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Journal of Virology, November 2004, p. 12497-12507, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12497-12507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

ChimeriVax-West Nile Virus Live-Attenuated Vaccine: Preclinical Evaluation 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, Massachusetts,1 DynPort Vaccine Co. LLC, Frederick, Maryland,2 Tulane National Primate Research Center, Covington, Louisiana,3 Vaxin, Inc., Birmingham, Alabama4

Received 31 March 2004/ Accepted 9 July 2004


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ABSTRACT
 
The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the time West Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of the vaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live- attenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. The ChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes to deliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homology between the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to develop the 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 nonhuman primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenuating 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 vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.


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INTRODUCTION
 
Following isolation of West Nile (WN) virus in New York in 1999, the virus rapidly spread across North America, causing disease in wild birds, horses, and humans. The number of human cases increased dramatically in 2002 and 2003, when 4,145 and 8,977 cases were reported, respectively (7, 8). WN virus is transmitted principally between wild birds and Culex mosquitoes (7). Recently, WN virus has been isolated in the West Indies and serosurveys have identified neutralizing antibody-positive avian species in Mexico (14), Jamaica, and the Dominican Republic (13, 17). The rapid geographic expansion of the virus is attributed to movement by viremic birds during local and migratory flight behavior. To date, there is no effective drug treatment against WN virus infection and surveillance and mosquito control measures have not significantly influenced the number of human infections (27). A vaccine against WN virus represents an important approach to the prevention and control of this emerging disease.

The ChimeriVax technology has been successfully used to develop a live vaccine against Japanese encephalitis (JE) virus that is now in phase II trials (23). JE virus is a close genetic relative of WN virus (31), a fact that expedited use of this technology to develop multiple WN virus vaccine candidates. The ChimeriVax technology employs the yellow fever (YF) 17D vaccine capsid and nonstructural genes to deliver the envelope genes (prM and E) of other flaviviruses. In the work presented here, the envelope genes of YF 17D were replaced with the corresponding genes of the wild-type WN virus NY99 strain previously described by Lanciotti et al. (19). The resulting YF/WN chimera lacked the mouse neuroinvasive property of WN virus and is less neurovirulent than YF 17D vaccine in both mouse and monkey models. Because WN virus, like other flaviviruses in the genus, is neurotropic for mammals (21, 29), attenuating point mutations were later introduced in the envelope 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 by others (1, 3, 28, 32) pertaining to attenuation phenotypes in related flaviviruses: specifically JE and tick-borne encephalitis viruses. Site-directed mutations in the WN virus E gene of the chimeric prototype vaccine, ChimeriVax-West Nile01, (ChimeriVax-WN01) resulted in a significant reduction in virus neurovirulence. Here we discuss a vaccine in a YF vaccine backbone; the WN virus envelope (E) protein mutagenesis rationale; and the assessment of the safety, immunogenicity, efficacy, and genetic stability of these ChimeriVax-WN vaccine candidates in the mouse and macaque models.


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MATERIALS AND METHODS
 
YF/WN chimeric clones and molecular procedures for virus assembly. Chimeric flaviviruses were constructed with the ChimeriVax two-plasmid technology previously described (9). Briefly, the two-plasmid system provides plasmid stability in Escherichia coli by dividing the cloned YF backbone into two plasmids. This provides smaller plasmids that are more stable to manipulate the YF sequences facilitating replacement of the prM and E genes of the flavivirus target vaccine. The WN virus prM and E genes used were cloned from the WN flamingo isolate 383-99 sequence (GenBank accession no. AF196835; kindly provided 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 of the WN virus prM gene was cloned precisely at the 3' end of the YF 17D capsid gene by overlap-extension PCR using Pwo polymerase (Roche Applied Science, Indianapolis, Ind.). This cloning step maintained integrity of the cleavage/processing signal encoded at the 3' end of the YF capsid gene. The 3' end of the E gene 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 WN virus into the YF 17D backbone was described previously (4). Silent mutations were introduced in the sequences of prM and E to create unique BspEI and EagI restriction sites. Digestion of the two plasmids with these restriction nucleases generated DNA fragments that were gel purified and ligated in vitro to produce a full-length chimeric cDNA. The cDNA was linearized with XhoI to facilitate in vitro transcription by SP6 polymerase (Epicentre, Madison, Wis.).

