Phase I Trial of Recombinant Modiﬁed Vaccinia Ankara Encoding … · Clinical Studies Phase I Trial of Recombinant Modiﬁed Vaccinia Ankara Encoding Epstein–Barr Viral Tumor

Clinical Studies

Phase I Trial of Recombinant Modified VacciniaAnkara Encoding Epstein–Barr Viral Tumor Antigensin Nasopharyngeal Carcinoma Patients

Edwin P. Hui1, Graham S. Taylor2, Hui Jia2, Brigette B.Y. Ma1, Stephen L. Chan1, Rosalie Ho1,Wai-Lap Wong1, Steven Wilson3, Benjamin F. Johnson4, Ceri Edwards5, Deborah D. Stocken2,Alan B. Rickinson2, Neil M. Steven2, and Anthony T.C. Chan1

AbstractEpstein–Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a

high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclearantigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinantvaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity tothese antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma todetermine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primarytherapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating doselevels between 5� 107 and 5 � 108 plaque-forming unit (pfu). Blood samples were taken during prescreening,immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicitywas tested by IFN-g ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and knownepitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one tofour and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccineantigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccinedose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded thatMVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studiesexamining clinical benefit. Cancer Res; 73(6); 1676–88. �2012 AACR.

IntroductionEpstein–Barr virus (EBV) persists in most individuals as a

life-long asymptomatic infection, with both lytic virus repli-

cation in the oropharynx and latent growth-transforminginfections in the B-lymphoid system being kept in check byimmune T-cell surveillance (1). Despite the usually asymptom-atic nature of EBV carriage, the virus has potent growth-transforming ability in vitro and oncogenic potential in vivo,causing fatal B-lymphoproliferative disease lesions in theimmunosuppressed and being strongly linked to several malig-nancies; these include endemic Burkitt lymphoma, a subset ofHodgkin lymphomas, certain aggressive T/NK cell lymphomas,and an epithelial tumor, nasopharyngeal carcinoma (NPC; ref.2). Of these, nasopharyngeal carcinoma is the most importantin world health terms, occurring worldwide but at particularlyhigh incidence throughout South East Asia, especially amongChinese people (3); thus, the Hong Kong Cancer Registry 2009data report a male age-standardized incidence of 14.6/100,000,ranking nasopharyngeal carcinoma the 6th most commoncancer in men in that region. Despite radiotherapy and (inmeta-analyses) additional chemotherapy improving out-comes, 2- and 5-year disease-free survival rates are still only63% and 52%, respectively, and distant metastases account formore than 40% of recurrences (4), at which point the prognosisis very poor (5, 6). New treatment modalities are thereforeneeded in both the adjuvant and salvage settings (7), partic-ularly approaches designed for populations at highest risk.

Authors' Affiliations: 1Department of Clinical Oncology, State Key Lab-oratory in Oncology in South China, Sir YK Pao Center for Cancer, HongKong Cancer Institute and Li Ka Shing Institute for Health Sciences, TheChinese University of Hong Kong, Shatin, Hong Kong; 2Cancer ResearchUK Centre, School of Cancer Sciences, University of Birmingham; 3HealthProtection Agency, West Midlands Public Health Laboratory, Heart ofEngland Foundation Trust, Birmingham; 4Section of Virology, ImperialCollege, London; and 5Cancer Research UK Drug Development Office,London, United Kingdom

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

E.P. Hui and G.S. Taylor contributed equally as first authors.

Corresponding Authors: Neil M. Steven, School of Cancer Sciences,University of Birmingham, Vincent Drive, Birmingham, B15 2TT, UnitedKingdom. Phone: 44-121-414-4474; Fax: 44-121-414-4486; E-mail:[email protected]; and Anthony Chan, The Chinese University ofHong Kong, Department of Clinical Oncology State Key Laboratory inOncology in South China, Sir YK Pao Center for Cancer Hong KongCancer Institute and Li Ka Shing Institute for Health Sciences, HongKong. Phone: 852-26322144; Fax: 852-26497426; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-2448

�2012 American Association for Cancer Research.

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Published OnlineFirst January 24, 2013; DOI: 10.1158/0008-5472.CAN-12-2448

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The consistent presence of EBV in all nasopharyngeal car-cinoma cells opens up the possibility of an immunotherapeuticapproach, exploiting the potential of the immune T-cell systemto recognize tumor cells through their expression of viralantigens. In that regard, most nasopharyngeal carcinomasexpress just 2 latent cycle proteins, the EB nuclear antigen 1(EBNA1), a sequence-specific DNA-binding protein involved inmaintenance of the episomal virus genome, and latent mem-brane protein 2 (LMP2), a membrane-signaling protein withgrowth-promoting activity in epithelial cells (2, 8, 9). From thestudy of healthy individuals, these proteins do not constitutedominant targets of the T-cell response induced by naturalEBV infection; nevertheless, they do contain a number ofsubdominant CD8 target epitopes (mainly in LMP2) andseveral CD4 epitopes (mainly in EBNA1), presented by indi-vidual HLA I andHLA II alleles, respectively (1). Indeed, there isalready some preliminary evidence both from a dendritic cell–based vaccine trial (10) and from subsequent adoptive T-celltherapy trials (11–15) that boosting CD8 immunity to LMP2epitopes may be of clinical benefit in nasopharyngeal car-cinoma. The present approach is distinct from the above andis based on vaccination with a modified vaccinia Ankara(MVA) recombinant vector expressing the tumor-associatedviral antigens; unlike the above cell-based protocols, such aprocedure is not limited to specialized centers but has thepotential for delivery on a large scale. The vaccine virus,MVA-EL, was constructed using sequences cloned from atypical Chinese EBV strain and encodes a functionallyinactive fusion protein containing the C-terminal half ofEBNA1 and full-length LMP2A (16). Note that the C-terminalEBNA1 fragment includes almost all known epitopes in thisprotein (1) but lacks the gly-ala repeat domain that partlyimpairs presentation of cis-linked sequences to CD8þ T cells(17–19).To dateMVAor similar poxvirus-based therapeutic vaccines

