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. 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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.

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	<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>
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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.

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 ...

production-purification-non-replicative-canine-adenovirus-type-2

Research

JoVE Journal

Immunology and Infection

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. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

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

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

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

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

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

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 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

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

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

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

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

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

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

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

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

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

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

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

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 setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

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 ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.