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


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  		TABLE 1. Switch oligonucleotides used for site mutagenesis

Viruses and cell lines. The wild-type WN virus used in animal challenge studies is the NY99 strain (NY99-35262-11 flamingo isolate, a homolog of the virus used to build chimeras) obtained from the Centers for Disease Control and Prevention, Fort Collins, Colo. (CDC stock designation B82332W) with two additional passages in Vero E6 cells to produce a master virus bank. YF 17D is a commercial vaccine (YF-VAX; Aventis Pasteur, Swiftwater, Pa.) used after reconstitution 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 prepared by RNA transfection (P1 virus) of Vero E6 cells (ATCC origin, CIDVR University of Massachusetts Medical Center cell bank, Worcester, Mass.). Research master seeds (RMS) were prepared by additional amplifications (either passage 2 or 3 at a 0.001 multiplicity of infection [MOI]) in Vero E6 cells. Vero E6 cells were maintained in minimal essential medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (HI-FBS) (HyClone, Logan, Utah). Preparation of pre-master seeds (PMS) for manufacture of the vaccine was initiated by RNA transfection of serum-free Vero (SF-Vero) cells obtained from a cell bank that had been manufactured and controlled to meet current Food and Drug Administration guidelines for cell culture vaccines. (The cells were obtained from an ATCC strain predating 1980, and the cell bank was made by Baxter/Immuno, Orth, Austria.) Progeny virus from the transfection step was amplified by a single passage in the same SF-Vero cell line to produce P2, which was designated the PMS for subsequent manufacture of clinical-grade vaccine. The SF-Vero cell line is propagated and maintained in a serum-free, animal protein-free medium formulation, VT-Media (Baxter/Immuno). Viruses for animal experiments were diluted in M199 with HEPES buffer (Invitrogen) and 20% HI-FBS (HyClone) unless otherwise indicated. Plaque assays to verify the titer of virus inoculi were performed in a Vero cell substrate as previously described (24).

Mouse studies. Protocols for mouse experiments were approved by the Institutional Animal Care and Use Committees at both University of Massachusetts Medical Center (Worcester, Mass.) and Acambis, Inc. (Cambridge, Mass.). Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles set forth in the Guide for the Care and Use of Laboratory Animals (27a). Female ICR mice (Taconic, Germantown, N.Y., or Harlan Sprague-Dawley, Indianapolis, Ind.) were inoculated intraperitoneally (i.p.) with 100 to 200 µl of wild-type WN virus NY99 for neuroinvasion tests or postvaccination challenge experiments (titers of inoculated viruses are indicated in the Results 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.) on the right side of the brain as previously described (24) and using a 20-µl volume of YF 17D or chimeric YF/WN constructs for neurovirulence testing (titers of inoculated viruses are indicated in the Results section and in the tables presented).

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

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

Nonhuman primate studies. Neurovirulence tests in rhesus macaques were performed according to World Health Organization (WHO) guidelines for testing YF vaccine (36) and as described previously for safety tests of ChimeriVax-JE vaccine (24). Animals were inoculated with specific virus candidates by inoculation of the frontal lobe of the brain (see Table 7). Blood samples were obtained daily for the first 10 days following inoculation, and serum viremia was measured by plaque assay on Vero cells. Animals were observed daily for clinical signs of encephalitis and associated symptoms such as fever or tremors. Animals were euthanized 30 days after infection, and the brain and spinal cord tissues were removed for histopathology. Slides were prepared from tissues of the frontal and temporal cortex, basal ganglia/thalamus (two levels), midbrain, pons, cerebellum (two levels of the nuclei and cortex), medulla oblongata, and six levels of each of cervical and lumbar enlargements of the spinal cord. Sections were stained with gallocyanine. Histological lesions were analyzed and scored for pathology relative to that of the YF 17D according to the criteria for evaluation of neurovirulence in rhesus monkeys proposed by the current WHO requirements. Mean lesion scores for individual monkeys were calculated for "target" (substantia nigra) and "discriminator" (basal ganglia/thalamus and the spinal cord) areas individually and for the target and discriminator areas combined.


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

A second neurovirulence test was performed with cynomolgus monkeys and inoculation with the YF/WNFVR vaccine candidate (ChimeriVax-WN02) production virus seed (P4). The study was conducted according to good laboratory practices (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 evaluated for changes in clinical signs (twice daily), body weight (weekly), and food consumption (daily). Clinical signs were assigned scores according to a clinical scoring system, based on the WHO requirements for YF vaccine (36). Blood samples were collected preinoculation on day 1 and on days 3, 5, 7, 15, and 31 for clinical pathology analysis (serum chemistry and hematology parameters). Additional blood samples were collected preinoculation on day 1 and on days 2 to 11 for viremia analysis, and on days 1 (predose) and 31 for antiviral antibody titer analyses.

To determine immunogenicity, rhesus monkeys were inoculated by the subcutaneous (s.c.) route with a single 0.5-ml dose containing ~4 log10 PFU of chimeric vaccine. Control animals received undiluted YF-VAX containing 4.49 log10 PFU in a 0.25-ml volume. Each vaccine dose was back titrated following immunization. Serum viremia was measured daily by plaque assay through day 10 after vaccination. Neutralizing antibody levels were measured by PRNT50 on days 14, 30, and 63 after vaccination. Animals were challenged 64 days after vaccination by i.c. inoculation of 125 µl containing 2.4 x 105 PFU of wild-type WN NY99 suspended in M199 with HEPES buffer (Invitrogen) and 10% sorbitol (Sigma). Monkeys were observed for viremia, clinical illness, and antibody response; severely ill animals were euthanized. The i.c. challenge model closely followed the model established during the development of ChimeriVax-JE vaccine (24, 26).

