Guirakhoo et al ChimVx-Den2

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[Human Vaccines 2:2, e1-e8, EPUB Ahead of Print: http://www.landesbioscience.com/journals/vaccines/abstract.php?id=2555; March/April 2006]; ©2006 Landes Bioscience

e1 Human Vaccines 2006; Vol. 2 Issue 2

Farshad Guirakhoo1*Scott Kitchener2,†

Dennis Morrison3

Remi Forrat4

Karen McCarthy2

Richard Nichols1

Sutee Yoksan5

Xiaochu Duan1

Thomas H. Ermak1

Niranjan Kanesa-Thasan1

Philip Bedford1

Jean Lang4

Marie-Jose Quentin-Millet4

Thomas P. Monath1

1Acambis, Inc..; Cambridge, Massachusetts USA

2Acambis Research Limited; Peterhouse Technology Park; Cambridge, UK

3Bio-Kinetic Clinical Applications; Springfield, Missouri USA

4Sanofi Pasteur; Campus Mérieux; Marcy-L'étoile, France

5Center for Vaccine Development; Institute of Sciences and Technology for Research& Development; Mahidol University; Nakhonpathom, Thailand

†Present address: Centre for Military and Veterans Health; Mayne Medical School;Herston, 4006 Australia

*Correspondence to: Farshad Guirakhoo; Acambis, Inc.; 38 Sidney Street; Cambridge,Massachusetts 02139 USA; Tel.: PLEASE PROVIDE; Fax: PLEASE PROVIDE; Email:[email protected]

Received 11/14/05; Accepted 12/15/05

This manuscript has been published online, prior to printing for Human Vaccines,Volume 2, Issue 2. Definitive page numbers have not been assigned. The current cita-tion is: Human Vaccines 2006; 2(2):http://www.landesbioscience.com/journals/vaccines/abstract.php?id=2555Once the issue is complete and page numbers have been assigned, the citation willchange accordingly.

KEY WORDS

Live attenuated, Chimeric yellow fever-dengue 2,Phase 1 trial, Safety, Immunogenicity

ACKNOWLEDGEMENTS

See page e8.

Research Paper

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (ChimeriVax™-DEN2) Vaccine: Phase I Clinical Trial for Safety and ImmunogenicityEffect of Yellow Fever Pre-immunity in Induction of Cross NeutralizingAntibody Responses to All 4 Dengue Serotypes

ABSTRACTA randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability,

and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adultsrandomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaqueforming units [PFU] or low dose, 3 log PFU) or YF-VAX® by the subcutaneous route (SC).To determine the effect of YF preimmunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjectspreviously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as anopen-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderateintensity, with no serious side-effects. One hundred percent and 92.3% of YF naïvesubjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively,seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX®

seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DENserotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with eitherChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculatedwith ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels ofneutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after1 year. These data demonstrated that (1) the safety and immunogenicity profile of theChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and (2) preimmunity toYF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a longlasting and cross neutralizing antibody response to all 4 DEN serotypes. The latterobservation can have practical implications toward development of a dengue vaccine.

INTRODUCTIONDengue, a disease caused by DEN virus with four distinct species (serotypes 1–4) is the

most important vector-borne viral disease of human kind. Approximately 100 millionpersons are affected by DEN viruses annually in tropical and subtropical regions of theworld.1,2 Severe and potentially lethal forms of the disease, dengue hemorrhagic fever(DHF) and dengue shock syndrome (DSS), are increasing in geographic distribution andincidence, and are currently affecting up to 500,000 individuals. These factors havespurred intensive efforts to construct a safe and effective DEN vaccine, but despite manyefforts spanning 50 years, no commercially available vaccine against DEN virus has beendeveloped. The development of a vaccine against DEN is considered a high priority by theWorld Health Organization.3 The pathogenesis of DHF drives the design of DENvaccines. DHF is an immunopathological disease, which occurs primarily in individualswho have sustained a prior infection with one of the DEN serotypes and are exposed toanother, heterologous serotype.4 Infection with any of the four serotypes of DEN providesdurable immunity to that specific serotype, based on neutralizing antibodies. However,following infection with one DEN serotype, immunity to other heterologous serotypes, ifoccurring at all, is of very short duration.5 A secondary infection with a heterologousserotype may be enhanced by infection of target cells (monocyte macrophages) bearing Fcreceptors with immune complexes of virus with binding antibody. The infected cells arethen cleared by DEN-specific T cell responses. The results of immune clearance in the faceof a high viral load are complement activation and the release of chemokines and cytokinesthat alter vascular permeability and elicit other pathogenic events associated with DHF.6

www.landesbioscience.com Human Vaccines e2

Phase 1 Study of Live Attenuated ChimeriVaxTM-DEN2 Vaccine

Given the immunopathogenesis of DHF, it is obvious thata successful vaccine against DEN must have two characteristics:(1) it must induce immunity against all 4 DEN virus serotypessimultaneously, and (2) it must induce long-lasting neutralizingantibody responses (titers should not fall to a level that leavesthe subject sensitized to immunopathological events but notprotected against future infections). These requirements, partic-ularly the latter, can only be met with a live, attenuated vaccinethat induces durable immunity. Empirical efforts to developlive, attenuated vaccine candidates have demonstrated thatachieving a balance between sufficient attenuation (safety) andimmunogenicity is a very complicated task. Attempts tocombine monovalent live vaccines into one vaccine uncoveredsignificant interactions between the four virus strains, resulting ininterference effects.7

ChimeriVax™-DEN2 is a live, attenuated, genetically engineeredvirus prepared by replacing the genes encoding the two structuralproteins, premembrane (prM) and envelope (E), of the YF 17Dvaccine virus with the corresponding genes of the DEN2 virus(strain PUO-218, isolated from a case of classical DF, Bangkok,Thailand).8 It is a monovalent component of a tetravalent vaccineformulation9 currently being investigated in a Phase I study.Preclinical studies have demonstrated that the ChimeriVax™-DEN2 virus was is not neurovirulent when administered to adultmice via the intracerebral (IC) route, is genetically stable in cell culture,induces low levels of viremia, and protects 100% of monkeys againstwt DEN2 heterologous challenge upon a single SC immunization.10

We present results of a proof of principle study for safety, tolera-bility and immunogenicity of ChimeriVax™-DEN2 and YF-VAX®

(as a control) in a randomized, double-blind, single-center outpatientstudy. In order to determine if YF immunity precludes vaccinationwith ChimeriVax™-DEN2, a group of YF-immune subjects has beenincluded in the study to receive ChimeriVax-DEN2 as an open-labelvaccine.

