ChimeriVax-West Nile Virus Live-Attenuated Vaccine

JOURNAL OF VIROLOGY, Nov. 2004, p. 12497–12507 Vol. 78, No. 220022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.22.12497–12507.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

ChimeriVax-West Nile Virus Live-Attenuated Vaccine: PreclinicalEvaluation of Safety, Immunogenicity, and Efficacy

Juan Arroyo,1,2 Chuck Miller,1 John Catalan,1 Gwendolyn A. Myers,1 Marion S. Ratterree,3

Dennis W. Trent,1,4 and Thomas P. Monath1*Acambis, Inc., Cambridge, Massachusetts1; DynPort Vaccine Co. LLC, Frederick, Maryland2; Tulane National

Primate Research Center, Covington, Louisiana3; and Vaxin, Inc., Birmingham, Alabama4

Received 31 March 2004/Accepted 9 July 2004

The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the timeWest Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of thevaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live-at-tenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. TheChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes todeliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homologybetween the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to developthe WN vaccine. Here we discuss preclinical studies with the ChimeriVax-WN virus in mice and macaques.ChimeriVax-WN virus vaccine is less neurovirulent than the commercial YF 17D vaccine in mice and nonhu-man primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenu-ating mutations in the YF 17D virus backbone and three point mutations introduced to alter residues 107, 316,and 440 in the WN virus E protein gene. The safety, immunogenicity, and efficacy of the ChimeriVax-WN02 vaccinein the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.

Following isolation of West Nile (WN) virus in New York in1999, the virus rapidly spread across North America, causingdisease in wild birds, horses, and humans. The number ofhuman cases increased dramatically in 2002 and 2003, when4,145 and 8,977 cases were reported, respectively (7, 8). WNvirus is transmitted principally between wild birds and Culexmosquitoes (7). Recently, WN virus has been isolated in theWest Indies and serosurveys have identified neutralizing anti-body-positive avian species in Mexico (14), Jamaica, and theDominican Republic (13, 17). The rapid geographic expansionof the virus is attributed to movement by viremic birds duringlocal and migratory flight behavior. To date, there is no effec-tive drug treatment against WN virus infection and surveil-lance and mosquito control measures have not significantlyinfluenced the number of human infections (27). A vaccineagainst WN virus represents an important approach to theprevention and control of this emerging disease.

The ChimeriVax technology has been successfully used todevelop a live vaccine against Japanese encephalitis (JE) virusthat is now in phase II trials (23). JE virus is a close geneticrelative of WN virus (31), a fact that expedited use of thistechnology to develop multiple WN virus vaccine candidates.The ChimeriVax technology employs the yellow fever (YF)17D vaccine capsid and nonstructural genes to deliver theenvelope genes (prM and E) of other flaviviruses. In the workpresented here, the envelope genes of YF 17D were replacedwith the corresponding genes of the wild-type WN virus NY99strain previously described by Lanciotti et al. (19). The result-ing YF/WN chimera lacked the mouse neuroinvasive propertyof WN virus and is less neurovirulent than YF 17D vaccine in

both mouse and monkey models. Because WN virus, like otherflaviviruses in the genus, is neurotropic for mammals (21, 29),attenuating point mutations were later introduced in the en-velope of the YF/WN chimera to further reduce its virulence.Mutation sites were targeted only to regions of the envelope(E) protein gene and were based on previous observations byothers (1, 3, 28, 32) pertaining to attenuation phenotypes inrelated flaviviruses: specifically JE and tick-borne encephali-tis viruses. Site-directed mutations in the WN virus E geneof the chimeric prototype vaccine, ChimeriVax-West Nile01,(ChimeriVax-WN01) resulted in a significant reduction in virusneurovirulence. Here we discuss a vaccine in a YF vaccine back-bone; the WN virus envelope (E) protein mutagenesis ratio-nale; and the assessment of the safety, immunogenicity, effi-cacy, and genetic stability of these ChimeriVax-WN vaccinecandidates in the mouse and macaque models.

MATERIALS AND METHODS

YF/WN chimeric clones and molecular procedures for virus assembly. Chi-meric flaviviruses were constructed with the ChimeriVax two-plasmid technologypreviously described (9). Briefly, the two-plasmid system provides plasmid sta-bility in Escherichia coli by dividing the cloned YF backbone into two plasmids.This provides smaller plasmids that are more stable to manipulate the YFsequences facilitating replacement of the prM and E genes of the flavivirus targetvaccine. The WN virus prM and E genes used were cloned from the WN fla-mingo isolate 383-99 sequence (GenBank accession no. AF196835; kindly provid-ed by John Roehrig, Centers for Disease Control and Prevention, Fort Collins,Colo.). Virus prME sequence cDNA was obtained by reverse transcription-PCR(RT-PCR) (XL-PCR kit; Applied Biosystems, Foster City, Calif.). The 5� end ofthe WN virus prM gene was cloned precisely at the 3� end of the YF 17D capsidgene by overlap-extension PCR using Pwo polymerase (Roche Applied Science,Indianapolis, Ind.). This cloning step maintained integrity of the cleavage/pro-cessing signal encoded at the 3� end of the YF capsid gene. The 3� end of the Egene was cloned at the 5� end of the YF NS1 protein coding sequence by overlap-extension PCR. The two-plasmid system used to clone the prME region of WNvirus into the YF 17D backbone was described previously (4). Silent mutationswere introduced in the sequences of prM and E to create unique BspEI and EagIrestriction sites. Digestion of the two plasmids with these restriction nucleases

* Corresponding author. Mailing address: Acambis, Inc., 38 SidneySt., Cambridge, MA 02139. Phone: (617) 761-4200. Fax: (617) 494-1741. E-mail: [email protected].

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generated DNA fragments that were gel purified and ligated in vitro to producea full-length chimeric cDNA. The cDNA was linearized with XhoI to facilitate invitro transcription by SP6 polymerase (Epicentre, Madison, Wis.).

Point mutations were introduced into various E gene codons to producevariants of the original chimera coding for wild-type WN virus prME genes(Transformer site-directed mutagenesis kit; Clontech, Palo Alto, Calif.). Table 1shows the mutation target sites and the oligonucleotide sequences used to createall of the YF/WN chimeras. Site mutations were confirmed by sequencing of theenvelope proteins (prME region) of the resulting viruses. Virus cDNA templatesfor sequencing originated from RNA extraction of virus containing infected Verocell supernatants (Trizol LS; Invitrogen, Carlsbad, Calif.) followed by RT-PCR(XL-PCR kit; Applied Biosystems) and sequencing with a CEQ 2000XL nucleicacid sequencer (Beckman-Coulter, Fullerton, Calif.).