Genetic stability (in vivo and in vitro passage) and sequencing. The chimeric YF/WN virus containing unmodified, wild-type WN virus prME sequence (designated ChimeriVax-WN01) was passed six times in Vero E6 cells followed by six passages in suckling mice by the i.c. route. The chimeric YF/WN virus containing three mutations introduced by site-directed mutagenesis (designated ChimeriVax-WN02) at the P2 level (PMS) and P3 level (RMS) were passed 12 and 10 times, respectively, in serum-free, protein-free SF-Vero cell substrate. All in vitro virus passages were performed with an initial MOI of 0.01 PFU/cell followed by harvest of the virus on the third day after infection. Passages in vivo were performed by initial i.c. inoculation of 105 PFU; brain tissue from ICR mice (Taconic) was harvested 3 days after inoculation and homogenized, and the clarified homogenate was used for passage to a new group of mice. Virus titers at each passage were determined by plaque assay. Neurovirulence of the passaged viruses was determined by i.c. inoculation of adult or suckling mice (see Tables 13 and 14). Sequencing of viral RNA was performed with Superscript II reverse transcriptase and XL-PCR; products were purified by QIAGEN gel extraction (QIAGEN, Valencia, Calif.). Sequencing reactions were prepared and analyzed using the standard Beckman CEQ 2000XL protocol and equipment (Beckman-Coulter). For virus passages, at least two independent sequencing reactions were executed per RT-PCR product strand sequenced; sense and antisense strands were sequenced each time. Mutation acceptance criteria needed a positive identity in at least three of four sequencing reactions analyzed; in addition, two independent operators read sequence chromatographs.


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  		TABLE 13. Neurovirulence of YF/WNFVR RMS (P4 and P11)a and PMS (P2 and P10)a in 2-day-old ICR strain mice relative to YF 17Db


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  		TABLE 14. Neurovirulence of small- and large-plaque viruses isolated from ChimeriVax-WN02 P5 vaccine in 8-day-old ICR mice inoculated i.c.a


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RESULTS
 
Virulence phenotype of chimeric YF/WN containing wild-type WN prME genes relative to YF 17D (YF-VAX). The initial WN virus chimera encoded the envelope and premembrane protein genes of the WN NY99 wild-type strain (designated ChimeriVax-WN01). This chimeric virus did not cause encephalitis after i.p. inoculation at doses of 106 PFU in 3- to 4-week-old adult ICR mice (Table 2). Encephalitis was assessed by daily observations for illness, paralysis, and death. ChimeriVax-WN01 resembles YF 17D vaccine (33) in being nonneuroinvasive in adult mice. In contrast, the WN NY99 wild-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 encephalitis after i.c. inoculation, a property consistent with that of YF 17D virus (10). We estimated the i.c. 50% lethal dose (LD50) of ChimeriVax-WN01 to be between 103 and 105 PFU. The neurovirulence phenotype of ChimeriVax-WN01 is lower than that of YF 17D virus, for which the i.c. LD50 is between 101 and 102 PFU (Table 3).


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


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

Evaluation of the multisite mutagenesis for attenuation. Amino acids in the envelope protein previously established as genetic determinates of virulence for ChimeriVax-JE were changed to reduce the virulence of YF/WN chimeras. The strategy for this mutagenesis approach was to design a safe attenuated WN vaccine; this strategy was first discussed in an earlier publication (4). Briefly, the selection of specific amino acid residues for mutagenesis was defined by previous studies of the attenuating mutations in a vaccine strain of JE virus (SA14-14-2) (3). Since the wild-type JE and WN viral E gene sequences are identical at the residues implicated in attenuation of JE (SA14-14-2) vaccine, with one exception at residue 176, we postulated that introduction of mutations at the majority of these sites into wild-type WN virus prME genes would result in a similar attenuation of the WN phenotype. Amino acid residues mapping to the wild-type WN envelope (E) gene positions 107, 138, 176, and 280 were all mutated in a single construct to encode amino acid residues F, K, V, and M, respectively. The new chimeric virus was identified as YF/WNFKVM. Chimeras were constructed in which each amino acid residue in the FKVM group was individually mutated to produce single-site mutants and to assess their individual roles in neurovirulence (Table 4). The dissection of the FKVM group into single site mutations identified only residue 107 as reducing virulence significantly (0% mortality in three mouse neurovirulence tests presented). 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 in minimal reduction of virulence (~60% mortality), while a mutation at residue 176 showed no impact. The neurovirulence of the multisite YF/WNFKVM construct resulted in 0 to 20% mortality. In later studies, amino acid residues 316 and 440 were mutated to V and R, respectively, based on previous data indicating mutations in the E protein which mapped to these regions thought to function in the biology of the E protein third domain (1, 32). Changes in neurovirulence of these mutants with respect to parental ChimeriVax-WN01 were evaluated in the mouse model as for the previous groups above (Table 5). A single mutation at residue 316 resulted in a greater attenuation (~30% mortality) than residue 440 but not as significant as residue 107. The single mutation at residue 440 resulted in a greater level of attenuation over those at residues 138 and 176, but only in two of the three independent tests performed (i.e., ~40% mortality observed with a mutation at residue 440). In summary, neurovirulence of the YF/WN chimeras in which modified amino acids were inserted in the E protein at residues 107, 316, and 440 were the most important contributors to neurovirulence. Based on this information, a multisite YF/WN107F316V440R construct was selected as our vaccine candidate (ChimeriVax-WN02).