expressing cellular target antigens have been tested in certainnonvirus-associated cancers; these trials almost all involveCaucasian patient cohorts and most focus only on CD8þT cell responses, indeed often on responses restricted throughjust one common Caucasian HLA I allele, HLA-A�02:01 (20, 21).In contrast, the present trial has several distinct features. First,the vaccine antigens are viral proteins against which patientswere likely to have some (albeit low) preexisting immunity (22,23) Second, the analysis focuses on both CD4þ and CD8þ T cellresponses to these antigens, as both arms of the T-cell systemare likely to be important for effective tumor control in thelonger term (24–26). Third, the vaccine is specifically designedfor use in the high-risk Chinese population, with its owndistinctive array of HLA I and HLA II alleles that will determinetarget epitope choice (23, 27–29). Thus, testing MVA-EL inpatients with nasopharyngeal carcinoma is important not onlybecause it addresses an urgent clinical need but also because ithas the potential to provide lessons of general relevance fortumor vaccination strategies.Here, we report the results of a first trial of MVA-EL

vaccination in Chinese patients with nasopharyngeal carcino-ma, focusing on patients who were in remission followingstandard therapy. The objectives were first to determine

tolerability of the vaccine across a range of doses, and secondto determine its capacity to induce EBNA1- and/or LMP2-specific T-cell responses, using natural fluctuations in immu-nity against irrelevant viral targets as an internal control. In theabsence of safety concerns, the scale, specificity, and diversityof vaccine responses were the critical factors determining doseselection and whether to proceed to further investigation.

Materials and MethodsPatients

This was a phase I dose-escalation trial of MVA-EL vacci-nation for patients with EBV-associated nasopharyngeal car-cinoma. Eligible patients had histologically poorly differenti-ated nasopharyngeal carcinoma, either confirmed as EBV-positive by in situ hybridization for the noncoding EBER RNAsor with characteristically raised serum immunoglobulin A(IgA) to EBV capsid antigen. All patients 12 weeks or morefollowing completion of first-line treatment, were in remissionand satisfied the following inclusion criteria: 18 years or more;free from toxicities >grade 1; using adequate birth control;performance status 0 to 1; life expectancy more than 4months;alkaline phosphatase and alanine aminotransferase (ALT)<1.5 � ULN; creatinine clearance >50 mL/min, hemoglobin>10.0 g/dL; lymphocytes >1.0 � 109/L; neutrophils �1.5 �109/L; platelets�100� 109/L; lacking known active hepatitis B,hepatitis C, or HIV infection, autoimmune or skin diseaserequiring therapy, active infection, severe egg allergy, splenicdysfunction, previous myeloablative, or current immunosup-pressive therapy.

The protocol (clinicaltrials.gov NCT01256853) was approvedby the Institutional Review Board. Conduct was ICH-GCP-compliant and all patients gave written informed consent.

ProceduresPatients received 3 intradermal vaccinations of MVA-EL

(Impfstoffwerk Dessau-Tornau) delivered at 3-week intervals.Sequential cohorts of 3 patients received doses of either5 � 107, 1 � 108, 2 � 108, or 3.3 � 108 plaque-forming units(pfu) per vaccination (dose levels 1–4, respectively). To gainadditional immune data, the highest dose cohort, dose level05 receiving 5� 108 pfu per vaccination, comprised 6 patients.Throughout the text, patients are identified by the code xxyy,in which xx is the vaccine dose level from 01 to 05 and yy isthe patient number from 01 to 18. Toxicity was graded accord-ing to the National Cancer Institute Common TerminologyCriteria for Adverse Events (CTCAE) Version 3.0 and using aprotocol specific grading of local injection site reactions.Blood was collected at prescreening, immediately before eachvaccination (day1; cycle 1, 2, or 3), 7 days after each vaccina-tion (day 8; cycle 1, 2, or 3), and at weeks 10, 11, 14, and 6 and12 months following the start of the vaccine course. Peripheralblood mononuclear cell (PBMC) and plasma samples werecryopreserved on each occasion, and T-cell response assays onan individual patient were conducted on simultaneouslythawed PBMC samples. ELIspot assays of T-cell responseswere conducted either against overlapping 15-mer peptidespools (pepmixes) spanning an antigen's primary sequence or

Therapeutic Vaccination of Epstein–Barr Virus Malignancies

www.aacrjournals.org Cancer Res; 73(6) March 15, 2013 1677




against defined epitopes peptides. Antigen-specific recogni-tion was defined as numbers of spot-forming cells (sfc)against a viral antigen �10 sfc/well and �2-fold greaterthan that against a negative control (actin) pepmix, epitope-specific recognition as a reading �10 sfc/well and �2-foldgreater than against the dimethyl sulfoxide (DMSO) solventcontrol. Mean spot counts for negative control wells (actinor DMSO) were subtracted from those for test wells (viralantigen pepmix or peptides, respectively) to generate meanadjusted readings. Vaccine responses were defined as anti-gen or epitope recognition for which the adjusted countspostvaccine cycle 2 or 3 were �2-fold higher than forprevaccination samples. EBV DNA loads in plasma wereassayed by quantitative PCR (qPCR); EBNA1 and vaccinia-specific antibodies were measured before and after vacci-nation using standard assays. Full details of all proceduresare provided in Supplementary Data.

Design and statistical analysisThe primary objectives were (i) to determine safety and to

characterize the toxicity profile of MVA-EL vaccine, and (ii)to describe changes in the frequency of functional T-cellresponses to EBNA1 and LMP2. The secondary objective wasto assess changes in levels of EBV genomes in plasma.

In all ELIspot assays, criteria to define antigen recognitionand vaccine response were predetermined and systemati-cally applied. Exploratory analysis of pepmix assay readingswas undertaken across all patients using repeated measuresANOVA to test whether mean adjusted readings at the 3time points differed significantly. For antigens in whichmean adjusted readings at the 3 time points were signifi-cantly different, adjusted readings at each later time pointwere compared with those for prevaccination samples usingthe Dunnett multiple comparison test. The relationshipbetween size of response and vaccine dose was analyzedby linear regression. All statistical testing was undertakenusing GraphPad Prism 5.

