We thank the team of the DKFZ virus production and development unit, in particular Marcus M&uuml;ller, Silvia M&uuml;nstermann, Barbara Liebetrau, and Mandy Roscher. This study has been partly supported by grants from the Federal Ministry of Education and Research (BMBF) and the Helmholtz Association in the framework of the Deutsches Krebsforschungszentrum / Canc&eacute;rop&ocirc;le du Grand-Est joint Programme in Applied Tumour Virology.

Note that a laboratory with a biosafety level 2 is required for the production of recombinant parvoviruses (recPV).
The protocol is subdivided in two main parts. The first part (production of recPV via transfection) is required to produce the minimal amount of recPVs that serves as inoculum in the second part of the protocol (production of recPVs via infection). Once a small amount of recPVs is produced, the first part of the protocol can be omitted, and the recombinant parvovirus can be amplified only via infection providing the gene encoding for the parvovirus capsid proteins through the adenovirus helper (see below).
1. Production of recPVs via Transfection
1.1 Viral DNA transfection
It is possible to use any established DNA transfection method in this section of the protocol either based on cationic lipids or calcium phosphate. We generally use Fugene HD Transfection Reagent. To control transfection efficiency, we recommend to transfecting additional plate with a plasmid expressing enhanced Green Fluorescent Protein (e.g. pEGFP-N1, Clontech). Transfection is considered efficient and optimal for virus production when at least 50% of the cell population results EGFP-positive 24 hours after transfection.
<ol>
	<li>1 day before transfection, seed 2 million HEK293T cells in a 10 cm plate, to obtain 50% cellular confluency at the day of transfection. Grow cells in 10 ml of Dulbecco's Modified Eagle Medium (DMEM) with addition of 10% foetal bovine serum, 2 mM L-glutamine, 100 U per ml Penicillin and 100 &mu;g/ml Streptomycin.</li>
	<li>Prepare virus-making mixture (1:2:1 molar ratio) in a 1.5 ml tube:
	<ol class="lalpha">
		<li>3 &mu;g of recPV vector harbouring the transgene of interest (e.i. phH-1-GFP6).</li>
		<li>6 &mu;g of vector pCMV/VP5 harbouring the parvovirus capsid gene unit</li>
		<li>8 &mu;g of adenovirus-derived helper plasmid pXX67.</li>
	</ol>
	</li>
	<li>Dilute the plasmids mix with serum-free medium up to 850 &mu;l (final concentration of 20 ng/&mu;l).</li>
	<li>Add 42.5 &mu;l Fugene HD (2.5:1 ratio &mu;l Fugene: &mu;g DNA). Do not touch side of tube when adding Fugene to the medium.</li>
	<li>Vortex gently for 5-10 seconds.</li>
	<li>Incubate the mixture at room temperature for 30 minutes.</li>
	<li>Add the transfection mixtures drop-wise to the cells. Shake the plate gently to evenly distribute the mixture throughout.</li>
</ol>
1.2 Virus production
<ol>
	<li>Incubate the plate at 37 &deg;C with 92% humidity and 5% CO2 for 72 hours before harvest. During this period progeny parvovirus particles are formed from the parvovirus plasmids.</li>
</ol>
1.3 Virus harvest
<ol>
	<li>In a tissue culture hood, scrape the cells within their medium, aspirate and transfer the suspension from the plate into a 15 ml plastic tube.</li>
	<li>Centrifuge the tube at 250 x g for 5 min at room temperature.</li>
	<li>Remove 95% of the supernatant.</li>
	<li>Vortex the tube gently to resuspend the pellet into the remaining supernatant (about 0.5 ml).</li>
	<li>Freeze the tube at -80 &deg;C. It is possible to stop the production at this stage or to continue with the protocol.</li>
	<li>Defrost the suspension and bring it at room temperature.</li>
	<li>Freeze the suspension in a liquid nitrogen bath and thaw in warm water at 37 &deg;C. Vortex the tubes vigorously for 15 sec and repeat the freeze-thaw cycle further two times.</li>
	<li>Centrifuge the tubes at 16,000x g for 15 min at 4 &deg;C.</li>
	<li>Collect the supernatant into a fresh tube. The tube at this stage contains the crude virus extract preparation.</li>
	<li>Proceed with the virus titration (see below). For the recPV carrying the GFP gene, a typical production should give 1-3 x 107 viral infectious units corresponding to about 1-3 x 1010 viral genome containing particles.</li>
</ol>
1.4 Virus storage
<ol>
	<li>For long-term storage freeze the tube containing the crude virus extract at -80 &deg;C. It is possible to use crude viral extracts as inoculum in the second part of the protocol.</li>
</ol>
2. Production of recPVs via Infection
Before starting with the recPV production via infection, produce and purify Adenovirus 5 carrying the parvovirus VP gene (Ad-VP helper, described in5) according to standard protocol8. It is also necessary to have a minimal amount of recPVs carrying the transgene of interest (e.g., produced via transfection as described above).
Below, we describe the recPV production in 175 cm2 flask (T175) format. Further amplification of the virus stock produced in this way can be conducted in 10 multilayer CellSTACK culture chambers (Corning) with minor adjustments of the protocol proposed.
2.1 Infection
<ol>
	<li>1 day before infection, plate 10 million NB324K cells in a T175 flask to obtain 60-80% cellular confluency at the day of infection. Prepare a second flask containing the same number of cells as a control (non-infected cells). Each T175 should have 20 ml of Minimum Essential Medium (MEM) as a final volume, supplemented with 5% foetal bovine serum, 2 mM L-glutamine, 100U per ml Penicilin and 100 &mu;g/ml Streptomycin.</li>
	<li>The next morning prepare virus mixture in a 2 ml plastic tube consisting of:
	<ol class="lalpha">
		<li>Ad-VP-helper5 at multiplicity of infection (MOI, infectious units/cell) = 10 (corresponding to 1 x 108 infectious units);</li>
		<li>recPVs at MOI = 0.5 (corresponding to 5 x 106 infectious units). Complete to 1.5 ml with MEM.</li>
	</ol>
	</li>
	<li>Apply the entire virus mixture in one of the T175 flask.</li>
	<li>Incubate the flask for 2 hr at 37 &deg;C, 92% humidity, 5% CO2 gently shaking every 15 min.</li>
	<li>Incubate for further 20-24 hr at 37 &deg;C, 92% humidity, 5% CO2.</li>
	<li>Replace the cultural medium with fresh medium.</li>
</ol>
2.2 Virus production
Incubate the flasks at 37 &deg;C with 92% humidity and 5% CO2 for 48 hours. As indication of efficient viral production, clear signs of cytotoxicity should be observed in the flask containing viral infected cells, starting 36-48 hours post-infection.
2.3 Virus harvest
<ol>
	<li>In a tissue culture hood, aspirate and transfer the medium supernatant from the flask into a 50 ml centrifuge tube.</li>
	<li>Wash the cells twice with 1.5 ml PBS.</li>
	<li>Trypsinize the cells with 1.5 ml of 0.25 % Trysin-EDTA. Add the cell suspension to the removed medium.</li>
	<li>Centrifuge the cells and the supernatant at 5,000 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant.</li>