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  		TABLE 4. Neurovirulence of ChimeriVax-WN01 (YF/WN) site-directed mutagenesis variants at E protein residues 107->F, 138->K, 176->V, 280->M, tested by i.c. inoculation in adult micea


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  		TABLE 5. Neurovirulence of ChimeriVax-WN01 site-directed mutagenesis variants at E protein residues 107->F, 316->V, and 440->R tested in adult micea

Neurovirulence studies in mice and in rhesus and cynomolgus macaques. Neurovirulence of viruses with single or multisite mutations in the YF/WN virus E gene was measured in 21-day-old mice inoculated by the i.c. route with doses between 104 and 105 PFU. This assessment identified only residues 107 and 280 (Table 4) and the combination of residues 316 and 440 (Table 5) as the dominant attenuating mutations as measured by mortality and AST. The chimera selected as our vaccine candidate had mutations F, V, and R at residues 107, 316, and 440, respectively, and was avirulent for the adult mouse (Table 5). However, this virus was neurovirulent for a 2-day-old suckling mouse (data not shown). Because mice become resistant to flavivirus infection in an age-dependent manner, the suckling mouse is the most sensitive host for determining subtle differences in neurovirulence. Preliminary studies with ChimeriVax-WN02 virus in suckling mice of various ages showed that mice 8 days of age were able to discriminate differences in neurovirulence, whereas younger mice were too susceptible to differentiate the attenuation phenotype of the ChimeriVax-WN02 vaccine candidate.

A GLP study was undertaken to characterize the neurovirulence of the good manufacturing practice (GMP) manufactured ChimeriVax-WN02 production virus seed (P4) and a vaccine lot (P5) prepared for clinical trials. Four litters (32 mice) of 8-day-old suckling mice were inoculated by the i.c. route with 20 µl containing 103, 104, or 105 PFU of either production virus seed (P4) or vaccine (P5) virus. Control animals of the same age received either 103or 105 PFU of YF-VAX. Negative controls were inoculated with diluent. The results are shown in Table 6. There were no differences across dose groups in the mortality ratios, and therefore data from dose groups for each test article were combined for statistical analysis. There was no difference in the mortality ratio of animals infected with P4 or P5. Both the production virus seed (P4) and the vaccine (P5) were highly attenuated compared to YF-VAX. The neurovirulence profile of the WN vaccine is therefore similar to that of the ChimeriVax-JE vaccine, which is currently in phase II clinical trials (25).


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  		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)

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 flavivirus antibodies by hemagglutination-inhibition (HI) test (kindly performed by Robert Shope, University of Texas Medical Branch, Galveston, Tex.). Groups of three young adult rhesus monkeys were inoculated by the i.c. route with 5 log10 PFU of ChimeriVax-WN01 or 4.4 log10 PFU of commercial YF 17D vaccine (YF-VAX) (Table 7). Monkeys inoculated with the chimera had a mean peak viremia titer of 1.85 ± 0.9 log10 PFU/ml with a mean duration of 4.5 days. Monkeys inoculated with YF-VAX had a similar viremia profile (mean peak viremia titer of 2.65 ± 0.1 log10 PFU/ml and a mean duration of 4.5 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 inflammatory, predominantly small perivascular infiltrates. The vast majority of them were scored as grade 1 on a scale of 1 to 4. No involvement of neurons was seen. The lesions were located mostly 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 and distribution of lesions did not reveal any noticeable difference.

On a second neurovirulence study, cynomolgus monkeys were inoculated with YF/WNFVR vaccine candidate (ChimeriVax-WN02) production virus seed (P4). These macaques were screened and found negative for flavivirus antibodies by HI test (kindly performed by Robert Shope). Eleven monkeys were inoculated i.c. with 4.74 log10 PFU of YF/WNFVR production virus seed (P4), 11 reference control monkeys received 5.34 log10 PFU of YF-VAX, and 5 negative control monkeys received diluent. The monkeys were evaluated for changes in clinical signs (twice daily), body weight (weekly), and food consumption (daily). Clinical signs were assigned scores according to a clinical scoring system based on the WHO requirements for YF vaccine (36).