This is the first study of a chimeric YF-DEN vaccine in humansthat shows that the vaccine is safe and immunogenic, and thatpreimmunity to YF does not interfere with ChimeriVaxTM-DEN2vaccination, but induces high levels of cross-reactive and long-lastingneutralizing antibodies against other DEN serotypes.

MATERIALS AND METHODSVaccines. ChimeriVax™-DEN2. The vaccine virus was manufactured

by BioReliance Corp. (Rockville, MD) in accordance with current GoodManufacturing Practices (cGMP). The virus was grown in Vero (Africangreen monkey kidney) cells from cell banks that have been tested for adven-titious agents, according to US Food and Drug Administration (FDA)guidelines for mammalian cell culture derived products. Supernatant fluidfrom Vero cell cultures containing vaccine virus was harvested, clarified fromcellular debris by filtration, and treated with nuclease enzyme (Benzonase®)to digest nucleic acids derived from host cells. The nuclease-treated bulkvirus was then concentrated by ultrafiltration and purified by diafiltration.The vaccine (Lot# 01H01, titer 8.1 log10 PFU/ml) was formulated with2.5% Human Serum Albumin (HSA) and 7.5% lactose in minimal essentialmedium Eagle’s salt (MEME) without phenol red or L-glutamine (vaccinediluent). Prior to inoculation, the vaccine was diluted with vaccine diluent(Lot# 01H02) to contain 5.0 (high dose) and 3.0 (low dose) log10 PFU/0.5 ml,respectively, and administered to subjects by SC injection into the deltoidregion.

YF-VAX®. Commercial YF 17D vaccine (YF-VAX®, Batch# UB 102AAand UB 132AA) was purchased from Sanofi-Pasteur (Swiftwater, PA). Thelyophilized vaccine contained sorbitol and gelatin as stabilizers, and was

reconstituted with sterile diluent provided (sodium chloride injection USP,containing no preservative, Batch# UB054AA and UB054AC). The formu-lated vaccine contained not less than 5.04 log10 PFU/0.5 ml, according tothe manufacturer’s brochure.

Clinical study. A randomized, double-blind, single-center outpatientstudy was performed. The clinical trial protocol and informed consentforms were approved by an Institutional Review Board (IRB) according toFDA regulations, as outlined in the US Code of Federal Regulations. Thestudy was conducted under Good Clinical Practices and an InvestigationalNew Drug application approved by the FDA. Written informed consent wasobtained from each subject in accordance with the ethical principles in theDeclaration of Helsinki at a screening visit, prior to entry into the study.The objectives of this study were to determine: (1) safety, tolerability, andviremia levels following administration of ChimeriVax™-DEN2 at doses of3.0 and 5.0 log10 PFU, respectively, and a control vaccine (YF-VAX®)administered by the SC route to healthy adult male and female subjects,(2) the neutralizing antibody response against wt DEN2 virus in subjectsvaccinated with ChimeriVax™-DEN2 at doses of 3.0 and 5.0 log10 PFU,respectively, (3) the effect of prior YF immunity on the immune response toChimeriVax™-DEN2, and (4) the duration of the neutralizing antibodyresponse in all subjects up to 1 year post vaccination.

After screening, 42 healthy adults 18–49 years of age, without priorimmunity to YF, Japanese encephalitis (JE) or tick-borne encephalitis (TBE),were randomized equally to 3 groups receiving a single vaccination on studyDay 1 with either low or high dose ChimeriVax™-DEN2 (3.0 or 5.0 log10PFU, respectively) or YF-VAX®. In addition, 14 subjects previously vacci-nated against YF (27–29 months prior) received high dose ChimeriVax™-DEN2 as an open-label vaccine (Table 1). Subjects returned to the clinic onDays 2-11, 21 and 31 for safety assessments, viremia, and antibody responses,and 6 and 12 months post-vaccination to measure durability of antibodyresponse. T cell responses were also measured on Days 1 and 31.

Safety was assessed based on adverse events (AEs; as assessed by sponta-neous reporting, structured interview, and subject diary), vital signs, physicalexamination, and routine laboratory investigations (haematology, bloodchemistry, and urinanalysis) at screening and on Days 11, 21 and 31 aftervaccination. Treatment-emergent AEs were defined as those starting orincreasing in severity after vaccination. Three physicians (Data MonitoringCommittee) independent of the trial reviewed serious AEs and other trialsafety issues.

Measurement of viremia. On Days 2–11, sera were collected and frozenbelow -60˚C for viremia testing. ChimeriVax™-DEN2 and YF-VAX®

viremia titers were measured by direct plaquing in Vero cell monolayergrown in 12-well plastic tissue culture plates. Plaques were enumerated bystaining Vero cell monolayers with crystal violet. The mean duration, meanpeak, and AUC of viremia induced by ChimeriVax™-DEN2 and YF-VAX®

were evaluated.Measurement of immune response. Responses against DEN2 and YF

17D viruses. Antibody responses were measured on Day 1 (prevaccination),Day 31, and 6 and 12 months post vaccination. Two different neutralizationmethods were performed at Acambis Inc. (Cambridge, MA): (1) PRNT50(50% plaque reduction neutralization titer, constant virus with varyingserum dilution) in Vero cells against ChimeriVax™- DEN2 and wt strains