Viruses and cell lines. The wild-type WN virus used in animal challengestudies is the NY99 strain (NY99-35262-11 flamingo isolate, a homolog of thevirus used to build chimeras) obtained from the Centers for Disease Control andPrevention, Fort Collins, Colo. (CDC stock designation B82332W) with twoadditional passages in Vero E6 cells to produce a master virus bank. YF 17D isa commercial vaccine (YF-VAX; Aventis Pasteur, Swiftwater, Pa.) used afterreconstitution of the lyophilized product or after one passage (P1) in Vero E6(American Type Culture Collection [ATCC] origin; Acambis, Inc., cell bank,Cambridge, Mass.). Chimeric YF/WN (i.e., ChimeriVax-WN) viruses were pre-pared by RNA transfection (P1 virus) of Vero E6 cells (ATCC origin, CIDVRUniversity of Massachusetts Medical Center cell bank, Worcester, Mass.). Re-search master seeds (RMS) were prepared by additional amplifications (eitherpassage 2 or 3 at a 0.001 multiplicity of infection [MOI]) in Vero E6 cells. VeroE6 cells were maintained in minimal essential medium (Invitrogen) containing10% heat-inactivated fetal bovine serum (HI-FBS) (HyClone, Logan, Utah).Preparation of pre-master seeds (PMS) for manufacture of the vaccine wasinitiated by RNA transfection of serum-free Vero (SF-Vero) cells obtained froma cell bank that had been manufactured and controlled to meet current Food andDrug Administration guidelines for cell culture vaccines. (The cells were ob-tained from an ATCC strain predating 1980, and the cell bank was made byBaxter/Immuno, Orth, Austria.) Progeny virus from the transfection step wasamplified by a single passage in the same SF-Vero cell line to produce P2, whichwas designated the PMS for subsequent manufacture of clinical-grade vaccine.The SF-Vero cell line is propagated and maintained in a serum-free, animalprotein-free medium formulation, VT-Media (Baxter/Immuno). Viruses for an-imal experiments were diluted in M199 with HEPES buffer (Invitrogen) and 20%HI-FBS (HyClone) unless otherwise indicated. Plaque assays to verify the titer ofvirus inoculi were performed in a Vero cell substrate as previously described (24).

Mouse studies. Protocols for mouse experiments were approved by the Insti-tutional Animal Care and Use Committees at both University of MassachusettsMedical Center (Worcester, Mass.) and Acambis, Inc. (Cambridge, Mass.). Re-search was conducted in compliance with the Animal Welfare Act and otherfederal statutes and regulations relating to animals and experiments involvinganimals and adhered to principles set forth in the Guide for the Care and Use ofLaboratory Animals (27a). Female ICR mice (Taconic, Germantown, N.Y., orHarlan Sprague-Dawley, Indianapolis, Ind.) were inoculated intraperitoneally (i.p.)with 100 to 200 �l of wild-type WN virus NY99 for neuroinvasion tests or post-vaccination challenge experiments (titers of inoculated viruses are indicated in theResults section and in the tables presented). ICR strain adult (3 to 4 weeks of age)and suckling (2 and 8 days of age) mice were inoculated intracerebrally (i.c.) onthe right side of the brain as previously described (24) and using a 20-�l volume ofYF 17D or chimeric YF/WN constructs for neurovirulence testing (titers of inocu-lated viruses are indicated in the Results section and in the tables presented).

Mice were observed daily for 21 days following inoculation to determinesurvival ratio and average survival time (AST) after virus challenge.

Neutralizing antibody titers were determined by a constant virus-serum dilu-tion 50% plaque reduction neutralization assay test (PRNT50) in Vero cells, aspreviously described (24). An equal volume (0.1 ml) of virus suspension contain-ing 700 PFU/ml and serial twofold dilutions of heat-inactivated serum wereincubated overnight at 4°C, and the serum-virus mixture was inoculated ontoVero cell monolayers grown in 12-well plates. An overlay of methylcellulose inminimal essential medium was added before incubation of the cultures at 37°Cfor 3 to 4 days prior to fixation and crystal violet staining for plaque count de-termination. The endpoint neutralization titer was the highest dilution of serumthat reduced plaques by 50% compared to a mouse hyperimmune serum control.

Nonhuman primate studies. Neurovirulence tests in rhesus macaques wereperformed according to World Health Organization (WHO) guidelines for test-ing YF vaccine (36) and as described previously for safety tests of ChimeriVax-JEvaccine (24). Animals were inoculated with specific virus candidates by inocula-tion of the frontal lobe of the brain (see Table 7). Blood samples were obtaineddaily for the first 10 days following inoculation, and serum viremia was measuredby plaque assay on Vero cells. Animals were observed daily for clinical signs ofencephalitis and associated symptoms such as fever or tremors. Animals wereeuthanized 30 days after infection, and the brain and spinal cord tissues wereremoved for histopathology. Slides were prepared from tissues of the frontal andtemporal cortex, basal ganglia/thalamus (two levels), midbrain, pons, cerebellum(two levels of the nuclei and cortex), medulla oblongata, and six levels of each ofcervical and lumbar enlargements of the spinal cord. Sections were stained withgallocyanine. Histological lesions were analyzed and scored for pathology rela-tive to that of the YF 17D according to the criteria for evaluation of neuroviru-lence in rhesus monkeys proposed by the current WHO requirements. Meanlesion scores for individual monkeys were calculated for “target” (substantianigra) and “discriminator” (basal ganglia/thalamus and the spinal cord) areasindividually and for the target and discriminator areas combined.

A second neurovirulence test was performed with cynomolgus monkeys andinoculation with the YF/WNFVR vaccine candidate (ChimeriVax-WN02) produc-tion virus seed (P4). The study was conducted according to good laboratorypractices (GLP) standards (14a). Eleven monkeys were inoculated i.c. with YF/WNFVR production virus seed (P4), 11 positive control monkeys received YF-VAX, and 5 negative control monkeys received diluent. The monkeys were eval-uated for changes in clinical signs (twice daily), body weight (weekly), and food con-sumption (daily). Clinical signs were assigned scores according to a clinical scoringsystem, based on the WHO requirements for YF vaccine (36). Blood sampleswere collected preinoculation on day 1 and on days 3, 5, 7, 15, and 31 for clinicalpathology analysis (serum chemistry and hematology parameters). Additional bloodsamples were collected preinoculation on day 1 and on days 2 to 11 for viremiaanalysis, and on days 1 (predose) and 31 for antiviral antibody titer analyses.

To determine immunogenicity, rhesus monkeys were inoculated by the sub-cutaneous (s.c.) route with a single 0.5-ml dose containing �4 log10 PFU ofchimeric vaccine. Control animals received undiluted YF-VAX containing 4.49log10 PFU in a 0.25-ml volume. Each vaccine dose was back titrated followingimmunization. Serum viremia was measured daily by plaque assay through day 10after vaccination. Neutralizing antibody levels were measured by PRNT50 on days14, 30, and 63 after vaccination. Animals were challenged 64 days after vaccinationby i.c. inoculation of 125 �l containing 2.4 � 105 PFU of wild-type WN NY99suspended in M199 with HEPES buffer (Invitrogen) and 10% sorbitol (Sigma).Monkeys were observed for viremia, clinical illness, and antibody response;se-verely ill animals were euthanized. The i.c. challenge model closely followed themodel established during the development of ChimeriVax-JE vaccine (24, 26).

Genetic stability (in vivo and in vitro passage) and sequencing. The chimericYF/WN virus containing unmodified, wild-type WN virus prME sequence (des-ignated ChimeriVax-WN01) was passed six times in Vero E6 cells followed bysix passages in suckling mice by the i.c. route. The chimeric YF/WN virus

TABLE 1. Switch oligonucleotides used for site mutagenesis

E protein position and residuea Primerb Marker site

107L3F 5�-CAACGGCTGCGGATTTTTTGGCAAAGGATCCATTGACACATGCGCC-3� BamHI138E3K 5�-GAAAGAGAATATTAAGTACAAAGTGGCCATTTTTGTCC-3� SspI*176V 5�-GCCCTCGAGCGGCCGATTCAGCATCACTCCTGCTGCGCCTTCAGTCACAC-3�*176Y 5�-GCCCTCGAGCGGCCGATTCAGCATCAC-3�280K3M 5�-GCAACACTGTCATGTTAACGTCGGGTCATTTG-3� HpaI316A3V 5�-CTTGGGACTCCCGTGGACACCGGTCACGGCAC-3� AgeI440K3R 5�-GGGGTGTTCACTAGTGGTTGGGCGGGCTGTCCATCAAGTG-3� SpeI

a Primers indicated with an asterisk are cloning primers used in fragment subcloning. One incorporates a change to valine as indicated.b Primers for site-directed mutagenesis to create YF/WN chimeric viruses. Nucleotide changes that switch to a new amino acid are indicated in bold. Silent restriction

(marker) sites introduced are underlined.

