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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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암 연구학

从尿囊液中生产高滴度重组新城疫病毒

Published: May 25, 2022
doi:
10.3791/63817
, , , , , ,
1Department of Pathobiology,University of Guelph

Summary

在这里，我们提供了生产，纯化和定量高滴度重组新城疫病毒的详细程序。该方案始终产生>6×109 个斑块形成单位/ mL，提供适合体内动物研究 的 病毒量。描述了确保 体内 安全性的其他质量控制测定。

Abstract

新城疫病毒（NDV），也称为禽原原变异病毒血清型-1，是一种阴性单链RNA病毒，已被开发为溶瘤病毒和病毒载体疫苗。NDV是一种有吸引力的治疗和预防药物，因为它具有良好的反向遗传学系统，有效的免疫刺激特性和出色的安全性。当作为溶瘤病毒或病毒载体疫苗给药时，NDV会引起强大的抗肿瘤或抗原特异性免疫反应，激活免疫系统的先天性和适应性臂。

鉴于这些理想的特征，NDV已在许多临床试验中进行了评估，并且是研究最充分的溶瘤病毒之一。目前，有两项涉及NDV的注册临床试验：一项评估用于SARS-CoV-2（NCT04871737）的重组NDV载体疫苗，另一项评估编码白细胞介素-12的重组NDV与抗PPD-L1抗体Durvalumab（一种抗PD-L1抗体（NCT04613492）的组合。为了促进这种非常有前途的病毒载体的进一步发展，需要简化用于生成高滴度， 体内级重组NDV（rNDV）的方法。

本文描述了在特定无病原体（SPF）胚胎化鸡蛋中扩增rNDV和从尿囊液中纯化rNDV的详细程序，并改进以减少纯化过程中的损失。还包括推荐的质量控制测定的描述，应进行这些测定以确认缺乏污染物和病毒完整性。总体而言，该详细程序能够合成、纯化和储存高滴度、 体内级、重组、致慢和中生 NDV，用于临床前研究。

Introduction

新城疫病毒，也称为禽原原病毒-1，是一种包膜性禽副粘液病毒，有可能用作溶瘤病毒或病毒载体疫苗1，2，3，4，5，6，7。最近，设计用于表达SARS-CoV-2的刺突蛋白的NDV已被描述为小鼠和仓鼠挑战模型7，8，9中的有效鼻内疫苗。当用作癌症免疫疗法时，它导致先天免疫细胞的募集，特别是自然杀伤细胞，产生I型干扰素，并产生抗肿瘤特异性T细胞10，11，12，13。除了这些有效的免疫刺激特性外，NDV还具有很强的安全性，并且具有完善的反向遗传学系统14，15。这些理想的特征促使在众多临床前和人体临床试验（NCT04871737，NCT01926028，NCT04764422）16，17中评估NDV。为了进一步发展这种非常有前途的免疫刺激病毒载体，需要详细的方法来生产和纯化可以在 体内安全施用的高滴度超纯NDV。

由于NDV是一种禽副粘病毒，因此最常在胚胎鸡蛋中扩增。虽然有基于细胞的系统可用于繁殖NDV，但大多数系统无法产生类似于胚胎鸡蛋18中达到的滴度。然而，在鸡蛋中生产NDV存在一些缺点，包括基于鸡蛋的生产冗长且不易扩展，采购大量SPF鸡蛋可能会有问题，并且存在鸡蛋过敏原污染的可能性13，18，19，20.最近，一组研究表明，在无血清培养基中悬浮液中生长的Vero细胞可以支持NDV的复制，其滴度与纯化21之前在鸡蛋中达到的滴度相当。然而，这需要病毒的连续传代以使病毒适应Vero细胞，并且仍然需要优化从悬浮液Vero细胞纯化NDV的方法21。

如前所述，用于纯化高滴度 体内级病毒的方法因问题22中的病毒而异。有一个完善的反向遗传学系统可用于产生重组NDV。该过程涉及使用cDNA克隆，辅助质粒和表达T7 RNA聚合酶的帮助病毒，先前已详细描述15，23。该方案可应用于致慢性或中源性 NDV。该方案中描述的病毒是一种重组中观NDV，编码来自 维多利亚 水母的绿色荧光蛋白（GFP），作为单独的转录单元插入病毒P和M基因之间，因为这以前被描述为外来转基因插入24的最佳位点。

封闭的方法根据其大小（范围为100至500nm）和密度15概述了NDV的纯化。这使得从接收卵到最终滴度，在大约3周内产生 体内级高滴度的NDV储液。描述了大规模生产基于鸡蛋的病毒时经常使用的技术，例如切向流过滤，深度过滤和密度梯度超速离心，从而使这些方法能够转化为更大规模的生产。前面描述的用于纯化NDV的技术已经通过掺入病毒稳定缓冲液，在密度梯度超速离心期间使用碘克沙醇以及描述各种质量控制措施来确保 体内级质量15而得到改进。这使得 体内级NDV的纯化达到3×10 10 PFU / mL从0.8至1.0L 的尿囊流体中达到3×PFU / mL。

Protocol

所有涉及使用动物的工作均由圭尔夫大学动物护理委员会根据加拿大动物护理委员会批准。所有工作均在加拿大的生物安全2级（BSL2）实验室进行，其中中胚层NDV是风险组2病原体。为了安全和无菌目的，NDV扩增和纯化所涉及的所有步骤都应在IIA型生物安全柜中进行。 1. 使用指定的无病原体胚胎鸡蛋扩增NDV 接种SPF胚胎鸡蛋注意：通常，八打SPF胚胎化的鸡…

Representative Results

收获尿囊液由于尿囊液是从胚胎鸡蛋中收获的，它应该看起来清晰透明。如果流体显示为不透明和黄色，则表示存在污染物。在纯化过程中加入这种尿囊流体会阻碍纯化过程，因为压力会迅速上升并超过10 psi，导致病毒剪切和传染性病毒的丢失。出现血性的尿囊液表明卵子接种了过多的病毒PFU。这批卵子的产量将大大低于预期，并且这种病毒不太可能 在体内使用。 接种有?…

Discussion

在临床前研究中用作治疗剂的病毒必须高度纯化，以避免在体内施用时的毒性15。如果不除去外来剂或污染物，这可导致严重的不良反应，否定病毒剂28的治疗效果。由于NDV是在胚胎化的鸡蛋中产生的，因此在临床前或临床模型29中，在体内使用之前必须去除几种污染的鸡蛋蛋白，例如卵清蛋白。即使经过强化纯化，卵清蛋白仍被?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

J.G.E.Y是安大略省兽医学院博士奖学金和安大略省研究生奖学金的获得者。这项工作由加拿大自然科学和工程研究委员会资助SKW（资助#304737）和LS（资助#401127）。

Materials

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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Tags

Keywords: Newcastle Disease VirusRecombinant VirusVirus PurificationTangential Flow FiltrationAllantoic FluidDepth FiltrationBuffer ExchangePressure ControlVirus Concentration
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Yates, J. G. E., Leacy, A., Pham, P. H., Zielinska, N., Tusnadi, E. A., Susta, L., Wootton, S. K. Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid. J. Vis. Exp. (183), e63817, doi:10.3791/63817 (2022).
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