ResultsTable 1 gives the clinical history of the 18 Chinese patients

with nasopharyngeal carcinoma (15 male and 3 female), whoparticipated in the trial between November 2006 and April2009. All had received prior radiotherapy and 14 of 18 alsochemotherapy for their disease. The median duration fromlast treatment to first vaccination was 20 weeks (range, 14–42 weeks) and all patients were clinically disease-free onentry into the trial. Each of these 18 individuals completedtheir 3 vaccinations at the planned dose and the trialproceeded through the 5 levels of increasing vaccine dose(5 � 107 to 5 � 108 pfu/vaccination) with no evidence ofdose-limiting toxicity. As detailed in Supplementary TableS1, related adverse events mainly involved injection sitereactions, seen at grade 1 in most patients but extendingto grade 2 in 2 cases, and transiently to grade 3 in 1 indi-vidual. Fatigue, flu-like symptoms, and arthralgia wereobserved occasionally at all vaccine doses, whereas myalgiawas only reported for patients on dose level 4 to 5.

The vaccine-coded EBNA1/LMP2 fusion protein, showndiagrammatically in Fig. 1A, contains a 280-amino acidsequence from the EBNA1 C-terminus and the full 497 aminoacids of LMP2A. To look for vaccine responses, PBMC samplescryopreserved from individual patients before vaccination, 1week after the second vaccination (cycle 2; day 8) and within4 weeks after the third vaccination (cycle 3; usually day 22)were tested in ELIspot assays against separate pepmixesencompassing the primary sequences of EBNA1 and LMP2.As controls, these assays tested the same PBMCs againstseparate pepmixes covering the sequences of actin (a self-antigen used to assess assay background), of EBNA3A (an EBVlatent protein not encoded by the vaccine) and of theinfluenza matrix and nucleoproteins (FLU, an unrelatedvirus against which patients are expected to have preexistingimmunity). Note that these assays on total PBMCs detect thesum of both CD4þ and CD8þ T-cell responses to epitopepeptides within each pepmix. Figure 1B shows examples ofthe results. Patients 0516 and 0410 were typical of manyvaccinees in that the prevaccine sample revealed low butdetectable preexisting memory responses to both EBNA1and LMP2, as well as responses to EBNA3A and FLU.Importantly, the responses to EBNA1 and LMP2 were clearlyincreased after the second and third vaccinations, whereasthose to the control antigens were not. In contrast, patient0206 was one of a minority in which we were unable todetect any significant prevaccine response to EBNA1 orLMP2 (nor, in this case, to EBNA3A). Here, vaccination againinduced a response to both EBNA1 and LMP2 but not to theEBNA3A control; interestingly, this patient also provided arare example in which the preexisting FLU response wascoincidentally raised in 1 of 2 postvaccine samples.

The numbers of patients showing significant increases inresponsiveness following vaccination at the different dose levelsare shown in Table 2. Responses to EBNA1 were seen in 12patients after 2 and 3 vaccine cycles, whereas LMP2 responseswere seen in 10 patients after 2 and in 9 patients after 3 cycles. Incontrast, only 4 of 18 patients showed a coincident increase inresponses to one or other of the control antigens (EBNA3A orFLU) and in all but one case the increase was only seen in 1 of 2postvaccine samples. This clear difference between vaccine-coded and control antigen responses was observed despite thefact that, before vaccination (see hatched columns; Supplemen-tary Fig. S1), the incidence of patients with low-level preexistingimmunity to EBNA1 (15 of 18) and LMP2 (12 of 18) was notdissimilar to the numbers with preexisting immunity toEBNA3A (9 of 18) or FLU (14 of 18). The individual pepmixassay results from all 18 patients are shown as histograms inSupplementary Fig. S1, those responses that are significantlyraised above prevaccine levels after 2 (gray columns) or 3 (blackcolumns) being identified by asterisks. Vaccination was asso-ciated with increased responses to both EBNA1 and LMP2 in 11patients, to EBNA1 only in 3 patients, and to LMP2only in 1 case.Overall, therefore, 15 of 18 patients responded to 1 or othervaccine-coded antigen, in most cases with responses beingapparent both after 2 and after 3 vaccine cycles.

About the absolute size of these vaccine-induced changes,Fig. 2A combines data from all 18 patients and shows,

Hui et al.

Cancer Res; 73(6) March 15, 2013 Cancer Research1678




for each pepmix antigen, the mean difference [�95% con-fidence intervals (CI)] in ELIspot counts per well after 2 and3 vaccine cycles and the corresponding prevaccine value.Clearly, there were statistically significant increases in reac-

tivity to the vaccine-coded EBNA1 and LMP2 antigens but nosignificant change in reactivity to the control EBNA3A andFLU antigens. It is also instructive to express these increasesrelative to the preexisting levels of EBNA1-specific and

Table 1. Patients, diagnoses, and previous treatments

Patient No.a Age, y Sex Stageb Radiotherapy Chemotherapyc

Time sincetreatment,wks

0101 41 M I (T1N0M0) 6,600 cGy in 33 fractions,(NP boost by intubation)1,800 cGy in 4 fractions

NA 15

0102 57 F II (T2bN0M0) 6,600 cGy in 33 fractions,(right PP boost) 1,400 cGyin 7 fractions

NA 16

0103 55 M III (T2bN2M0) 6,600 cGy in 33 fractions,(bilateral PP boost) 1,000cGy in 5 fractions, (left PPboost) 400 cGy in 2 fractions

Cisplatin (6 cycles) 15

0204 59 F II (T2bN1M0) 6,600 cGy in 33 fractions,(NP boost) 800 cGy in4 fractions

NA 22

0205 46 M III (T2bN2M0) 6,600 cGy in 33 fractions,(NP boost) 800 cGy in4 fractions


0206 46 M IV (T2bN3M0) 6,600 cGy in 33 fractions,(left PP boost) 1,400 cGy in7 fractions


0307 53 M III (T3N2M0) 6,600 cGy in 33 fractions,(NP boost) 4 fractions 800cGy


0308 40 M III (T2bN2M0) 6,600 cGy in 33 fractions,(NP boost) 800 cGy in4 fractions, (LN boost)750 cGy in 2 fractions


0309 47 M IV (T4N1M0) 7,000 cGy in 35 fractions Inductioncarboplatinþpaclitaxel(2 cycles), cisplatin (7 cycles)

22

0410 50 M IV (T4N0M0) 7,000 cGy in 35 fractions Induction cisplatinþgemcitabine (2 cycles),cisplatin (6 cycles)