	<li>Add 1 ml of TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.7) to the cell pellet and gently vortex the tube to resuspend the cells.</li>
	<li>Freeze the cell suspension in a liquid nitrogen bath and thaw in warm water at 37 &deg;C. Vortex the tube vigorously for 15 sec and repeat the freeze-thaw cycle three times.</li>
	<li>Treat the cell suspension with 50 U/ml Benzonase Nuclease for 30 min at 37 &deg;C in order to digest the cell genomic and non-packaged viral DNAs.</li>
	<li>Centrifuge the tube at 5,000 x g for 20 min at 4 &deg;C.</li>
	<li>Collect the supernatant into a fresh tube. The supernatant at this stage contains the crude virus extract preparation. Typical yields of recPV-GFP produced following this protocol should be in the range of 1-10 x 108 viral infectious units.</li>
</ol>
2.4 Virus storage
<ol>
	<li>For short term storage (maximum 2 months), store virus at 4 &deg;C.</li>
	<li>For long term storage (longer than 2 months), store virus at -80 &deg;C.</li>
</ol>
3. RecPV Purification
<ol>
	<li>For the purification of recPVs from the crude lysates, we recommend to run an Iodixanol discontinous gradient according to Zolotukhin et al. 9. We recommend using a minimum of 5 ml of crude viral extract (obtained from production in five T175 flasks) for efficient virus purification.</li>
</ol>
4. Recombinant Parvovirus Titration
<ol>
	<li>Perform recPV titration as described in El-Andaloussi et al.5</li>
</ol>
5. Quality Controls
<ol>
	<li>Use the protocol described in El-Andaloussi et al.5 to check for the presence of undesired replication competent viruses (RCV) carrying out parvovirus plaque assay in NB324K indicator cells.</li>
</ol>
6. Representative Results
An example of recPVs production via transfection in the presence or absence of adenovirus genomic elements is shown in Figure 3. Cells were transfected with pCMV/VP (plasmid carrying the gene encoding for the VP parvovirus capsid proteins) together with phH-1-GFP (a recPV harbouring the GFP gene) or phH-1-luciferase (a recPV harbouring the firefly luciferase gene), with (+ pXX6) or without (-pXX6) the pXX6 plasmid (carrying the adenovirus E2A, E4(orf6) and VA RNA genes). Equal volume of crude cell extracts were applied to NB324K cells and GFP transduction or luciferase assays were performed as reported in El-Andaloussi et al.5. A clear increase in the recPVs production was obtained in the presence of pXX6 with production increasing from 0.3 GFP transductional units (TU)/cell obtained according to conventional protocols to approximately 5 TU/cell obtained following our method (Fig. 3A, B). A significant increase (about 24-fold) of recPV production was also observed in the case of phH-1-luciferase (Fig. 3C).These results indicate that the genetic material contained in pXX6 is able to boost parvovirus production.
A representative example of recPVs production via infection is shown in Figure 4. NB324K cells were co-infected with various recPVs (as indicated in the figure) and Ad-VP-helper (harbouring the gene encoding for the PV VP capsid proteins). Ad-VP helper further enhanced recPV production up to &gt;70 TU per seeded cell (Fig. 4A) without increasing the occurrence of undesirable replication competent viral particles (Fig. 4B).
<img alt="Figure 1" src="/files/ftp_upload/3518/3518fig1.jpg" /> 
Figure 1. Vectors based on autonomous parvoviruses. (A) Top: The transgene replaces part of the VP-coding genes and is under the control of the viral promoter P38. The genes for NS1/2 are retained and their expression is controlled by the viral promoter P4. The vector genome is flanked by the parvoviral ITRs, which contain cis-acting elements that are required for replication and packaging of the recombinant genome. Bottom: A plasmid carrying the VP-gene under either a heterologous (e.g. CMV), or autologous (e.g. P38) promoter (Px), is supplied in trans during recombinant parvovirus production in order to compensate for the disruption of the structural genes in the recombinant genome. ITR, inverted terminal repeat. Figure adapted from 4. (B) Schematic view of the classical protocol used for the production of recPVs. HEK293T cells are transiently transfected with viral DNA (vector and helper plasmids) and after three days, cells are collected and viruses harvested.
<img alt="Figure 2" src="/files/ftp_upload/3518/3518fig2.jpg" /> 
Figure 2. Production of recPVs with the help of the Ad-VP. (A) Schematic maps of recPV and Ad-VP genomes. The recPV contains a heterologous transgene which replaces part of the VP region. The Ad-VP harbors the parvovirus VP gene. (B) Schematic view of the protocol described in this manuscript. NB324K cells are co-infected with recPV and Ad-VP viruses. After three days cells are harvested and recPV particles recovered from cell lysate.
<img alt="Figure 3" src="/files/ftp_upload/3518/3518fig3.jpg" /> 
Figure 3. Stimulation of recPV production by adenovirus-based plasmids pXX6 . HEK293T cells, seeded in 10 cm dishes, were transfected with pCMV/VP in combination with phH-1-GFP (A and B) or phH-1-luciferase (C) to produce recPVs. Simultaneously, cells were co-transfected with the adenovirus-derived helper plasmids pXX6 or not, as indicated. Three days post-transfection, cells were harvested and lysed by three freeze and thaw cycles. Equal volumes of crude virus extracts were applied to NBK indicator cells, and transduction assays were carried out. (A) Representative micrographs showing GFP positive cells within confluent NB324K monolayers. (B) Quantification of the GFP transduction assays expressed in transduction unit (TU) per seeded cell. (C) Quantification of the luciferase activity expressed as relative luciferase units (RLU). The luciferase assay was performed as described in El-Andaloussi et al.5. Columns represent average values from three replicates with standard deviation bars. Number on top of the + pXX6 column, in (B and C), indicates the fold increase in the recPV virus titers, obtained in the presence of pXX6 versus without.
<img alt="Figure 4" src="/files/ftp_upload/3518/3518fig4.jpg" /> 
Figure 4. Stimulation of recPV production by means of recombinant Ad-VP helper virus . (A) NB324K cells, were infected with purified Ad-VP helper virus at MOI of 10 (Ad-X unit/cell, titrated with Adeno-X Rapid Titer Kit), and then superinfected with either of the following recombinant parvovirus Chi-hH-1-EGFP11 (0.1 TU/cell) or H-1-GFP (0.5 TU/cell)5. One day post-infection, medium was changed and two days later, cells were collected and lysed via three freeze-and-thaw cycles. Crude cell extracts were used to determine virus titers by transduction assay according to El-Andaloussi et al.5. TU, transduction unit. (B) Viral batches produced in the presence of absence of Ad-VP were analyzed for their content of replication competent viral particles (RCV) by plaque assay on NB324K indicator cells.

