We thank past and present members in JCT and LM-S laboratories for the development and improvement of the arenavirus reverse genetics techniques and plasmids. We also thank Snezhana Dimitrova for technical support. The monoclonal antibody directed to JUNV NP (SA02-BG12) was obtained from BEI Resources (NIAID Biodefense and Emerging Infections Research Resources Repository). BYHC was supported by Grant Number GM068411 from Institutional Ruth L. Kirschstein National Research Service Award. EO-R is a Fullbright-Conicyt (BIO 2008) and a Rochester Vaccine Fellowship (2012) recipient. EO-R current support is provided by a postdoctoral Fellowship for Diversity &amp; Academic Excellence from the Office for Faculty Development &amp; Diversity at the University of Rochester. Research in LM-S laboratory is funded by the NIH grants RO1 AI077719, R21NS075611-01, R03AI099681-01A1, the NIAID Centers of Excellence for Influenza Research and Surveillance (HHSN266200700008C), and The University of Rochester Center for Biodefense Immune Modeling (HHSN272201000055C).
Research at JCT laboratory was supported by grants RO1 AI047140, RO1 AI077719, and RO1 AI079665 from NIH.

1. Arenavirus Rescue Transfection
Our rescue system was based on the use of both polymerase II protein expression plasmids encoding the nucleoprotein (NP) and RNA-dependent-RNA-polymerase (L), the viral trans-acting factors required for RNA replication and gene expression of the arenavirus genome 7, 19 , and plasmids capable to direct intracellular synthesis, via the cellular RNA polymerase I (pol-I), of the S and L antigenome RNA species 7 . In our studies we use the pCAGGs protein expression plasmid, which uses the chicken &beta;-actin promoter and polyadenylanation (pA) signal sequences, and the human pol-I plasmid, which uses the human polymerase I promoter and murine terminator sequences (Figure 2). The S and L RNA segments are cloned into the hpol-I plasmid in an antigenomic orientation to allow for the generation of genomic RNA segments upon transcription by hpol-I. For the rescue of wild-type recombinant LCMV (rLCMV) and Candid#1 (rCandid#1) the pCAGGs NP and L from each virus were co-transfected together with their respective human pol-I S and L RNA segments (Figure 3). Generation of recombinant trisegmented LCMV (r3LCMV) and Candid#1 (r3Candid#1) followed a similar protocol but the S segment was split into two different pol-I S plasmids, each encoding a distinctive reporter gene: in one, the NP open reading frame (ORF) was replaced with the Green Fluorescent Protein (GFP) reporter gene (hpol-I S GFP/GP) and, in the other, the viral glycoprotein precursor (GPC) ORF is replaced with the Gaussia luciferase (Gluc) reporter gene (hpol-I S NP/Gluc). A schematic representation of the protocol is illustrated in Figure 4. The following transfection and infection protocol has been established for 6-well-plates.
<ol>
	<li>OptiMEM-Lipofectamine 2000 (LPF2000) mixture: Prepare 250 &mu;l of OptiMEM media and, depending on the virus rescue, 10 - 12 &mu;g of LPF2000 (1 &mu;g/&mu;l) per transfection (Table 1). A ratio of 2.5 &mu;g LPF2000 per &mu;g of DNA is recommended. Incubate for 5 - 10 min at RT. Meanwhile, prepare the plasmid transfection mixture in a separate microcentrifuge tube (step 1.2).</li>
	<li>Plasmid DNA transfection mixture: Prepare the plasmid transfection cocktail in a microcentrifuge tube using the recommended amounts for each virus rescue (see Table 1). Bring the final volume to 50 &mu;l with OptiMEM.</li>
	<li>OptiMEM-LPF2000-DNA plasmid mixture: Add 250 &mu;l of the OptiMEM-LPF2000 mixture (step 1.1) into the plasmid DNA transfection mixture (step 1.2). Incubate this mixture for 20 - 30 min at RT. Meanwhile prepare and count 1 x 106 Vero cells per transfection reaction. Vero cells will be transfected in suspension.</li>