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

ChimeriVax-WN vaccine virus was detected in the sera of 10 of 11 monkeys inoculated with ChimeriVax-WN02 vaccine production 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 between treatment groups (P = 0.4067; analysis of variance [AVOVA]). A higher proportion of monkeys (91%) was viremic on the first day after inoculation than that seen in the YF-VAX group (27%). On days 2 to 3 after inoculation, the proportion of viremic monkeys (82%) was the same as for YF-VAX. The mean peak viremia was 2,097 ± 1,845 PFU/ml. Although the mean peak viremia 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 titers for ChimeriVax-WN vaccine remained within acceptable group and individual monkey specifications, based upon WHO requirements for YF 17D vaccine (36). The WHO specifications stipulate that no individual monkey will have a viremia exceeding 500 i.c. adult mouse LD50/ml and that no more than 10% of the animals will have a viremia exceeding 100 i.c. mouse LD50/ml. We have determined that these limits correspond to 20,000 Vero PFU/0.03 ml and 4,000 PFU/0.03 ml, respectively, in the case of YF-VAX (an LD50 for ChimeriVax-WN02 cannot be determined). The monkey viremias observed following ChimeriVax-WN02 do not exceed the limits set for YF vaccine.

There were no abnormalities in hematology or clinical chemistry values associated with treatment. A complete necropsy was performed on day 31, and tissues were prepared for histopathology. There were no ChimeriVax-WN02 production seed (P4)-related histopathologic changes in kidney, heart, liver, adrenal glands, or spleen.

Histopathology of the brain and spinal cord was performed according 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 of 11 and 10 of 11 of YF-VAX-treated and ChimeriVax-WN02 vaccine-treated monkeys, respectively, and there were no CNS lesions in the vehicle control monkeys. Inflammatory lesions induced by both viruses in the meninges and the brain and spinal cord matter were minimal to mild (grades 1 or 2) and composed of scanty, mostly perivascular infiltrates of mononuclear cells. There was no involvement of neurons in any of the ChimeriVax-WN02- or YF-VAX-treated monkeys. Summary data are presented in Table 8. ChimeriVax-WN production virus seed (P4) was significantly less neurovirulent (P < 0.05) than the reference article, YF-VAX, in the target, discriminator, and combined mean lesion scores. All monkeys developed high titers of neutralizing antibodies to the respective virus with which they were inoculated (data not shown).


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  		TABLE 8. Summary of CNS histopathologic lesion scores in cynomolgus monkeys inoculated by the i.c. route with ChimeriVax-WN02 production virus seed (P4), YF-VAX, or negative control

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


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  		TABLE 9. Neutralizing antibody titers (PRNT50) and protective activity of ChimeriVax-WN candidate vaccines in adult ICR mice challenged by the i.p. routea

In mice, ChimeriVax-WN02 vaccine elicited antibody titers that were approximately 10-fold lower than those elicited by ChimeriVax-WN01 virus, reflecting the greater attenuation of this virus. However, when mice were challenged i.p. with 1,000 LD50 of wild-type WN NY99, mice that had been immunized with 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 protected only 40% of the animals.

Young adult rhesus monkeys seronegative for WN neutralizing antibodies were vaccinated by the s.c. route with three different chimeric vaccines: (i) a chimera containing the E107 (L->F) single-site mutation (YF/WNF); (ii) a chimera containing two mutations at E316 (A->V) and E440 (K->R) (YF/WNVR); and (iii) ChimeriVax-WN02 containing all three mutations.

Viremia in the monkeys immunized with the different ChimeriVax-WN viruses following s.c. inoculation was longer relative to YF-VAX in some animals, although the levels detected at later time points were very low (Table 10). Viremias in monkeys receiving the ChimeriVax-WN vaccines ranged from 1.0 to 2.3 log10 PFU/ml, with a mean duration of 3.5 to 5 days. The mean peak titers of the viremia in monkeys given YF-VAX were approximately the same as those receiving the WN vaccines. Among the ChimeriVax-WN vaccines, the viremia titers measured suggest an inverse relationship between the number of attenuating mutations in the chimera and the peak titer of viremia (Table 10), but small sample size precludes definitive characterization of these differences.


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

The immunogenicities of vaccine candidates with one, two, or three attenuating mutations were similar (Table 11). Neutralizing antibody titers ranged from 40 to >640 depending upon the vaccine. There were no significant differences in neutralizing antibody response between treatment groups (Table 11). High titers of neutralizing antibodies (>100 PRNT50) were present 30 and 63 days after vaccination. The observation that monkeys developed neutralizing antibodies by day 14 indicates that ChimeriVax-WN02 elicits rapid onset of protective immunity.


