Name: production and purification of non replicative canine adenovirus type 2 derived vectors
Uploaded: 2013-12-03T13:50:00
Description: Watch this Scientific Journal Video about Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors at JoVE.com

This work was supported financially by grants from INSERM, the CNRS, the Fondation pour la Recherche M&#233;dicale and the Agence Nationale pour la Recherche (ANR- 10-Blanc-1322) to D.G-D; The European Commission&#39;s Seventh Framework Program to B.K.; M.S. was supported by a postdoctoral fellowship from FRM (programme &#34;Equipe FRM 2009&#34;); C.B. was supported by a postdoctoral fellowship from UE, under grant agreement number 245266 (Orbivac).

1. Construction of a Nonreplicative CAV2 Genome Plasmid
Note: all plasmids described in this protocol have been described previously7 and are available upon request.
<ol>
	<li>Clone the GOI into the pShuttle plasmid, using appropriate restriction enzymes, to generate pShuttle-GOI (Figure 1A).</li>
	<li>Digest 2 &#956;g of pShuttle-GOI by AscI/PacI. The digestion releases the Shuttle-GOI fragment (~2.9 kbp + GOI ORF sequen...

<ol>
	<li>Kaufmann, J. K., Nettelbeck, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+chimeras+for+gene+therapy,+vaccination,+and+oncolysis:+adenoviruses+and+beyond.">Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond.</a> Trends Mol. Med. 18, 365-376 (2012).</li><li>Curr, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topics Microbiol. Immunol. 343, 195-224 (2010).</li><li>Zak, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merck+Ad5/HIV+induces+broad+innate+immune+activation+that+predicts+CD8(+)+T-cell+responses+but+is+attenuated+by+preexisting+Ad5+immunity.">Merck Ad5/HIV induces broad innate immune activation that predicts CD8(+) T-cell responses but is attenuated by preexisting Ad5 immunity.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3503-3512 (2012).</li><li>Paillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+non-human+adenoviruses+versus+human+adenoviruses.">Advantages of non-human adenoviruses versus human adenoviruses.</a> Hum. Gene Ther. 8, 2007-2009 (1997).</li><li>Ackermann, A., Guelzow, T., Staeheli, P., Schneider, U., Heimrich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+viral+dissemination+in+the+mouse+nervous+system,+using+a+green+fluorescent+protein-expressing+Borna+disease+virus+vector.">Visualizing viral dissemination in the mouse nervous system, using a green fluorescent protein-expressing Borna disease virus vector.</a> J. Virol. 84, 5438-5442 (2010).</li><li>Bru, T., Salinas, S., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+canine+adenovirus+type+2+and+its+vectors.">An update on canine adenovirus type 2 and its vectors.</a> Viruses. 2, 2134-2153 (2010).</li><li>Bouet-Cararo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+adenoviruses+elicit+both+humoral+and+cell-mediated+immune+responses+against+rabies+following+immunisation+of+sheep.">Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep.</a> Vaccine. 29, 1304-1310 (2011).</li><li>Soudais, C., Skander, N., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+transduction+of+neurons+throughout+the+rat+CNS+using+novel+helper-dependent+CAV-2+vectors.">Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors.</a> FASEB J. 18, 391-393 (2004).</li><li>Deinhardt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5+and+Rab7+control+endocytic+sorting+along+the+axonal+retrograde+transport+pathway.">Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway.</a> Neuron. 52, 293-305 (2006).</li><li>Soudais, C., Laplace-Builhe, C., Kissa, K., Kremer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+transduction+of+neurons+by+canine+adenovirus+vectors+and+their+efficient+retrograde+transport+in+vivo.">Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.</a> FASEB J. 15, 2283-2285 (2001).</li><li>Peltekian, E., Garcia, L., Danos, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotropism+and+retrograde+axonal+transport+of+a+canine+adenoviral+vector:+a+tool+for+targeting+key+structures+undergoing+neurodegenerative+processes.">Neurotropism and retrograde axonal transport of a canine adenoviral vector: a tool for targeting key structures undergoing neurodegenerative processes.</a> Mol. Ther. 5, 25-32 (2002).</li><li>Chartier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+recombinant+adenovirus+vectors+by+homologous+recombination+in+Escherichia+coli.">Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.</a> J. Virol. 70, 4805-4810 (1996).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> Am. J. Hyg. 27, 493-497 (1938).</li><li>Segura, M. M., Puig, M., Monfar, M., Chillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography+purification+of+canine+adenoviral+vectors.">Chromatography purification of canine adenoviral vectors.</a> Human Gene Ther. Methods. 23, 182-197 (2012).</li><li>Segura, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+PCR+assay+for+quantification+of+canine+adenoviral+vectors.">A real-time PCR assay for quantification of canine adenoviral vectors.</a> Journal of virological. 163, 129-136 (2010).</li><li>Fernandes, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocess+development+for+canine+adenovirus+type+2+vectors.">Bioprocess development for canine adenovirus type 2 vectors.</a> Gene Ther. , (2012).</li><li>Rinne, A., Littwitz, C., Bender, K., Kienitz, M. C., Pott, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+delivery+of+short+hairpin+RNA+(shRNA)+mediates+efficient+gene+silencing+in+terminally+differentiated+cardiac+myocytes.">Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.</a> Methods Mol. Biol. 515, 107-123 (2009).</li><li>Huang, B., Kochanek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+silencing+of+huntingtin+expression+by+shRNA.">Adenovirus-mediated silencing of huntingtin expression by shRNA.</a> Hum. Gene Ther. 16, 618-626 (2005).</li><li>Tordo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+adenovirus+based+rabies+vaccines.">Canine adenovirus based rabies vaccines.</a> Dev. Biol. 131, 467-476 (2008).</li></ol>

