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and we tested the effect of a single immunization. Having established that low doses of DNA were effective in the mouse model, we then tested the prophylactic effect of a low-dosage combination of these DNAs in the guinea pig vaginal-infection model. Genital disease in guinea pigs closely resembles that in humans (38, 39), and the model has been widely used to test potential vaccines (5, 6) and antiviral chemotherapies (40) for genital herpes.

MATERIALS AND METHODS

Viruses and Cells. Vero, baby hamster kidney (BHK)-21, and rhabdomyosarcoma (RD) cells, and HSV-2 strain MS were obtained from the American Type Culture Collection. HSV-2 strain Curtis was obtained from Andre Nahmias (Emory University, Atlanta). Virus was routinely prepared by infection of nearly confluent Vero or BHK cells with a multiplicity of infection of 0.1 at 37°C in a small volume of cell culture medium without serum. After 1 hr, virus inoculum was removed and cultures were re-fed with high glucose DMEM (BioWhittaker) supplemented with 2% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 25 mM Hepes, 50 units/ml penicillin, and 50 μg/ml streptomycin (all GIBCO/ BRL). Incubation was continued until cytopathic effect was extensive; usually for 24–48 hr. Cell-associated virus was collected by centrifugation at 1800×g for 10 min at 4°C. Supernatant virus was clarified by centrifugation at 640×g for 10 min at 4°C, and stored at −70°C.

Mice and Guinea Pigs. Female BALB/c mice (Charles River Breeding Laboratories) and female Duncan Hartley guinea pigs (Harlan Sprague Dawley) were maintained and used in accordance with the Institutional Animal Care and Use Committee approved protocols.

Cloning and DNA Preparation. HSV-2 strain Curtis DNA, used as template for polymerase chain reactions (PCR), was prepared from nucleocapsids isolated from infected Vero cells (41). An 1182-bp fragment encoding the gD precursor gene was amplified by PCR (Perkin-Elmer/Cetus) using synthetic oligonucleotide primers (Midland Certified Reagent, Midland, TX), which corresponded to 5′ and 3′ end-flanking sequences for the HSV-2 gD gene and contained BglII restriction sites. A 2121-bp sequence encoding the amino terminal 707 aa of HSV-2 gB was amplified by PCR. Primers corresponding to the 5′ flanking sequence, and complementary to nucleotides 2110–2121 (42) were used to generate BglII restriction sites flanking the coding sequence, and to add the termination codon TAA immediately after nucleotide 2121. BglII-digested PCR-amplified fragments were ligated into vectors V1J or V1Jns (36, 37). Escherichia coli DH5α (GIBCO/ BRL) was transformed according to the manufacturer’s specifications. Candidate plasmids were characterized by restriction mapping, and the vector-insert junctions were sequenced using the Sequenase DNA sequencing kit, version 2.0 (United States Biochemical). The gD-coding sequence, originally cloned in V1J, was subcloned into V1Jns. For simplicity, the final gD and truncated gB plasmid constructions were designated gD-2 and ΔgB-2, respectively. Large-scale DNA preparations were essentially as described (36).

Expression of Recombinant Proteins. Plasmid DNA was precipitated onto RD cells (ATCC CCL136) by the calcium phosphate method using Pharmacia CellPhect Kit reagents according to the manufacturer’s instructions, except that 5 or 15 μg of DNA per well were used. After 48 hr, cell lysates were resolved by electrophoresis and then transferred to nitrocellulose membranes. Immunoblots were processed with an anti-HSV gD monoclonal antibody (Advanced Biotechnologies, Columbia, MD) or sheep anti-HSV-2 antiserum (ViroStat, Portland, ME) and developed with an enhanced chemiluminescence detection kit (Amersham).

