Viral vectors and cells
Oncolytic VACVs were derived from the Copenhagen strain and are deleted of both the thymidine kinase (J2R) gene and the large subunit of ribonucleotide reductase (I4L) gene. TG6002 expressing the fusion gene FCU1 (DI4LDJ2R/FCU1 VACV) was constructed as previously described15. The same methods were used to generate the double-deleted VACV expressing GFP, designated VVTG17990, by homologous recombination between the previously described DJ2R/GFP VACV and the pDI4L shuttle plasmid containing the selection cassette encoding the guanine phosphoribosyltransferase surrounded by the flanking sequences of VACV I4L gene 57. TG6002 and VVTG17990 were amplified in chicken embryonic fibroblasts (CEF).
Primary CEF were used for recombination, amplification, and production of viral vectors. CEF cells were prepared and maintained as previously described 58.
Laboratory dogs
Four healthy adult male beagle dogs (Harlan Laboratories, Gannat, France) were used. All dogs were acclimatized for seven days and were under the care of a veterinarian. The dogs were housed individually in inox-steel bar boxes with a resin soil substrate and a softwood chips litter. The room temperature was 19°C (+/- 2°C) with a humidity greater than 35%, and the day/night cycle was 12:12 hours. Dogs were fed daily with a commercial diet and given potable water ad libitum.
Study design and treatments
The first part of the study aimed to determine the maximum tolerated dose (MTD) in three dogs, with each dog receiving an increasing dose of TG6002 by intravenous injection (Fig. 1). The second part of the study assessed tolerability of several intravenous injections of TG6002 at the identified MTD in one dog (Fig. 1). In the first part of the study, three dogs were treated on day 0 with a single intravenous injection of TG6002. TG6002 was diluted in 100 mL of NaCl 0.9% and perfused, under sedation, in the cephalic vein for one hour. Dogs were sedated by intravenous administration of 0.2 mg/kg butorphanol (Torbugesic, Zoetis, Malakoff, France) and 10 µg/kg medetomidine (Domitor, Orion Corporation, Espoo, Finland). The doses chosen in this study were similar to the doses previously described in the safety study by intramuscular route 24. Dog 1 received 1 x 105 PFU/kg, Dog 2 received 1 x 106 PFU/kg, Dog 3 received 1 x 107 PFU/kg. To detect any side effects, injections were performed early in the morning to allow for observations and administration between each dog were spaced by 7 days. Dogs were evaluated daily by a physical examination for 14 days after the injection of TG6002. Complete blood counts and biochemistry analyses were performed before TG6002 injection (day 0) and at 7 and 14 days afterward.
To measure viral shedding, blood samples were collected before injection (day 0), one hour after injection, and at days 3, 7, 10 and 14; saliva, urine and feces were collected at days 0 (before injection), 7 and 14.
The MTD was defined as the highest dose of TG6002 that did not cause major side effects.
One dog (Dog 4) was used for evaluating the tolerability of multiple injections. Dog 4 received three intravenous injections of TG6002 at days 0, 7 and 14 at the defined MTD. TG6002 was administered according to the same procedure. Dog 4 was evaluated daily by a physical examination for 35 days. Complete blood counts and biochemistry analyses were performed at days 0, 7, 14, 21, 28 and 35.
Blood samples were collected at days 0 (before first injection), 3, 7 (before second injection), 14 (before third injection), 17, 21, 24, 28, 31, 35 and one hour after each intravenous virus administration (days 0, 7, 14). Saliva, urine and feces were collected at days 0 (before first injection), 7 (before second injection), 14 (before third injection), 21, 28 and 35.
At the end of the study, dogs were euthanized to assess virus biodistribution. The dogs were anesthetized with an intravenous administration of 0.2 mg/kg of butorphanol (Torbugesic, Zoetis, Malakoff, France), 3 mg/kg of ketamine (Ketamine 1000, Virbac, Carros, France) and 15 µg/kg of medetomidine (Domitor, Orion Corporation, Espoo, Finland). After each dog was sedated, an intravenous injection of 180 mg/kg of sodium pentobarbital solution (Dolethal, Vetoquinol, Magny Vernois, France) was given. Death was confirmed by the inability to hear respiratory sounds and heartbeat using a stethoscope. Samples of heart, liver, mesenteric and pre-scapular lymph nodes, kidneys, spleen, lungs, and testicles were collected from all dogs and stored at -80 °C until analysis.
Adverse events
Adverse events were monitored by daily physical examination, complete blood count and biochemistry analyses and graded according to the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events guidelines 59.