Table 1 Treatment schedule

Group No. Subjects YF-immune Vaccine Dose log10 PFU in 0.5 mL

Double-blind, randomized1 14 No ChimeriVax™-DEN2 5.02 14 No ChimeriVax™-DEN2 3.03 14 No YF-VAX® =≥5.04Open4 14 Yes ChimeriVax™-DEN2 5.0



of DEN2 (PR159, isolated in Puerto Rico in 1964, representing theAmerican genotype I; and JaH, isolated in Jamaica in 1982, representing theAmerican genotype II), and (2) LNI (log neutralization index, constant serumwith varying virus dilution; routinely used for measurement of responses tomany live viral vaccines including YF 17D) in Vero cells against YF 17Dvirus. The titer is defined as LNI, which is the log10 virus titer differencebetween serum-virus mixture for the test sample and a negative controlserum.11,12

For PRNT, sera were heat-inactivated (at 56˚C for 30 minutes), seriallydiluted (2-fold), mixed with an equal volume of DEN virus (approximately50 PFU/well), and used to inoculate duplicate wells of confluent Vero cells.After 1 hour adsorption, cells were overlaid with methylcellulose andincubated for 4 days (37˚C, 5% CO2 atmosphere), at which time they werefixed with formaldehyde. Plaques were stained by incubation with 3H5(anti-DEN2) monoclonal antibody solution, and sequentially incubatedwith goat anti-mouse IgG-alkaline phosphatase conjugate, followed by5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (BCIP/NBT)substrate solution. The titer (PRNT50) was calculated from the highest dilutionof test serum reducing the mean plaque count (of duplicate wells) by ≥50%,compared to the mean value of a standard normal control serum.13 A serumis considered to be positive for the presence of neutralizing antibodies whenthe neutralizing antibody titer thus determined is at least superior or equalto 1:10. For LNI, 10-fold dilutions of YF17 D virus (commercial YF-VAX®

vaccine passaged once in Vero cells) were mixed with heat inactivated testsera. The mixture was used to inoculate Vero cells. Plaques were visualizedby staining the monolayer with crystal violet.11,12

Responses against prototype strains of DEN 1- 4. This PRNT wasperformed in LLC-MK2 cells at the Center for Vaccine Development,Institute of Sciences and Technology, Mahidol University (Thailand). TheDEN 1-4 strains selected are those currently used to standardize the DENneutralization test under a research program sponsored by the World HealthOrganization. Wild type (wt) strains included: DEN1 (strain 16007, isolatedin Thailand in 1964), DEN2 (strain 16681, isolated in Thailand in 1964),DEN3 (strain 16562, isolated in the Philippines in 1964), and DEN4(strain 1036, isolated in Indonesia in 1967). The test was performed accordingto Russell et al.14 Sera were heat-inactivated (at 56˚C for 30 minutes),serially diluted (4-fold), mixed with an equal volume of DEN viruses

(approximately 50 PFU/well), and used to inoculatetriplicate wells of confluent LLC-MK2 cells. After12 hours adsorption, cells were overlaid withmethylcellulose, and incubated for 7 days at 37˚C,5% CO2 atmosphere, at which time they were fixedwith formaldehyde and plaques were visualized bystaining with crystal violet. Plaques were thencounted, and the PRNT50 is was determined byusing log probit analysis. The percent reduction ofplaques at each dilution level was plotted to determinethe 50% reduction titer: plaque reduction pointsbetween 15% and 85% were used. Results wereexpressed as reciprocal of dilution. A serum wasconsidered to be positive for the presence of neutral-izing antibodies when the neutralizing antibodiestiter thus determined is ≥1:10.

T-cell studies. Collection, processing and cultureof peripheral blood mononuclear cells (PBMC).The T cell response was evaluated on Days 1 and 31by measuring the production of IFNγ by PBMCstimulated in culture with inactivated ChimeriVax™-DEN2 virus antigen produced by glutaraldehydefixation as previously described.15 Whole blood wascollected in Vacutainer cell preparation tubes (CPT,BDBiosciences) and sent to Acambis, Inc., forisolation and cryopreservation of PBMC. Cellswere washed in RPMI 1640, cryopreserved in heat-inactivated human AB serum (SeraCare, OceansideCA) containing 10% DMSO, stored in liquid

nitrogen, and thawed immediately before testing. For measuring IFNγproduction, PBMC were cultured in RPMI 1640 containing 10% heat-inactivated human AB serum in 96-well flat bottom plates at 1.5 x 105 cellsper well for 7 days at 37˚C with glutaraldehyde-inactivated Chimeri-Vax™-DEN2 virus-infected Vero cell antigen. Controls consisted of inactivatedmock-infected Vero cells. Inactivated viral antigen or control Vero cell antigenwas added at a concentration of 1:100.15 PBMC were also stimulated with1 µg/ml ConA as an assay positive control. IFNγ production was determinedby ELISA using culture supernatants collected on Day 7.

IFNγ ELISA. Culture supernatant samples from Day 7 were analyzed forIFNγ content at three dilutions (1:2, 1:10, 1:50) by an indirect ELISA assay(OptEIATM human IFNγ Kit, BDBiosciences, San Diego, CA) according tothe manufacturer’s instructions (assay range 5–300 pg/ml). Plates were readat 450 nm (reference wavelength 570 nm) on a VersaMax Spectrophotometer(Molecular Devices Corp., Sunnyvale, CA). Standard curves were generated,and IFNγ concentrations were calculated using Softmax Pro software.

Statistical methods. Analysis population. All subjects who received atleast one vaccination were included in the safety population. The PerProtocol (PP) population was defined as subjects who received at least onevaccination with double-blind study medication, who provided serumsamples at least up to Day 31, and who had no significant protocol deviationsidentified prior to unblinding.