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containing three mutations introduced by site-directed mutagenesis (designatedChimeriVax-WN02) at the P2 level (PMS) and P3 level (RMS) were passed 12and 10 times, respectively, in serum-free, protein-free SF-Vero cell substrate. Allin vitro virus passages were performed with an initial MOI of 0.01 PFU/cellfollowed by harvest of the virus on the third day after infection. Passages in vivowere performed by initial i.c. inoculation of 105 PFU; brain tissue from ICR mice(Taconic) was harvested 3 days after inoculation and homogenized, and theclarified homogenate was used for passage to a new group of mice. Virus titersat each passage were determined by plaque assay. Neurovirulence of the pas-saged viruses was determined by i.c. inoculation of adult or suckling mice (seeTables 13 and 14). Sequencing of viral RNA was performed with Superscript IIreverse transcriptase and XL-PCR; products were purified by QIAGEN gelextraction (QIAGEN, Valencia, Calif.). Sequencing reactions were prepared andanalyzed using the standard Beckman CEQ 2000XL protocol and equipment(Beckman-Coulter). For virus passages, at least two independent sequencingreactions were executed per RT-PCR product strand sequenced; sense andantisense strands were sequenced each time. Mutation acceptance criterianeeded a positive identity in at least three of four sequencing reactions analyzed;in addition, two independent operators read sequence chromatographs.

RESULTS

Virulence phenotype of chimeric YF/WN containing wild-type WN prME genes relative to YF 17D (YF-VAX). Theinitial WN virus chimera encoded the envelope and premem-brane protein genes of the WN NY99 wild-type strain (desig-nated ChimeriVax-WN01). This chimeric virus did not causeencephalitis after i.p. inoculation at doses of 106 PFU in 3- to4-week-old adult ICR mice (Table 2). Encephalitis was as-sessed by daily observations for illness, paralysis, and death.ChimeriVax-WN01 resembles YF 17D vaccine (33) in beingnonneuroinvasive in adult mice. In contrast, the WN NY99wild-type virus was lethal for mice when inoculated by the i.p.route with as few as 1 to 4 PFU (5; unpublished results).ChimeriVax-WN01 retained the ability to cause lethal en-cephalitis after i.c. inoculation, a property consistent withthat of YF 17D virus (10). We estimated the i.c. 50% lethaldose (LD50) of ChimeriVax-WN01 to be between 103 and 105

PFU. The neurovirulence phenotype of ChimeriVax-WN01 islower than that of YF 17D virus, for which the i.c. LD50 isbetween 101 and 102 PFU (Table 3).

Evaluation of the multisite mutagenesis for attenuation.Amino acids in the envelope protein previously establishedas genetic determinates of virulence for ChimeriVax-JEwere changed to reduce the virulence of YF/WN chimeras.

The strategy for this mutagenesis approach was to design a safeattenuated WN vaccine; this strategy was first discussed in anearlier publication (4). Briefly, the selection of specific aminoacid residues for mutagenesis was defined by previous studiesof the attenuating mutations in a vaccine strain of JE virus(SA14-14-2) (3). Since the wild-type JE and WN viral E genesequences are identical at the residues implicated in attenua-tion of JE (SA14-14-2) vaccine, with one exception at residue176, we postulated that introduction of mutations at the ma-jority of these sites into wild-type WN virus prME genes wouldresult in a similar attenuation of the WN phenotype. Aminoacid residues mapping to the wild-type WN envelope (E) genepositions 107, 138, 176, and 280 were all mutated in a singleconstruct to encode amino acid residues F, K, V, and M, respec-tively. The new chimeric virus was identified as YF/WNFKVM.Chimeras were constructed in which each amino acid residue inthe FKVM group was individually mutated to produce single-sitemutants and to assess their individual roles in neurovirulence(Table 4). The dissection of the FKVM group into single sitemutations identified only residue 107 as reducing virulencesignificantly (0% mortality in three mouse neurovirulence testspresented). Residue 280 followed with 0% mortality after a 105

viral dose; however, inconsistency of this attenuated phenotype(i.e., mortality ratios of 40 to 89%) was observed in the lower-viral-dose groups tested. A mutation at residue 138 resulted inminimal reduction of virulence (�60% mortality), while a mu-tation at residue 176 showed no impact. The neurovirulence ofthe multisite YF/WNFKVM construct resulted in 0 to 20% mor-tality. In later studies, amino acid residues 316 and 440 weremutated to V and R, respectively, based on previous data in-dicating mutations in the E protein which mapped to theseregions thought to function in the biology of the E proteinthird domain (1, 32). Changes in neurovirulence of thesemutants with respect to parental ChimeriVax-WN01 wereevaluated in the mouse model as for the previous groupsabove (Table 5). A single mutation at residue 316 resulted in agreater attenuation (�30% mortality) than residue 440 but not

TABLE 2. Neuroinvasiveness of ChimeriVax-WN01 relative toYF 17D based on dose response in ICR micea

Test article i.p. Back titration dose(log10 PFU)

% Mortality(no. dead/no. tested)d

ChimeriVax-WN01 (P2)b 0.89 0 (0/5)2.23 0 (0/5)3.24 0 (0/5)4.06 0 (0/5)5.45 0 (0/5)6.51 0 (0/5)

YF17D (ATCC) 2.78 0 (0/3)4.48 0 (0/3)

Negative control NAc 0 (0/3)

a Harlan-Sprague, ICR strain (3 to 4-week-old female mice).b P2 indicates a second-generation passage virus on Vero cells. West Nile virus

strains are typically neuroinvasive after i.p. inoculation as shown by others (5).c NA, not applicable.d AST was not determined.

TABLE 3. Neurovirulence of ChimeriVax-WN01 relative toYF 17D based on dose response in ICR micea

Test article i.c.Back

titration dose(log10 PFU)b

% Mortality(no. dead/no.

tested)

AST(days)

ChimeriVax-WN01 (P2) �2 0 (0/5)�0.30 0 (0/5)

0.89 20 (1/5) 112.23 0 (0/5)3.24 20 (1/5) 104.06 60 (3/5) 95.45 20 (1/5) 9

YF17D (ATCC) 0 20 (1/5) 90 60 (3/5) 10.30.9 100 (5/5) 9.20.98 100 (5/5) 8.22.78 100 (5/5) 8

Negative control NAc 0 (0/3)

a Harlan-Sprague, ICR strain (3 to 4-week-old female mice).b Actual dose delivered i.c. assumed to be 20 �l for the back titration calcu-

lations shown.c NA, not applicable.

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as significant as residue 107. The single mutation at residue 440resulted in a greater level of attenuation over those at residues138 and 176, but only in two of the three independent testsperformed (i.e., �40% mortality observed with a mutation atresidue 440). In summary, neurovirulence of the YF/WN chi-meras in which modified amino acids were inserted in the Eprotein at residues 107, 316, and 440 were the most importantcontributors to neurovirulence. Based on this information, amultisite YF/WN107F316V440R construct was selected as ourvaccine candidate (ChimeriVax-WN02).

