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 sequence) from the plasmid backbone (2.8 kbp).</li>
	<li>Purify the Shuttle-GOI fragment after agarose gel electrophoresis, using NucleoSpin Gel and PCR Clean-up kit.</li>
	<li>In parallel, digest 1 &#956;g of pCAV2 genomic plasmid by SwaI. The pCAV2 plasmid contains a unique SwaI site located within the E1 region (Figure 1B). Heat-inactivate this restriction enzyme at 65 &#176;C for 20 min.</li>
	<li>Transform competent E. coli BJ5183 bacteria with 500 ng of purified Shuttle-GOI fragment and 100 ng of linearized pCAV2 plasmid for homologous recombination (Figure 1B). Spread on LB-Amp plates. Incubate plates at 37 &#176;C for at least 20 hr.</li>
	<li>Perform PCR analysis directly on bacterial colonies using EmeraldAmp master mix with forward (5&#39;-CACGAGGCCCTTTCGTCTTCAA-3&#39;) and reverse (5&#39;-GCGGTAGTTTATCACAGTTAAATTGC-3&#39;) primers, under the following cycling conditions: 10 min at 95 &#176;C (for one cycle), 95 &#176;C for 1 min; 58 &#176;C for 1 min; 72 &#176;C for 1 min/kbp (for 39 cycles), and finally 5 min at 72 &#176;C. A product of 1 kbp + GOI ORF sequence is amplified from pCAV-GOI positive clones, whereas no PCR product can be detected with negative clones.</li>
	<li>Inoculate at least two positive colonies overnight each in 5 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm).</li>
	<li>Extract plasmid DNAs using Nucleospin plasmid kit. Transform competent E. coli DH5&#945; bacteria with an aliquot of each DNA prep. Spread bacterial suspension on LB-Amp plates and incubate overnight at 37 &#176;C.</li>
	<li>Inoculate two colonies of each positive clone overnight in 3 ml LB-amp medium at 37 &#176;C in a shaker (220 rpm). Extract plasmid DNA using Nucleospin plasmid kit.</li>
	<li>Digest the resulting DNA and the parental pCAV2 plasmid (as a control) with NdeI. The restriction pattern of parental pCAV2 is 14.8 kbp, 9 kbp, 7.9 kbp, and 1.6 kbp. The GOI expression cassette is inserted within the 7.9 kbp fragment and contains at least one NdeI site, i.e. one in the cassette and the potential sites present in the GOI.</li>
	<li>Select positive constructions and proceed to step 2 with at least 2 independent clones.</li>
</ol>
2. Transfection of Recombinant CAV2 Genome Plasmid into DK-E1 Cells
Note: CAV2-derived non replicative vectors are genetically modified organisms classified as Biosafety Level 2 (BSL-2), whereas their use on animal models is classified as BSL-1. Please use proper containment and waste handling measures, including personal protective equipment, and work under BSL-2 laminar flow hoods.
<ol>
	<li>A day before transfection, seed DK-E1 cells on a 6-well plate. Grow cells at 37 &#176;C and 5% CO2, in Dulbecco&#39;s Modified Eagle Medium (DMEM) with high glucose, containing 7% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, and 100 U/ml penicillin/100 &#956;g/ml streptomycin. Cells should be 70-80% confluent for transfection.</li>
	<li>Digest 2 &#956;g of pCAV-GOI plasmid with AscI, to release the nonviral sequence of the plasmid. Check the resulting restriction pattern by 0.8% agarose gel electrophoresis. Digestion should yield a ~31 kbp fragment containing the recombinant genome and a 2 kbp fragment corresponding to the plasmid backbone.</li>
	<li>Mix digested DNA with 200 &#956;l of Jet Prime Buffer by vortexing for 10 sec. Add 4 &#956;l of Jet Prime and mix by vortexing for 10 sec. Spin briefly to remove droplets and incubate for 10 min at room temperature.</li>
	<li>Add the transfection mix dropwise onto cells. Gently shake plates to evenly distribute the mix and incubate at 37 &#176;C.</li>
</ol>
3. Propagation of Recombinant CAV2
<ol>
	<li>One week after transfection, collect cells and culture medium in a 15 ml polypropylene tube. Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C) and clear the lysate by centrifugation for 10 min at 1,800 x g.</li>
	<li>Collect supernatant and store at -20 &#176;C for subsequent virus propagation.</li>
	<li>Infect an 80-90% confluent monolayer of DK-E1 cells grown in one well of 6-well plate with 0.5 ml of the virus-containing supernatant. Incubate at 37 &#176;C for 1 hr, under mild agitation. Remove inoculum and replace it with 1.5 ml of complete DMEM containing 5% heat-inactivated FCS.</li>
	<li>Incubate cells at 37 &#176;C for 3-4 days. Cells should be monitored daily for appearance of a cytopathic effect (CPE, Figures 2A-B). If no clear CPE is visible, harvest cells after 4 days of culture and repeat steps 3.1-3.4.</li>
	<li>When a clear CPE affects the majority of cells, usually after 2-4 viral amplification rounds, collect cells with culture medium and freeze-thaw 3x as described above. Infect 80-90% confluent DK-E1 cells grown in three 10 cm diameter tissue culture dishes, using each time 0.5 ml of virus-containing supernatant.</li>
	<li>After 3-4 days (when CPE is complete), harvest cells from these 10 cm dishes, again disrupt them by three freeze-thaw cycles, clear the lysate by centrifugation for 10 min at 1,800 x g, collect supernatant and store at -20 &#176;C for large-scale CAV2 amplification.</li>
