INTRODUCTION
West Nile virus (WNV), a mosquito-borne flavivirus (family Flaviviridae), recently emerged as a public, veterinary, and wildlife health threat in North America.1 Between 1999 and 2001 in the United States alone, WNV has been responsible for 142 human meningoencephalitis cases,2 814 equine encephalitis cases,3–5 and 11 932 avian deaths (mostly crows and related species of birds in the family Corvidae).3,4,6 These numbers increased during 2002. Certain birds are the primary vertebrate reservoir hosts,7 but incidental infections occur in other birds and mammals. WNV-associated mortality in birds was not observed in nature prior to the late 1990s.
The emergence of WNV disease has stimulated efforts to develop vaccines for humans,8 horses,9 and birds.10 One vaccine candidate that two of the authors (JA and TPM) have been investigating is comprised of the virus backbone from the yellow fever (YF) 17D vaccine strain in which the surface glycoprotein pre-membrane (prM)and envelope (E) genes are replaced with that of WNV such that a YF-WN recombinant chimera (termed ChimeriVax-WN) is formed.11 Initial studies indicate that ChimeriVax-WN generates protective humoral immunity in hamsters.12 However, the ability of ChimeriVax-WN to replicate or generate a protective immune response in birds is unknown.
Determining the ability of this chimeric vaccine candidate to replicate in birds would be important information, in part because of its potential use in vaccinating certain valuable birds, and in part to understand its potential to enter a natural transmission cycle in birds that could lead to unintentional infection of other vertebrates. Thus, we inoculated chickens and fish crows with the chimera (and also chickens with YF-17D virus) to determine the susceptibility of birds to infection. Furthermore, we challenged vaccinated crows with a virulent strain of WNV to determine vaccine efficacy.
MATERIALS AND METHODS
Virus strains.
The NY99-4132 strain of WNV was isolated from the brain of an American crow and passaged twice in Vero cell culture. The attenuated 17D strain of YF virus was acquired from the Centers for Disease Control and Prevention’s reference collection. The recombinant ChimeriVax-WN virus (hereafter termed the vaccine) was derived from YF-17D and a NY99 strain of WNV as described5 and passaged three times in Vero E6 cells.
Experimental infection.
Birds seronegative for WNV, as determined in a plaque-reduction neutralization test (PRNT),13 were used in infection studies. Dekalb Delta hens (>15 weeks old) were obtained from Hudson Pullet Farms (Fort Lupton, CO). Adult fish crows (Corvus ossifragus) were provided by the Maryland Department of Natural Resources (Annapolis, MD). All birds were held in standard chicken cages with food and water provided ad libitum. Birds were inoculated by subcutaneous injection of the vaccine suspension in 0.1 mL of BA-1 diluent (Hanks M-199 salts, 0.05M Tris, pH 7.6, 1% bovine serum albumin, 0.35 g/L of sodium bicarbonate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 1 μg/mL of Fungizone). To determine viremia, blood samples (0.4 mL) were collected for seven days postinoculation into Microtainer serum separator tubes (Becton Dickinson and Company, Franklin Lakes, NJ). After coagulation (30 minutes at ambient temperature) and centrifugation (5,200 × g for three minutes), serum was frozen at −70°C. The detection and titration of viruses in serum samples was performed using a plaque assay as described,13 and modified as follows: the overlay was 0.5% SeaKem® LE agarose (Bio-Whittaker Molecular Applications, Rockland, ME) with M199 nutrients and antibiotics as in the BA-1 diluent. Neutral red was added to a second overlay one day prior to plaque formation. Each sample was titrated in duplicate using serial 10-fold dilutions in BA-1 diluent. Postinoculation serum samples were assayed for specific antibodies using the PRNT. The serum samples were titrated using two-fold serial dilutions to verify 90% and 80% endpoint neutralization titers.
RESULTS
To determine the optimal immunization dose of ChimeriVax-WN for avian vaccination, four groups of two hens were inoculated with 200 plaque-forming units (PFU), 2,000 PFU, 20,000 PFU, and 240,000 PFU, respectively, and bled daily for seven days for determination of viremia. All eight hens, including one control hen, failed to develop a detectable viremia by plaque assay (threshold of detection = 50 PFU/mL of serum). The chickens were bled on 14 and 21 days postimmunization to test for vaccine-specific neutralizing antibodies. None of the hens displayed neutralizing antibodies to WNV or the vaccine at 14 and 21 days postimmunization. These results suggested that the vaccine could not replicate in chickens.
To determine if chickens were permissive for YF-17D virus replication, we inoculated 15 hens with 2,000 PFU of the YF-17D vaccine. They were bled daily for seven days to determine levels of detectable viremia, but none developed detectable levels of YF-17D virus in their blood. The hens were bled on 14 and 27 days postinoculation for detection of YF-17D-specific neutralizing antibodies. None of the chickens developed neutralizing antibodies to YF-17D virus. These results suggested that YF-17D virus replication did not occur in chickens.