39

0411 57 F III (T3N0M0) 7,000 cGy in 35 fractions Cisplatin (6 cycles) 170412 57 M III (T3N0 M0) 7,000 cGy in 35 fractions Cisplatin (4 cycles) 140513 62 M II (T2bN1M0) 7,000 cGy in 35 fractions Cisplatin (5 cycles) 140514 42 M IV (T4N1M0) 7,000 cGy in 35 fractions,

(LN boost) 750 cGy in 2fractions


0515 55 M II (T2bN1M0) 7,000 cGy in 35 fractions Cisplatin (6 cycles) 210516 36 M II (T2bN0M0) 7,000 cGy in 35 fractions NA 180517 63 M IV (T4N1M0) 7,000 cGy in 35 fractions Cisplatin (6 cycles) 420518 55 M II (T2bN1M0) 7,000 cGy in 35 fractions Cisplatin (6 cycles) 27

Abbreviations: NP, nasopharynx; PP, parapharynx; LN, lymph node.aPatient identifiers are denoted by the prefix xx–yy, in which xx is the dose level from01 to 05 and yy is the patient number from01 to 18.bAccording to Unio Internationale Contra Cancrum/American Joint Committee on Cancer (UICC/AJCC) 1997 cancer staging manual.cCisplatin 40 mg/m2 was given weekly concurrently with radiotherapy (except as induction chemotherapy).






LMP2-specific immunity seen in the prevaccine pepmixcounts. Again, combining data from all 18 patients, reactivityto EBNA1 was increased by means of 3.5-fold (range, 0.0–8.8)and 3.2-fold (0.5 to 8.2) after 2 and 3 vaccine cycles respec-

tively, and reactivity to LMP2 by means of 3.7-fold (0.0–10.5)and 3.9-fold (0.4–16.6) over the same period. It is alsoapparent from Table 2 that the proportion of patientsmounting significant responses to EBNA1 and/or LMP2 was

A

B

GA repeat1 642

Prevaccination C2D8

363

1 497

LMP2

EBNA1

EBNA1/LMP2Y

F

Y

F

Patient 0516

Patient 0410

Patient 0206

C3D22

Prevaccination

FLU

EBNA3A

LMP2

EBNA1

Actin(-ve control)

FLU

EBNA3A

LMP2

EBNA1

Actin(-ve control)

FLU

EBNA3A

LMP2

EBNA1

Actin(-ve control)

C2D8 C3D22

Prevaccination C2D8 C3D22

Figure 1. T-cell responses inpatients with nasopharyngealcarcinoma stimulated by MVA-ELvaccination. A, design of theEBNA1 LMP2 chimeric gene insertin the MVA-EL vaccine. DNAencoding the carboxy terminal halfof EBNA1 (dark gray) was fused tothe full-length LMP2 gene (lightgray) to generate the fusion geneinserted into MVA. Locations ofdefined CD8 and CD4 T-cellepitopes studied in ELIspot assaysare shown as black or white lines,respectively. Two tyrosine residueswithin the LMP2 part of the fusionwere mutated to phenylalanine toabrogate LMP2 signaling function.B, representative results from threepatients using the whole-antigenpepmix ELIspot assay. PBMCsamples taken at three differenttime points (screening, C2D8, andC3D22) were each stimulated withEBNA1 and LMP2 pepmixes intriplicate wells. Actin pepmixserved as the negative controlindicating background T-cellactivity in the assay.Cellswerealsotested with EBNA3A and influenzapepmixes; these antigens are notpresent in the MVA-EBNA1/LMP2vaccine and therefore measurenonspecific amplification of T-cellresponses. In all three patients, theT-cell response to EBNA1 andLMP2 is increased at C2D8following 2 cycles of vaccine.These responses increased furtherat C3D22 for patients receivinglower doses of vaccine (patients0206 and 0410), whereas no furtheramplification was seen for thepatient receiving the highest dose(patient 0513). Note that for patient0206, the influenza response isincreased at C3D22 but isunchanged at the earlier time point,possibly indicating seasonalinfection with influenza. Across thetrial, only two patients had alteredinfluenza T-cell responsecompared with 15 patients withincreased EBNA1/LMP2responses.

Hui et al.





greater at dose level 5 than at the lower dose levels 1 to 4,implying a dose–response relationship. This was furtherexplored by plotting response size (the difference in adjustedreadings between pre- and postvaccination levels) against

vaccine dose. The results, shown in Fig. 2B, indeed suggesteda linear relationship for both EBNA1 and LMP2 responsesfollowing cycle 2 and for LMP2 responses following cycle 3(Fig. 2B).

Table 2. Numbers of patients with �2-fold increases in antigen-specific responders across vaccinationaccording to vaccine dose level

Following cycle 2 vs. prevaccination Following cycle 3 vs. prevaccination

EBNA1 LMP2 EBNA3A FLU EBNA1 LMP2 EBNA3A FLU

2-Fold amplification ofimmune response

Dose level 1 1/3 0/3 0/3 0/3 1/3 1/3 0/3 0/3Dose level 2 1/3 1/3 0/3 0/3 2/3 1/3 0/3 1/3Dose level 3 3/3 1/3 0/3 0/3 2/3 1/3 0/3 0/3Dose level 4 1/3 3/3 0/3 0/3 2/3 2/3 0/3 0/3Dose level 5 6/6 5/6 0/6 1/6 5/6 4/6 2/6 1/6All patients 12/18 10/18 0/18 1/18 12/18 9/18 2/18 2/18

NOTE: Cryopreserved PBMC from three time points were thawed and tested in ELIspot assays against overlapping peptides coveringthe whole sequence of actin, EBNA1, LMP2, EBNA 3, and influenza antigen (FLU) at 3� 105/well. Adjusted spot counts were the spotcounts for the viral antigensminus the spot count for actin. Postvaccination, adjusted readings�2-fold higher than for prevaccinationsamples defined an immune response.

