Evaluation of the Efficacy, Potential for Vector Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep

Animals.Institutional animal care and use committees from the Canadian Food Inspection Agency or the University of Wyoming approved all animal studies. Forty conventional mixed-breed sheep were vaccinated with RVFV MP-12 in four separate studies, while 32 randomly selected cohorts served as nonvaccinated controls (Table 1). All sheep were purchased from reputable breeders and housed under insect-proof biological safety level (BSL)-2 (experiments 1 to 3) or BSL-3 Ag (experiment 4) conditions. Four mice were immunized with MP-12 to generate polyclonal antibodies for use in competitive enzyme-linked immunosorbent assays (cELISAs).

Virus and vaccine strains.The RVFV MP-12 strains were provided by the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), designated uMP-12 (vaccine used in previously published studies), and, when available, by the vaccine manufacturer (Zoetis, Florham Park, NJ), designated zMP-12. The manufacturer-prepared MP-12 only became available for experiments 3 and 4. uMP-12 was propagated 1 time in fetal lung fibroblast MRC-5 cell cultures (American Tissue Culture Collection [ATCC], Manassas, VA) in medium 199 with Earle's salts (M199-E [Sigma-Aldrich, St. Louis, MO]; 100 U/ml penicillin and 100 μg/ml streptomycin sulfate [Sigma-Aldrich]) with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA) and was titrated using a plaque assay as described previously (39). The manufacturer-provided zMP-12 was received as a master seed stock cell culture material (7.4 log 50% tissue culture infective dose [TCID₅₀]/ml) for experiment 3 and as a lyophilized vaccine for experiment 4. The wild-type RVFV strain ZH501 (40), originally provided by Heinz Feldmann, National Microbiology Laboratory, Winnipeg, Canada, was propagated in Aedes albopictus C6/36 cells (ATCC) for use in animal infections and titrated in Vero E6 cells (ATCC) as previously described (41). The C6/36 cells were maintained in 47% ESF-921 (Expression Systems, Davis, CA)-47% Eagle's minimal essential medium (EMEM)-2.5% FBS (Wisent, St-Bruno, QC, Canada)-2.5% HEPES (25 mM final)-1% sodium pyruvate (1 mM final) (Sigma-Aldrich) at 28°C in phenolic style cap or plug seal cap flasks (Corning, Corning, NY).

Experimental design.Four experiments were performed using sheep vaccinated with RVFV MP-12, and mock-vaccinated cohorts served as controls. Experiments 1 and 2 used the MP-12 vaccine strain used in previously published studies (uMP-12). The manufacturer-prepared vaccine (zMP-12) became available and was used for experiments 3 and 4. Sheep numbers and treatments are listed in Table 1. All vaccinations were administered subcutaneously at the shoulder.

(i) Experiment 1.A short-term experiment was conducted to test for the potential to detect vaccine virus in tissues postvaccination and for the potential to infect mosquitoes by feeding on vaccinated sheep. Four ewes were vaccinated with 1 ml of uMP-12 containing 2.9 ×10⁶ PFU/ml diluted in phosphate-buffered saline (PBS) immediately prior to use, and 2 control ewes received diluent only. Wool was shaved from the axillary region to allow for mosquito feeding. Aedes aegypti and Culex quinquefasciatus in mesh-top containers were allowed to feed on all sheep for 20 min on 2, 3, and 4 days postvaccination (dpv), held for 10 to 14 days, and tested for RVFV by reverse transcription (RT)-PCR. Blood samples were collected from sheep prior to vaccination and daily until necropsy at 3 or 4 dpv, when liver, spleen, and blood samples were collected and frozen at −20°C until testing by RT-PCR and virus isolation within 2 weeks of sampling.

(ii) Experiment 2.Ten vaccinated and 8 control 2-month-old lambs were treated as in experiment 1. Aedes taeniorhynchus and Culex tarsalis were allowed to feed on 5 each of the vaccinated and control sheep 2 to 4 dpv as described for experiment 1. Blood samples were collected on 1 to 7, 14, 21, and 28 dpv and tested for uMP-12 by virus isolation and RT-PCR. Serum was tested for neutralizing antibodies by a plaque reduction assay. Sheep were observed daily for normal activity and appetite, and rectal temperatures were taken prior to vaccination and at 1 to 5 dpv.

