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Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields > 6 × 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.
Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system.
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed.
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.
Newcastle Disease Virus, also known as Avian Orthoavulavirus-1, is an enveloped avian paramyxovirus with the potential to be used both as an oncolytic virus or a viral-vectored vaccine1,2,3,4,5,6,7. Most recently, NDV engineered to express the spike protein of SARS-CoV-2 has been characterized as an effective intranasal vaccine in mouse and hamster challenge models7,8,<....
All work involving the use of animals was approved by the University of Guelph Animal Care Committee in accordance with the Canadian Council on Animal Care. All work is performed in a BioSafety Level 2 (BSL2) laboratory in Canada where mesogenic NDV is a Risk Group 2 Pathogen. All steps involved in the amplification and purification of NDV should be performed in a Type IIA biological safety cabinet for safety and sterility purposes.
1. Amplification of NDV using specified pathogen-free embryonated chicken eggs
Harvesting allantoic fluid
As allantoic fluid is harvested from embryonated chicken eggs, it should appear clear and transparent. If the fluid appears opaque and yellow, this indicates the presence of contaminants. Inclusion of this allantoic fluid during purification will impede the purification process, as the pressure will quickly rise and surpass 10 psi, resulting in the shearing of the virus and loss of infectious virus. Allantoic fluid that appears bloody suggests that the eggs were inoculate.......
Viruses used as therapeutic agents in preclinical studies must be highly purified to avoid toxicity when administered in vivo15. If adventitious agents or contaminants are not removed, this can lead to severe adverse reactions negating the therapeutic effect of the viral agent28. As NDV is produced in embryonated chicken eggs, there are several contaminating egg proteins, such as ovalbumin, that must be removed prior to its use in vivo in preclinical or cl.......
The authors have no conflicts of interest to declare.
J.G.E.Y was the recipient of an Ontario Veterinary College PhD Scholarship and an Ontario Graduate Scholarship. This work was funded by Natural Sciences and Engineering Research Council of Canada Discovery Grants to SKW (grant #304737) and LS (grant #401127).
....Name | Company | Catalog Number | Comments |
0.25% Trypsin | HyClone | SH30042.02 | |
1 mL Slip-Tip Syringe | BD | 309659 | |
10 mL Luer-Lok Syringe | BD | 302995 | |
10% Povidone Iodine Solution | LORIS | 109-08 | |
15 mL Conical Tubes | Thermo-Fisher | 14955240 | |
18G x 1 1/2 in Blunt Fill Needle | BD | 305180 | |
18G x 1 1/2 in Precision Glide Needle | BD | 305196 | |
25 G x 5/8 in Needle | BD | 305122 | |
2-Mercaptoethanol | Thermo-Fisher | 03446I-100 | |
30% Acrylamide/Bis Solution 37.5:1 | BioRad | 1610158 | |
4% Paraformaldehyde-PBS | Thermo-Fisher | J19943-K2 | |
5 mL Luer-Lok Syringe | BD | 309646 | |
96 Well Tissue Culture Plate - Flat Bottom | Greiner Bio One | 655180 | |
Acetic Acid, Glacial | Thermo-Fisher | A38-212 | |
Agarose | Froggabio | A87-500G | |
Alexa-Fluor 488 Goat-Anti-Mouse | Invitrogen | A11001 | |
Allegra X-14 Centrifuge | Beckman Coulter | B08861 | |
Ammonium Persulfate | BioRad | 161-0700 | |
Bleach (5%) | Thermo-Fisher | 36-102-0599 | |
Broad, unserrated tipped forceps | Thermo-Fisher | 09-753-50 | |
Bromophenol Blue | Sigma-Aldrich | 114405-25G | |