<ol>
	<li>Cotmore, S. F., Tattersall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvoviral+host+range+and+cell+entry+mechanisms.">Parvoviral host range and cell entry mechanisms.</a> Adv. Virus. Res. 70, 183 (2007).</li><li>Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+parvoviruses+as+cancer+therapeutics.">Oncolytic parvoviruses as cancer therapeutics.</a> Cytokine Growth Factor Rev. 21, 185 (2010).</li><li>Hristov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+Its+Nonstructural+Protein+NS1,+Parvovirus+H-1+Induces+Apoptosis+via+Accumulation+of+Reactive+Oxygen+Species.">Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species.</a> J. Virol. 84, 5909-5909 (2010).</li><li>Cornelis, J. J., Salome, N., Dinsart, C., Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+based+on+autonomous+parvoviruses:+novel+tools+to+treat+cancer.">Vectors based on autonomous parvoviruses: novel tools to treat cancer.</a> J. Virol. 6, 193-193 (2004).</li><li>El-Andaloussi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adenovirus-based+helper+system+to+support+production+of+recombinant+parvovirus.">Novel adenovirus-based helper system to support production of recombinant parvovirus.</a> Cancer Gene Ther. 18, (2011).</li><li>Kestler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis+requirements+for+the+efficient+production+of+recombinant+DNA+vectors+based+on+autonomous+parvoviruses.">cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.</a> Hum. Gene. Ther. 10, 1619-1619 (1999).</li><li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J. Virol. 72, 2224 (1998).</li><li>He, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+system+for+generating+recombinant+adenoviruses.">A simplified system for generating recombinant adenoviruses.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 2509 (1998).</li><li>Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+purification+using+novel+methods+improves+infectious+titer+and+yield.">Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.</a> Gene. Ther. 6, 973 (1999).</li><li>Maxwell, F., Harrison, G. S., Maxwell, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+production+of+recombinant+AAV+by+transient+transfection+of+NB324K+cells+using+electroporation.">Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation.</a> J. Virol. Methods. 63, 129 (1997).</li><li>Wrzesinski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+and+pseudotyped+parvoviruses+minimize+the+contamination+of+recombinant+stocks+with+replication-competent+viruses+and+identify+a+DNA+sequence+that+restricts+parvovirus+H-1+in+mouse+cells.">Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.</a> J. Virol. 77, 3851 (2003).</li></ol>