	<li>Preparation of Vero cells: Vero cells are cultured in 100 mm dishes. Before starting, bring the 1x PBS, DMEM 10% FBS 1% PS media, and trypsin-EDTA mixture to 37 &deg;C.
	<ol>
		<li>Wash cells, twice, with 5 ml of 1x PBS.</li>
		<li>Trypsinize cells with 1 ml of trypsin-EDTA and wait until cells detach (approximately 5 min). Gentle tapping maybe required to fully detach the cells from the dishes.</li>
		<li>Carefully, resuspend the cells in 10 ml of DMEM 10% FBS 1% PS in a 15 ml centrifuge tube.</li>
		<li>Centrifuge the cells for 5 min at 1,000 rpm in a centrifuge.</li>
		<li>Resuspend the cells in 10 ml of DMEM 10% FBS 1% PS and count the cells using a hemocytometer and adjust concentration to 1 x 106 cells/ml. It was observed that 0.5 - 1 x 106 Vero cells per transfection produced the best results.</li>
	</ol>
	</li>
	<li>Mix LPF2000/DNA and cells: After 20 - 30 min room temperature incubation (step 1.3), add 1 ml of Vero cells (1 x 106) (step 1.4) to the OptiMEM-LPF2000-DNA plasmid mixture and incubate for 5 min at room temperature.</li>
	<li>Seed LPF2000/DNA-cell mix into 6-well-plates: Add the DNA-LPF2000-Vero mixture (step 1.5) into the wells of the 6-well plate. Gently shake the 6-well-plate and let the transfection incubate overnight in the incubator at 37 &deg;C and 5% CO2.</li>
	<li>Change transfection medium: Approximately 16 - 24 hr post-transfection, remove the transfection media, add 2 ml of infection media and incubate transfected cells for an additional 48 hr.</li>
	<li>Cell passage: After 48 hr of incubation in infection media, remove the tissue culture supernatant (TCS) and pass the transfected cells into a 100 mm dish. The TCS rarely contains infectious virus because the transfected cells need additional incubation to generate virus particles. For the cell passage:
	<ol>
		<li>Wash cells, twice, with 2 ml of 1x PBS.</li>
		<li>Trypsinize cells with 0.5 ml of trypsin-EDTA and wait until cells detach.</li>
		<li>Resuspend the cells in 1 ml of DMEM 10% FBS 1% PS in a 1.5 ml microcentrifuge tube.</li>
		<li>Centrifuge the cells for 5 min at 5,000 rpm, 4 &deg;C in a microcentrifuge.</li>
		<li>Carefully resuspend the cells in 1 ml of infection media and transfer the cells to a 100 mm dish. Bring up the total volume in the plate, with infectious media, to 8 ml, a sufficient volume to prevent drying of the cells as well as for concentrating the virus.</li>
	</ol>
	</li>
	<li>Gently shake the 100 mm dish and continue the incubation of Vero cells in the incubator at 37 &deg;C and 5% CO2 for 72 hr.</li>
	<li>Collection of TCS: After 72 hr incubation, collect the TCS in a 15 ml centrifuge tube. Remove cell debris by centrifuging for 5 min at 2,500 rpm in a centrifuge. Infectious arenavirus is found in the TCS, so remove from the cell debris pellet and store the TCS at -80 &deg;C.</li>
</ol>
2. Confirmation of Recombinant Arenavirus Rescue
Detection of a successful rescue of rLCMV and rCandid#1 is achieved via a focus forming unit (FFU) immunofluorescence assay (IFA). For wild-type virus rescue we use antibodies specific for the viral NP. The r3LCMV and r3Candid#1 encode two reporter genes (GFP and Gluc), thus allowing viral detection and titration without the need of antibodies by fluorescence microscopy (GFP) and/or luminescence (Gluc).
<ol>
	<li>Confirm wild-type recombinant arenavirus rescue: The day before titration, wash Vero cells twice with PBS, trypsinize and prepare 96-well plates to reach 80 - 90% confluence next day (4 x 104 cells/well). Gently shake the plates by hand to get a uniformed distribution of the cells. Culture the cells, overnight, in the 37 &deg;C incubator with 5% CO2. Check the cells under the microscope to confirm a monolayer, before proceeding with the infection:
	<ol>
		<li>Serially dilute (10 fold dilutions) the virus-containing TCS recovered from the transfected cells in the 100 mm dish (step 1.10) in OptiMEM.</li>
