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/WN
FKVM. 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 10
5 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/WN
FKVM 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-WN
01 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/WN
107F
316V
440R construct was selected as our vaccine candidate (ChimeriVax-WN
02).
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 10
4 and 10
5 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-WN
02 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-WN
02 vaccine candidate.
A GLP study was undertaken to characterize the neurovirulence of the good manufacturing practice (GMP) manufactured ChimeriVax-WN
02 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 10
3, 10
4, or 10
5 PFU of either production virus seed (P4) or vaccine (P5) virus. Control animals of the same age received either 10
3or 10
5 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).
In a pilot monkey neurovirulence study, the ChimeriVax-WN
01 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 log
10 PFU of ChimeriVax-WN
01 or 4.4 log
10 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 log
10 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 log
10 PFU/ml and a mean duration of 4.5 days). Histological scores induced by ChimeriVax-WN
01 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/WN
FVR vaccine candidate (ChimeriVax-WN
02) 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 log
10 PFU of YF/WN
FVR production virus seed (P4), 11 reference control monkeys received 5.34 log
10 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-WN
02 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-WN
02 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 LD
50/ml and that no more than 10% of the animals will have a viremia exceeding 100 i.c. mouse LD
50/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 LD
50 for ChimeriVax-WN
02 cannot be determined). The monkey viremias observed following ChimeriVax-WN
02 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-WN
02 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-WN
02- 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).
Immunogenicity and efficacy studies in mice and rhesus monkeys.
The immunogenicity of ChimeriVax-WN
01 and ChimeriVax-WN
02 was evaluated in adult ICR mice inoculated by the s.c. route. Serum neutralizing antibodies were measured by PRNT
50 4 weeks after vaccination with a single dose, and titers were expressed as the geometric mean titer (GMT) (Table
9).
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 log
10 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.
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 PRNT
50) were present 30 and 63 days after vaccination. The observation that monkeys developed neutralizing antibodies by day 14 indicates that ChimeriVax-WN
02 elicits rapid onset of protective immunity.
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.
Postchallenge viremias (∼10
2 to 10
3 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-WN
01 or the YF/WN
FVR chimeric vaccine candidate (ChimeriVax-WN
02) 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-WN
01, no mutations were selected relative to the wild-type sequence of the prM and E genes in the ChimeriVax-WN
01 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/WN
FVR virus in Vero E6 cells. In a separate study, in vitro passage of YF/WN
FVR 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-log
10 PFU dose of viruses including E313 and E336 mutations showed no increase in virulence relative to YF/WN
FVR 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-WN
02 vaccine, no reversions at these critical residues were detected.
The GMP manufactured ChimeriVax-WN
02 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 log
10 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 log
10 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.