Figure 2. Summary of whole-antigenpepmix responses for all patients inthe trial. A, the mean change inELIspot readings for all patients thatoccurred after two (diamond) or three(cross) cycles of vaccination areshown along with the 95% CIs.Results are expressed as sfc/3 � 105

PBMC. First, a repeated measuresANOVA was used to test whether themean pepmix ELIspot readings(adjusted by subtracting backgroundcounts against actin) at the three timepoints were significantly different fromeach other: EBNA 1, P ¼ 0.0001;LMP2, P¼ 0.002; EBNA 3A,P¼ 0.40;FLU, P ¼ 0.27. Using the Dunnettmultiple comparison test, statisticallysignificant increases (P < 0.05 asshown) were observed for vaccine-encoded antigens EBNA1 and LMP2but not for control antigens EBNA3Aor FLU. B, higher vaccine doses elicitstronger EBNA1 and LMP2 T-cellresponses. Responses obtained inwhole-antigen pepmix assays from all18 vaccinated patients are plottedagainst vaccine dose. The change inthe adjusted spot count/3 � 105

PBMC in the prevaccination samplesversus the samples after cycle 2and 3, respectively, is plotted againstthe vaccine dose given in pfu. Thelinear regression lines with 95% CIsare shown with an estimate ofgoodness of fit of the line to the data(R2) and the P value testing the nullhypothesis that the slope of the linedoes not differ significantly from 0.

B

A

-80

Cycle 2 Day 8

EBNA1

LMP2

Cycle 3 Day 22–29

-40

0

40

80

120

EBNA1 LMP2 EBNA 3A FLU

Ch

ang

e in

EL

Isp

ot

read

ing

s fr

om

pre

vacc

inat

ion

sam

ple

s

P < 0.05 P < 0.05 P < 0.05 P < 0.05

-100

0

100

200

300

-100

0

100

200

300

-100

0

100

200

300

2.0×108 4.0×108 6.0×108

2.0×108 4.0×108 6.0×108 2.0×108 4.0×108 6.0×108

2.0×108 4.0×108 6.0×108

Vaccine dose (pfu)

R 2 = 0.35P = 0.01

R 2 = 0.12P = 0.16

R 2 = 0.26P = 0.03

R 2 = 0.48P = 0.03

Vaccine dose (pfu)

Vaccine dose (pfu) Vaccine dose (pfu)

-100

0

100

200

300






While vaccine dose clearly seemed to influence both theincidence and size of responses to the MVA-EL vaccine, weconsidered 2 other possible factors that could be involved.The first factor was regulatory T cells (Treg). Multiparametricflow-cytometric analysis of PBMCs from 6 representativepatients showed the vaccine-induced increases in EBNA1-and/or LMP2-specific T-cell responses seen in ELIspot assaysoccurred in patients regardless of whether Treg frequencieswere normal or elevated in their peripheral blood (Supple-mentary Fig. S2). These data also showed that for 3 of 6patients Treg frequency was unchanged by vaccination; asmall increase was observed in 2 patients and a smalldecrease occurred in another. Focusing on 2 patients, 0517and 0518, for whom long-term (6 month) immune data wasavailable and who received the same vaccine dose, we notedit was patient 0518, who had the lower frequency of Tregs, inwhom the EBNA1- and LMP2-specific T-cell responses weresustained. Indeed, the EBNA1-specific response in thispatient was readily detectable 6 months after vaccination(Supplementary Table S3). The second factor was the priorstatus of the patient with respect to vaccinia exposure fromsmallpox vaccination. We therefore screened all 18 patientsand identified 4 individuals who were negative for antivac-cinia antibodies; these included patients receiving the MVA-EL vaccine across a range of doses from level 1 (patient 0101),through 3 (patient 0308) to level 5 (patients 0513 and0514). Figure 3A shows the size of vaccine-induced T-cellresponses to EBNA1 and LMP2 pepmixes in these 4 patientsversus the 14 patients with serologic evidence of priorvaccinia exposure. No significant differences were apparent.Note that nonreplicating MVA-based vaccines such as MVA-EL are primarily designed to induce T cell rather thanantibody responses to the vaccine-coded antigen; neverthe-less, such vaccines would be expected to induce a serologicresponse to structural proteins of the MVA virion. Indeed,we confirmed that antivaccinia antibodies were induced inthe 4 previously vector-na€�ve recipients and were boosted inthose with preexisting immunity (data not shown). Interest-ingly, as shown in Fig. 3B, we also noted that the mean anti-EBNA1 antibody titer in the vaccine recipients was raisedslightly above the prevaccine mean, although the increaseonly achieved statistical significance in the sample takensoon after completing the vaccine course, and not 6 or 12months later. Again, there was no apparent difference in theantibody response to EBNA1 in vaccinia-na€�ve and vaccinia-immune individuals.

We then sought to further analyze the vaccine-induced T-cell responses seen in the pepmix assays. Previous work hasidentified a number of peptide epitopes within the primarysequences of EBNA1 and LMP2 recognized by CD4þ andCD8þ T cells (1, 23, 27–29). Figure 1A shows the location of 6known CD4 epitopes and 12 known CD8 epitopes lyingwithin the EBNA1-LMP2 vaccine antigen; their coordinates,amino acid sequences, and where available, their HLArestrictions are detailed in Supplementary Table S2. Addi-tional PBMC aliquots were tested in ELIspot assays againstepitope peptides relevant to their HLA I and HLA II types,either using the peptides individually, or where cell numbers

were limiting, against small pools of relevant CD4 or CD8epitopes.

Figure 4A shows the epitope-specific ELIspot assay platesfrom one such patient, 0206 (HLA-A�11:01, A�02:03 and HLA-DP5-positive), who had already shown evidence of vaccine-induced EBNA1 and LMP2 responses in the pepmix assays(see Fig. 1 and Supplementary Fig. S1). In line with thatLMP2 pepmix response, this patient gave evidence ofresponses to 2 known LMP2-derived CD8 epitopes, SSC andLLS, that are restricted by the A�11:01 and A�02:03 alleles,respectively; likewise, in line with the EBNA1 pepmixresponse, this patient responded to a CD8 epitope, VLK/A�02:03, and a CD4 epitope, VFLQ/DP5, both derived fromEBNA1. These results, and corresponding data from epitopepeptide screening on 2 further vaccine recipients, patients0513 and 0516, are shown as graphs in Fig. 4B, below

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Figure 3. Preexisting immunity to the vector does not affect the size ofimmune response to the vaccine-coded EBV antigens. A, the differencebetweenwhole-antigen pepmix responsemeasured by ELIspot after twocycles of vaccination and that, at prevaccination, is plotted againstseropositivity for vaccinia-specific immunoglobulin before MVA-ELvaccination. The means are shown as horizontal bars and are expressedas sfc/3 � 105 PBMC. B, EBNA1 antibody responses before and afterMVA-EL vaccination. Using data from all patients, prevaccination anti-EBNA1 IgG titers were compared with those measured using samplesfrom C3 D29, 6 and 12 months postvaccination. Patients who werevaccinia seronegative before MVA-EL vaccination are represented byopen symbols. The mean values (horizontal bars) were significantlydifferent (P ¼ 0.025 repeated measures ANOVA). Compared with theprevaccination control sample, the mean difference in reading wassignificant at the 5% level only for the samples taken at C3 D29 but not at6 and 12 months (the Dunnett multiple comparison test).