Centramate Cassette Holder | PALL | CM018V | |
ChemiDoc XRS+ | BioRad | 1708265 | |
CO2 Incubator | Thermo-Fisher | ||
Coomassie Brilliant Blue R-259 | Thermo-Fisher | BP101-50 | |
DF1 Cells | ATCC | CRL-12203 | |
Diet Gel Recovery | ClearH2O, INC | 72-01-1062 | |
Digital 1502 Sportsman Egg Incubator | Berry Hill | 1502W | |
D-Mannitol | Sigma-Aldrich | M4125-500G | |
Egg Candler | Berry Hill | A46 | |
Ethanol (70%) | Thermo-Fisher | BP82031GAL | |
Ethylenediaminetetraacetic acid (EDTA) solution, pH 8.0, 0.5 M in H2O | Thermo-Fisher | BP2482-500 | |
Female Threaded Tee fittings, nylon, 1/8 in NPT(F) | Cole-Parmer | 06349-50 | |
Fetal Bovine Serum | Gibco | 12483-020 | |
Fine Point High Precision Forceps | Thermo-Fisher | 22-327379 | |
Fluorescent Microscope | ZEISS AXIO | Not necessary if not performing IFA or if NDV does not encode a fluorescent protein | |
Freeze Dry System Freezone 4.5 | LABCONCO | ||
GiBOX Gel Imager | Syngene | Imaging of Agarose Gels | |
Glycerol | Thermo-Fisher | G33-1 | |
Glycine | Thermo-Fisher | BP381-5 | |
High Capacity cDNA Reverse Transcriptase Kit | Thermo-Fisher | 4368814 | |
High Glucose Dulbecco's Modified Essential Medium | Cytiva | SH30022.01 | |
Humidity Kit | Berry Hill | 3030 | |
Iodixanol | Sigma-Aldrich | D1556 | 60% (w/v) solution of iodixanol in water (sterile) |
L-Lysine Monohydrochloride | Sigma-Aldrich | 62929-100G-F | |
Male and Female Luer-Lok a 1/8 in national pipe thread, NPT | Cole-Parmer | 41507-44 | |
Masterflex L/S Digital Drive | Cole-Parmer | RK-07522-20 | Peristaltic Pump with digital display |
Masterflex L/S Easy Load Pump Head for Precision Tubing | Cole-Parmer | RK-07514-10 | |
Masterflex Silicon tubing (Platinum) L/S 16 | Cole-Parmer | 96420-16 | BioPharm Platinum-Cured Silicone |
MC Pro 5 Thermocycler | Eppendorf | EP950040025 | |
Methanol | Thermo-Fisher | A412-4 | |
Mini Protean Tetra Cell | BioRad | 1658000EDU | SDS-PAGE cast and running appartus |
Mouse-Anti-NDV | Novus Biologicals | NBP2-11633 | Clone 6H12 |
Normal Goat Serum | Abcam | AB7481 | |
NP-40 | Thermo-Fisher | 85124 | |
Omega Membrane LV Centramate Cassette, 100K | PALL | OS100T02 | |
Optima XE-90 Ultracentrifuge | Beckman Coulter | A94471 | |
OWL Easycast B1A Mini Gel Electrophoresis System | Thermo-Fisher | B1A | |
PBS 10X Solution | Thermo-Fisher | BP399-20 | |
Poly(Ethylene Glycol) Average Mn 20,000 | Sigma-Aldrich | 81300-1KG | |
PowePac 300 | BioRad | Model 1655050 - for Agarose gel electrophoresis | |
Q5 High Fidelity 2X Master Mix | New England Biolabs | M0492S | |
QIA Amp Viral RNA Mini Kit | Qiagen | 52904 | |
RedSafe | Thermo-Fisher | 50999562 | |
Slide-a-lyzer Dialysis Cassette (Extra Strength), 10,000 MWCO 0.5-3 mL | Thermo-Fisher | 66380 | |
Sodium Dodecyl Sulfate | Thermo-Fisher | BP166-500 | |
Sodium Hydroxide (Pellets) | Thermo-Fisher | S318-10 | |
Specific pathogen free eggs | CFIA | NA | Supplier will vary depending on location |
Sucrose | Thermo-Fisher | S5-3 | |
Supracap 50 Depth Filter | PALL | SC050V100P | |
Surgical Scissors | Thermo-Fisher | 08-951-5 | |
Sw41Ti Rotor | Beckman Coulter | 331362 | Used in protocol step 2.3.1, 2.3.6, 2.3.7 |
SX4750 Rotor | Beckman Coulter | 369702 | |
SxX4750 Adaptor for Concial-Bottom Tubes | Beckman Coulter | 359472 | |
TEMED | Invitrogen | 15524-010 | |
Thin-Wall Ultraclear centrifuge tubes (9/16 in x 3 1/2 in) | Beckman Coulter | 344059 | |
Tris Base | Thermo-Fisher | BP152-5 | |
Tubing Screw Clamp | PALL | 88216 | |
Tween 20 | Sigma-Aldrich | P1379-1L | |
Utility Pressure Gauges | Cole-Parmer | 68355-06 |
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