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

Rhesus monkeys vaccinated with YF-VAX developed neutralizing antibodies against YF 17D with GMTs of 380 on day 14 postvaccination and 2,153 by day 63, which was 1 day before challenge with the virulent WN NY99 virus.

Monkeys immunized with ChimeriVax-WN single, double or triple mutants were uniformly protected against lethal i.c. challenge with WN NY99 (Table 12). It is noteworthy that 50% of the animals vaccinated with ChimeriVax-WN developed fever after challenge, with an average duration of 5 days postchallenge, suggesting that they sustained subclinical infections. An i.c. challenge with WN virus is extremely aggressive and is the only route of challenge tested to induce WN virus disease in naïve rhesus monkeys. It is likely that virus replication occurs in brain tissue after i.c. inoculation and before a specific immune response in the brain can be recruited for clearance of the virus. In the case of a human peripherally challenged by a mosquito bite, preexisting immunity would rapidly neutralize the virus and fever is unlikely to occur. However, none of the ChimeriVax-WN-immunized animals developed detectable viremia after challenge, none developed signs of illness (aside from fever), and none died. Vaccinated animals showed an increase in antibody levels postchallenge (Table 11), suggesting that viral replication and antigenic stimulation occurred without associated illness.


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  		TABLE 12. Viremia and clinical outcome in rhesus monkeys immunized with ChimeriVax-WN or YF-VAX and challenged 63 days later by the i.c. route with 5.38 log10 PFU of wild-type WN NY99 virus

Postchallenge viremias (~102 to 103 PFU/ml) were detected in the control monkeys that had previously been immunized with YF-VAX (Table 12). Two out of four monkeys vaccinated with YF-VAX (M017 and R286) developed a high fever and signs of encephalitis: muscle tremors, anorexia, and spasticity. These two animals were euthanized between days 9 and 11 after challenge. The other two YF-VAX-vaccinated animals developed fever and survived i.c. challenge with WN NY99 strain without any clinical symptoms; this finding is attributed to cross-protection across the two flaviviruses.

Two monkeys without any prior vaccination were also challenged with WN NY99 virus. The two challenge control animals developed fever between days 5 and 9 postchallenge, with slight tremors progressing to ataxia and spasticity between days 10 and 11, and were euthanized between days 10 and 12.

Genetic stability. In vitro and in vivo substrate-passage studies with ChimeriVax-WN01 or the YF/WNFVR chimeric vaccine candidate (ChimeriVax-WN02) were conducted to determine genetic stability of the constructs when grown in stationary cell cultures and in brain tissue. After six in vitro Vero E6 cell passages of the virus followed by six in vivo ICR adult mouse brain passages of ChimeriVax-WN01, no mutations were selected relative to the wild-type sequence of the prM and E genes in the ChimeriVax-WN01 construct nor was there an increase in mouse neurovirulence (data not shown). A heterozygous mutation in the E protein at position E336 resulting in a cysteine-to-serine change was identified following 10 in vitro passages of the YF/WNFVR virus in Vero E6 cells. In a separate study, in vitro passage of YF/WNFVR in SF-Vero cells (manufacturing substrate) resulted in selection of a mutation at position E313 that changed the amino acid at that position from glycine to arginine. Neurovirulence of these passaged viruses for the 2-day-old suckling mice (n = 10) inoculated with a nominal 2-log10 PFU dose of viruses including E313 and E336 mutations showed no increase in virulence relative to YF/WNFVR PMS (Table 13). During all serial passages of the virus in Vero cells or brain tissue, no reversions were detected at target E protein amino acid residues 107F, 316V, or 440R, the attenuation markers for the vaccine candidate. Additionally, during scale-up manufacturing of the ChimeriVax-WN02 vaccine, no reversions at these critical residues were detected.

The GMP manufactured ChimeriVax-WN02 production virus seed (P4) was used for inoculation of large-scale Vero-SF cultures grown on microcarrier beads in 100-liter bioreactors. An additional mutation (L->P) occurred in the vaccine at position 66 in the M protein. This mutation was associated with production of slightly smaller plaque size. The vaccine lot (P5) contained equal ratios of small and large plaques. Virus populations with and without the M66 mutation were isolated by plaque purification and compared to the PMS (no detectable mutations) 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 µl containing 2, 3, or 4 log10 PFU of either large-plaque or small-plaque virus and observed for 21 days for signs of illness and death. For comparative purposes, litters of mice were inoculated with similar doses of the PMS (P2) and vaccine lot (P5) viruses. Mice of the same age were also inoculated with 2 log10 PFU of YF-VAX. Negative controls were inoculated with diluent (Table 14). There were no differences in mortality ratios across dose groups, and data were combined for analysis. Since the mortality ratio across all treatment groups differed (P < 0.0001), pairwise comparisons were performed. The M66 mutation had no effect on mouse neurovirulence.