(iii) Experiment 3.Ten 4-month-old lambs were vaccinated using zMP-12 master seed stock with titer determined in PFU as 1.35 × 10⁶ PFU/ml, diluted according to company recommendations at 1:100 in Dulbecco's modified Eagle medium (DMEM) (Gibco, Carlsbad, CA) immediately prior to use and given as a 1-ml dose. Ten negative-control lambs received diluent only. Four vaccinates received a second dose 40 days after the primary vaccination. Aedes taeniorhynchus and Culex tarsalis were allowed to feed on 5 each of the vaccinated and control sheep on 2 to 4 dpv as described above. Activity and appetite were monitored, rectal temperatures were taken daily prior to vaccination and at 1 to 7 dpv, and blood samples were collected on days 0, 2, 3, 4, 7, and 10 and weekly to 60 dpv. The sheep were then moved to outdoor pens and maintained to 24 months postvaccination with blood samples collected every 60 days for testing for neutralizing antibodies by a plaque reduction assay.

(iv) Experiment 4.Sixteen 3-month-old lambs received lyophilized zMP-12 vaccine diluted to 3 × 10³ PFU/2.5 ml with stabilizer (20% lactose, 20% gelatin, 16% N-Z-Amine, extender [HALS], 45 mg/ml gentamicin). The dosage and preparation were based on the directions provided by the manufacturer. Four cohorts received diluent only and served as negative controls housed with the vaccinates. All lambs were challenged with virulent virus 21 dpv. The rectal temperature was monitored daily, prior to vaccination, postvaccination, and postchallenge. Blood samples from all sheep were collected prior to vaccination and at 1, 7, 14 and 21 dpv to monitor neutralizing antibodies in serum by a plaque reduction assay. All animals were challenged at 21 dpv with 1 ml of RVFV ZH501 (2 × 10⁷ PFU/ml) with the dosage verified by back titration on Vero E6 cells. Blood samples were collected prior to challenge and then daily until 6 days postinfection (dpi) when animals were euthanized, and liver and spleen were collected for virus isolation and virus RNA detection by RT-PCR.

Mosquitoes.Early studies with MP-12 found transient low-titered viremia (<10³ PFU/ml) in some vaccinates, a level thought to be insufficient to establish an infection in mosquitoes (31, 33). We further tested this assumption by allowing mosquitoes to feed on vaccinates or controls on days 2 to 4 postvaccination. Mosquitoes then were held for 10 to 14 days to allow for the clearance of the blood meal and amplification of the virus. Mosquito legs and bodies were tested separately by plaque assays on Vero cells to determine the extent of an infection. If an infection was established but failed to cross the midgut barriers, only the body would test positive, but legs and body would both be positive in a disseminated infection (13).

The mosquito species and numbers used in the study are listed in Table 2. The sources of the mosquitoes were as follows: Aedes aegypti, the Liverpool strain from a colony temporarily maintained at the Arthropod-Borne Animal Diseases Research Unit (ABADRU), Manhattan, KS; Culex quinquefasciatus, from the Center for Medical, Agricultural and Veterinary Entomology (CMAVE), Gainesville, FL; Culex tarsalis, from an ABADRU colony originally established in California; and Aedes taeniorhynchus, obtained as eggs from CMAVE. Mosquitoes were raised to adults and maintained at 24 ± 2°C on a 10% sucrose solution with a 12-h photoperiod. Prior to feeding on sheep, mosquitoes were anesthetized using CO₂ for 10 to 15 s and placed in Plexiglas containers topped with fine-mesh fabric screens that allowed them to feed on the exposed skin of vaccinated sheep. Postfeeding, blood-fed females were sorted and maintained for 10 to 14 days, prior to individual testing for MP-12 by RT-PCR. Positive and negative controls included mosquito pools spiked with MP-12 and mosquitoes fed on the nonvaccinated sheep.

To determine the dose of zMP-12 in blood that was infectious to mosquitoes, Culex tarsalis and Aedes taeniorhynchus were fed defibrinated sheep blood spiked with 3, 4, and 5 log₁₀ PFU/ml of zMP-12 using the Hemotek feeding system (Discovery Workshops, Accrington, United Kingdom). Five to 10 blood-fed mosquitoes of each species at each zMP-12 dose were frozen at −80°C on the day of feeding as positive controls. The remaining blood-fed mosquitoes (n = 86) were sorted and maintained for 14 days and then tested individually for the presence of virus by a plaque assay.

Antibody detection assays.Serum neutralization antibodies were measured using 70% plaque reduction titer (PRNT₇₀) tests as previously described (31). Briefly, sera were heat treated for 30 min at 56°C and diluted 1:10, and then serial 4-fold dilutions were made in complete medium. Diluted sera were then incubated with an equal volume of medium with 100 PFU of virus for 60 min at 37°C prior to inoculation onto monolayers of Vero cells. After 1 h of incubation at 37 C for adherence, cells were overlaid with complete medium containing 0.5% SeaKem ME agarose (Sigma-Aldrich) and incubated for 3 days prior to overlay with complete medium with agarose containing 0.1 mg/ml neutral red dye and incubation for an additional 2 days. Plaques were counted, and titers were calculated as the reciprocal of the endpoint dilution that reduced the plaque count by ≥70% of the negative control. Experiments 1 to 3 were performed in BSL-2 against the attenuated uMP-12 strain, and experiment 4 was performed in BSL-3 against wild-type ZH501. Neutralization titers obtained against the virulent virus ZH501 and attenuated MP-12 were compared using serum samples from experiment 4 collected at 21 dpv and 6 dpi.