Hui et al.





A

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Vaccina�on Vaccina�on Vaccina�on

Figure 4. MVA-EL stimulates CD8 and CD4 immune responses to multiple EBNA1 and LMP2 epitopes within an individual. A, shown are results obtainedtesting PBMCs frommultiple time points from a single patient (0206) in an ELIspot assay using definedEBNA and LMP2 epitope peptides. Epitope sequencesare denoted as being restricted through MHC class I (3 residue codes) and MHC class II (4 residue codes). DMSO solvent was included as a negativecontrol and phytohemagglutinin (PHA) as a positive control. For most time points, sufficient cells were available for testing with each peptide or control intriplicate; to aid clarity only one representative well is shown in the figure. Following vaccination, epitope specific T-cell responses can be detected within8 days and increase in magnitude, peaking 8 days after the third dose of vaccine (C3D8) and still detectable up to a further 50 days later (C3D51).Note that the EBNA1 and LMP2-specific T-cell responses detected in thewhole-antigen ELIspot assay (see Fig. 1A)map to knownCD8 epitopes in LMP2 andto known CD8 and CD4 epitopes in EBNA1. B, MVA-EL stimulates broad immune responses in multiple patients. Top, responses observed for threepatients to whole antigens (pepmix); actin control (open boxes), EBNA1 (gray) and LMP2 (black) using samples obtained at three time points. The remainingpanels show the results of ELIspot assays against epitope peptides within LMP2 and EBNA1, respectively, across a wider range of time points. These panelsshow the adjusted readings, for example, the difference between the epitope peptide and DMSO control spot counts at the same time points and areexpressed as sfc/3�105 PBMC. For patients 0206 and 0513, pools of two LMP2peptideswere used (Supplementary Table S3); subsequent assays assignedT-cell responses to the individual epitope peptides indicated on the figure.






histograms of the same patients' pepmix responses.Patients 0513 and 0516 had been identified as respondingto LMP2 in pepmix assays; both individuals were HLA-A�24:02–positive and indeed both responded to 2 LMP2-derived CD8 epitopes restricted by this allele, PYL and TYG.Likewise, both patients had shown an EBNA1 pepmixresponse and epitope peptide screening confirmed thatboth responded to one or more relevant EBNA1-derivedCD4 epitopes.

The detailed results obtained from all patients screenedin epitope peptide assays are recorded in SupplementaryTable S3. Overall 11 of 18 patients gave evidence of epitoperesponses appropriate to their HLA type in the periodduring the vaccine course (up to, but not including, week14). Responses were again seen most consistently in indi-viduals receiving the highest vaccine dose. Six-month fol-low-up samples were available for 12 patients, who had anEBNA1 and/or LMP2 response during vaccination allowingthe durability of these responses to be measured. Persis-tence of previously identified vaccine responses was notobserved for the 6 patients vaccinated at dose levels 1 to 4

but were identified in 3 of 6 patients at the highest doselevel.

All patients were also screened by qPCR for the presence ofEBVDNA in plasma; this not only provides a surrogatemeasureof tumor burden in nasopharyngeal carcinoma (30) but alsoshows a transient increase as tumor cells lyse in vivo imme-diately following radio/chemotherapy (31). In this trial, allpatients were clinically disease-free at the time of vaccination,and so, not surprisingly, most patients' plasma samples provedto be EBV DNA-negative both before and 1 month aftervaccination. However, 1 patient (0517) did have low butdetectable prevaccination levels of plasma EBV DNA. Inter-estingly, as shown in Fig. 5, this patient made a significant T-cell response to both EBNA1 and LMP2 pepmixes (and to atleast 1 EBNA1 CD4 epitope) during the course of vaccination,though this seemed to fall subsequently. Immediately followingthe peak of this immunologic response, EBV genome levels inplasma increased to a peak at 10 weeks and then decreased toundetectable levels in the 6- and 12-month samples. After 19months, however, this patient experienced nodal relapse, withplasma EBV DNA again detectable.

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Figure 5. Time course of EBV genomes in plasma in relation to immune responses for patient 0517. Events during screening, on and after trialparticipation are shown. Top, whole-antigen pepmix ELIspot data showing sfc/3 � 105 PBMC recognizing EBNA1 (open triangles) and LMP2 (closedtriangles) pepmixes. Middle, ELIspot data showing sfc/3 � 105 PBMC recognizing a pool of three EBNA1 CD4þ T-cell epitopes (PQCRþLRVLþNPKF).Bottom, EBV genome copies/mL plasma, the lower limit of detection of the assay is 500 EBV genomes/mL plasma (shown as horizontal brokenline). Times of vaccination are shown as arrows below the x-axis and the timescale shown on this axis applies to all three graphs. Low but stable levels ofEBV DNA in the patient's plasma then increased to a peak immediately after vaccination before decreasing to undetectable levels This peakwas preceded by vaccine-stimulated increases in EBNA1 and LMP2 immune responses. Much later (67 weeks), this patient experienced a parotidlymph node relapse (arrow). At time of relapse, the EBV level was 1,637 copies/mL but is not plotted because it was measured in a different center usinga different assay.

Hui et al.





