DNA Vaccine for West Nile Virus Infection in Fish Crows

Materials and Methods

The plasmid DNA, pCBWN, codes for the prM and E glycoproteins of WNV. The plasmid was purified from Escherichia coli XL-1 blue cells with EndoFree Plasmid Giga Kits (QIAGEN, Inc., Santa Clarita, CA) and suspended in 10 mM Tris buffer, pH 8.5, at a concentration of 10.0 mg/mL. For IM vaccination, the DNA vaccine was formulated in phosphate-buffered saline (PBS), pH 7.5, at a concentration of 1.0 mg/mL. For oral exposure, the drymicroencapsulated DNA was suspended in PBS, pH 7.5, at a concentration of 2.0 mg/mL.

The method for microencapsulating DNA was adapted from procedures previously described for virus and sub-unit vaccines and isolated proteins.^[14,15,16] We performed all steps with sterile reagents and aseptic technique. Two 10-mg aliquots of WNV cDNA were transferred to separate test tubes with enough water to make 9-mL volumes. Resulting suspensions were mixed on a clinical rotator until solution was complete; 1 mL of 0.6% w/v aqueous sodium alginate (Fluka Chemical Co., Ronkonkoma, NY) solution was added to each tube, and the contents of each were gently inverted 20 times. Each DNA/alginate solution was pumped at 1.2 mL/min through a 76-µm orifice in a 1-mm internal diameter glass tube against the side of which a 20-KHz laboratory sonicator probe was firmly pressed. The emerging train of droplets was directed into a modified T-tube, through which a recirculated 40 mL of 0.25% w/v neutral aqueous spermine hydrochloride (Sigma-Aldrich Corp., St. Louis, MO) solution was pumped at 10 mL/min. A placebo microcapsule formulation was prepared by using alginate reagent without DNA. Resulting microcapsule suspensions were allowed to equilibrate for 30 min, pelleted at 500 x g for 20 min, and washed three times by decanting, suspending, and repelleting. Wash liquids were reserved for measuring the DNA that escaped encapsulation. Placebo and vaccine formulations and washes were frozen at -20°C and lyophilized overnight, then suspended in 5 mL of PBS to produce a final concentration of 2 mg/mL of the encapsulated DNA.

Fish crows (C. ossifragus) were captured with a rocket-propelled net at various locations in Maryland. Birds were transported to a biosafety level 3 laboratory at the U.S. Army Medical Research Institute of Infectious Diseases, allocated into four groups, and placed in stainless steel cages (3-4 birds/cage); blood was collected for evidence of antibodies against flaviviruses. Birds were provided a mixture of cat and dog food ad libitum and water. This diet was supplemented with hardboiled eggs as well as vitamin supplements.

Serial 10-fold dilutions of the blood samples from each crow were made in standard diluent (10% heat-inactivated fetal bovine serum in medium 199 with Earle's salts, NaHCO₃, and antibiotics). These samples were tested for infectious virus by plaque assay on Vero cells in 6-well plates (Costar, Inc., Cambridge, MA) as previously described,^[17] except that the second overlay, containing neutral red stain, was added 2 or 3 days after the first overlay.

Serum samples were assayed for WNV-specific antibodies by using the plaque-reduction neutralization test (PRNT), as previously described.^[18] Briefly, each serum sample was diluted 1:10 in standard diluent (as above) and mixed with an equal volume of BA1 (composed of Hanks' M-199 salts, 1% bovine serum albumin, 350 mg/L of sodium bicarbonate, 100 U/mL of penicillin, 100 mg/L of streptomycin, and 1 mg/L of fungizone in 0.05 M Tris, pH 7.6) containing a suspension of WNV (NY99-4132 strain) at a concentration of approximately 200 plaque-forming units (PFU)/0.1 mL, such that the final serum dilution was 1:20 and the final concentration of WNV (the challenge dose) was approximately 100 PFU/0.1 mL. After 1-h ncubation at 37°C, we added the serum/virus mixtures onto Vero monolayers in 6-well plates, 0.1 mL per well in duplicate. We determined the mean percentage of neutralization for each specimen by comparing the number of plaques that developed (see Plaque Assay section) relative to the number of plaques in the challenge dose, as determined by back titration. Preliminary samples were screened for antibodies to WNV in the same manner, as well as for neutralizing antibodies to St. Louis encephalitis virus, a closely related flavivirus that may cross-react serologically with WNV^[19] and may partially protect against WNV infection.^[20]

The crows were placed in four groups: 1) those inoculated IM with vaccine, 2) those that had oral vaccine, 3) positive controls (i.e., those that received placebo inoculation and viral challenge), and 4) room controls (i.e., those that received placebo inoculation and placebo challenge). After an acclimatization period of approximately 1 month, the 10 crows in group 1 (9 fish crows and 1 American crow [C. brachyrhynchos]) were inoculated IM with 0.5 mg of the DNA vaccine in a total volume of 0.5 mL (0.25 mL in each breast). The 9 crows in group 2 (8 fish crows and 1 American crow) were given 0.5 mg of the encapsulated DNA vaccine orally in 0.25 mL of PBS, and 20 fish crows (groups 3 and 4) were each inoculated and orally exposed as above except that a placebo was used in place of the vaccine. Blood was collected weekly from the jugular vein and the serum tested for neutralizing antibodies to WNV. Six weeks after vaccination, all birds in groups 1, 2, and 3 were inoculated subcutaneously with 0.1 mL of a suspension containing 10⁵ PFU (10⁶ PFU/mL) of the 397-99 strain of WNV, which had been isolated from the brain of an American crow that died in New York City during the fall of 1999 and passaged once in Vero cells before use in this study. The crows in group 4 were inoculated with 0.1 mL of diluent. Three or four crows in each group were bled (0.1 mL) from the jugular vein each day; each bird was bled every third day. Blood samples were added to 0.9 mL of diluent + 10 U of heparin/mL. Blood samples were frozen at -70°C until tested for infectious virus by plaque assay.

Comments

Commenting is limited to medical professionals. To comment please Log-in.

Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.