Safety evaluation. Adverse events. Treatment-emergent AEs were codedaccording to the MedDRA (Medical Dictionary for Regulatory Activities)version 4.0, and tabulated by system organ class, preferred term, and severityfor each treatment group. The incidence of all treatment-emergent AEs andtreatment-emergent AEs considered related to the study drug (i.e., ratedpossibly, probably or definitely related) were described between treatmentgroups. A symptom index was calculated for each subject as the sum of themaximal duration x severity scores for fever/chills, myalgia/arthralgia,rash/pruritus, eye pain/conjunctivitis, headaches and malaise.

Viremia. For analysis purposes, the absence of detectable virus in serumwas designated as value “0”, even though the lowest detectable value basedon the test method used was 10 PFU/mL. Mean duration, mean peak, andAUC of viremia induced by ChimeriVax™-DEN2 and YF-VAX® werecalculated per group. The difference between treatment groups for eachmeasure of viremia (peak, duration, and AUC) was compared using an

Table 2 Incidence of treatmenttreatment-related AEs, in terms of no. (%)of subjects in each treatment group

ChimeriVaxTM- ChimeriVaxTM- YF-VAX® ChimeriVaxTM-DEN2 5. DEN2 3.0 DEN2 5.0

0log10 PFU log10 PFU log10 PFU YF-Immune

N 14 14 14 14Any 9 (64.3) 10 (71.4) 11 (78.6) 12 (85.7)AE in ≥ 2 subjects in any groupFatigue 4 (28.6) 2 (14.3) 1 (7.1) 4 (28.6)Malaise 2 (14.3) 3 (21.4) 0 (-) 2 (14.3)Pyrexia 1 (7.1) 0 (-) 4 (28.6) 1 (7.1)Rigors 1 (7.1) 1 (7.1) 1 (7.1) 4 (28.6)Arthralgia 0 (-) 0 (-) 1 (7.1) 3 (21.4)Myalgia 2 (14.3) 1 (7.1) 3 (21.4) 5 (35.7)Headache 9 (64.3) 5 (35.7) 8 (57.1) 6 (42.9)Photophobia 0 (-) 0 (-) 2 (14.3) 0 (-)Diarrhea 1 (7.1) 1 (7.1) 0 (-) 2 (14.3)Pharyngo- laryngeal pain 1 (7.1) 2 (14.3) 0 (-) 1 (7.1)Rash 0 (-) 1 (7.1) 0 (-) 3 (21.4)Injection site mass 0 (-) 0 (-) 2 (14.3) 0 (-)Injection site pain 0 (-) 0 (-) 2 (14.3) 0 (-)Symptom Index (mean ± SD) 2.9 ± 4.0 2.4 ± 2.8 2.6 ± 2.7 4.4 ± 4.8



Analysis of Variance (ANOVA) model.A residual analysis was performed inorder to check that the underlyingassumptions of normality and equalityof variance were met within the model.If the assumptions were violated, suitabletransformation of the data or alternativenonparametric modeling methods wereconducted. The Fisher’s Exact Test wasused to compare the proportion ofsubjects viremic on one or more studydays between treatment groups.

Immunogenicity evaluation. Sero-conversion rates on Day 31 to therespective strain were comparedbetween treatment groups for each of thestrains using Fisher’s Exact Test. ForGMT calculation, antibody level <10was converted into the whole numberminus one (i.e., 9). GMTs on Days 1,31, 6 and 12 months were summarizedby treatment group. The difference inGMTs on Days 1 and 31 were comparedbetween treatment groups using anANOVA model. The seroconversion ratewas defined as the proportion of subjectswithout neutralizing antibody atbaseline (Day 1) who developedPRNT50 of ≥1:10 at each timepoints (forresponse against DEN antigen) or LNIof ≥0.7 day31 minus day1 (for responseagainst YF antigen).

RESULTSSubject population. Fifty-six subjects,

14 per treatment group, were randomized, vaccinated, and completed the31-day follow-up. All 56 subjects were included in the safety population; 53subjects (14 in the 5.0 log10 PFU YF-immune group and 13 in each of theother treatment groups) were included in the PP population. Three subjectswere excluded from the PP population (one from each YF naïve group) dueto prior YF immunity (i.e., LNI ≥0.7 on Day 1).

Overall and within each group, the majority of subjects were female(41/56, 73.2%) and Caucasian (50/56, 89.3%). The treatment groups weresimilar in respect to mean age and body mass index (BMI).

Safety. No serious AEs (SAEs) were reported during the study. Overall,the most frequent individual treatment-emergent AEs (in any group) wereheadache (62.5%), myalgia (39.3%), and fatigue (28.6%). Table 2 presentsthe incidence of treatment-related AEs and symptom index in the differentgroups. The profile of AEs following vaccination with ChimeriVax™-DEN2 was similar to that with YF-VAX®. The incidence of related AEs wasslightly higher in YF-immune than YF-naïve subjects vaccinated withChimeriVax™-DEN2 high dose (85.7 vs. 64.3%, respectively), with ahigher incidence of myalgia, arthralgia, rash, and rigors, and a lower incidenceof headache. Six subjects (1 YF-naïve, 1 YF-immune subject in theChimeriVax™-DEN2 high dose group, and all 4 YF-naïve subjects vaccinatedwith YF-VAX®) presented pyrexia considered related to the study treatment.Body temperature remained lower below 38˚C. Most AEs were of mild tomoderate intensity. Two subjects, one in the ChimeriVax™-DEN2 lowdose and one in the YF-VAX® group, experienced severe headache possiblyrelated to the vaccine for one day. Most AEs resolved within 1–4 days. Thesymptom index was low among different groups. There were no obviouspatterns for shifts in haematology, blood chemistry, or urinanalysis variables.Ten subjects (4 YF-immune subjects (29%) in the ChimeriVax™-DEN2high dose group and 2 YF-naïve (14%) each in the three treatment groups)had abnormal laboratory values reported as AEs. The most commonly

reported laboratory abnormality was increased Creatine Kinase (CK) in fivesubjects, which was noted to be coincidental with muscle strain or injuryassociated with recreational activities in all incidents. All elevations of liverenzymes were minimal and unrelated to significant clinical symptoms.