		<li>Remove media from the seeded Vero cells, wash twice with 50 &mu;l of 1x PBS, and infect cells with 50 &mu;l of the serially diluted virus (step 2.1). Infect cells for 1.5 hr at 37 &deg;C and 5% CO2.</li>
		<li>After infection, remove the virus inoculum, add 100 &mu;l of infection media/well and incubate the cells for 16 - 18 hr. Viral re-infection may occur after 18 hr incubation, which could result in over-estimation of the viral titers.</li>
		<li>At 16 - 18 hr post-infection, remove the TCS from each well and fix the cells with 4% formaldehyde diluted in 1x PBS, for 15 min at RT.</li>
		<li>Next, remove the fixation solution and permeabilize the cells with 0.1% triton X-100 diluted in 1x PBS, for 10 min at room temperature.</li>
		<li>Aspirate the permeabilization solution, then wash cells 3 times with 1x PBS.</li>
		<li>Block the cells with 2.5% albumin bovine serum (BSA) in 1x PBS (blocking solution) for 1 hr at RT. Alternatively, the cells can be blocked overnight at 4 &deg;C and continued with antibody incubation the following day.</li>
		<li>Meanwhile prepare the primary antibody. Dilute the LCMV- and Candid#1-specific primary antibodies in blocking solution, and then centrifuge the antibody/blocking solution for 15 min at 3,500 rpm. The monoclonal anti-LCMV NP antibody clone 1.1.3 (1:30 dilution) 16 and the monoclonal anti-JUNV NP antibody SA02-BG12 from BEI Resources (1:500 dilution) can be used for the detection of rLCMV and rCandid#1, respectively.</li>
		<li>After a 1 hr incubation, aspirate the blocking solution and add 50 &mu;l of the primary antibody to the cells and incubate for 1 hr at 37 &deg;C.</li>
		<li>At this time, dilute the secondary antibody in blocking solution, and then centrifuge the antibody/blocking solution for 15 min at 3,500 rpm. The polyclonal rabbit anti-mouse IgG FITC secondary antibody for the detection of primary monoclonal antibodies was used to obtain images observed in Figure 5.</li>
		<li>After 1 hr incubation, aspirate the primary antibody, wash 3 times with 1x PBS, and add the secondary antibody for 30 min at 37 &deg;C.</li>
		<li>Following 30 min incubation, aspirate the secondary antibody and wash 3 times with 1x PBS. The cells are now ready to be observed using fluorescence microscopy to both determine the success of viral rescue and titration by counting the focus forming units per ml (FFU/ml).</li>
	</ol>
	</li>
	<li>r3LCMV and r3Candid#1 viral rescue: Since these viruses express two reporter genes, their rescues can be monitored by fluorescence microscopy to detect GFP expression or Gluc expression in TCS. Rescue can be also confirmed by expression of Gluc in TCS. To titrate the trisegmented viruses, we follow steps 2.1 to 2.1.4, however cells do not need to be fixed to determine a successful viral rescue because of GFP expression. The titration of the virus will be determined by counting the FFU/ml. To determine successful viral rescue by Gluc expression, Gluc expression from TCS can be measured using a Gluc assay kit (Biolux Gaussia Luciferase Assay kit, New England Biolabs). To that end:
	<ol>
		<li>Pipet 100 &mu;l of TCS into a 96-well white plate (Flat bottom microtiter plates, Fisher Scientific).</li>
		<li>Set up the luminometer (LumiCount, Packard Biosciences).</li>
		<li>Add 50 - 100 &mu;l Gluc Assay solution (as recommended by the manufacturer) to each sample and measure the Gluc reporter gene expression with the luminometer. As a negative control, Gluc expression of the TCS from the wild-type viruses infected cells was measured.</li>
	</ol>
	</li>
</ol>
3. Passage of Tissue Culture Supernatants
Viral rescue depends on transfection efficiencies. Vero cells have been shown to have lower transfection efficiencies than other cell lines 14 . If virus titers in the TCS are low, infect fresh Vero cells at a multiplicity of infection (MOI) of 0.01 (LCMV) or 0.1 (Candid#1) for 72 hr to amplify the virus.