</ol>
4. Large-scale CAV2 Purification
<ol>
	<li>Infect 80-90% confluent monolayers of DK-E1 cells grown in forty 10 cm diameter tissue culture dishes. For each dish, use 0.1 ml of virus-containing supernatant diluted in 1 ml of complete DMEM without FCS. Incubate cells at 37 &#176;C for 3 to 4 hr under mild agitation. Add 5 ml of complete DMEM supplemented with 5% FCS.</li>
	<li>Incubate for 3 days. Harvest infected cells from the forty 10 cm dishes in 50 ml polypropylene tubes. Pellet cells at 1,200 x g for 10 min at 4 &#176;C and resuspend them in 15 ml of DMEM medium.</li>
	<li>Disrupt cells by three freeze-thaw cycles (-80 &#176;C/37 &#176;C). Remove cell debris by centrifuging at 1,800 x g for 10 min and store supernatant at -20 &#176;C prior to purification of viral particles.</li>
	<li>Prepare a discontinuous CsCl gradient in a 14 ml Ultra-Clear tube. First, pour 2 ml of CsCl solution (1.40 g/ml&#160;in phosphate buffered saline) at the bottom of the tube. Then, add slowly 2 ml of CsCl solution (1.25 g/ml&#160;in phosphate buffered saline) on top of the first solution.</li>
	<li>Load carefully the viral supernatant on top of the CsCl gradient. Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 1.5 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, two white bands are clearly visible at the interface between the 2 CsCl solution layers (Figure 2C). Using a 21 G needle and a syringe, collect by side puncture the lower band containing mature CAV2 particles. Try as much as possible to avoid collecting the upper band, which corresponds to empty viral particles.</li>
	<li>Prepare a continuous CsCl gradient in a 14 ml transparent tube by mixing the collected viral particles with a CsCl solution (1.34 g/ml&#160;in phosphate buffered saline). Fill the tube with mineral oil up to 2-3 mm from the top. Centrifuge for 18 hr at 130,000 x g and 18 &#176;C in a swinging SW40 rotor.</li>
	<li>After centrifugation, collect the white CAV2-containing band by side puncture with a syringe as described above. Again, try to avoid collecting the remaining upper band. This should be easier in this continuous gradient, which separates the two bands better. The total volume of collected suspension should not exceed 2 ml.</li>
	<li>Equilibrate a Sephadex G-25 PD-10 column with 30 ml of PBS.</li>
	<li>Load the viral suspension and discard the flow-through.</li>
	<li>Elute with fractions of 500 &#956;l of PBS. Collect fractions 5-7, which usually contain the desalted CAV2 particles. The virus-containing fractions can easily be identified because they are opalescent. Add 150 &#956;l of glycerol to the 1.5 ml of CAV2 suspension and store in aliquots at -80 &#176;C.</li>
</ol>
5. Titration of CAV2 by End-point Dilution
<ol>
	<li>Thaw an aliquot of purified virus on ice and perform 10-fold serial dilutions (ranging from 10-2-10-12) in serum-free DMEM.</li>
	<li>Add 50 &#956;l of each viral dilution in 5 wells of a 96-well plate. Add 1.5 x 104 DK-E1 cells in each well. Incubate the plate at 37 &#176;C and 5% CO2 for 5 days.</li>
	<li>At day 5 post-infection, monitor CPE appearance by microscopic observation (Figures 2A-B). Infectious titers are determined as median tissue culture infectious doses (TCID50), using the Reed and Muench statistical method13.</li>
</ol>

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Authors have nothing to disclose.

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...

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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 ...

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Immunology and Infection

7.0K ビュー.  Université Toulouse 3. These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro 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.

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

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.

組換えアデノ随伴ウイルスベクターの産生と力価測定

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

免疫・感染学

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).

アデノウイルス由来のシステムの助けを借りて効率的な組換えパルボウイルスの生産

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.

骨髄由来マクロファージの生産

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

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

アデノウイルス血清型5ベクターワクチンアプローチの開発のための抗原キャプシド取り込み戦略を活用

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

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.

アデノウイルスに感染したヒト正常細胞からのウイルス複製コンパートメントに富むサブ核画分の単離

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.

イヌ好中球細胞外トラップリリースの定量化のためのシンプルな蛍光アッセイ

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.

実験的自己免疫性脳脊髄炎研究のためのシンプルで効率的な生産とマウスミエリンオリゴデンドロサイト糖タンパク質の精製

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

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

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.

2型糖尿病と関連した非アルコール性脂肪性肝炎のための高度なマウスモデル

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.

ウイルスから離れた高収率細胞外小胞製剤の精製

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 ...