Viremia. Magnitude, duration, and AUC of viremia in subjects duringthe 11 days after vaccination are shown in Table 3. More YF-naïve subjectsvaccinated with ChimeriVax™-DEN2 than with YF-VAX® developedviremia on one or more study days: 8 (57.1%) in the ChimeriVax™-DEN25.0 log10 PFU group and 9 (64.3%) in the ChimeriVax™-DEN2 3.0 log10PFU group, compared with 2 (14.3%) in the YF-VAX® group. Peak viremia(p = 0.038) and AUC (p = 0.039) were significantly higher, and duration ofviremia (p = 0.022) was significantly shorter in subjects vaccinated withYF-VAX® than with ChimeriVax™-DEN2 3.0 log10 PFU. There was nostatistically significant difference between the two dose groups ofChimeriVax™-DEN2 with respect to peak (p = 0.084), duration (p = 1.000),or AUC (p = 0.98) of viremia. Slightly higher numbers of YF-immunesubjects, compared with YF-naïve subjects, developed viremia followingvaccination with ChimeriVax™-DEN2 5.0 log10 PFU (11/14, p = 0.472).Most subjects developed viremia within 8 days after vaccination.

The number of subjects with viremia (p = 0.472), viremia mean peak(p = 0.148), duration of viremia (p = 0.236), and AUC (p = 0.091) tendedto be higher in YF-immune subjects following ChimeriVax™-DEN25.0 log10 PFU than in YF-naïve subjects. However, none of these valueswere significant in this small sample size. The higher level of viremia inYF-immune subjects raised concerns about an increased risk of AEs; it didnot, however, exceed the viremia level from YF vaccination.

Immunogenicity. Response 30 days after vaccination. All (100%) and12/13 (92.3%) of YF-naïve subjects inoculated with 5.0 and 3.0 log10PFUof ChimeriVax™-DEN2, respectively, seroconverted to wt DEN2 strain16681 (Table 4) with similar GMT (Table 5). Seroconversion rates and

Table 3 Summary of Viremia

Treatment YF-VAX® 5.0 log10 ChimeriVaxTM- ChimeriVaxTM- ChimeriVaxTM-Group PFUa YF-naïve DEN2 3.0 log10 DEN2 5.0log10 DEN2 5.0 log10

PFU YF-naïve PFU YF-naïve PFU YF-immune

No. subjects 14 14 14 14No. (%) subjects viremic 2 (14.3) 9 (64.3) 8 (57.1) 11 (78.6)Peak (PFUa/mL) [SD] 20.0 [51.44] 11.4 [12.31] 12. 1 [16.72] 29.3 [38.72]Duration (Days) [SD] 0.4 [1.16] 1.2 [1.42] 1.4 [1.65] 1.9 [1.23]AUCb (PFU/mL) [SD] 44.3 [116.86] 20.0 [33.74] 20.7 [32.04] 50.4 [67.61]

a PFU, plaque-forming units, measured in Vero cell cultures. b Area under the curve.

Table 4 Seroconversion rate (%) by treatment group, Day 31

Virus Used in YF-VAX® ChimeriVaxTM- ChimeriVaxTM - ChimeriVaxTM

Neutralization YF-naïve DEN2 3.0 log10 DEN2 5.0 log10 -DEN2 5.0 log10Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune

N = 13 N = 13 N = 14

DEN2: strain 16681 0% 92.3% 100% 100%DEN2: ChimeriVax-D2 0 100 100 100DEN2: strain PR-159 0 84.6 92.3 100DEN2: strain JaH 0 92.3 92.3 100DEN1: strain 16007 0 23.1 23.1 100DEN3: strain 16562 0 23.1 15.4 100DEN4: strain 1036 0 0 0 100YF 17D strain 92.3% 0 0 ND

ND: Not determined



GMTs against other DEN2 strains were the highest in the YF-immunegroup. Ninety-two (92.3%) of subjects inoculated with YF-VAX® serocon-verted to YF 17D virus. None of YF naïve subjects inoculated with Chimeri-Vax™-DEN2 seroconverted to YF 17D virus. In the YF-immune groupwho received ChimeriVax™-DEN2, 2 subjects had a boost in YF antibodies.Low seroconversion rates (not higher than 23.1%) to heterologous DENserotypes 1, 3, and 4 were observed in YF-naïve subjects inoculated withChimeriVax™-DEN2 at high or low dose. In contrast, 100% of YF-immune subjects inoculated with ChimeriVax™-DEN2 seroconverted toall heterologous DEN serotypes. GMTs against heterologous DEN

serotypes on Day 31 were significantlyhigher in YF-immune subjects vaccinatedwith ChimeriVax™-DEN2 than in YF-naïve subjects. For DEN1, GMTs inYF-immune subjects, and YF-naïvesubjects vaccinated with either 5.0 or3.0 log10 PFU ChimeriVax™-DEN2were 79.2 vs. 10.1 and 12, respectively(p < 0.0001). Similarly, for DEN3, titerswere 73.2 vs. 13.2 and 11.8 (p < 0.0001)(Table 5). None of the YF-naïve subjectsseroconverted to DEN4. GMT to DEN4in YF-immune subjects was 57.3.

Response 6 and 12 months aftervaccination. One hundred percent ofsubjects vaccinated with Chimerivax™-DEN2 were seropositive against wtDEN2 strain 16681 at 6 and 12months after vaccination (Tables 6and 8). At these timepoints, GMTremained high, with levels between183.3 and 744.1 at 12 months (Tables7 and 9). Seropositivity rates and GMTagainst DEN2 strains PR-159 and JAHdecreased steadily until 1 year aftervaccination, and were the highest in theYF-immune group. These two strainsare from the Americas, and belong totwo distinct variant groups (America Iand II, respectively).

