Custer, B

Custer, B. antibodies that not only neutralized ANDV but also cross-neutralized other HPS-associated hantaviruses, including Sin Nombre virus. To determine if the antibodies elicited in the monkeys could confer protection, we performed a series of passive-transfer experiments using a recently described lethal HPS animal model (i.e., adult Syrian hamsters develop HPS and die within 10 to 15 days after challenge with ANDV). When injected into hamsters 1 day before challenge, sera from the vaccinated monkeys either provided sterile protection or delayed the onset of HPS and death. When injected on day 4 or 5 5 after challenge, the monkey sera protected 100% of the hamsters from lethal disease. These data provide a proof of concept for a gene-based HPS vaccine and also demonstrate the potential value of a postexposure immunoprophylactic to treat individuals after exposure, or potential exposure, to these highly lethal hantaviruses. Hantaviruses are rodent-borne, enveloped RNA viruses in the family High Fidelity DNA polymerase (Invitrogen): one 3-min cycle at 94C followed by 30 cycles of 94C for 30 s and 68C for 8 min. The PCR product was cut with BL21(DE3) (Novagen, Inc.) and purified by affinity chromatography on Ni-nitrilotriacetic acid columns (Qiagen). Endpoint titers were determined as the highest dilution that had an optical density greater than the mean optical density of serum samples from negative control wells plus 3 standard deviations. The SEOV N antigen was used to detect HTNV N-specific antibodies, and the PUUV N was used to detect ANDV N-specific antibodies. PRNT. Neutralization assays were performed as previously described (11). HTNV, ANDV, and BCCV plaque reduction neutralization tests (PRNT) were stained with neutral red after 1 week, and SNV PRNT were stained after 9 days. Plaques were counted (at 37C) 2 to 3 3 days after staining. IFAT. Indirect fluorescent antibody tests (IFAT) were a modification of a previously described procedure (12). COS cells grown on 15-mm-diameter glass coverslips in 12-well cell culture plates were transfected with 1 g of pWRG/AND-M DNA using Fugene6 (Boehringer Mannheim) as described by the manufacturer. Two days after transfection, the coverslips were rinsed once with phosphate-buffered saline (PBS; pH 7.4) and fixed with acetone for 10 min at room temperature. The slides were rinsed three times in PBS and blocked for 10 BAY 80-6946 (Copanlisib) min in PBS containing 5% fetal bovine serum and 3% goat serum. Hamster sera were diluted 1:100 in blocking buffer and then incubated on the transfected cells for 1 h at 37C. The coverslips were rinsed three times with PBS and incubated for 1 h at 37C with biotin-labeled goat anti-hamster immunoglobulin G and immunoglobulin M antibodies (Pharmingen). The coverslips were rinsed as before and incubated for 30 min at 37C with fluorescein-labeled streptavidin (Kirkegaard & Perry Laboratories). Hoechst stain (1 g per ml) was included in the streptavidin solution as a counterstain. The coverslips were BAY 80-6946 (Copanlisib) rinsed three times with PBS and once with deionized water and then placed on a drop of fluorescent BAY 80-6946 (Copanlisib) mounting medium (DAKO) on glass slides. The cells were observed with a Nikon E600 fluorescence microscope. Gene gun vaccinations. Cartridges for the gene gun were prepared as described previously (11, 13). Plasmid DNA was precipitated onto gold beads (3 g of DNA per mg of gold), and then tubing was coated with the DNA-coated gold beads. Gene gun cartridges consisting of Neurog1 0.5 mg of gold coated with 0.75 g of plasmid DNA were prepared and stored desiccated at 4C until they were used. Syrian hamsters were vaccinated with the XR1 particle-mediated epidermal delivery device (gene gun) (Powderject Vaccines, Inc., Madison, Wis.), four administrations per vaccination, at nonoverlapping sites on the shaved abdominal epidermis using 400 lb of helium pressure/in2. Female rhesus macaques (for 20 min, and the supernatant was collected. Monkey serum from DNA-vaccinated monkeys and human serum (PEL-FREEZ Biologics, Rogers, Ariz.) were heat inactivated (56C; 30 min). One milliliter of serum or plasma was injected intraperitoneally into hamsters using a 1-ml syringe with a 25-gauge 5/8-in. needle. Challenge with hantaviruses. Adult female Syrian BAY 80-6946 (Copanlisib) hamsters (= 0.0569). Hamsters that survived challenge were rechallenged with ANDV to ensure that they were exposed to virus. At least one animal (no. 504) had no antibody response after two successive challenges but was clearly protected from lethal disease. Open in a separate window FIG. 1. Evaluation of HFRS DNA vaccine in ANDV-hamster lethal-disease model. Hamsters were vaccinated with pWRG/HTN-M(x) or the negative control plasmid pWRG7077 and then challenged with ANDV. For animals that succumbed, the day.