCTL assays (Fig. 4). In infected mice, vaccinia virusreplicates after inoculation (2), but the replication-de-ficient adenovirus cannot (10), which means that in vivothe differences in the level of the SNV N protein ex-pression is even larger. Therefore the amount of theprotein produced is not the reason why rAd-SNV-N in-duced a much higher number of IFN-�–producing cells

RECOMBINANT ADENOVIRUS VECTOR VACCINE AND CTL RESPONSES

than the Vac-SNV N. There is a report of a vacciniavirus–induced defect in the presentation of influenzaantigens, which can be overcome by enhancing thedegradation of the influenza protein in infected cells(21); but the vaccinia virus vector is often used verysuccessfully in CTL assays to express antigenic pro-teins (3). The adenovirus vector that we used in this re-port lacks the E1 and E3 genes (14), the products ofwhich can block antigen presentation by MHC class Imolecule (18). We measured the surface expression ofmouse MHC class I molecules, H-2Kb and H-2Db, andwe did not observe any difference in the surface ex-pression level of these two molecules after infection ofcells with either the Vac-SNV N or the rAd-SNV N in-fection (data not shown). After intraperitoneal inocu-lation, these two viruses may infect different antigen-presenting cells and/or may have different effects onthe infected antigen-presenting cells. An amino acid se-quence homology search was performed using the Na-tional Center for Biotechnology Information’s pro-tein–protein BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/), and we found that the five SNV N–spe-cific epitopes analyzed in this study were not homolo-gous to any peptide sequences in the proteins that ade-novirus type 5 encoded.

In conclusion, recombinant adenovirus induced a muchhigher number of epitope-specific, interferon-�–produc-ing cells to the inserted gene product than the prime-boostimmunization combining recombinant vaccinia virus andplasmid DNA carrying the same insert, despite the factthat the recombinant adenovirus produces less proteinfrom the inserted gene than the recombinant vacciniavirus. It is important to determine what factors make re-combinant adenovirus more immunogenic for epitope-specific T cells, for the purpose of developing more ef-ficacious vaccines.

ACKNOWLEDGMENTS

We thank Raymond M. Welsh of University of Mass-achusetts Medical School for kindly providing the L-Db and Jurkat-Kb cell lines, and Patrick C. Stocktonand Thomas G. Ksiazek of the Centers for Disease Con-trol and Prevention for the rabbit anti-SNV serum. Wealso thank Anita M. Leporati and Destin Heilman fortechnical assistance, and Alan L. Rothman, TomokoToyosaki-Maeda, and Anuja Mathew for advice andsuggestions.

This work was supported by grant U19-AI-057319from the National Institute of Allergy and Infectious Dis-eases, the National Institutes of Health.

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FIG. 4. Protein expression from recombinant adenovirusand recombinant vaccinia virus. The SNV N protein was de-tected by rabbit anti-SNV serum (A). Arrow indicates band cor-responding to SNV N protein. The same membrane was washedand then stained with SimpleBlue SafeStain (Invitrogen) to vi-sualize all proteins transferred (B).

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Address reprint requests to:Dr. Masanori Terajima

Center for Infectious Disease and Vaccine ResearchUniversity of Massachusetts Medical School

55 Lake Avenue NorthWorcester, MA 01655

E-mail: Masanori.Terajima@umassmed.edu

Received March 30, 2005; accepted August 9, 2005.

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