Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Summary

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.

Abstract

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.

Introduction

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 vaccine^{1,2,3,4,5,6,7}. 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 models^7,8,<....

Protocol

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

1. Inoculation of SPF embryonated chicken eggs
   NOTE: Typically, eight dozen SPF embryonated chicken....

Results

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.......

Discussion

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.......

Materials

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