At 6 and 12 months, 100% ofYF-immune subjects inoculated withChimeriVax™-DEN2 remained sero-positive to DEN1 and DEN3, while64.3% and 28.6% were seropositive toDEN4 at 6 and 12 months, respectively.In these subjects, GMTs against DEN1and 3 decreased steadily but remainedrelatively high at 12 months (89.2and 71.8 against DEN1 and DEN3,respectively).

T cell response. T cell responses wereevaluated in cell culture by IFNγproduction in response to inactivatedviral cell lysate, which has been shownto generate primarily CD4+ T cellresponses to the vaccine.16

IFNγ cytokine production. IFNγcytokine production was compared onDays 1 and 31 of the study (beforevaccination and on Day 30 after vacci-nation) by testing the response toinactivated ChimeriVaxTM-DEN2 virusantigen. Comparisons between vaccina-tion groups were made using the differ-ence between values on Days 31 and 1

(Fig. 1). All groups responded to the inactivated antigens (IFNγ responsesranged from undetected to over 7,500 pg/ml). Subjects who received 3.0 or5.0 log10 PFU of ChimeriVaxTM-DEN2 vaccine had equivalent IFNγ levels(geometric mean IFNγ response of 161 and 168 pg/ml), and Chimeri-VaxTM-DEN2 vaccinated subjects had slightly greater responses than YFvaccinated subjects (geometric mean IFNγresponse of 96 pg/ml [not signif-icant, p = 0.565 vs. 3.0 log10 PFU or p = 0.505 vs. 5.0 log10 PFU]).YF-immune subjects who received the 5.0 log10 PFU dose vaccine had anincreased number of IFNγ responders and increased geometric mean IFNγlevels (geometric mean IFNγ response of 578 pg/ml) relative to naïve

Table 5 GMTs for DEN PRNT tests and Mean titers for YF LNI by treatment group, Day 31

Virus Used in YF-VAX® ChimeriVaxTM- ChimeriVaxTM- ChimeriVaxTM- Neutralization YF-naïve DEN2 3.0 log10 DEN2 5.0 log10 DEN2 5.0 log10Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune

N = 13 N = 13 N = 14

DEN2: strain 16681 <10 365.0 358.6 383.3DEN2: ChimeriVax-D2 <10 570.0 921.3 975.4DEN2: strain PR-159 <10 313.8 218.3 724.5DEN2: strain JaH <10 227.8 240.3 463.9DEN1: strain 16007 <10 12.0 10.1 79.2DEN3: strain 16562 <10 11.8 13.2 73.2DEN4: strain 1036 <10 <10 <10 57.3YF17D strain <11.968 <10.025 -0.064 0.676

Table 6. Proportion Seropositive (%) by treatment group, 6 months

Virus Used in YF-VAX® ChimeriVaxTM- ChimeriVaxTM- ChimeriVaxTM-Neutralization YF-naïve DEN2 3.0 log10 DEN2 5.0 log10 DEN2 5.0 log10Test N = 13 PFU YF-naïve PFU YF-naïve PFU YF-immune

N = 13 N = 13 N = 14

DEN2: strain 16681 0% 100% 100% 100DEN2: ChimeriVax-D2 0 100 100 100DEN2: strain PR-159 0 84.6 76.9 92.9DEN2: strain JaH 0 76.9 69.2 92.9DEN1: strain 16007 0 30.8 23.1 100DEN3: strain 16562 0 23.1 15.4 100DEN4: strain 1036 0 7.7 7.7 64.3

Table 7. GMTs by treatment group, 6 months


N = 13 N = 13 N = 14

DEN2: strain 16681 <10 568.6 285.1 870.2DEN2: ChimeriVax-D2 <10 606.8 303 672DEN2: strain PR-159 <10 55.1 49.5 160DEN2: strain JaH <10 29.0 24.7 72.5DEN1: strain 16007 <10 14.4 <10 285.1DEN3: strain 16562 <10 <10 <10 268.1DEN4: strain 1036 <10 <10 <10 23.8



subjects receiving either 3.0 log10 PFU(not significant, p = 0.3231) or 5.0log10 PFU of ChimeriVaxTM-DEN2 (notsignificant, p = 0.1827).

About 65% (9/14) of the Chimeri-VaxTM-DEN2TM and YF vaccinatedsubjects had a positive IFNγ response tothe administered vaccine as test antigen,whereas about 90% (13/14) of YF-immune subjects vaccinated withChimeriVaxTM-DEN2 had a positiveresponse. Positive response was definedas 5-fold background or ≥ 50 pg/ml onDay 30, if Day 1 was less than 10 pg/ml(below sensitivity of the ELISA assay).

DISCUSSIONThis was the first clinical trial for

the evaluation of safety and tolera-bility of ChimeriVax™-DEN2 as asingle dose vaccine of 5.0 or 3.0 log10

PFU in both YF-naïve and YF-immune, healthy subjects. There isa theoretical risk of sensitization ofsubjects to DHF on exposure to aheterologous DEN serotype afterimmunization with a monovalentvaccine. This risk is mitigated byconducting the trial in the Midwestof the United States, which is notendemic for dengue. Trials of liveattenuated monovalent denguevaccines have been previouslyperformed in the USA, with thesame theoretical concern regarding sensitization of subjects to DHF.They were also conducted in adults, who have a lower risk forDHF/DSS than children living in endemic areas. To reduce riskfurther, the trial excluded subjects who frequently travel to dengueendemic areas. Subjects were advised of the potential risk ofenhanced disease if exposed to DEN viruses in the future.