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DISCUSSION
 
The original YF/WN chimeric virus constructed by insertion of the prME genes from a wild-type WN virus strain was attenuated with respect to the parental YF 17D virus vector, but retained a degree of neurovirulence for adult mice. To develop a vaccine candidate with a wider margin of safety, we selectively introduced mutations in the donor WN virus. Mutations introduced into the E protein of the WN donor virus utilized a strategy based on the previous construction of ChimeriVax-JE vaccine, which contained donor prME genes from an attenuated vaccine strain of JE (SA14-14-2 virus) (3, 4, 28). The SA14-14-2 virus contains mutations at six amino acid residues (E107, E138, E176, E279, E315, and E439) that play a role in neurovirulence (3). The WN and JE wild-type gene sequences are conserved at most of these residues (except 176), suggesting that mutations introduced at these sites in WN virus could have the same attenuating effect as they did in the case of JE SA14-14-2. As predicted, we found that mutagenesis of the WN E residues E107, E280 (corresponding to E279 in JE virus), and E316 (corresponding to E315 in JE virus) caused attenuation of the YF/WN virus chimera. Surprisingly, while an E138 mutation, E->K, was associated with a marked attenuation of JE virus (3, 34), a corresponding mutation in the WN gene did not reduce the neurovirulence of the YF/WN virus to the expected 0% mortality by the mouse neurovirulence test. Mutation of the E protein at E440 (corresponding to E439 in JE virus) from K->R, a conservative residue change, also reduced neurovirulence for mice. A construct with the three mutations of F, V, and R at positions E107, E316, and E440, respectively, was designated ChimeriVax-WN02 and was selected as the candidate for manufacture of the vaccine for clinical studies. ChimeriVax-WN02 was not neuroinvasive compared to WN NY99 virus and had reduced neurovirulence compared to YF 17D vaccine virus. Attenuation of this virus was conferred by the mutation at E107, which maps to the fusion peptide in the second domain as predicted in the crystal structure of the E protein (1, 12, 32). This amino acid is thought to reduce virulence by altering the function of the fusion peptide in the natural cycle of the virus replication. The additional ChimeriVax-WN02 mutations at positions E316 and E440 map in domain III on the crystal structure of the E protein. Residue E316 is thought to be involved in binding of tick-borne encephalitis virus to the virus receptor on the cell plasma membrane (1, 32) and thus may play a role in WN virus cell entry. Residue E440 is in the transmembrane region of the E protein and is believed to be involved in anchoring the E protein during its translation in the endoplasmic reticulum; hence, a mutation at E440 may be altering the natural association of the E protein with prM (2). The K-to-M mutation at position E280 that attenuated neurovirulence for mice was not included in the final vaccine because it appeared unstable, similar to the corresponding residue in JE virus E protein sequence (i.e., E279) shown to be unstable during in vitro passage. A reversion to K at position 279 in the JE virus E protein occurred after less than five passages of the virus in MRC-5 cells (22). Mutation of residue E176 from Y in the WN virus sequence to either V or I, as seen in JE strains, did not suggest a significant change in neurovirulence; therefore, position E176 was not changed in the final vaccine candidate sequence (unpublished results). This observation contrasts to the previously published results linking a mutation from I to V at position E176 in the JE virus envelope protein to neurovirulence (3, 28). Other approaches to flavivirus chimeras employed an attenuated dengue virus genome backbone to produce chimeric dengue virus vaccine candidates against the four major serotypes (15); similarly, a dengue virus has been used to deliver the prM and E genes of WN virus, producing an attenuated vaccine candidate shown protective in a nonhuman primate model (30). This dengue/WN virus chimeric construct was attenuated by virtue of the chimeric nature and as a result of 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-type WN virus; (ii) introduction of three site-directed mutations in two E protein domains, each independently associated with attenuation; and (iii) conservation of the FVR mutations after in vitro passage in manufacturing-related substrates.

The safety of ChimeriVax-WN02 was evaluated in a sensitive 8-day-old suckling mouse model and in rhesus monkeys and in cynomolgous macaques inoculated by the i.c. route. In all host-virus pairings, the chimeric virus proved to be significantly less neurovirulent than the licensed YF-VAX vaccine. The monkey safety test was performed as prescribed by current regulations applicable to YF vaccines (36) and showed that the vaccine was significantly less virulent than YF-VAX. The nonhuman primate model has been previously used to assess the safety of other chimeric vaccines against JE and dengue virus (6, 18, 24).