Immunization. In all cases, DNA dose refers to the total amount of DNA injected per animal per round of immunization; one-half of the total was delivered to each injection site. Mice were anesthetized by i.p. injection of a mixture of 2 mg ketamine HCl (Aveco, Fort Dodge, IA) and 0.2 mg xylazine (Mobley, Shawnee, KS) in saline. The hind legs were shaved with electric clippers and washed with 70% ethanol. Each quadriceps muscle was injected with 50 μl of DNA diluted into sterile saline just prior to use. Control animals were shamimmunized with saline or vector DNA. Mice were 5–6 weeks old at the time of the first immunization. Guinea pigs, weighing 400–550 g at the time of the first immunization, were anesthetized by subcutaneous injection of 22 mg ketamine plus 5 mg xylazine/kg. The hind legs were washed with 70% ethanol and each quadriceps muscle was injected i.m. with 100 μl of DNA or saline.

Serology. Sera were assayed for HSV-specific responses in ELISAs using either HSV glycoproteins partially purified from HSV-2 Curtis-infected BHK cell lysates (mouse sera) or recombinantly expressed gD and ΔgB purified from recombinant baculovirus-gD-and baculovirus-gB-infected SF21 cultures (guinea pig sera). Recombinant viruses were constructed using the BacPAK Baculovirus Expression System (CLONTECH) pBacPAK8 transfer vector and Bsu361-digested BacPAK6 virus and gD and ΔgB coding sequences from gD-2 and ΔgB-2, respectively. Glycoproteins from HSV-2 or baculovirus-gD-infected cultures were purified by Lentil Lectin Sepharose chromatography (Pharmacia) essentially as described (43). Truncated gB was purified from clarified culture medium adjusted to 0.1 mM MnCl2, 0.5% Nonidet P-40, batch adsorbed at room temperature to Lentil Lectin Sepharose 4B, and eluted as described. For the ELISA, glycoproteins were diluted to 5 μg/ml total protein in 50 mM carbonate buffer (pH 9.5), 100 μl per well was applied to Maxi-sorb 96-well plates (Nunc) and allowed to absorb at 4°C overnight. All subsequent incubations were carried out in 100 μl volumes for 1 hr at room temperature and plates were washed four times with phosphate buffered saline (PBS, pH 7.2), with or without one distilled water wash between steps. Dilution buffer (920 mM Tris·HCl, pH 7.5/137 mM NaCl/2.7 mM KCl/0.5% gelatin/0.05% Tween 20) was used as a blocking agent, as well as for the serial dilution of immune sera and the dilution of alkaline phosphatase-labeled goat anti-mouse (Boehringer Mannheim) or goat anti-guinea pig (Accurate Chemicals) IgG. The ELISA was developed with 1 mg/ml p-nitrophenylphosphate in 10% diethanolamine (pH 9.8), 0.5 mM MgCl·6 H20 at 37°C, and optical absorbance was read at 405 nm. Serum dilutions were scored as positive if the OD405 signal exceeded by more than 3 SD the mean OD405 signal (six replicates) of sera from sham-immunized mice at the same dilution, or if the OD405 signal exceeded by >0.1 OD unit, the signal of the guinea pig’s preimmune serum at the same dilution. The reciprocal of the last sample dilution scored positive was taken as the endpoint titer. Individual endpoint titers were used to calculate geometric mean titers (GMT). For purposes of calculation, sera negative at the lowest dilution tested were assigned endpoint titers equal to the reciprocal of the next lower dilution if the dilution series had been extended. ELISA titers are HSV-specific as originally shown by lack of measurable ELISA titer in sera from naive or saline-immunized animals, and by the lack of reaction of immune sera with antigen prepared from mock-infected BHK cell lysates.

Neutralization Assays. Sera from DNA- or saline-immunized animals were heat inactivated at 56°C for 30 min prior to serial dilution in DMEM/2% heat-inactivated FBS. Fifty microliters of each dilution were delivered to duplicate wells in a sterile polypropylene, 0.5 ml 96-well plate (Marsh Biomedical Products, Rochester, NY). HSV-2 stocks were diluted to 4000 plaque forming units (pfu)/ml and 50 μl of virus was added to sample wells and the plate incubated



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