African green monkeys (AGM) were feral caught and pre-screened to ensure sero-negativity against RSV and SeV. Twelve AGM were housed for vaccinations at BioQual (Rockville, MD) under BSL2 conditions as specified by the Association for Assessment and Accreditation for Laboratory Animal Care (AAALAC) guidelines. Prior to sample collections, vaccinations or challenges, animals were anesthetized with ketamine hydrochloride (Putney Inc., Portland, ME). Clinical assessments, sample collections, vaccinations and necropsies were conducted at Bioqual. Blood chemistries were conducted at the Idexx Research Laboratory (Totowa, NJ). The RSV challenge stock was pre-tested in two additional AGM that were feral caught, pre-screened to ensure sero-negativity, and housed at the Tulane National Primate Research Center (TNPRC). All animal housing, care and research was performed in compliance with the National Research Council Guide for the Care and use of Laboratory Animals, guidelines at TNPRC (accredited by AAALAC) and in accordance with the Animal Welfare Act guidelines. Protocols were IACUC approved.
The previously described SeV Enders isolate was grown in the allantoic fluid of hen’s eggs [7
]. The SeVRSV vaccine was produced by reverse genetics by cloning the full-length RSV-F gene from the A2 strain (ATCC, VR-1302) into a T7-driven, full-length, modified SeV genome, between F and HN genes [12
]. To rescue virus, 293T cells were infected with a UV-inactivated T7 RNA polymerase-expressing recombinant vaccinia virus (vTF7.3) for 1 h at 37°C. Cells were then co-transfected with the cDNA plasmid containing the recombinant genome described above, and three supporting plasmids expressing the NP, P and L genes of SeV. Cells were cultured for two days in trypsin-supplemented growth medium. Virus in cell supernatants was harvested and expanded in the allantoic fluid of hens’ eggs. After stocks of SeV and SeVRSV were prepared, the viruses were purified on sucrose gradients and stored in 0.1% human serum albumin at −80°C.
Immunizations and challenge
On day −3 relative to vaccination, sera, nasal washes, throat swabs and bronchoalveolar lavages (BAL) were collected. To collect nasal washes, a 6 ml syringe and 18 G plastic needle were used to administer 4 ml saline into nasal cavities. Samples were collected into a sterile 15 ml tube. To collect BAL, a #14 French red rubber tube was placed in the trachea and advanced to the bronchi. A 12 ml syringe was used to administer 4 ml saline which was then retracted. Nasal washes, throat swabs and BAL samples were aliquoted immediately after sampling, and quick frozen.
On day 0, four animals received PBS by intranasal (i.n.) and intratracheal (i.t.) routes (1 ml by each route). Four additional animals received SeV, 1 × 106 EID50 by the intranasal (i.n.) route and 1 × 106 EID50 by the intratracheal (i.t.) route. A third group of four animals received SeVRSV, at a 1 × 106 EID50 dose by the intranasal (i.n.) route and 1 × 106 EID50 by the intratracheal (i.t.) route. Additional collections of sera, nasal washes, throat swabs and BAL were on days 3, 5, 7, 10, 14 and 25. On day 28, all animals were challenged with 1.4 × 106 pfu RSV A2 from infected Hep2 supernatants, in 1 ml by the i.n. route. This was considered day 0 for challenge. Samples were then taken on days 3, 5, 7, 10, and 14 and necropsy was performed on Day 20. The RSV A2 challenge stock virus (ATCC, Rockville, MD) had been confirmed infectious by prior i.n. inoculations of two dedicated AGM at the Tulane National Primate Research Center.
SeV and SeVRSV measurements
For SeV or SeVRSV detection, samples were serially diluted in DMEM, 0.5 mg/ml gentamicin, 2mM L-glutamine and 5 μg/ml acetylated trypsin, and added to 96-well plates of LLC-MK2 cell monolayers in 100 μl. After an overnight incubation, cultures were supplemented with 100 μl DMEM, 0.5 mg/ml gentamicin, 2mM L-glutamine, and 0.15% BSA. After an additional 4 day incubation, 50–100 μl supernatants from wells were used for standard hemagglutination (HA) assays with an equal volume of chicken red blood cells for 30–60 min. TCID50 were calculated using the Reed-Muench formula.