The high dose of ChimeriVax™-DEN2 used in the current trial(5.0 log10 PFU) corresponds to the routinely administered standarddose of YF-VAX®, and was based on the expectation thatChimeriVax™-DEN2 would have a similar replication efficiency inhumans than the YF 17D virus used as live vector for DEN prM-Egenes. The lower dose (3.0 log10 PFU) was selected based onpreclinical data in nonhuman primates: a tetravalent formulationcontaining all 4 chimeric viruses required a 3-log dose ofChimeriVax™-DEN2 and a higher dose of the other 3 viruses toavoid possible interference effects.9

Adverse events were anticipated to resemble those associated withChimeriVax™-JE. Data from Phase 1 and 2 trials of this vaccineindicated that it was well tolerated and highly immunogenic at alldose levels tested (5.8 to 1.8 log10 PFU).17 We found no unexpectedsafety concerns with ChimeriVax™-DEN2 vaccinations. Theprofile of AEs observed following ChimeriVax™-DEN2-vaccinationwas similar to that observed after vaccination with YF-VAX®. Therewas some evidence suggesting a dose-response relationship forChimeriVax™-DEN2 tolerability, with a higher incidence ofheadache and fatigue. However, most were mild to moderate inintensity and transient, generally resolving within 1–4 days. The

most commonly reported laboratory abnormality was increased CKrelated to muscle injury. Other liver enzyme elevations (AST andALT) were minor and unrelated to clinical syndromes.

The transient low level of viremia following administration ofChimeriVax™-DEN2 vaccine was not closely associated with symp-toms or signs of ill health. No differences were observed betweenviremia induced with either dose of ChimeriVax™-DEN2 viruses.

Table 8. Proportion seropositive (%) by treatment group, 12 months


N = 13 N = 13 N = 14

DEN2: strain 16681 0% 100% 100% 100DEN2: ChimeriVax-D2 0 100 100 100DEN2: strain PR-159 0 69.2 69.2 92.9DEN2: strain JaH 0 61.5 53.8 85.7DEN1: strain 16007 0 30.8 23.1 100DEN3: strain 16562 0 23.1 7.7 100DEN4: strain 1036 0 0 7.7 28.6

Table 9. GMTs by treatment group, 12 months


N = 13 N = 13 N = 14

DEN2: strain 16681 <10 368.9 183.3 744.1DEN2: ChimeriVax-D2 <10 272.7 272.7 320.0DEN2: strain PR-159 <10 42.2 30.6 72.5DEN2: strain JaH <10 18.0 14.5 32.8DEN1: strain 16007 <10 13.1 10.1 89.2DEN3: strain 16562 <10 <10 <10 71.8DEN4: strain 1036 <10 <10 <10 <10

Figure 1. IFNγ responses to vaccine (study Day 31 minus Day 1). YF:YF-VAX®. CVx3: ChimeriVax-DEN2 low dose group (3.0 log10 PFU). CVx5:ChimeriVax-DEN2 high dose group (5.0 log10 PFU). YF-CVx5: YF-immune,ChimeriVax-DEN2 high dose group. Bars represent the geometric mean.Values ≤ 10 pg/ml (the sensitivity of the IFNγ ELISA assay) were plotted as5 pg/ml.



More subjects vaccinated with ChimeriVax™-DEN2 than withYF-VAX® developed viremia. However, the magnitude and AUC(but not duration) of viremia in these subjects were lower than withYF-VAX® (not significant). The viremia profiles of the two groups(YF-naive and YF-immune) receiving 5.0 log10 PFU of Chimeri-Vax™-DEN2 clearly indicated that preexisting YF immunity didnot hinder the replication of vaccine virus, and even the number ofsubjects with viremia as well as viremia levels tended to be higher inYF-immune subjects. However, this trend was not associated with anincreased risk of AEs. Similarly, the preimmunity to YF 17D did notinterfere with ChimeriVax™-JE vaccination, In sum, Chimeri-Vax™-DEN2 vaccine induced a low level of viremia with a transientduration in all subjects.

Immunogenicity was assessed based on the neutralizing antibodyresponse, represented as the proportion of subjects seroconverted towt strain 16681 (ChimeriVax™-DEN2 groups) or YF 17D virus(YF-VAX® group) by PRNT50, 30 days after vaccination. Bothdoses of ChimeriVax™-DEN2 demonstrated high levels of neutral-izing antibodies against DEN2 (i.e., homologous strain), similar tothe level of neutralizing antibodies induced by ChimeriVax™-JEagainst JE virus17 and the level of neutralizing antibodies induced byChimeriVax™-WN against WN virus (data not shown). Themagnitude was such that dose-response was not evident even againstother wt strains of DEN2 virus (e.g., PR-159 and JaH) or fromassessment of GMTs. The fact that this vaccine was highly effectiveagainst several important genotypes of DEN2 is very encouraging.Cross-reactivities with other DEN serotypes were limited in YF naïvesubjects. However, when subjects were primed with YF-VAX®, thelevel of cross-reactive neutralizing antibodies against the other 3 DENserotypes was increased and lasted up to one year (the last samplingtime). The highest and lowest cross-reactive neutralizing antibodieswere directed against DEN1 and DEN 4 viruses, respectively. TheGMT against DEN4 virus in YF-immune subjects was still in thepositive range (23.8) when measured at 6 months post immunization,but it dropped to <10 after 1 year. The anamnestic response amongYF-immune subjects could have great implications for the developmentof an immunization strategy for a tetravalent vaccine by avoiding thepotential interference problem that presenting a single YF-DENchimeric virus poses to the host. In this instance, the induction ofinterferon occurs in concert with virus replication and is modulatedeffectively by the YF nonstructural proteins18,19 to allow sufficientreplication required for immunization. Since the initial immunizingagent (YF) is incapable of sensitizing the subject to DHF, there willbe no danger that the first (priming) inoculation would leave thesubject vulnerable to this disease if the second injection was delayedor not administered. Across YF vaccines, the sharing of genescontrolling T cell immunity and the chimeric DEN vaccine containingan YF vector backbone might be the postulate for the unique immu-nizing capacity of the sequential immunization regimen.

There are precedents for sequential immunization. In YF-immune individuals who received inactivated TBE vaccine,anti-TBE IgG antibodies appeared earlier and in higher titers thanin nonYF-immune subjects. In addition, a broad spectrum flavivirushemagglutination inhibition (HI) response in all subjects and lowtiter of neutralizing response against DEN2 in some subjects weredeveloped.20 Price et al.21 previously described a method for sequen-tial flavivirus immunization, comprising a series of three immunizationswith DEN2 and two heterologous viruses (YF and JE). However,unlike the present study, the sequence of YF followed byDEN2, without the addition of JE immunization, failed to confercross-protective immunity.