Adult fish crows, paired into four groups, were inoculated with four different doses of the vaccine (Table 1) and subsequently bled daily for determination of viremia for seven days. All eight crows, plus one control crow, failed to develop detectable viremia. The crows were bled approximately once a week to monitor the homologous antiviral antibody response. At 15 days postinoculation, one crow (crow 12, which received the highest dose of vaccine) circulated low levels of neutralizing antibodies (~80% neutralization at a 1:10 serum dilution), which persisted for at least 120 days. At 48 days postinoculation, the seven crows that failed to develop neutralizing antibodies were reinoculated with a high dose of the vaccine (~100 000 PFU). None of the revaccinated birds developed detectable viremias during the seven days postinoculation. At 36 days after revaccination, none of these birds circulated significant WNV-neutralizing antibodies. All eight vaccinated crows were given booster immunizations with ~100,000 PFU at 52 days after the second vaccination. Twenty days after the booster immunization, three of the eight crows circulated antibodies that neutralized ~70% of the vaccine (serum dilution = 1:10) and only one crow (crow 12) showed this level of WNV neutralization. On the 72nd day after the second vaccination, all eight crows (and four control crows) were then challenged with 2,000 PFU of wild-type, virulent WNV. The resulting viremia data are shown in Table 1. The mean peak titers (log10 PFU/mL of serum) in the vaccinated crows versus the controls were 5.6 and 5.3, respectively. The mean duration of viremia was 5.4 days versus 4.5 days. Although none of the four control birds died of the infection, two of the eight vaccinated birds eventually succumbed prior to termination of the study 14 days postchallenge. These data demonstrate no reduction of either morbidity or viremia in the eight vaccinated birds compared with the four unvaccinated birds. At termination, all four controls and the six surviving vaccinees circulated high levels of WNV-neutralizing antibodies.
DISCUSSION
We evaluated the immunogenicity and protective efficacy of a recombinant YF-WNV chimera in chickens and fish crows. Both avian models were refractory to vaccine replication at low inoculation doses. Fish crows only developed antibodies to the vaccine when inoculated with a high dose (20,000 PFU) or a high booster dose (100,000 PFU). The weak B cell response suggests that the vaccine replicated poorly, if at all, in the bird species studied. Although we did not evaluate T cell responses, these are not as important as B cell responses in protecting vertebrate hosts from WNV infection and disease.14,15
In the vaccinated group, two crows succumbed to infection when challenged with wild-type WNV. One of these crows circulated low-level homologous antibodies to WNV prior to challenge. The fatal outcome in these birds could have been due to antibody-dependent enhancement in which incomplete or partial immunity leads to enhanced infection and/or disease. However, the fatal outcomes in the vaccinated group were not statistically significant (P = 0.5, by Fisher’s exact test), nor were the differences in mean peak viremias (P = 0.5, by Student’s t-test) or duration of viremia (P = 0.2, by Wilcoxon rank sum test).
To further understand the susceptibility of birds to infection by the vaccine, we inoculated chickens with YF-17D to look at the replication efficiency of this virus in birds. None of the 15 chickens showed any evidence of detectable viremia during the acute phase, or developed YF-17D-specific neutralizing antibodies, indicating an inability of the virus to replicate and to stimulate an immune response, a finding consistent with other studies of YF infections in birds.16 Chickens may be susceptible to infection by higher doses of YF-17D. However, we sought to determine whether YF naturally replicates in chickens; thus, we chose a natural dose that mosquitoes typically inoculate.
The ChimeriVax-WN vaccine failed to induce protective immunity to WNV infection in chickens and fish crows. These results were consistent with the data suggesting YF-17D did not replicate in birds, because the vaccine is a YF-17D construct with only the envelope protein genes of WNV inserted in the genome. The inability of the vaccine to replicate in birds is unlikely due to the WNV portion of the construct because WNV is known to replicate in many bird species, including both fish crows7 and chickens.17,18 An important implication of our study was that if the vaccine is used in people or horses, it will not likely amplify in competent avian hosts and establish a natural transmission cycle.
ChimeriVax-WN vaccination doses in 12 fish crows and post-challenge viremia data
| Crow sample no. | Status | 1° Vaccination (PFU) | 2° Vaccination (PFU) | Booster (PFU) | Maximum viremia* | Viremia duration (days)† |
|---|---|---|---|---|---|---|
| * Viremia is expressed in log plaque-forming units (PFU) per milliliter of serum. | ||||||
| † Days during which viremia was at least 50 PFU/mL. | ||||||
| 1 | Control | 0 | 0 | 0 | 4.6 | 5 |
| 2 | Control | 0 | 0 | 0 | 5.5 | 5 |
| 3 | Control | 0 | 0 | 0 | 5.5 | 4 |
| 4 | Control | 0 | 0 | 0 | 4.7 | 4 |
| 5 | Vaccinee | 15 | 105 | 105 | 5.6 | 6 |
| 6 | Vaccinee | 15 | 105 | 105 | 4.5 | 6 |
| 7 | Vaccinee | 95 | 105 | 105 | 3.9 | 4 |
| 8 | Vaccinee | 95 | 105 | 105 | 4.8 | 7 |
| 9 | Vaccinee | 1,250 | 105 | 105 | 4.8 | 5 |
| 10 | Vaccinee | 1,250 | 105 | 105 | 4.5 | 4 |
| 11 | Vaccinee | 20,000 | 105 | 105 | 6.4 | 6 |
| 12 | Vaccinee | 20,000 | 0 | 105 | 4.7 | 5 |
Authors’ addresses: Stanley A. Langevin and Nicholas Komar, Centers for Disease Control and Prevention, PO Box 2087, Fort Collins, CO 80522, Telephone 970-221-6496, Fax: 970-221-6476, E-mail: nck6@cdc.gov. Juan Arroyo and Thomas P. Monath, Acambis, Inc., 38 Sidney Street, Cambridge, MA 02139, Telephone: 617-494-1339, Fax: 617-494-1741, E-mail: juan.arroyo@acambis.com.
Acknowledgments: We thank Autumn Blesh, Eric Edwards, and Jason Velez for providing technical assistance; Richard Bowen (Colorado State University, Fort Collins, CO) for providing animal care facilities; and Michel Bunning for arranging the acquisition of the fish crows used in this study.
Financial support: This study was partially supported by the American Bird Conservancy (Washington DC) and by Acambis, Incorporated, through a Cooperative Research and Development Agreement.
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