DiscussionVirus-associated malignancies offer an important model

for the development of T cell–based cancer therapies, and theexample of nasopharyngeal carcinoma is particularly interest-ing in several respects. On the one hand, nasopharyngealcarcinoma cells consistently carry the EBV genome and inmost cases are also positive for both HLA I and HLA IImolecules at the cell surface (29, 32, 33), implying visibility toboth CD4þ and CD8þ T cells. On the other hand, virus antigenexpression in nasopharyngeal carcinoma cells is limited toEBNA1 and LMP2 (and in a minority of cases, also LMP1) andnone of these proteins are dominant targets of the naturalvirus-induced T-cell response (1). Nevertheless, most patientswith nasopharyngeal carcinoma do have low levels of CD4 andCD8 memory T cells specific for EBNA1 and/or LMP2 in thecirculation (22, 23), and indeed low numbers of CD8þ T cellswith relevant reactivities have been detected in at least sometumor biopsies (29, 34). Furthermore, adoptive transfer ofautologous T cells, expanded in vitro to enrich for EBNA1/LMP2–specific reactivities, has given early indications thatnasopharyngeal carcinoma may be accessible to attack byappropriately activated, antigen-specific cells (11–15). Thepresent work set out to explore whether one might be ableto reactivate patients' immunity to the EBNA1 and LMP2antigens by vaccination rather than by adoptive T-cell transfer,thereby opening up the possibility of a second immunother-apeutic strategy for nasopharyngeal carcinoma, indeed onethat would be deliverable to large numbers of patients withoutrequiring specialist laboratory support.The results of this first vaccine trial carried out on patients

with nasopharyngeal carcinoma in disease remission followingconventional therapy, show that the MVA-EL vaccine is bothsafe and immunogenic. On the issue of safety, vaccine doses upto 5� 108 pfu, given intradermally on 3 successive occasions at3 weekly intervals, were well tolerated even in patients whowere recovering from potentially immunosuppressive multi-cycle chemotherapy 4 to 10 months previously. Adverse eventswere limited to mainly mild injection site and transient sys-temic reactions, similar to reported patterns for other recom-binant MVA vaccines up to 109 pfu (21, 35–37; SupplementaryTable S1). This safety profile bodes well for future developmentof theMVA-EL–based vaccine approach as an immunotherapyfor nasopharyngeal carcinoma.About immunogenicity, from the evidence of pepmix assays

the vaccine specifically increased circulating T-cell responsesto EBNA1 and/or LMP2 in 15 of 18 patients. In most cases,responses to these antigens were detectable prevaccination,albeit at low levels, indicating that the vaccine was largelyboosting rather than priming immunity. Therewere occasionalexamples of EBNA1 and/or LMP2 responses in the apparentabsence of significant preexisting immunity (see patients 0308and 0412; Supplementary Fig. S1), but it may well be thatmemory cell numbers in these individuals before vaccinationwere simply below the level of detection of the pepmix assay.Combining data from all 18 patients, mean levels of bothEBNA1- and LMP2-specific T-cell numbers in the blood wereincreased 3- to 4-fold by vaccination. This effect was specific asidiosyncratic increases in preexisting immunity to control

antigens (EBNA3A and FLU) were observed only occasionally,in 4 of 18 patients, and overall there was no significant changein their mean response levels. About the absolute size ofresponses to the vaccine-coded antigens, the mean increasesin EBNA1 and LMP2 pepmix counts (Fig. 2A), recalculated as230 and 156 sfc/106 PBMC, respectively, compare well againstthose observed with other MVA recombinant vaccines, beingsimilar to those for the oncofetal antigen 5T4 in patientswith colorectal cancer with concurrent chemotherapy (38)and exceeding those seen for other MVA cancer vaccines(21, 39). Higher frequency responses have been observed withMVA vaccines against malaria or tuberculosis; however,those studies used primed healthy volunteers and focused onrecall responses to some of those organism's most immuno-genic proteins (40, 41), not (as in the present work) recentchemotherapy recipients responding to naturally subdomi-nant EBV antigens. Perhaps, more importantly, the size ofMVA-EL–induced responses in the blood was similar or higherthan measured for LMP2-specific effector frequencies follow-ing adoptive transfer of in vitro-expanded T-cell preparationsinto patients with nasopharyngeal carcinoma and Hodgkinlymphoma (11, 14, 42).

An important trial objective was to determine the dose forsubsequent efficacy trials. Selection of 5 � 108 pfu is sup-ported by several pieces of evidence. Thus, a greater pro-portion of responders in pepmix assays were observed at thehighest than the lower 4 doses and the size of response alsoincreased with dose (Table 2, Fig. 2B). Likewise, definedepitope-specific responses were detected in only 5 of 12patients receiving dose levels 1 to 4 but were present,typically at higher levels, in all 6 patients at the highestdose (Supplementary Table S3). Although the small sampleprevented multivariate analysis for all possible factors influ-encing responsiveness (e.g., HLA type, age, nasopharyngealcarcinoma treatment, and prevaccine immunity), univariateregression modeling also suggested a relationship betweenvaccine dose and immune response. Interestingly, this dose/response relationship was more apparent after 2 rather than3 vaccine cycles, raising the possibility that vaccine dose maybe less critical with repeated exposure. It is still a question ofdebate whether preexisting antibodies to the vaccinia vectoritself, particularly virus-neutralizing antibodies, might limitthe effectiveness of MVA-based vaccination. Our data on thispoint are limited as only 4 of 18 vaccine recipients wereantivaccinia antibody-negative. However, we saw no evi-dence that immune responses to the vaccine-encodedEBNA1 and LMP2 antigens were significantly different invector-na€�ve versus vector-immune individuals (Fig. 3).

The pepmix assays provide an important overall measure ofT-cell immunity to a specific antigen but do not discriminatebetween CD4þ and CD8þ responses and give no informationon specific epitope choice. In a second set of assays, therefore,we took advantage of existing epitope maps of the EBNA1 andLMP2 antigens (1) to re-examine the vaccine-inducedresponses in ELIspot assays using defined epitope peptidesrelevant to patients' HLA types. This showed that overallresponses detected in pepmix assays often included reactiv-ities to known CD8 epitopes in LMP2 or EBNA1 epitopes plus