Neurovirulence studies in mice and in rhesus and cynomolgusmacaques. Neurovirulence of viruses with single or multisite mu-tations in the YF/WN virus E gene was measured in 21-day-oldmice inoculated by the i.c. route with doses between 104 and 105

PFU. This assessment identified only residues 107 and 280 (Ta-ble 4) and the combination of residues 316 and 440 (Table 5)as the dominant attenuating mutations as measured by mor-tality and AST. The chimera selected as our vaccine candidatehad mutations F, V, and R at residues 107, 316, and 440, respec-tively, and was avirulent for the adult mouse (Table 5). However,this virus was neurovirulent for a 2-day-old suckling mouse (datanot shown). Because mice become resistant to flavivirus infectionin an age-dependent manner, the suckling mouse is the mostsensitive host for determining subtle differences in neuroviru-lence. Preliminary studies with ChimeriVax-WN02 virus in suck-ling mice of various ages showed that mice 8 days of age were ableto discriminate differences in neurovirulence, whereas youngermice were too susceptible to differentiate the attenuation phe-notype of the ChimeriVax-WN02 vaccine candidate.

A GLP study was undertaken to characterize the neuroviru-

lence of the good manufacturing practice (GMP) manufac-tured ChimeriVax-WN02 production virus seed (P4) and avaccine lot (P5) prepared for clinical trials. Four litters (32mice) of 8-day-old suckling mice were inoculated by the i.c.route with 20 �l containing 103, 104, or 105 PFU of eitherproduction virus seed (P4) or vaccine (P5) virus. Control ani-mals of the same age received either 103or 105 PFU of YF-VAX. Negative controls were inoculated with diluent. Theresults are shown in Table 6. There were no differences acrossdose groups in the mortality ratios, and therefore data fromdose groups for each test article were combined for statisticalanalysis. There was no difference in the mortality ratio ofanimals infected with P4 or P5. Both the production virus seed(P4) and the vaccine (P5) were highly attenuated compared toYF-VAX. The neurovirulence profile of the WN vaccine istherefore similar to that of the ChimeriVax-JE vaccine, whichis currently in phase II clinical trials (25).

In a pilot monkey neurovirulence study, the ChimeriVax-WN01 construct was compared to that of the YF 17D vaccine.Rhesus macaques were screened and found negative for flavi-virus antibodies by hemagglutination-inhibition (HI) test(kindly performed by Robert Shope, University of Texas Med-ical Branch, Galveston, Tex.). Groups of three young adult

TABLE 4. Neurovirulence of ChimeriVax-WN01 (YF/WN)site-directed mutagenesis variants at E protein residues 1073F,

1383K, 1763V, 2803M, tested by i.c. inoculation in adult micea

Test article(Vero passage)b

Target dose(log10 PFU)

Backtitration dose(log10 PFU)


tested)

AST(days)

ChimeriVax-WN01 (P3) 4 4.87 100 (5/5) 8.605 6.09 60 (3/5) 9

YF/WN107F (P2) 4 4.22 0 (0/5)4 4.42 0 (0/8)5 4.99 0 (0/5)

YF/WN138K (P3) 4 4.26 60 (3/5) 10.334 4.41 63 (5/8) 11.405 5.48 60 (3/5) 9.33

YF/WN176V (P3) 4 4.42 80 (4/5) 12.505 5.54 80 (4/5) 11

YF/WN280M (P3) 4 4.14 40 (2/5) 94 4.55 89 (7/8) 11.865 5.14 0 (0/5)

YF/107F138K280M (P2) 4 3.70 0 (0/5)5 4.81 0 (0/5)

YF/107F138K176V280M (P3) 4 4.13 0 (0/5)5 5.10 20 (1/5) 7

YF-VAX 3 2.77 100 (5/5) 9

WN NY99 4 3.90 100 (5/5) 5

a Taconic, ICR strain (3 to 4-week-old female mice).b P2 and P3 indicate second and third generation virus passage on Vero cells,

respectively.

TABLE 5. Neurovirulence of ChimeriVax-WN01 site-directedmutagenesis variants at E protein residues 1073F,

3163V, and 4403R tested in adult micea

Test article i.c.(Vero passage)

Backtitration dose(log10 PFU)


tested)

AST(days)

ChimeriVax-WN01 (P3) 4.11 83 (10/12) 9.204.74 60 (3/5) 10.334.83 100 (8/8) 10.63

YF/WN316V (P3) 4.09 25 (3/12) 12.334.67 38 (3/8) 10.674.57 38 (9/24) 11.22

YF/WN440R (P3) 4.17 83 (10/12) 9.224.60 38 (3/8) 10.334.35 56 (14/25) 11.21

YF/WN316V440R (P3) 3.90 17 (2/12) 16.54.12 40 (2/5) 133.71 36 (9/25) 12

YF/WN107F316V440R (P4) 3.72 0 (0/12)5.54 0 (0/12)

a Taconic, ICR strain, 3 to 4-week-old female mice. Results of independentexperiments are shown.

TABLE 6. Comparative neurovirulence of the ChimeriVax-WN02(YF/WN107F316V440R) vaccine candidate (P5), production virus seed

(P4), and YF-VAX in 8-day-old suckling ICR mice (GLP study)

Test article % Mortality(no. dead/no. tested)a

Negative control 0 (0/32)ChimeriVax-WN02 P4 production virus seed 1 (1/96)ChimeriVax-WN02 P5 vaccine lot 02K01 4 (4/96)YF-VAX 98 (63/64)

a Statistical significance was determined by Fisher’s exact test (two sided).P � 0.0001 for ChimeriVax-WN02 P5 versus YF-VAX, and P � 0.3684 forChimeriVax-WN02 P4 versus P5.



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rhesus monkeys were inoculated by the i.c. route with 5 log10

PFU of ChimeriVax-WN01 or 4.4 log10 PFU of commercial YF17D vaccine (YF-VAX) (Table 7). Monkeys inoculated withthe chimera had a mean peak viremia titer of 1.85 0.9 log10

PFU/ml with a mean duration of 4.5 days. Monkeys inoculatedwith YF-VAX had a similar viremia profile (mean peak vire-mia titer of 2.65 0.1 log10 PFU/ml and a mean duration of4.5 days). Histological scores induced by ChimeriVax-WN01

were lower than those of a higher dose of YF-VAX (Table 7).Histological lesions in all six monkeys were mildly inflamma-tory, predominantly small perivascular infiltrates. The vast ma-jority of them were scored as grade 1 on a scale of 1 to 4. Noinvolvement of neurons was seen. The lesions were locatedmostly in YF vaccine discriminator centers (the basal ganglia/thalamus areas and both enlargements of the spinal cord).Comparison of the two groups of monkeys for the severity anddistribution of lesions did not reveal any noticeable difference.

On a second neurovirulence study, cynomolgus mon-keys were inoculated with YF/WNFVR vaccine candidate(ChimeriVax-WN02) production virus seed (P4). These ma-caques were screened and found negative for flavivirus anti-bodies by HI test (kindly performed by Robert Shope). Elevenmonkeys were inoculated i.c. with 4.74 log10 PFU of YF/WN-FVR production virus seed (P4), 11 reference control monkeysreceived 5.34 log10 PFU of YF-VAX, and 5 negative controlmonkeys received diluent. The monkeys were evaluated forchanges in clinical signs (twice daily), body weight (weekly),and food consumption (daily). Clinical signs were assignedscores according to a clinical scoring system based on theWHO requirements for YF vaccine (36).

YF 17D vaccine virus was detected in the sera of 10 of 11monkeys inoculated with YF-VAX. The mean peak viremia standard deviation (SD) was 357 579 PFU/ml, and the meannumber of viremic days was 2.45 1.13. Monkey viremia titerswere below the 500 and 100 YF-VAX mouse i.c. LD50 values,which are the maximum acceptable titers for individual mon-key and group viremia titers (i.e., present in no more than 10%of the monkeys), respectively, as established under the WHOrequirements for YF 17D vaccine.