Complete blood count and biochemistry analysis
Complete blood counts were performed using a Procyte Hematology analyzer (IDEXX Laboratory Inc, Westbrook, ME). Biochemistry analyses were performed using a Catalyst Biochemistry analyzer (IDEXX Laboratory Inc, Westbrook, Maine, United States).
Sample collection for viral shedding
Five milliliters of blood was collected in an EDTA tube, saliva samples were taken with buccal swabbing (Universal viral transport kit, Becton Dickinson, Franklin Lakes, New Jersey, United States), 5 ml of urine was collected in a sterile Falcon tube, and one gram of feces was transferred to a sterile Falcon tube. Samples were stored at -80 °C until analysis.
q-PCR
Quantitative polymerase chain reaction was used to detect TG6002 genomes in whole blood, saliva, urine, feces and organ samples. DNA was extracted from 100 µl of whole blood, saliva and urine. For organs and feces, 30 milligrams of each organ sample and one gram of feces were transferred in GentleMACS M–type tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 600 µL of PBS and were dissociated using a GentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). DNA was extracted from one hundred microliters of lysates. q-PCR was performed as previously described.24 Samples were measured in triplicate. The limit of detection for analysis was 15 copies/100 µl for whole blood samples, 30 copies/100 µl for urine, 3,600 copies/g for feces and 400 copies/30 mg for organ samples. The limit of detection for saliva could not be set due to the small amount collected.
Enzyme-linked immunosorbent assay (ELISA)
For determination of anti-VACV antibody response, TG6002 was inactivated 20 minutes under UV lamp on ice. Ninety-six well plates (Corning, Corning, New York, United States) were coated with carbonate-bicarbonate buffer (Sigma, Saint Quentin, Fallavier, France) containing inactivated TG6002 (3 x 106 PFU/100 µl/well) and allowed to attach overnight at 4°C. Wells were washed with PBS and binding sites were blocked at room temperature for one hour with PBS containing 0.05% Tween 20 and 5% non-fat dry milk, followed by adding a 2-fold dilution series of the serum. After a two hours incubation at room temperature, plates were washed with PBS and 100 µl of recombinant peroxidase-conjugated, protein A/G (A/G-HRP protein, Thermo Fisher Scientific, Waltham, Maine, United States) diluted 20,000-fold was added. Plates were incubated for one hour at room temperature and washed with PBS. A volume of 100 µL of TMB solution (Sigma, Saint Quentin, Fallavier, France) was added for 30 minutes. The color reaction was stopped by 100 µL of 1 M H2SO4 solution. The absorbance was read at 450 nm on a spectrophotometric plate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland). A 1/3,000 diluted rabbit polyclonal VACV antibody (B65101R, Interchim Inc, Montlucon, France) was used as positive control. End-point-titers were determined as the highest dilution with an absorbance value greater than the absorbance value from normal dog sera. Results were given as log values and a log titer value of 2 or below was considered negative.
To determine the anti-FCU1 antibody response, ELISA was performed as described above except that plates were coated overnight with carbonate-bicarbonate buffer containing FCU1 peptide at 7.6 ng/well. A 1/3,000 diluted rabbit polyclonal anti-FCU1 peptide was used as positive control 58.
Neutralizing antibodies
Two-fold serial dilutions from 1/40 to 1/5,120 of serum samples were incubated with 2 x 103 PFU of VVTG17990 in Corning tubes for one hour at 37°C and incubated with CEF for 3 days at 37°C with 5% CO2. Plates were examined using fluorescence microscopy (Stereoscopic microscope Nikon SMZ18 and epi-fluorescence light source Nikon Intensilight C-HGFI) to score GFP positive plaques. The percent neutralization was calculated relative to the number of GFP plaques in the absence of serum. The neutralizing antibody titer was defined as the highest serum dilution resulting in a 50% reduction in the number of plaques. Samples were measured in triplicate and mean neutralization titers for dogs were plotted ± standard deviation.
Ethical approval
This study was conducted in accordance with European legislation and French regulations on the protection of animals used for scientific purposes (Directive 2010/63/EU, 2010; Code rural, 2018; Décret 2013-118, 2013) and complied with the recommendations of the “Charte nationale portant sur l’éthique en expérimentation animale” established by the “Comité National de Réflexion Ethique sur l’Expérimentation Animale” (Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation—Ministère de l’Agriculture et de l’Alimentation). The protocol (n°1431_v2) was approved by the VetAgro Sup Ethical Committee (C2EA No. 18) and the Ministry of National Education, Higher Education and Research.