known CD4 epitopes in EBNA1. This ability to stimulate bothCD4þ and CD8þ T cells may reflect the EBNA1/LMP2 fusionprotein's ability to access both HLA class I and class IIprocessing pathways within MVA-EL–infected antigen-pre-senting cells (16). Certainly, this coinduction of CD4 and CD8responses by the vaccine is encouraging, as both T-cell subsetsare likely to be required for effective vaccine-induced tumorsurveillance (24–26). In this regard, we noted that varyingresponse kinetics were observed for different epitopes; theseincluded (i) stable responses postcycle 1, (ii) early responsesdiminishing immediately before cycle 2 and boosted on cycle 2and 3, and (iii) responses rising and falling with successivevaccinations (Fig. 4). These different patterns in the bloodmayreflect the complex interplay between boosting responses,mobilization of T lymphocytes from the circulation into tissue,and possibly effector cell exhaustion, all factors that remain tobe dissected in the context of cancer vaccination. It was alsointeresting to note occasional examples of patients withEBNA1 and/or LMP2 pepmix responses that could not bemapped in the epitope peptide assays (see patient 0101 forboth antigens, patient 0514 for LMP2); this might be becausenot all potentially relevant epitopes could be included in allassays, but may equally well indicate that new target epitopesremain to be discovered in these proteins. Combining bothpepmix and defined epitope screenings would therefore seemto be the best strategy for therapeutic vaccine trials, particu-larly where these involve patients not selected by HLA typeand/or not of Caucasian origin. Variation in HLA alleles andsuballeles, both within and between human populations, candictate novel patterns of epitope choice that are distinct frompreviously mapped reactivities. Using as an example the dif-ferent distribution of A2 suballeles prevalent in Chinese versusCaucasian populations, in the present work vaccine-inducedreactivities were more often seen against recently identifiedHLA-A�02:03–restricted peptides in EBNA1 (VLK) and LMP2(LLS) than to the better knownLMP2 epitopes presented by theA�02:01 allele.

About the therapeutic potential of this vaccine, we wouldstress that patients in the current trial were in remission and itwas not the purpose to measure clinical effect. However, it wasinteresting to observe that the 1 patient who had a detectableload of EBVDNA in plasma as amarker of tumor burden beforevaccination (patient 0517) showed a transient spike in EBVlevels following the development of a vaccine-induced T-cellresponse (Fig. 5A). A similar observation wasmade in a patientwith nasopharyngeal carcinoma treated with EBV-specific Tcells (11), and transient increases in EBV DNA are well recog-nized as an indicator of tumor cell killing in patients receivingconventional therapies (31). The example of patient 0517 is alsointeresting because this patient suffered a late recurrence oftumor some time after the vaccine-induced T-cell responsehad subsided. In fact, only 3 of 6 patients given the highestvaccine dose (patients 0515, 0516, and 0518) showed a sus-tained increase in circulating EBNA1 and/or LMP2-specificmemory cell numbers present in the postvaccine bleeds at 6months. While an immediate boosting of tumor-specificimmunity by MVA-EL vaccination may be valuable in a ther-apeutic context, achieving a durable increase in these

responses is an important long-term goal. Extending the vaccinecourse, either using the same vector or alternating with aheterologous vector expressing the EBNA1/LMP2 fusion anti-gen, would be one approach in that regard. Another possiblefactor determining long-term vaccine-induced immunity couldbe Tregs. In agreement with a previous study (44), we found thefrequency of Tregswas raised in the blood of somepatientswithnasopharyngeal carcinoma. Interestingly, upon comparing 2patients for whom Treg frequencies and long-term immunedatawere available (andwho received the samedose of vaccine),we found differences in the durability of the vaccine-stimulatedimmune response inversely correlated with Treg frequency.EBNA1- and LMP2-specific T-cell responses were sustained inpatient 0518 (with a vaccine-boosted EBNA1-specific responsebeing detectable 6 months after vaccination) but were tran-sient in patient 0517 in whom the frequency of Tregs washigher. However, confirming whether Tregs are indeedresponsible for the differences in durability of the vac-cine-induced T-cell response we observed, this will requirea larger number of patients to be analyzed.

In summary, these findings and another trial of MVA-EL inUnited Kingdom patients (article in preparation), clearly showthe immunogenicity of the vaccine and point to its potential asan adjuvant treatment of nasopharyngeal carcinoma whencombined with conventional therapies. The work has alsoallowed the highest and most consistently immunogenic vac-cine dose, 5� 108 pfu, to be chosen for an ongoing phase II trialdetermining the clinical response rate toMVA-EL in the settingof relapsed or metastatic disease. Demonstration of the vac-cine's immunogenicity in the context of nasopharyngeal car-cinoma opens up its potential applicability to a wider range ofEBV-associated tumors. Thus, EBV-positive Hodgkin lympho-ma expresses both EBNA1 and LMP2 (as well as LMP1; ref. 2),whereas EBV-positive T/NK lymphoma, another malignancyseen most commonly in South East Asia, expresses EBNA1and a shortened LMP2, which, though lacking N-terminalsequences, retains all of the currently known T-cell targetepitopes (43). In each of these settings, the vaccine might beused to maintain remission following primary treatment oras treatment of relapsed disease, either alone or in combina-tion with adoptive T-cell therapy or other modalities.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: E.P. Hui, G.S. Taylor, A.B. Rickinson, N.M. Steven, A.T.C. ChanDevelopment of methodology: E.P. Hui, G.S. Taylor, H. Jia, N.M. Steven, A.T.C.ChanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.P. Hui, G.S. Taylor, H. Jia, B.B.Y. Ma, S.L. Chan, R. Ho,W.L. Wong, S. Wilson, B.F. Johnson, A.T.C. ChanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.P. Hui, G.S. Taylor, H. Jia, B.F. Johnson, D.D.Stocken, A.B. Rickinson, N.M. Steven, A.T.C. ChanWriting, review, and/or revision of the manuscript: E.P. Hui, G.S. Taylor,B.B.Y. Ma, S.L. Chan, R. Ho, W.L. Wong, C. Edwards, D.D. Stocken, A.B. Rickinson,N.M. Steven, A.T.C. ChanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): E.P. Hui, B.B.Y. Ma, S.L. Chan, R. Ho,W.L. Wong, A.T.C. Chan

Hui et al.





Study supervision: E.P. Hui, G.S. Taylor, C. Edwards, A.B. Rickinson, N.M.Steven, A.T.C. Chan

Grant SupportThis study was supported by grants from Cancer Research UK,

Research Grant Council of Hong Kong (CUHK 460708), and Hong KongCancer Fund.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received June 25, 2012; revised October 31, 2012; accepted November 23, 2012;published OnlineFirst January 24, 2013.

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2013;73:1676-1688. Published OnlineFirst January 24, 2013.Cancer Res Edwin P. Hui, Graham S. Taylor, Hui Jia, et al. Patients

Barr Viral Tumor Antigens in Nasopharyngeal Carcinoma−Epstein Phase I Trial of Recombinant Modified Vaccinia Ankara Encoding

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