ChimeriVax-WN vaccine virus was detected in the sera of 10of 11 monkeys inoculated with ChimeriVax-WN02 vaccine pro-duction seed bank (P4). The duration of viremia was 1 to 5 days(mean, 2.9 1.38) with peak titers ranging from 180 to 6,400

PFU/ml. The number of viremic days did not differ betweentreatment groups (P � 0.4067; analysis of variance [AVOVA]).A higher proportion of monkeys (91%) was viremic on the firstday after inoculation than that seen in the YF-VAX group(27%). On days 2 to 3 after inoculation, the proportion of viremicmonkeys (82%) was the same as for YF-VAX. The mean peakviremia was 2,097 1,845 PFU/ml. Although the mean peak vire-mia titers for ChimeriVax-WN02 production virus seed (P4)were higher than that of the reference YF-VAX vaccine (P �0.0073; ANOVA), individual monkey and group viremia titersfor ChimeriVax-WN vaccine remained within acceptable groupand individual monkey specifications, based upon WHO require-ments for YF 17D vaccine (36). The WHO specifications stip-ulate that no individual monkey will have a viremia exceeding500 i.c. adult mouse LD50/ml and that no more than 10% of theanimals will have a viremia exceeding 100 i.c. mouse LD50/ml.We have determined that these limits correspond to 20,000 VeroPFU/0.03 ml and 4,000 PFU/0.03 ml, respectively, in the caseof YF-VAX (an LD50 for ChimeriVax-WN02 cannot be deter-mined). The monkey viremias observed following ChimeriVax-WN02 do not exceed the limits set for YF vaccine.

There were no abnormalities in hematology or clinical chem-istry values associated with treatment. A complete necropsywas performed on day 31, and tissues were prepared for his-topathology. There were no ChimeriVax-WN02 productionseed (P4)-related histopathologic changes in kidney, heart,liver, adrenal glands, or spleen.

TABLE 7. Pilot study with rhesus monkeys of neurovirulence of ChimeriVax-WN01 relative to YF-VAX basedon neuropathological evaluation at 30 days post-i.c. inoculations

Test article Monkey Sexa Back titration dose(log10 PFU)

Individual histopathological score

Target area Discriminator area Sum of areas

YF-VAX G211 M 4.40 0.5 0.64 0.59P417 F 4.40 0 0.43 0.28N555 F 4.40 1.5 0.66 0.94

Mean SD 0.67 0.76 0.58 0.13 0.60 0.33

ChimeriVax-WN01 N525 M 5.07 1.0 0.58 0.72D402 M 4.99 0.5 0.48 0.48C358 F 5.06 0 0.42 0.28

Mean SD 0.50 0.50 0.49 0.08 0.49 0.22

a M, male; F, female.

TABLE 8. Summary of CNS histopathologic lesion scores incynomolgus monkeys inoculated by the i.c. route with ChimeriVax-

WN02 production virus seed (P4), YF-VAX, or negative control

Treatment group n

Mean SD lesion scores

Targetareas

Discriminatorareas

Combinedscore

Negative control 5 0 0 0ChimeriVax-WN02

production virusseed (P4)

11 0.12 0.11 0.13 0.13 0.13 0.09

YF-VAX 11 0.5 0.22 0.54 0.23 0.52 0.2P-valuea 0.000476 0.000357 0.000122

a The Kruskall-Wallis test was used for comparison of the ChimeriVax andYF-VAX groups.



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Histopathology of the brain and spinal cord was performedaccording to the methods described by Levenbook et al. (20)and incorporated into the WHO requirements for YF vaccine(36). Central nervous system (CNS) lesions were noted in 11 of11 and 10 of 11 of YF-VAX-treated and ChimeriVax-WN02

vaccine-treated monkeys, respectively, and there were no CNSlesions in the vehicle control monkeys. Inflammatory lesionsinduced by both viruses in the meninges and the brain andspinal cord matter were minimal to mild (grades 1 or 2) andcomposed of scanty, mostly perivascular infiltrates of mononu-clear cells. There was no involvement of neurons in any of theChimeriVax-WN02- or YF-VAX-treated monkeys. Summarydata are presented in Table 8. ChimeriVax-WN productionvirus seed (P4) was significantly less neurovirulent (P � 0.05)than the reference article, YF-VAX, in the target, discrimina-tor, and combined mean lesion scores. All monkeys developed

high titers of neutralizing antibodies to the respective viruswith which they were inoculated (data not shown).

Immunogenicity and efficacy studies in mice and rhe-sus monkeys. The immunogenicity of ChimeriVax-WN01 andChimeriVax-WN02 was evaluated in adult ICR mice inoculatedby the s.c. route. Serum neutralizing antibodies were measuredby PRNT50 4 weeks after vaccination with a single dose, and titerswere expressed as the geometric mean titer (GMT) (Table 9).

In mice, ChimeriVax-WN02 vaccine elicited antibody titersthat were approximately 10-fold lower than those elicited byChimeriVax-WN01 virus, reflecting the greater attenuation ofthis virus. However, when mice were challenged i.p. with 1,000LD50 of wild-type WN NY99, mice that had been immunizedwith either ChimeriVax-WN01 or -02 were protected in a dose-dependent manner. A vaccine dose of 105 PFU of ChimeriVax-WN02 protected all animals, whereas a dose of 103 PFU pro-tected only 40% of the animals.

Young adult rhesus monkeys seronegative for WN neutral-izing antibodies were vaccinated by the s.c. route with three dif-ferent chimeric vaccines: (i) a chimera containing the E107 (L3F) single-site mutation (YF/WNF); (ii) a chimera containing twomutations at E316 (A3V) and E440 (K3R) (YF/WNVR); and(iii) ChimeriVax-WN02 containing all three mutations.

Viremia in the monkeys immunized with the differentChimeriVax-WN viruses following s.c. inoculation was longerrelative to YF-VAX in some animals, although the levels de-tected at later time points were very low (Table 10). Viremiasin monkeys receiving the ChimeriVax-WN vaccines rangedfrom 1.0 to 2.3 log10 PFU/ml, with a mean duration of 3.5 to 5days. The mean peak titers of the viremia in monkeys givenYF-VAX were approximately the same as those receiving theWN vaccines. Among the ChimeriVax-WN vaccines, the vire-mia titers measured suggest an inverse relationship between

TABLE 9. Neutralizing antibody titers (PRNT50) and protectiveactivity of ChimeriVax-WN candidate vaccines in

adult ICR mice challenged by the i.p. routea

Vaccine s.c. dose(log10 PFU)

PRNT50GMT SD(4 wk post-s.c. vaccine)

Wild-typeWN NY99challengei.p. dose

(log10 PFU)

% Survival(no. live/

total)

ChimeriVax-WN01 3.48 197 93 3 100 (8/8)

ChimeriVax-WN02 2.64 20 0 3 40 (4/10)5.01 37 45 3 100 (9/9)

Negative control NAb 0 3 0 (0/5)

a Mice were challenged 4 weeks after s.c. vaccination (challenge titer was notback titrated).

b NA, not applicable.

