Name: generation of recombinant arenavirus for vaccine development in fda-approved vero cells
Uploaded: 2013-08-01T13:00:00
Description: Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

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 pCAGG...

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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.

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 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.
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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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