Recombinant virus products

Over the last two decades, the use of eukaryotic cells for expression of recombinant proteins has become the preferred choice for many applications. This is primarily the case when posttranslational modifications and correct disulfide-bond formation are necessary for protein folding and activity. Among the eukaryotic expression systems, the baculovirus-infected insect cell platform has gained particular attention, resulting in the development and implementation of multiple strategies for protein expression. Here, we present baculovirus-infected insect cells as an efficient expression system for eukaryotic proteins. We demonstrate a simplified and a shortened procedure for recombinant virus production that is sufficient for large-scale production of proteins in insect cells. 

An important safety issue of viral vectors is whether or not the recombinant viruses are able to replicate in the infected cells. Replication of viral vectors is unwanted in most gene-therapy approaches. Therefore, replication-defective vectors have been designed, which are able to perform only one initial infectious cycle within the target cell. In addition, replication-competent vectors have been designed, which are able to productively infect the target cell and to spread in the target tissue. 

The first hurdle encountered during the development of alfalfa as a recombinant protein production system was the relative inefficiency of the available expression cassettes. A study in which a tomato proteinase inhibitor I transgene was expressed in tobacco and alfalfa under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed that 3-4 times more protein accumulated in tobacco leaves compared to alfalfa leaves [5]. Despite the low efficiency of the CaMV 35S promoter in alfalfa, bio-pharmaceutical production using this system has been reported in the scientific literature. Such reports include expression of the foot and mouth disease virus antigen [6], an enzyme to improve phosphorus utilization [7] and the anti-human IgG C5-1 [8]. In this last work, the C5-1 antibody accumulated to 1% total soluble protein [8]. 

The lack of human pathogenicity of plant viruses rules out the risks of human infection by exposure in the field or in food products to a plant virus. However, biological containment of the virus expression vector remains a primary safety concern as it can be considered a risk to the environment. This includes the spread of recombinant viruses to weeds... 

PaUvizumab, humanized monoclonal IgGi against F protein of respiratory syncytial virus (RSV), recombinant DNA product... 

Other protocols involve cell lines with integrated rep/cap cassettes (Clark et al., 1995 Gao et al., 1998 Liu et al., 1999 Chadeuf et al., 2000 Mathews et al., 2002 Qiao et al., 2002a,b) infected with adenovirus or, alternatively, a recombinant herpesvirus system has been used to provide both helper virus function and rep/cap (Conway et al., 1997, 1999). In a switch away from using mammalian cell and helper virus production systems, rAAV vectors have been made in insect cells where the AAV genes are expressed under the control of insect promoters and the traditional helper virus gene products are not required (Urabe et al., 2002). Stable producer cell... 

Chadeuf, G. et al. (2000). Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome. J. Gene Med. 2, 260-268. 

Conway, J. E. et al. (1999). High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex vims type I vector expressing AAV-2 Rep and Cap. Gene Ther. 6, 986-993. 

Salvetti, A. et al. (1998). Factors influencing recombinant adeno-associated virus production. Hum. Gene Ther. 9, 695-706. 

Transient expression Production of recombinant viruses Transient and stable expression Production of recombinant viruses Transient and stable expression Vaccine production Transient and stable expression Stable expression... 

Blasey HD, Lundstrom K, Tate S, Bernard AR (1997), Recombinant protein production using the Semliki Forest Virus expression system, Cytotechnology 24 65-72. 

Wu J, Ruan Q, Peter Lam FfY (1998), Evaluation of spent medium recycle and nutrient feeding strategies for recombinant protein production in the insect cell-baculo-virus process, J. Biotechnol. 66 109-116. 

Wang P, Granados RR, Shuler ML (1992), Studies on serum-free culture of insect cells for virus propagation and recombinant protein production, J. Invertebr. Pathol. 59 46-53. 

Simplifications caused by recent advances in technology of producing recombinant baculoviruses combined with the ability to infect simultaneously with several different recombinant viruses allows for the production and analysis of multisubunit proteins. 

The experiments in this section describe the procedures to obtain a high-titer recombinant virus stock either through recombination of RT-PCR product with RT-deleted proviral DNA or PRO-PCR product with PRO deleted proviral DNA. The susceptibility testing is outlined in Subheading 4. The following steps are described (Fig. 1) ... 

Although this is the most crucial step in the recombinant virus assay, the procedure is easy and simple. In general, 10 pg of proviral plasmid and 2 pg of PCR are used for recombination. In most cases, less PCR product will also perform well, but it may take somewhat longer before the virus breaks through. In difficult cases, more PCR product can be used, but one should be aware that, in these cases, the recombinant virus might not be representative for the virus population in the patient. The virus obtained can be directly used for stock titration as described in the protocol for the MT-4/MTT assay in Subheading 4.I.3.I. 

Rabinowitz JE, Xiao W, Samulski RJ. 1999. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus. Virology 265 274-85... 

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