TABLE 10. Viremia in rhesus monkeys inoculated by the s.c. route with YF-VAX, ChimeriVax-WN virus constructs with single ordouble mutations, and the ChimeriVax-WN02 vaccine candidate

Vaccine andmonkey

Vaccine dose(log10 PFU)

Viremia (log10 PFU/ml) at day postinoculationa: Mean peaktiter SD

Mean duration(days)1 2 3 4 5 6 7 8 9 10

YF-VAXM017 4.49 0 1.0 2.1 2.9 2.4 0 0 0 0 0 2.4 0.5 3.5B101 4.49 0 1.6 2.0 1.9 0 0 0 0 0 0R286 4.49 0 1.8 2.8 2.6 0 0 0 0 0 0T081 4.49 1.3 1.0 1.5 2.0 0 0 0 0 0 0

YF/WN107FN313 4.19 1.6 2.0 1.0 1.3 1.0 0 0 0 0 0 2.2 0.2 5P367 4.19 0 1.7 1.6 1.8 2.3 1.6 0 0 1.3 0T087 4.19 2.3 2.3 1.3 1.3 0 0 0 0 0 0AE81 4.19 2.3 2.1 1.6 1.3 0 0 0 1.0 0 0

YF/WN316V440RR918 4.0 0 2.0 1.5 1.7 0 0 0 0 0 0 1.8 0.2 3.5N577 4.0 1.0 1.9 1.5 1.0 0 1.0 0 0 0 0M233 4.0 0 0 1.0 1.0 0 0 0 1.0 1.6 1.8T757 4.0 0 0 0 1.6 0 0 0 0 0 0

YF/WNFVRJ729 3.92 1.0 0 0 1.0 1.3 1.0 0 1.0 0 1.0 1.4 0.2 4.5T445 3.92 1.0 1.6 1.5 0 0 1.0 0 0 0 1.0T086 3.92 1.0 0 1.3 1.3 0 0 0 0 0 0T491 3.92 0 1.5 0 1.0 0 0 1.0 0 1.0 0

a No virus was detected in the assay at day 0 (preinoculation); 1.0 log10 PFU/ml is the assay lower limit.



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the number of attenuating mutations in the chimera and thepeak titer of viremia (Table 10), but small sample size pre-cludes definitive characterization of these differences.

The immunogenicities of vaccine candidates with one, two,or three attenuating mutations were similar (Table 11). Neu-tralizing antibody titers ranged from 40 to 640 dependingupon the vaccine. There were no significant differences inneutralizing antibody response between treatment groups (Ta-ble 11). High titers of neutralizing antibodies (100 PRNT50)were present 30 and 63 days after vaccination. The observationthat monkeys developed neutralizing antibodies by day 14 in-dicates that ChimeriVax-WN02 elicits rapid onset of protectiveimmunity.

Rhesus monkeys vaccinated with YF-VAX developed neu-tralizing antibodies against YF 17D with GMTs of 380 on day14 postvaccination and 2,153 by day 63, which was 1 day beforechallenge with the virulent WN NY99 virus.

Monkeys immunized with ChimeriVax-WN single, double ortriple mutants were uniformly protected against lethal i.c. chal-

lenge with WN NY99 (Table 12). It is noteworthy that 50% ofthe animals vaccinated with ChimeriVax-WN developed feverafter challenge, with an average duration of 5 days postchal-lenge, suggesting that they sustained subclinical infections. Ani.c. challenge with WN virus is extremely aggressive and is theonly route of challenge tested to induce WN virus disease innaïve rhesus monkeys. It is likely that virus replication occursin brain tissue after i.c. inoculation and before a specific im-mune response in the brain can be recruited for clearance ofthe virus. In the case of a human peripherally challenged by amosquito bite, preexisting immunity would rapidly neutralizethe virus and fever is unlikely to occur. However, none of theChimeriVax-WN-immunized animals developed detectableviremia after challenge, none developed signs of illness (asidefrom fever), and none died. Vaccinated animals showed anincrease in antibody levels postchallenge (Table 11), suggestingthat viral replication and antigenic stimulation occurred with-out associated illness.

Postchallenge viremias (�102 to 103 PFU/ml) were detectedin the control monkeys that had previously been immunizedwith YF-VAX (Table 12). Two out of four monkeys vaccinatedwith YF-VAX (M017 and R286) developed a high fever andsigns of encephalitis: muscle tremors, anorexia, and spasticity.These two animals were euthanized between days 9 and 11after challenge. The other two YF-VAX-vaccinated animalsdeveloped fever and survived i.c. challenge with WN NY99strain without any clinical symptoms; this finding is attributedto cross-protection across the two flaviviruses.

Two monkeys without any prior vaccination were also chal-

TABLE 11. Reciprocal neutralizing antibody titers (PRNT50)against ChimeriVax-WN virus, rhesus monkeys inoculated by thes.c. route with YF-VAX or ChimeriVax-WN vaccine candidates

Vaccine andmonkeya

Dose(log10 PFU)

PRNT50 on dayb:

Postimmunization Postchallenge

14 30 63 15 31–34

YF-VAXM017 4.49 �320 640 5,120 NAd NAB101 �320 640 2,560 1,280 2,560R286 �320 640 640 NA NAT081 640 640 2,560 10,240 5,120

GMT 380 640 2,153 3,620 3,620

YF/WN107FN313 4.19 160 640 640 2,560 5,120P367 �40 640 640 5,120 2,560T087 �40 640 640 2,560 1,280AE81 �40 640 160 10,240 20,480

GMT 57 640 453 4,305 4,305

YF/WN316V440R918 4.0 �40 320 640 1,280 2,560N577 �40 160–320c 320 1,280 2,560M233 �40 160–320c 320 640 1,280T757 �40 40 640 1,280 5,120

GMT 40 135 453 1,076 2,560

YF/WNFVRJ729 3.92 �40 320 80 5,120 5,120T445 80 640 160 640 5,120T086 160 320–640c 640 1,280 5,120T491 80 320 160 2,560 5,120

GMT 80 381 190 1,280 5,120

a For GMT, if an endpoint was not reached, the assay limit titer was used in thecalculation (e.g., 640 taken as 640 and �40 was taken as 40).

b PRNT50 was calculated after subtraction of the PRNT from day 0 serumsamples.

c PRNT50 calculation fell between the titers shown. The lower titer was usedfor the GMT calculation.

d NA, not applicable.

TABLE 12. Viremia and clinical outcome in rhesus monkeysimmunized with ChimeriVax-WN or YF-VAX and

challenged 63 days later by the i.c. route with5.38 log10 PFU of wild-type WN NY99 virus

Vaccine andmonkey

Viremia by day post-i.c.challenge (log10 PFU/ml)

No. of monkeys withoutcome/total (%)

1 2 3 4 5 Illness Death

Neg controlK396 2.0 3.1 2.6 2.4 0 2/2 (100) 2/2 (100)P500 2.0 3.1 2.6 2.2 1.0

YF-VAXM017 2.5 2.6 1.7 1.3 0 2/4 (50) 2/4 (50)B101 2.6 2.5 1.7 0 0R286 3.3 3.3 1.7 0 0T081 2.4 3.0 2.4 0.5 0

YF/WN107FN313 0 0 0 0 0 0/4 (0) 0/4 (0)P367 0 0 0 0 0T087 0 0 0 0 0AE81 0 0 0 0 0

YF/WN316V440RR918 0 0 0 0 0 0/4 (0) 0/4 (0)N577 0 0 0 0 0M233 0 0 0 0 0T757 0 0 0 0 0

ChimeriVax-WN02J729 0 0 0 0 0 0/4 (0) 0/4 (0)T445 0 0 0 0 0T086 0 0 0 0 0T491 0 0 0 0 0



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lenged with WN NY99 virus. The two challenge control ani-mals developed fever between days 5 and 9 postchallenge, withslight tremors progressing to ataxia and spasticity between days10 and 11, and were euthanized between days 10 and 12.