We have nothing to disclose.

We have shown that recPV production can be enhanced by the presence of adenoviral genomic elements. We have increased the recPV yields by more than 10 fold (from 0.3 to 5 TU/cell) by providing adenovirus genomic element via transfection and by more than 100 fold co-infecting the cells with Ad-VP-helper in combination with the recPV in comparison to conventional protocols. The protocol described here can be further optimized by determining the most appropriate timing for the delivery of the adenovirus genomic elements and...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM </td>
 <td>Sigma </td>
 <td>D5796</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>Sigma</td>
 <td>M4655</td>
 </tr>
 <tr>
 <td>Foetal Bovine Serum</td>
 <td>PAA</td>
 <td>A15-101</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Gibco</td>
 <td>25030-024</td>
 </tr>
 <tr>
 <td>Fugene</td>
 <td>Roche</td>
 <td>047097050001</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Invitrogen</td>
 <td>25300062</td>
 </tr>
 <tr>
 <td>Benzonase Nuclease</td>
 <td>Sigma</td>
 <td>E8263</td>
 </tr>
 <tr>
 <td>Adeno-X Rapid Titer Kit </td>
 <td>Clontech</td>
 <td>632250</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

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

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 ...

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10.9K Views.  German Cancer Research Center (DKFZ). 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).

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

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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 ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.