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No conflicts of interest declared.

Generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has become a widely used approach for the investigation of many different facets of arenavirus biology. Here we document a crucial improvement to the current system by performing arenavirus rescues in Vero cells, allowing for the potential generation of FDA-approved vaccine candidates against arenaviruses and vaccine vectors against other infectious diseases.
The experimental procedures involved are in gener...

Arenaviruses are enveloped viruses with a bisegmented, negative-stranded RNA genome 3 that belong to the Arenaviridae family. The arenavirus genome encodes four proteins in an ambisense fashion from two separate viral segments 3. The large (L) segment encodes the RNA-dependent RNA polymerase (L) and the small RING (Really Interesting New Gene) finger protein (Z) that serves as the main driving force of virus budding. The small (S) segment encodes the viral nucleoprotein (NP) and the surface glycoprotein (GP) (Figure 1).
Several members of the Arenaviridae family are responsible for lethal hemorrhagic fever (HF) in humans 3. Of principle concerns are Lassa virus (LASV) and Jun&iacute;n virus (JUNV), which are known to cause high mortality in hospitalized patients 8, 9 . Although these viruses are endemic to West Africa and rural Argentina, respectively, there are increasing concerns of importation of LASV and JUNV to non-endemic areas due to increased travel 10. Additionally, although not normally associated with severe disease in humans, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is considered a neglected pathogen as there have been cases of lethal infection in immunocompromised patients 6, 15 and is responsible for congenital birth defects and spontaneous abortions in pregnant women 2, 13 . Currently there are no US FDA-approved vaccines against arenaviruses and treatment is limited to the nucleoside analog ribavirin, which is only partially effective and often associated with significant side effects.
The introduction of plasmid-based reverse genetics 4 and generation of recombinant arenaviruses 7, 19 have greatly advanced the field of arenavirus research. Currently, rodent cells (such as BHK-21) are used for the generation of recombinant arenaviruses due to the species-specific murine RNA polymerase I (pol-I) promoter directing the initial transcription of the S and L segments. However virus rescue in BHK-21 cells are not an approved method for the generation of recombinant arenaviruses as potential vaccine seed candidates. Here we document the use of the human pol-I promoter for efficient rescue of the prototypic Old World (OW) LCMV and the New World (NW) JUNV Candid#1 strain in Vero cells. Using a similar methodology, we generated recombinant trisegmented LCMV (r3LCMV) and Candid#1 (r3Candid#1) arenaviruses that contain two additional foreign genes encoded in two different S RNA segments 5 . This new system not only follows a highly reproducible and simple protocol but can be immediately implemented in the generation of recombinant arenaviruses as potential vaccine or vaccine vector seeds.

<table border="1">
	<tbody>
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 <td></td>
 <td></td>
 <td></td>
 <td>Material and methods</td>
 </tr>
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 <td></td>
 <td></td>
 <td></td>
 <td>
 Cell lines 
Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &deg;C incubator with 5 % CO2 in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586). 
 
Plasmids 
All plasmids, with the exception of hpol-I L, can be grown at 37 &deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer's recommendations and stored at -20 &deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis. 
 
Viruses 
The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed. 
 
Tissue culture media and solutions 
DMEM 10 %FBS 1 %PS: 445 ml Dulbecco's modified Eagle's medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &deg;C. This media will be used for maintenance of Vero cells. 
 
Infectious Media: 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &deg;C. This media will be used during viral infections. 
 
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
2.5 % BSA: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &deg;C. This is used as a blocking solution for IFA.
</td>
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 </tbody>
 </table>

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.

Successful rescue of a recombinant wild-type arenavirus will be confirmed by the presence of viral antigens using IFA (Figure 5). In the case of recombinant trisegmented viruses, successful viral rescue can be assessed by observing GFP expression by fluorescence microscopy (Figure 6A). Successful rescue will be further confirmed by assessing Gluc expression (Figure 6B). Representative results illustrating the successful rescue of wild-type and trisegmented arenavirus wer...

Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

Generation of Recombinant Arenavirus for Vaccine Development 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 ...

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Research

JoVE Journal

Immunology and Infection

16.8K Views.  University of Rochester School of Medicine and Dentistry. 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 v...

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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.

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

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.