Genetic stability. In vitro and in vivo substrate-passage stud-ies with ChimeriVax-WN01 or the YF/WNFVR chimeric vac-cine candidate (ChimeriVax-WN02) were conducted to deter-mine genetic stability of the constructs when grown instationary cell cultures and in brain tissue. After six in vitroVero E6 cell passages of the virus followed by six in vivo ICRadult mouse brain passages of ChimeriVax-WN01, no muta-tions were selected relative to the wild-type sequence of theprM and E genes in the ChimeriVax-WN01 construct nor wasthere an increase in mouse neurovirulence (data not shown). Aheterozygous mutation in the E protein at position E336 re-sulting in a cysteine-to-serine change was identified following10 in vitro passages of the YF/WNFVR virus in Vero E6 cells.In a separate study, in vitro passage of YF/WNFVR in SF-Verocells (manufacturing substrate) resulted in selection of a mu-tation at position E313 that changed the amino acid at thatposition from glycine to arginine. Neurovirulence of these pas-saged viruses for the 2-day-old suckling mice (n � 10) inocu-lated with a nominal 2-log10 PFU dose of viruses includingE313 and E336 mutations showed no increase in virulence

relative to YF/WNFVR PMS (Table 13). During all serial pas-sages of the virus in Vero cells or brain tissue, no reversionswere detected at target E protein amino acid residues 107F,316V, or 440R, the attenuation markers for the vaccinecandidate. Additionally, during scale-up manufacturing of theChimeriVax-WN02 vaccine, no reversions at these critical res-idues were detected.

The GMP manufactured ChimeriVax-WN02 production vi-rus seed (P4) was used for inoculation of large-scale Vero-SFcultures grown on microcarrier beads in 100-liter bioreactors.An additional mutation (L3P) occurred in the vaccine atposition 66 in the M protein. This mutation was associated withproduction of slightly smaller plaque size. The vaccine lot (P5)contained equal ratios of small and large plaques. Virus pop-ulations with and without the M66 mutation were isolated byplaque purification and compared to the PMS (no detectablemutations) and the vaccine lot in the suckling mouse model.One litter (10 mice) of 8-day-old mice was inoculated by the i.c.route with 20 �l containing 2, 3, or 4 log10 PFU of either large-plaque or small-plaque virus and observed for 21 days for signsof illness and death. For comparative purposes, litters of micewere inoculated with similar doses of the PMS (P2) and vac-cine lot (P5) viruses. Mice of the same age were also inoculatedwith 2 log10 PFU of YF-VAX. Negative controls were inocu-lated with diluent (Table 14). There were no differences inmortality ratios across dose groups, and data were combinedfor analysis. Since the mortality ratio across all treatmentgroups differed (P � 0.0001), pairwise comparisons were per-formed. The M66 mutation had no effect on mouse neuroviru-lence.

DISCUSSION

The original YF/WN chimeric virus constructed by insertionof the prME genes from a wild-type WN virus strain wasattenuated with respect to the parental YF 17D virus vector,but retained a degree of neurovirulence for adult mice. Todevelop a vaccine candidate with a wider margin of safety, weselectively introduced mutations in the donor WN virus. Mu-tations introduced into the E protein of the WN donor virusutilized a strategy based on the previous construction ofChimeriVax-JE vaccine, which contained donor prME genesfrom an attenuated vaccine strain of JE (SA14-14-2 virus) (3,4, 28). The SA14-14-2 virus contains mutations at six aminoacid residues (E107, E138, E176, E279, E315, and E439) that

TABLE 13. Neurovirulence of YF/WNFVR RMS (P4 and P11)a

and PMS (P2 and P10)a in 2-day-old ICR strainmice relative to YF 17Db

Virus i.c. MutationBack

titration dose(log10 PFU)

% Mortality(no. dead/total)

AST(days)

ChimeriVax-WN02RMS

P4 None 1.73 80 (8/10) 14.5P11 E336C3S 2.08 60 (6/10) 13.67

ChimeriVax-WN02PMS

P2 None 2.10 60 (6/10) 13P10 E313G3R 1.88 70 (7/10) 13.67

YF-VAX NAc 1.90 100 (10/10) 10.6

Negative control NA 0 (0/10)

a Viruses were passed in a serum-free (SF-Vero) stationary-cell substrate.b Taconic, ICR strain, mice.c NA, not applicable.

TABLE 14. Neurovirulence of small- and large-plaque viruses isolated from ChimeriVax-WN02 P5 vaccine in8-day-old ICR mice inoculated i.c.a

Test article Mutation % Mortality(no. dead/no. tested)

P value forb:

Test article vs Large plaque vssmall plaqueNegative control YF-VAX

Sham (negative control) 0 (0/10) �0.0001PMS (P2) None 13 (4/30) 0.5558 �0.0001Vaccine lot (P5; large and small plaque) E313G3R, M66L3P 23 (7/30) 0.1612 �0.0001Large plaque E313G3R 3 (1/30) 1.000 �0.0001 0.3533Small plaque E313G3R, M66L3P 13 (4/30) 0.5558 �0.0001YF-VAX 100 (10/10) �0.0001

a All ChimeriVax-WN02 viruses used in the evaluation contained the site-directed attenuating mutations 107F316V440R. Additional mutations appearing during Verocell passage are shown in the table.

b Fisher’s exact test (two sided).



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play a role in neurovirulence (3). The WN and JE wild-typegene sequences are conserved at most of these residues (except176), suggesting that mutations introduced at these sites in WNvirus could have the same attenuating effect as they did in thecase of JE SA14-14-2. As predicted, we found that mutagenesisof the WN E residues E107, E280 (corresponding to E279 inJE virus), and E316 (corresponding to E315 in JE virus) causedattenuation of the YF/WN virus chimera. Surprisingly, whilean E138 mutation, E3K, was associated with a marked atten-uation of JE virus (3, 34), a corresponding mutation in the WNgene did not reduce the neurovirulence of the YF/WN virus tothe expected 0% mortality by the mouse neurovirulence test.Mutation of the E protein at E440 (corresponding to E439 inJE virus) from K3R, a conservative residue change, also re-duced neurovirulence for mice. A construct with the threemutations of F, V, and R at positions E107, E316, and E440,respectively, was designated ChimeriVax-WN02 and was se-lected as the candidate for manufacture of the vaccine forclinical studies. ChimeriVax-WN02 was not neuroinvasive com-pared to WN NY99 virus and had reduced neurovirulencecompared to YF 17D vaccine virus. Attenuation of this viruswas conferred by the mutation at E107, which maps to thefusion peptide in the second domain as predicted in the crystalstructure of the E protein (1, 12, 32). This amino acid isthought to reduce virulence by altering the function of thefusion peptide in the natural cycle of the virus replication. Theadditional ChimeriVax-WN02 mutations at positions E316 andE440 map in domain III on the crystal structure of the Eprotein. Residue E316 is thought to be involved in binding oftick-borne encephalitis virus to the virus receptor on the cellplasma membrane (1, 32) and thus may play a role in WN viruscell entry. Residue E440 is in the transmembrane region of theE protein and is believed to be involved in anchoring the Eprotein during its translation in the endoplasmic reticulum;hence, a mutation at E440 may be altering the natural associ-ation of the E protein with prM (2). The K-to-M mutation atposition E280 that attenuated neurovirulence for mice was notincluded in the final vaccine because it appeared unstable,similar to the corresponding residue in JE virus E proteinsequence (i.e., E279) shown to be unstable during in vitropassage. A reversion to K at position 279 in the JE virus Eprotein occurred after less than five passages of the virus inMRC-5 cells (22). Mutation of residue E176 from Y in the WNvirus sequence to either V or I, as seen in JE strains, did notsuggest a significant change in neurovirulence; therefore, po-sition E176 was not changed in the final vaccine candidatesequence (unpublished results). This observation contrasts tothe previously published results linking a mutation from I to Vat position E176 in the JE virus envelope protein to neuro-virulence (3, 28). Other approaches to flavivirus chimeras em-ployed an attenuated dengue virus genome backbone to pro-duce chimeric dengue virus vaccine candidates against the fourmajor serotypes (15); similarly, a dengue virus has been used todeliver the prM and E genes of WN virus, producing an atten-uated vaccine candidate shown protective in a nonhuman pri-mate model (30). This dengue/WN virus chimeric constructwas attenuated by virtue of the chimeric nature and as a resultof a 30-nucleotide deletion in the 3� end noncoding region(untranslated region) of the virus genome.