ELISAs were conducted to compare antibody levels at prescreen and on days 10, 14 and 25. Tests for SeV- and RSV-specific antibodies were each performed. For the SeV ELISA, purified virus was first disrupted using 10× disruption buffer (0.05 M Tris, pH 7.8, 0.6 M KCL and 0.5% Triton X-100), brought to a concentration of 10 μg/ml in PBS and added to ELISA plates at 50 μl/well. After overnight incubation at 4°C, plates were washed once with PBS and blocked with 3% BSA in PBS for 1–2 hr or overnight. Test samples were diluted in 3% BSA/0.1% Tween in PBS and added to washed plates for 1 hr at 37°C. Plates were washed 7X with 0.1% Tween in PBS. Plates were developed with Goat anti Monkey IgG conjugated to Alkaline Phosphatase (Rockland) in 3% BSA and 0.1% Tween in PBS for 1 hr 37°C followed by another 7X wash and p-nitrophenyl phosphate (Sigma) used at 1.0 mg/ml in diethanolamine buffer. Assays were read at O.D. 405 nm after 15–30 minutes on a microplate reader (Model 3550, BioRad). For RSV antibody measurements, ELISA plates were coated with purified RSV F. This antigen was prepared by collecting allantoic fluid from eggs infected with a SeV recombinant that expressed secreted RSV F, followed by purification of F protein on an a anti-RSV F (Synagis®/Palivizumab) affinity column. RSV ELISAs were conducted with diluted antibodies and developing reagents as described above.
SeV-specific microneutralization titers
One day prior to SeV microneutralization assays, LLC-MK2 cells were plated at 2 × 105 cells/ml in 96-well flat-bottomed tissue culture plates. On the following day, diluted serum samples were plated in replicate at 100 μl/well in a separate 96-well plate. Virus was then added at ~15 TCID50/well. After a 1 hour incubation at 37°C, 5% CO2, media were removed from LLC-MK2 cell culture plates and replaced with the antibody-virus mixtures. Cells were again incubated overnight. Supernatants were then removed and replaced with DMEM medium with 0.1% BSA, glutamine, gentamicin and 2–4 μg acetylated trypsin. After an additional three days of culture, plates were developed by ELISA. To conduct the ELISA, supernatants were aspirated and cells were fixed for one minute with 100 μl acetone/DPBS (80:20 volume ratio, DPBS from LONZA#17-513F). Wells were washed with DPBS. A mixture of two purified murine monoclonal anti-Sendai virus antibodies (S/2 and M57/1) conjugated to horse radish peroxidase was next added to wells in 1% BSA. After one hour at room temperature, wells were washed 6X with DPBS. Assays were developed with TMB Peroxidase and the color reaction was stopped by adding 4N H3PO4. Assays were read at OD 450 nm. ‘Max’ controls for the microneutralization assays were wells with virus only and negative controls were wells with medium only. For test wells, an OD reading of ≤50% the average ‘Max’ reading was considered positive for neutralization. The neutralization titer for a given test sample was scored as the highest dilution that supported neutralization in ≥50% wells.
RSV-specific microneutralization titers
RSV microneutralization assays were the same as described for the neutralization assay above, with a few exceptions. Hep2 cells served as targets for virus growth and were grown in EMEM with 10% FBS, glutamine and gentamicin (no trypsin was used). The RSV A2 virus was plated at 50–100 plaques per well. The developing antibody for the ELISA was horse radish peroxidase-conjugated anti-RSV (Synagis®) antibody.
For RSV measurements, serially diluted samples in EMEM medium supplemented with glutamine and gentamicin were added to Hep-2 cell monolayers in 12-well plates; after 1 h at 37°C and 5% CO2, the virus was removed and wells were overlaid with EMEM medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75% methylcellulose. After incubation for 5 to 6 days at 37°C and 5% CO2, the methylcellulose was removed, cells were washed with PBS and fixed with formalin phosphate, and the plates were stained with hematoxylin and eosin for enumeration of plaques.
Tissues were collected in 10% neutral buffered formalin (with fixative volume used at 15–20X tissue volume). Tissues were processed at St. Jude on an automated tissue processor (ThermoFisher Excelsior Processor). This involved six changes of graded alcohol, three changes of xylene and three changes of paraffin. Tissue was then embedded in paraffin (Richard-Allen Type 9) and sectioned on a rotary microtome (4 microns). Sections were mounted on positively charged glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) and stained using an automated stainer (ThermoFisher Gemini Stainer) with hematoxylin and eosin for evaluation by P. Vogel, an ACVP board-pathologist.