After s.c. inoculation of rhesus monkeys, viremias were more erratic and of longer duration in animals immunized with the ChimeriVax-WN vaccines than in animals given YF-VAX (Table 10). The mean peak titer viremia for YF-VAX-vaccinated monkeys was ~1 log higher than that for the ChimeriVax-WN02 (triple mutant) vaccine candidate. The longer viremia observed after immunization with the chimeric viruses suggests that the viruses replicate in different tissues had different reticuloendothelial clearance rates from the parental YF 17D virus or had different kinetics of immune response. We are currently studying the sites of replication of ChimeriVax-WN02 and YF-VAX in tissues of cynomolgus macaques and will report results in a future publication. In addition, future clinical trials will assess the magnitude and duration of viremia following ChimeriVax-WN02 and YF-VAX and establish correlations between viremia and adverse events. The low titer of the viremia observed in rhesus monkeys after s.c. vaccination with the chimeric vaccine candidates suggests that ChimeriVax-WN02 vaccine has an acceptable phenotype for trials in humans.

The triply mutated virus (ChimeriVax-WN02) vaccine appeared to be less immunogenic than the wild-type chimera in mice, but performed satisfactorily in nonhuman primates. ChimeriVax-WN02 vaccine rapidly elicited a neutralizing antibody response in all rhesus monkeys and provided solid protection against an aggressive i.c. challenge with 5 log10 PFU of WN NY99 virus.

A partially protective immune response was observed in two of the four rhesus monkeys immunized with YF 17D and subsequently challenged with wild-type WN virus. Previous observations by others have shown the cross-protective effect of prior exposure to phylogenetically related flaviviruses and concluded that potential for protective cross-reactivity is unlikely to prevent infection and only likely to prevent disease (16). Similarly, we observed that prior YF immunization of monkeys did not prevent infection (viremia) after WN virus challenge, but may have provided an element of protection against death. It should be pointed out that the interval between YF immunization and challenge was relatively brief and that cross-protection between heterologous flaviviruses often diminishes over time, probably due to affinity maturation of the antibody response and waning of T-cell immunity. It is highly unlikely that YF immunity would provide reliable cross-protection of humans and therefore a specific, homologous (WN) vaccination strategy must be pursued. This observation is similar to a previous report that hamsters vaccinated with YF 17D were somewhat cross-protected against WN virus challenge-induced disease (35). However, in the rhesus model, only those animals immunized with the WN vaccines and subsequently challenged with wild-type WN virus i.c. did not show postchallenge WN virus viremia. All of the surviving animals vaccinated with ChimeriVax-WN displayed increases in WN neutralizing antibodies after i.c. challenge indicating that the challenge virus had replicated in brain tissue (without causing illness) or that the challenge inoculum, which was quite large, provided sufficient antigen for stimulation of B cells. The experimental design did not allow a proper test of whether the preexisting immunity would have been "sterilizing " if the challenge inoculum had been delivered by a natural (parenteral) route instead of i.c. Sterile immunity could be tested by measuring the immune response to nonstructural proteins of the WN challenge virus. Others have reported that experimental WN vaccines elicit sterilizing immunity against parenteral challenge (11).

Since the first experimental vaccine construct with wild-type prME sequence was less neurotropic than commercial YF vaccine, the chimeric vaccine with three attenuating mutations has a wide margin of safety. To maintain this ultra-attenuated phenotype, the vaccine candidate must retain attenuating mutations FVR introduced to retain the level of attenuation required. Because of the quasispecies nature of RNA viruses, variations in the sequence are to be expected during vaccine manufacture. Therefore, quality of the product is carefully monitored during manufacture by tests for genotypic and phenotypic stability. Safety is ensured by the demonstration of conservation of the amino acid residues identified to play a role in attenuation (shown by direct sequencing release tests); for ChimeriVax-WN02, the required conserved residues are E protein 107F, 316V, and 440R. In addition, the attenuated virulence phenotype of the vaccine is tested by infant mouse neurovirulence test performed on seed viruses and each vaccine batch. Currently, the product specifications for sequence data have been expanded to include full genomic sequencing of each vaccine batch rather than confirmation only of the point mutations at residues 107, 316, and 440. When ChimeriVax-WN02 virus was passed in Vero cells, with at least twice the number of passages required for manufacture of the vaccine, the FVR mutations were maintained. Passage of the vaccine candidate, in vitro or in vivo, selected mutations in the vicinity of residue E316 (at E313 and at E336) without compromising the neurovirulence phenotype of ChimeriVax-WN02 and supporting our mutagenesis approach to ensure vaccine safety. When the vaccine was scaled up for manufacture of clinical material in 100-liter bioreactors, a mutation at M66 was detected. This mutation also did not affect neurovirulence or immunogenicity of the vaccine.


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

We would like to acknowledge contributions from the following: independent contributor Inessa Levenbook; from University of Massachusetts 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, Ken Draper; 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 Denise Goens for critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Acambis, Inc., 38 Sidney St., Cambridge, MA 02139. Phone: (617) 761-4200. Fax: (617) 494-1741. E-mail: tom.monath{at}acambis.com. Back


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Journal of Virology, November 2004, p. 12497-12507, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12497-12507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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