Safety of ChimeriVax WN02 (YF/WNFVR) is characterized

by three features: (i) loss of neuroinvasion relative to wild-typeWN virus; (ii) introduction of three site-directed mutations intwo E protein domains, each independently associated withattenuation; and (iii) conservation of the FVR mutations afterin vitro passage in manufacturing-related substrates.

The safety of ChimeriVax-WN02 was evaluated in a sensitive8-day-old suckling mouse model and in rhesus monkeys and incynomolgous macaques inoculated by the i.c. route. In all host-virus pairings, the chimeric virus proved to be significantly lessneurovirulent than the licensed YF-VAX vaccine. The monkeysafety test was performed as prescribed by current regulationsapplicable to YF vaccines (36) and showed that the vaccine wassignificantly less virulent than YF-VAX. The nonhuman pri-mate model has been previously used to assess the safety ofother chimeric vaccines against JE and dengue virus (6, 18, 24).

After s.c. inoculation of rhesus monkeys, viremias were moreerratic and of longer duration in animals immunized with theChimeriVax-WN vaccines than in animals given YF-VAX (Ta-ble 10). The mean peak titer viremia for YF-VAX-vaccinatedmonkeys was �1 log higher than that for the ChimeriVax-WN02 (triple mutant) vaccine candidate. The longer viremiaobserved after immunization with the chimeric viruses suggeststhat the viruses replicate in different tissues had different re-ticuloendothelial clearance rates from the parental YF 17Dvirus or had different kinetics of immune response. We arecurrently studying the sites of replication of ChimeriVax-WN02

and YF-VAX in tissues of cynomolgus macaques and willreport results in a future publication. In addition, future clin-ical trials will assess the magnitude and duration of viremiafollowing ChimeriVax-WN02 and YF-VAX and establish cor-relations between viremia and adverse events. The low titer ofthe viremia observed in rhesus monkeys after s.c. vaccinationwith the chimeric vaccine candidates suggests that ChimeriVax-WN02 vaccine has an acceptable phenotype for trials in hu-mans.

The triply mutated virus (ChimeriVax-WN02) vaccine ap-peared to be less immunogenic than the wild-type chimera inmice, but performed satisfactorily in nonhuman primates.ChimeriVax-WN02 vaccine rapidly elicited a neutralizing anti-body response in all rhesus monkeys and provided solid pro-tection against an aggressive i.c. challenge with 5 log10 PFU ofWN NY99 virus.

A partially protective immune response was observed in twoof the four rhesus monkeys immunized with YF 17D andsubsequently challenged with wild-type WN virus. Previousobservations by others have shown the cross-protective effectof prior exposure to phylogenetically related flaviviruses andconcluded that potential for protective cross-reactivity is un-likely to prevent infection and only likely to prevent disease(16). Similarly, we observed that prior YF immunization ofmonkeys did not prevent infection (viremia) after WN viruschallenge, but may have provided an element of protectionagainst death. It should be pointed out that the interval be-tween YF immunization and challenge was relatively brief andthat cross-protection between heterologous flaviviruses oftendiminishes over time, probably due to affinity maturation of theantibody response and waning of T-cell immunity. It is highlyunlikely that YF immunity would provide reliable cross-pro-tection of humans and therefore a specific, homologous (WN)vaccination strategy must be pursued. This observation is sim-



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ilar to a previous report that hamsters vaccinated with YF 17Dwere somewhat cross-protected against WN virus challenge-induced disease (35). However, in the rhesus model, only thoseanimals immunized with the WN vaccines and subsequentlychallenged with wild-type WN virus i.c. did not show postchal-lenge WN virus viremia. All of the surviving animals vaccinatedwith ChimeriVax-WN displayed increases in WN neutralizingantibodies after i.c. challenge indicating that the challengevirus had replicated in brain tissue (without causing illness) orthat the challenge inoculum, which was quite large, providedsufficient antigen for stimulation of B cells. The experimentaldesign did not allow a proper test of whether the preexistingimmunity would have been “sterilizing ” if the challenge inoc-ulum had been delivered by a natural (parenteral) route in-stead of i.c. Sterile immunity could be tested by measuring theimmune response to nonstructural proteins of the WN chal-lenge virus. Others have reported that experimental WN vac-cines elicit sterilizing immunity against parenteral challenge(11).

Since the first experimental vaccine construct with wild-typeprME sequence was less neurotropic than commercial YF vac-cine, the chimeric vaccine with three attenuating mutations hasa wide margin of safety. To maintain this ultra-attenuatedphenotype, the vaccine candidate must retain attenuating mu-tations FVR introduced to retain the level of attenuation re-quired. Because of the quasispecies nature of RNA viruses,variations in the sequence are to be expected during vaccinemanufacture. Therefore, quality of the product is carefullymonitored during manufacture by tests for genotypic and phe-notypic stability. Safety is ensured by the demonstration ofconservation of the amino acid residues identified to play arole in attenuation (shown by direct sequencing release tests);for ChimeriVax-WN02, the required conserved residues are Eprotein 107F, 316V, and 440R. In addition, the attenuatedvirulence phenotype of the vaccine is tested by infant mouseneurovirulence test performed on seed viruses and each vac-cine batch. Currently, the product specifications for sequencedata have been expanded to include full genomic sequencing ofeach vaccine batch rather than confirmation only of the pointmutations at residues 107, 316, and 440. When ChimeriVax-WN02 virus was passed in Vero cells, with at least twice thenumber of passages required for manufacture of the vaccine,the FVR mutations were maintained. Passage of the vaccinecandidate, in vitro or in vivo, selected mutations in the vicinityof residue E316 (at E313 and at E336) without compromisingthe neurovirulence phenotype of ChimeriVax-WN02 and sup-porting our mutagenesis approach to ensure vaccine safety.

When the vaccine was scaled up for manufacture of clinicalmaterial in 100-liter bioreactors, a mutation at M66 was de-tected. This mutation also did not affect neurovirulence orimmunogenicity of the vaccine.

ACKNOWLEDGMENTS

This work was funded by a NIAID R01 grant AI48297 and NIHgrant 5P51-RR00164-41.

We would like to acknowledge contributions from the following:independent contributor Inessa Levenbook; from University of Mas-sachusetts Medical School, Worcester, Sharone Green, Francis Ennis,and John Cruz; from the Centers for Disease Control and Prevention,John Roehrig; from Sierra Division, Charles River Laboratories, KenDraper; from University of Texas, Galveston, Amelia P. Travassos da

Rosa and Robert B. Tesh; and from Acambis, Inc., Rich Weltzin,Zheng-Xi Zhang, Jian Liu, and Rick Nichols. Thanks go to DeniseGoens for critical review of the manuscript.

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ChimeriVax-West Nile Virus Live-Attenuated Vaccine