For experiment 3, antibodies were also tested using cELISAs developed as potential diagnostic tools to detect antibodies to three in vitro expressed RVFV proteins: N-terminal half of glycoprotein GN (GNn), nucleocapsid protein (N), and nonstructural protein from the S segment (NSs). Four mice were immunized twice at a 4-week interval by intraperitoneal injections of 0.1 ml containing 2.4 × 10⁵ log uMP-12 in PBS. Blood samples were collected 4 weeks following the final booster, and polyclonal serum was separated and stored at −80°C until use in the cELISAs. Proteins used in the assay were produced by in vitro methods. The genetic sequence for GNn was acquired from the RVFV medium segment cloned into pWRG7077 plasmid, obtained from C. Schmaljohn (USAMRIID). The N and NSs protein coding sequences were acquired from the RVFV small gene segment cloned into pcDNA 3.1 and pQE-9, respectively, both obtained from F. Weber, Marburg University, Germany. Protein coding sequences were subcloned into the pET-30 Ek/LIC expression vector (Novagen, Darmstadt, Germany), and proteins were produced in BL21 cells. GNn and N proteins were purified by a nickel affinity column; the N-terminal His tag was cleaved using the enterokinase cleavage capture kit (Novagen) according to the manufacturer's directions, and NSs was purified by His-Trap chromatography with the Pharmacia fast protein liquid chromatography (FPLC) system (model AKTA P-920). The purified proteins were applied to the wells of 96-well plates at 0.5 to 1.0 pg/μl. Sheep serum samples from experiment 3 were diluted 1:10 and added to duplicate wells, incubated for 1 h at room temperature, and washed 3 times in wash buffer (PBS, 0.05% Tween 20, 0.1% bovine serum albumin) prior to the addition of 1:1,000 mouse anti-MP-12. After incubation at room temperature for 1 h, the wells were similarly washed and then incubated with 1:200 biotinylated goat anti-mouse horseradish peroxidase (HRP) and 1:200 streptavidin conjugates (Biogenex, Fremont, CA). Optical density (OD) measurement at 492 nm, following addition of o-phenylenediamine substrate, allowed calculation of the serum antibody percent inhibition (PI) titers. These were calculated as 1 − (test serum OD/negative serum OD) × 100.

Virus isolation and titrations.Virus isolation and titrations were performed using a plaque assay as previously described (38) to test for postvaccinal MP-12 virus in the sheep and mosquitoes (experiments 1 to 4) and for postchallenge wild-type ZH501 (experiment 4). Briefly, 400 μl/well of 10-fold serially diluted sample (virus stock, serum, or 10% [wt/vol] clarified tissue homogenates) in DMEM (Wisent) was applied to triplicate wells of confluent Vero E6 cells in 12-well plates. Plates were incubated at 37°C in 5% CO₂ for 1 h and then inoculum was replaced with complete medium with a 2% carboxymethyl cellulose overlay (Sigma-Aldrich). Plates were formalin (10%; Sigma-Aldrich) fixed and stained with crystal violet (0.5%, 80% ethanol, 20% PBS) after 4 days of incubation. Titers are reported as log₁₀ PFU.

RNA extraction and RT-PCR.For experiments 1 to 3, RNA was isolated from serum or 10% (wt/vol) tissue homogenates using the MagMAX-96 viral RNA isolation kit (Applied Biosystems, Foster City, CA), following the manufacturer's recommendations, with final elution in 50 μl of RNase-free water. Extracted RNA was used in an RT-PCR assay utilizing primers and probes targeting the L gene as described previously (42) with the minor modification of changing the forward primer's 14th nucleotide from G to A so it would exactly match the MP-12 sequence (43). For experiment 4, RNA was similarly isolated using TriPure reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Real time RT-PCR using the same protocol (42) was modified to a one-step procedure (39). Armored enterovirus RNA was used as an exogenous internal control, and a plasmid standard curve was set up to estimate the number of detected copies of viral RNA (39).

Statistics.Serum neutralization titers were calculated as geometric means by group for each time point. The Student t test (Excel 97, 2003) for independent samples was used for statistical comparisons between the groups, and differences with P values of <0.05 were considered significant. Comparisons of postchallenge RT-PCR copy numbers and infectious virus titers between vaccinates and controls in experiment 4 were tested using the Mann-Whitney U test (Social Science Statistics on-line calculator; http://www.socscistatistics.com/tests/mannwhitney/Default.aspx) for independent samples with nonnormal distribution.