The simple method described herein, adapted from the well-documented Ad5 technology, allows the efficient production of nonreplicative CAV2 vectors with a typical cloning capacity of ~4.2 kbp. Regarding time considerations, a recombinant CAV2 genome is usually generated in two weeks, whereas it will take an additional 5 weeks to amplify, purify and titer viral suspensions.
Although the production of the first batch of CAV2-derived vectors may appear time-consuming, it should be reminded that C...

Over the past decades, adenoviruses (Ads) derived vectors have proven their efficacy for gene therapy and vaccination, as well as in oncolytic virotherapy 1. Ads are nonenveloped icosahedral viruses of the Adenoviridae family, which have been isolated from mammals, birds, reptiles, amphibians and fish. Since their discovery in 1953, Ads have been intensively studied as models to study virus/cell interactions and, more recently, as vector-based gene delivery systems 2. Indeed, Ads-based vectors offer many advantages, such as their broad host range and well-characterized biology, together with the ease with which they can be genetically manipulated and amplified for large-scale production.
In order to overcome clinical difficulties related to pre-existing immunity in human populations towards vectors derived from human Ads 3, we and others began to derive vectors from nonhuman Ads 4. In addition, nonhuman adenoviral vectors appear to be more adapted for mass vaccination in veterinary medicine than the classically used adenovirus type 5 (Ad5), since they should be more compliant with safety and security requirements during the risk assessment by the national regulatory authorities. In the late 1990s, we described the first recombinant CAV2 as a nonhuman alternative to vectors derived from Ad5 5. Since then, numerous studies have confirmed the potential of CAV2 vectors for therapeutic or antigenic gene transfer (for reviews 6,7). Like other Ads, CAV2-derived vectors are stable and can be produced at high titers, which facilitates their in vivo use. They are also safe, since they are not integrative, although they allow long lasting transgene expression in vivo (&#62;1 year) 8. Remarkably, and better than human Ads serotypes, CAV2 vectors have been shown to be highly neurotropic, with a very efficient retrograde transport in axons 9,10. This intrinsic property brought the idea of using recombinant CAV2 vectors to transduce neurons of specific brain areas that are hardly accessible to lentiviral vectors, so far commonly used for gene transfer into the central nervous system 11.
Here, we describe a simple and classical protocol to construct, produce and purify nonreplicative CAV2 vectors derived from the Manhattan strain. Recombinant CAV2 genomes are constructed by cloning the desired gene of interest (GOI) in a cassette downstream of the cytomegalovirus (CMV) early promoter, which provides a ubiquitous expression. CAV2 vectors have a cloning capacity of ~4.2 kbp, which allows expressing large cDNA. In a second step, this expression cassette is inserted by homologous recombination in place of the E1 region of the CAV2 genome, leading to a nonreplicative virus (dlE1), as described initially by Chartier et al.12&#160;Finally, viral particles are produced upon transfection of the genomic plasmid into a CAV2-E1-expressing canine kidney cell line (DK-E1) and are serially amplified prior to concentration and purification of viral stocks. The method described here uses a two-step ultracentrifugation in cesium chloride (CsCl) gradients, which enables enrichment of infectious particles with a high final titer (usually over 1010 infectious particles per ml). The viral suspension is then desalted by chromatography through disposable Sephadex G-25 columns so that it is ultimately pure enough to be directly used in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Restriction enzymes</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:112px;">Various</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Jet Prime transfection kit</td>