Similar to our observation, boosted DEN heterologous responseswere observed in YF-immune subjects vaccinated with live attenuatedDEN vaccines.22 The short-term (to Day 30) antibody responses weredemonstrated with antibody assays including neutralization, but theauthors concluded that evidence for protection against subsequentDEN virus infection was inconclusive. Unlike the present study, theauthors could not demonstrate conclusively the prior timing orreceipt of YF vaccination, long-term broad neutralization antibodyresponses, or provide evidence for cross-reactive T cell responses toDEN.23 Simultaneous administration of attenuated YF and DENvaccines to humans seemed to have modified fever and generalsymptoms, and had increased antibody responses to both viruses.24

Moreover, initial vaccination with YF followed by natural DENinfection or administration of an attenuated DEN vaccine boostedantibodies to both viruses.25 Scott et al26 showed that subjects whowere previously immunized with YF and subsequently inoculatedwith a live, attenuated DEN2 vaccine had enhanced immuneresponses to DEN2, which were also more durable (lasting 3 years)than in subjects without previous YF immunity. However, becausethe immune responses had not been measured by neutralization test,27

which is the only test that predicts protective immunity, it is not clearwhether the enhanced response might have been due to enhancing(binding, nonneutralizing) antibodies elicited to DEN2 virus by thepreceding YF vaccination. In addition, Scott et al did not show thatYF followed by DEN2 vaccines elicited a long-lasting immuneresponse to the other three DEN serotypes (1, 3, or 4). In our study,YF-immune subjects had been inoculated with YF vaccine 24-29months prior to inoculation with Chimerivax™-DEN2. A shorterinterval between these 2 vaccinations could have the same positiveeffect: in a recent study, YF vaccination only 5 months prior toinoculation with a ChimeriVax™-DEN tetravalent vaccine alsoincreased the neutralizing antibody response to DEN viruses,compared to nonimmune subjects (unpublished data). While YFimmunity boosted the response to DEN viruses after ChimeriVax™vaccination, the reciprocal was not true (ChimeriVax™-DEN2 didnot boost antibodies against YF virus). Of 14 YF immune subjectswho received ChimeriVax™-DEN 2, only 2 (14%) had a boost inYF antibody, and one of these had a very marginal titer at baseline(LNI 0.65; data not shown).

Flavivirus cross-reactive epitopes have been located withindomain II (formerly A), whereas type- or subtype-specific epitopeshave been found in domains I and III (formerly C and B) of TBEvirus.28,29,30 Monoclonal antibodies recognizing epitopes withindomain II exhibited strong HI and neutralizing activities, whereasthose binding to epitopes within domains I and III exhibited weakHI and neutralizing activities.31 It has been shown that binding ofTBE monoclonal antibodies to epitopes within domains I and IIIenhanced (in unidirectional way) the binding of antibodies todomain II.28,32 It is possible that prior YF infection had created apopulation of B cells recognizing a broad array of cross-reactiveDEN epitopes. Enhancing antibodies were found in sera of YF-immune subjects taken before vaccination with a live attenuatedDEN2 vaccine. The levels of seroconversion to DEN2 were signifi-cantly higher in YF-immune subjects with enhancing antibodiescompared to nonimmune subjects.27 However, the opposite was nottrue, since prevaccination with DEN2 vaccine did not increase theseroconversion rate against YF vaccine.8,33,34

The T-cell responses in this clinical trial were consistent with theneutralizing antibody responses in that both doses of vaccine stimu-lated similar T cell immune responses, and prior immunity to YF



virus did not inhibit the T cell response to ChimeriVax™-DEN2.IFNγ responses were virtually the same for the 2 doses of Chimeri-Vax™-DEN2 (103 and 105 pfu). In addition, the IFNγ response toChimeriVax™-DEN2 was not diminished by prior vaccinationagainst YF virus and even higher numbers of responders were seen,suggesting a trend for enhanced T cell immunity in YF preimmunesubjects. The T cell response generated to inactivated virus antigenprobably represents CD4+ responses to both DEN structural and YFnonstructural proteins. Inactivated DEN viral lysate producesprimarily CD4+ responses in naturally infected subjects16 and insubjects who have received live attenuated DEN vaccines.35

However, this study did not determine the specific proteins againstwhich the immune response was generated. Recent studies inpatients with secondary DEN virus infection using overlappingDEN peptide sequences have shown that T cell responses weregenerated to both DEN structural and non-structural proteins.36

In conclusion, this single dose monovalent ChimeriVax™-DEN2 vaccine has been well tolerated, with a safety profile consistentwith that of YF-VAX®, and comparable immunogenicity to therespective target wt virus strains. Cross-reactivity to other DENserotypes was low in YF naïve subjects immunized with ChimeriVax™-DEN2. However, preimmunity to YF-VAX® enhanced the level ofdurable DEN cross-reactive neutralizing antibodies, which may beuseful in designing a strategy for a dengue vaccine. Nevertheless, thecurrent ChimeriVax™-DEN2 vaccine is destined to contribute tothe development of a tetravalent vaccine. For this purpose,ChimeriVax™-DEN2 is believed to be safe and immunogenic,warranting further development towards a market-ready DEN vaccine.

AcknowledgementsAuthors would like to thank all clinical and laboratory staff at

Acambis, Inc., Bio-Kinetic Clinical Application, Inc., and MahidolUniversity, Thailand, especially Alison Deary for clinical trialmanagement and Robert Schrader for serology tests. We also thankDr. Bruno Guy, Sanofi-Pasteur, for helpful discussion on T-cell studies.This work was supported by Sanofi-Pasteur, Marcy-L'Etoile, France.

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Guirakhoo et al ChimVx-Den2