Lentiviral vector production and titration
rSIV.F/HN lentiviral vector particles were produced essentially as described previously (8), via a five-plasmid transient transfection method using HEK293T cells grown in suspension. Briefly, the single ORF cDNA of palivizumab was configured using publicly available sequence (10) as described previously (11), and CpG-depleted, human codon-optimized, and synthesized using the GeneArt Gene Synthesis service (Thermo Fisher Scientific). The palivizumab, or a CpG-free Gaussia Luciferase (soGLux), cDNA was inserted via unique NheI and PsPOMI sites into a recombinant simian immunodeficiency virus (SIV) genome plasmid backbone under the transcriptional control of the hCEF promoter (18). Vectors were purified using anion exchange chromatography and tangential flow filtration and formulated into either FreeStyle293 media or TSSM buffer (tromethamine 20 mM, NaCl 100 mM, sucrose 10 mg/mL, and mannitol 10 mg/mL). The functional titre in Transducing Units per mL (TU/mL) of lentiviral vectors was determined based on the genomic integration of WPRE DNA sequence after transduction of HEK293F cells in vitro.
rAAV vector production and titration
Recombinant rAAV was produced as described (19). Briefly, the CpG-free palivizumab and Gaussia Luciferase (soGLux) cDNAs were inserted, via unique NheI and PsPOMI site, into a recombinant adeno-associated virus (AAV) genome plasmid backbone with AAV2 ITRs under the transcriptional control of the CASI promoter (6) . HEK293T cells were transfected with the plasmids pAdDeltaF6, pAAVRep2/Cap8, and prAAV2ITR with transgene using polyethylenimine (PEI; Polysciences Inc.). After 72 hours, cells were resuspended in lysis buffer (1M Tris(hydroxymethyl)aminomethane, 150 mM NaCl) and EDTA-free protease inhibitor cocktail and underwent four freeze-thaw cycles. Cell lysates were incubated (37ºC for 30 min) with Benzonase (50 U/mL final concentration) and clarified via centrifugation, and purified using iodixanol gradient fractionation and diafiltration into D-PBS using Amicon Ultra-15 100K MWCO filters. The number of Genome Copies (GC/mL) was determined by qPCR.
All procedures involving laboratory mice were carried out in accordance with UK Home Office approved project and personal licenses under the terms of the Animals (Scientific Procedures) Act 1986 (ASPA 1986), were approved by the University of Oxford or Imperial College Animal Welfare Ethical Review Body as appropriate, and are reported in compliance with the ARRIVE guidelines (https://arriveguidelines.org). Female BALB/c mice, 6-8 weeks old at the initiation of studies, were used. Animals were arbitrarily assigned to study groups using an open-label randomised block approach. Overall, 259 animals were used (Figure 2: n=12/group, 3 groups, n=11/group, 3 groups, n=22/group 1 group, n=16/group 1 group; Figure 3 & 4: n=16/group 7 groups; Figure 5; n=5/group, 8 groups). Group sizes reduced during the study as effect sizes became more predictable. Animals were euthanised at the end of each study by cervical dislocation or intraperitoneal injection of 100 μL pentobarbital.
Administration of vector and virus to mice
Anaesthesia was induced using inhalation of 4-4.5% isoflurane (Abbott, Maidenhead, UK) and maintained with 2.5-3.5% isofluorane. For delivery of lentiviral vector and for RSV infection, a total volume of 100 μL was administered by nasal sniffing as previously described (20) Adeno-associated viral vector was administered by injection into the gastrocnemius or quadriceps muscle using a 500 μL insulin syringe with 29G needle in a total volume of 40 μL.
Collection of samples from mice
Blood was collected from the tail vein of mice, stored overnight at 4oC and centrifuged to isolate serum. For collection of broncho-alveolar lavage fluid (BALF), mice were euthanised and dissected to expose the trachea. A small tear was made in the trachea and a 0.75 mm cannula used to infuse the lung with 1 mL BALF solution (PBS, 50 µM EDTA; 1% BSA was included except for leukocyte quantification) and then fluid collected by gentle aspiration. Flushing the lung was performed three times using the same (for studies with monoclonal antibodies), or fresh (for studies with GLux) BALF solution. For quantification of protein expression, the collected BALF was centrifuged to sediment cells, and the supernatant used for ELISA or luciferase assay as described below.
Quantification of protein expression
Palivizumab levels in the cell culture media, serum and BALF were measured using Human IgG ELISA kit (Bethyl, Cambridge, UK) according to the manufacturer’s instructions. GLux activity in the serum was measured using BioLux Gaussia Luciferase Assay Kit (NEB, Ipswich, USA) according to the manufacturer’s instructions. Expression levels in ELF were corrected for the dilution using lavage fluid collection urea assay as described previously (8).
Total and differential leukocyte quantification
The BALF was centrifuged at 3,500 rcf and the cell pellet resuspended in Ammonium-Chloride-Potassium buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) to lyse red blood cells. After 2 minutes, DMEM (Sigma-Aldrich) was added, the samples were centrifuged at 3,500 rcf and the pellet was resuspended in 500 µl of DMEM. For total leukocyte quantification, an aliquot of the cell suspension was mixed with 0.1% trypan blue and cells manually counted using a haemocytometer. For differential leukocyte quantification, 100 µL of the cell suspension was transferred onto a glass slide (Tharmac, Waldsoms, Germany) using Cytospin III Cytocentrifuge (Thermo Fisher Scientific) and allowed to air-dry. The slides were fixed and stained using Reastain Quick-Diff Kit (Reagena, Toivala, Finland) and visualised using a light microscope. At least 300 cells per slide were counted, differentiating between macrophages, lymphocytes and neutrophils based on their morphology.
Group sizes were initially projected from previous data (13), using IgG expression and RSV-induced weight loss as the primary endpoints and were reduced throughout the study as estimates of effect size became more robust. G*Power software v126.96.36.199 (21) was used to project group sizes and establish achieved power. Statistical analysis was performed using Prism software v8.4.2 for Mac (GraphPad Software). The non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons post-hoc test was used to compare experimental groups with the indicated negative control group throughout. For weight loss studies, area under the curve values for the 7 days post RSV challenge were first calculated for each animal prior to Kruskal-Wallis evaluation. Numerical values in text are presented as mean±standard deviation. Symbols and error bars in figures represent the mean and standard deviation unless stated otherwise. A calculated p-value of p<0.05 was deemed a significant difference and indicated on figures where appropriate. In figures, the symbols ns, *, **, *** and **** represent p>0.05, <0.05, p<0.01, p<0.001 and p<0.0001 respectively.