			<td style="width:188px;">Polyplus</td>
			<td style="width:112px;">114-01</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">DMEM</td>
			<td style="width:188px;">Gibco (Life Technologies)</td>
			<td style="width:112px;">10567014</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">E. coli BJ5183 bacteria</td>
			<td style="width:188px;">Agilent</td>
			<td style="width:112px;">200154</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">PD-10 desalting column</td>
			<td style="width:188px;">GE Healthcare</td>
			<td style="width:112px;">17-0851-01</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">EmeraldAmp polymerase</td>
			<td style="width:188px;">Takara</td>
			<td style="width:112px;">RR320A</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">Nucleospin plasmid kit</td>
			<td style="width:188px;">Macherey-Nagel</td>
			<td style="width:112px;">740558.250</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">NucleoSpin Gel and PCR Clean-up</td>
			<td style="width:188px;">Macherey-Nagel</td>
			<td style="width:112px;">740609.50</td>
			<td style="width:112px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:200px;">14 ml tubes, Ultra Clear</td>
			<td style="width:188px;">Beckman</td>
			<td style="width:112px;">344060</td>
			<td style="width:112px;"></td>
		</tr>
	</tbody>
</table>

production and purification of non replicative canine adenovirus type 2 derived vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

Production of recombinant CAV2 vectors relies on basic but important molecular biology techniques. Prior to the production of viral vectors, it is indeed necessary to construct a recombinant CAV2 genome bearing an expression cassette for the transgene. This construction involves two steps: first, the gene of interest (GOI) is cloned in a shuttle plasmid as an expression cassette (i.e.&#160;with promoter and polyadenylation signal). This functional element is flanked by two CAV2-derived genomic sequences, represe...

Watch this Scientific Journal Video about Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors at JoVE.com

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Produktion og oprensning af ikke replikativ Canine adenovirus type 2-afledte vektorer

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Research

JoVE Journal

Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

Produktion og Titering af rekombinant adeno-associeret virale vektorer

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Effektiv Rekombinant Parvovirus Produktion ved hjælp af adenovirusafledte Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Bone Marrow-derived Macrophage Production

Bone Marrow-afledt Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Isolering af Viral Replication Rum-berigede underentreprise nukleare fraktioner fra Adenovirus-inficerede Normal humane celler

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

A Simple Fluorescens Assay for Kvantificering af Canine Neutrofil Ekstracellulær Trap Frigivelse

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Enkel og effektiv produktion og oprensning af Mouse Myelin oligodendrocyt Glycoprotein for Eksperimentel Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

En avanceret murine model for alkoholfri steatohepatitis i forbindelse med type 2 diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Rensning af ekstracellulære vesikulære præparater med høj udbytte afstand fra virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...